Download by Matthew Patrick Mc Cusker B.A.(Mod) in Microbiology ,1995

Molecular studies of the LysR-type regulator, mexT, and its regulation of the
Pseudomonas aeruginosa outer membrane porin, OprD.
by
Matthew Patrick Mc Cusker
B.A.(Mod) in Microbiology ,1995.
Trinity College Dublin, Ireland.
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ABSTRACT
Pseudomonas
a e r u g i n o s a OprD is a specific porin which facilitates the uptake of basic
amino acids and imipenem, a carbepenem antibiotic with high potency against
P.aeruginosa.
Resistance to imipenem occurs frequently during antibiotic therapy of
patients with P. a e r u g i n o s a infections. This study was initiated to investigate the
regulatory mechanisms in P . a e r u g i n o s a for the regulation of o p r D . A putative LysR-type
regulator, designated mexT,
was cloned on a 1.2 kb PCR fragment. D N A sequencing
predicted a 304 amino acid mature MexT protein. It showed strong homology to other
LysR-type regulators. When overexpressed it induced expression of the
mexEF-oprN
efflux operon while repressing transcription of o p r D .
A second line of investigation was to try and identify conditions where this mexT
gene
might play a role in the regulation of its target genes in P . a e r u g i n o s a . Salicylate, an
aromatic weak acid known to reduce porin expression and induce low levels of multiple
antibiotic resistance in other bacteria, was found to reduce expression of oprD
via
transcription. No change in expression of any of the efflux operons was noted. Sodium
benzoate, another aromatic weak acid, was shown to reduce the levels of OprD in the
outermembranes of P . a e r u g i n o s a but the mechanism of reduction was found not to via
transcription. Neither iron or zinc concentrations were found effect expression of o p r D or
any of the efflux operons. Finally, expression of these genes during growth phase was
examined. No changes were noticed.
ii
T A B L E OF CONTENTS
ABSTRACT
ii
T A B L E OF CONTENTS
iii
LIST OF FIGURES
v
LIST OF T A B L E S
vi
LIST OF A B B R E V I A T I O N S
vii
ACKNOWLEDGEMENT
1
INTRODUCTION
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
2
'
1
Pseudomonas aeruginosa
Pseudomonas aeruginosa Outer Membrane
Porins and their role in P. aeruginosa
Antibiotic uptake across the outer membrane
Imipenem
Multiple-antibiotic Resistance in Pseudomonas aeruginosa
OprD and its regulation
Aims of this Study
METHODS A N D MATERIALS
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
3
ix
1
4
8
9
15
18
11
20
Strains, plasmids and growth conditions
Reagents
Genetic manipulations
D N A sequencing
Transfer of D N A into P.aeruginosa
Cloning strategy for the mexT gene
Cloning and analysis of the mexT gene
Overexpression of the mexT gene in P. aeruginosa
Membrane isolation and Protein analysis
Determination of Minimal Inhibitory Concentration (MIC) Assay
X y l E assays
:
Growth phase experiments
RESULTS
20
20
21
21
23
23
24
24
25
27
29
29
30
3.1.1
Molecular cloning of the mexT Gene
3.1.2
Nucleotide sequence of the mexT gene
3.1.3
MexT shows similarity to NahR and other LysR-type transcriptional
regulators
3.1.4
Overexpression of the me^rgene in P.aeruginosa
3.1.5
MexT reduces transcription of oprD
in
30
31
33
35
39
2
3.1.6
Summary
3.1.7
Introduction
3.1.8
Salicylate and sodium benzoate; effects on antibiotic susceptibilities and
outer membrane protein production in P. aeruginosa
3.1.9
Salicylate reduces transcription of the oprD gene in P.aeruginosa
3.1.10 Effect of iron and zinc concentrations on the antibiotic susceptibilities of
P. aeruginosa
3.1.11 Effect of growth phase on expression of the mex efflux operons of
P.aeruginosa
3.1.12 Summary
4
DISCUSSION
40
41
42
47
48
52
54
55
REFERENCES
67
iv
LIST OF FIGURES
Fig 1:
Schematic diagram of the cell envelope of Gram negative bacteria
Fig 2:
Structures of some antibiotics and amino acids that penetrate through the
OprD channel
Fig 3:
:
5
7
Schematic representation of the organization of the mex efflux operons in
14
P. aeruginosa
Fig 4:
Diagram of plasmid p M C l utilized for sequencing of the mexT gene
Fig 5:
Diagram of plasmid pMC2 utilized for the overexpression of the mexT
gene
26
28
Fig 6:
Physical map of the mexT gene
30
Fig 7:
Nucleotide and deduced amino acid sequence of the mexTgene
32
Fig 8:
Similarity of MexT to NahR
34
Fig 9:
SDS-PAGE demonstrating repression of OprD by overexpression of the
37
mexT gene
Fig 10: Western immunoblots of P.aeruginosa strains
38
Fig 11: Effect of salicylate and sodium benzoate on the outer membrane proteins
oi P. aeruginosa P A O l
Fig 12: Western immunoblots of outer membranes of P.aeruginosa grown in the
presence of 32 m M salicylate and 16 m M sodium benzoate
Fig 13: Outer membrane profiles of P.aeruginosa P A O l grown in MuellerHinton with different concentrations of zinc
Fig 14: Outer membrane profiles of P.aeruginosa PAOlduring growth phases
v
45
46
51
53
LIST OF TABLES
Table I: Bacterial strains
21
Table II: Plasmids used in study
22
Table III: Oligonucleotide primers used in study
27
Table IV:MICs for P.aeruginosa strains
36
Table V: Effect of MexT on an oprD::xylE chromosomal transcriptional fusion in
40
P.aeruginosa
Table VI: Antibiotic susceptibilities of P.aeruginosa P A O l in Mueller-Hinton in
the presence of salicylate and other structurally related compounds
44
Table VII: Effect of salicylate and sodium benzoate on an oprDr.xylE
chromosomal transcriptional fusion in P.aeruginosa
48
Table VIII: Effect of iron and zinc concentrations on the antibiotic susceptibilities
of P. aeruginosa
50
vi
LIST O F ABBREVIATIONS
MIC
minimum inhibitory concentration
OD
optical density
EDTA
ethylenediamine tetra acetate
MH
Mueller Hinton
LBNS
Luria broth, normal salt
LPS
lipopolysaccharide
Cm
chloramphenicol
Tet
tetracycline
Gent
gentamycin
Amp
ampicillin
Carb
carbenicillin
Im
imipenem
Cefp
cefpirome
Nor
norfloxacin
SDS
sodium dodecyl sulfate
SDS-PAGE
SDS polyacrylamide gel electrophoresis
mAb
monoclonal antibody
bp
base pair
kb
kilobase
kDa
kilodalton
PCR
polymerase chain reaction
vii
ORF
open reading frame
LTTR
LysR-type transcriptional regulator
HTH
helix-turn-helix
MCS
multiple cloning site
Mr
molecular mass
His
histidine
Arg
arginine
Ala
alanine
Glu
glutamate
viii
ACKNOWLEDGEMENT
I am grateful to my supervisor, Dr. Robert E. W. Hancock, for his guidance and
support during the time in his lab. A special thanks to Dr. Martina Ochs for her advice
and patience over the last two years. I also thank Susan Farmer and Manjeet Bains as
well as the rest of Hancock lab for their technical help and encouragement during this
project. I also acknowledge the help and support of my supervising committee, Drs.
Fernandez and Mohn. Last but not least a personal word of thanks to Dr. Tony Warren
for his emotional support during the first and very difficult year in the department.
The financial support of the Medical Research Council of Canada is gratefully
acknowledged.
ix
1
1.1
INTRODUCTION
Pseudomonas aeruginosa
Pseudomonas aeruginosa is an opportunistic Gram negative pathogen which is a major
cause of nosocomial (hospital-acquired) infections (Schimpff et al., 1970). These
infections usually occur in immunocompromised hosts such as burn victims and cancer
patients, or in people with cystic fibrosis. P.aeruginosa produces a variety of toxins,
enzymes and pigments, some of which undoubtedly contribute to the pathogenic
properties of the bacteria (Liu, 1974). P.aeruginosa is becoming a major clinical problem
and is one of the most difficult infections to treat due to its high intrinsic resistance to the
many of the most commonly used antibiotics including first and second generation
penicillins and cephalosporins, tetracycline, chloramphenicol, quinolones and their
derivatives (Bryan, 1979). This phenomenon has been partly attributed to the low outer
membrane permeability of P. aeruginosa. It has been shown that the permeability of-the
outer membrane of P. aeruginosa to (3-lactam antibiotics is from twelve to one hundred
fold lower than the permeability of the E.coli membrane to the same compounds (Nicas
and Hancock, 1983). This low outer membrane permeability, which springs from the
properties of the pore-forming proteins, porins, in the outer membrane
P.aeruginosa,
acts only to slow down the movement of the antibiotics into the cell (Nikaido and
Hancock, 1986). It does not itself cause resistance.
1
1.2
Pseudomonas aeruginosa Outer Membrane
The cell envelope of P.aeruginosa consists of two membranes separated by a layer of
peptidoglycan and a cellular compartment called the periplasm (Fig. 1). The inner or
cytoplasmic membrane is a phospholipid bilayer containing a large number of
polypeptides. The peptidoglycan is located under the outer membrane and is primarily
responsible for the mechanical strength of the cell wall (protecting the cell e.g. against
osmotic shock) and for maintaining cell shape (Oliver, 1987). The periplasm lies between
the inner and outer membranes. Proteins residing in the periplasmic space fulfill
important functions in the detection and processing of essential nutrients and their
transport into the cell (Oliver, 1987).
The outer membrane consists of an asymmetric bilayer (Fig. 1), in which the inner
monolayer is composed of phospholipid, while the outer monolayer
contains
lipopolysaccharide (LPS) (Nikaido and Nakae, 1979). LPS is a complex molecule
consisting of three covalently linked regions; lipid A , a core oligosaccharide region and
an O-specific chain (Rietschel et al., 1982). The lipid A region is conserved, consisting of
substituted B-linked glucosamine disaccharide, usually (1—>6)-linked. The disaccharide is
fully substituted with six or seven saturated or hydroxyl fatty acid residues, phosphate,
and the core oligosaccharide (Karunaratne et al., 1992). The core oligosaccharide is a
complex oligosaccharide usually linked to lipid A via 2-keto-3-deoxyoctonate, K D O . In
P.aeruginosa the core contains glucose, heptose, K D O , rhamnose, galactosamine and
alanine (Kropinski et al., 1979). The core may be capped with repeating tri- to
pentasaccharide units termed the O-antigen. This is the immunodominant part of the LPS
2
molecule in the intact bacterial cell. It has been shown that the O-antigen of P.aeruginosa
often contains such sugars as glucose, rhamnose, glucosamine, fucosamine and
quinavosamine (Rropinski et al., 1985).
The asymmetric distribution and chemical characteristics of LPS give the outer
membrane many of its unique barrier properties. The presence of a large amount of
phosphate in the core region of P.aeruginosa LPS results in the strong surface negative
charge (Sherbert and Lakshmi, 1973). The non-covalent cross-bridging of adjacent LPS
molecules with divalent cations (Mg
2+
or Ca ) (Rottem and Leive, 1977), and the
2+
hydrophobic interactions between the outer membrane proteins and Lipid A (Nikaido and
Vaara, 1985), also contribute to the stability of the LPS in the outer membrane. The
combination of these properties makes the P. aeruginosa highly resistant to hydrophobic
antibiotics, bile salts, detergents, proteases, lipases and lysozyme (Nikaido and Vaara,
1985).
The P.aeruginosa outer membrane also contains a number of "major" proteins (Fig. 1).
These include the murein lipoproteins, the major outer membrane protein, OprF, and
porins. The two lipoproteins of P.aeruginosa, OprL and OprI are inserted in the inner
phospholipid monolayer and are non-covalently associated with peptidoglycan (Hancock
et al., 1981). They have a structural role in stabilizing the architecture of the outer
membrane-peptidoglycan complex. OprF is also strongly but not covalently associated
with peptidoglycan and plays a role in outer membrane stability and cell shape
determination (Woodruff and Hancock, 1989). Porins are a group of proteins forming
trans-outer-membrane, water filled channels. In general, porins have monomer molecular
3
weights in the range of 28 kDa to 48 kDa, are present in the membrane as oligomers,
usually trimers and have a high content of R-sheet structure. In P.aeruginosa, OprB,
OprC, OprD, OprE, OprF, OprO and OprP have been identified as porins (for a review,
see Hancock et al., 1990).
1.3
Porins and their role in P. aeruginosa
Porins are generally divided into two classes: non-specific or general porins and specific
porins (Nikaido and Vaara, 1985). General porins form water-filled channels that permit
the passive diffusion of hydrophilic molecules below a certain molecular size, and thus
are responsible for the non-specific exclusion limit of the outer membrane. Specific
porins also produce water-filled channels which contain stereospecific substrate-binding
sites (Hancock, 1987). The diffusion of the specific substrate is accelerated when the
solute concentration is low, but is slowed down when the concentration is high.
General porins take up molecules based on size, electric charge and hydrophilicity. OprF,
the major outer membrane porin of P.aeruginosa is a non-specific porin (Nikaido and
Vaara, 1985). Its pore-forming properties have been shown in a model membrane system
and in intact cells (Benz and Hancock, 1981). The pore diameter was estimated to be
20 A, about twice the width of the E.coli porin channels, and can allow passage of
saccharides with molecular weights of approximately 3,000 (Nikaido and Hancock,
1986). However, only 400 of the estimated 200,000 OprF molecules per cell are proposed
4
Fig 1:
Schematic diagram of the cell envelope of Gram negative bacteria.
The cell envelope of Gram negative bacteria consists of two membranes, the cytoplasmic
and outer membrane which are separated by the periplasm. The outer membrane is an
asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids
in the inner leaflet. The periplasm separates the two membranes and contains the meshlike peptidoglycan layer. The inner membrane (cytoplasmic membrane) is a phospholipid
bilayer. Proteins shown are: a three-component resistance-nodulation-division efflux
system consisting of the integral outer membrane pore proteins, the periplasmic
membrane fusion proteins (MF) and the integral cytoplasmic R N D pump protein; an
outer membrane porin protein forming a diffusion channel and a peripheral periplasmic
penicillin binding protein (PBP) which functions in the synthesis of the peptidoglycan
layer.
5
to form such large channels. The rest appear to form small channels that are thought to be
impermeable to antibiotics (Woodruff et al., 1986). Two other porins, namely OprC and
OprE, are general porins with a small channel size and are anaerobically induced. It is
generally accepted that the low overall permeability of the channels of the general porins,
explains the low outer membrane permeability of P.aeruginosa compared to E.coli
(Angus et al., 1982; Nicas and Hancock, 1983). This in turn, was proposed to contribute
to the high intrinsic resistance of P.aeruginosa to antibiotics (Nikaido and Hancock,
1986).
This low outer membrane permeability may be an advantage for excluding antibiotics but
it means that the bacterium also excludes certain essential nutrients. To overcome this and
allow effective uptake of essential nutrients available at low concentrations in the
medium, several specific porins are present in the P.aeruginosa outer membrane. OprB
is induced in the presence of glucose and is involved in the uptake of glucose and xylose
(Trias et al, 1987). OprP is induced under phosphate limiting conditions (< 0.15 mM)
(Hancock et al., 1982). It is part of the phosphate specific transport (Pst) system of
P.aeruginosa (Poole and Hancock, 1986). OprO, another porin with high homology to
OprP, forms pyrophosphate-specific channels (Hancock et al., 1992). OprD was
discovered due to its role in the facilitated uptake of imipenem, a carbepenem which
shows excellent activity against P.aeruginosa (Trias and Nikaido, 1990a). The natural
substrate for OprD is not imipenem, but its structural analogues, basic amino acids and
small peptides containing those amino acids (Fig. 2) (Trias and Nikaido, 1990b).
6
Imipenem
COOH
COOH
NH
CH — C H - C H - C H - N H
2
2
2
2
2
2
Lysine
COOH
Arginine
NH .
.CH —CH
2
\
2
2
—NH—C-NH
^
J
2
II
NH
COOH
CH — N H \
II
Histidine
N
H
-
/ C H
2
. C H , - C H - N H ^
COOH
Fig 2:
Structures of some antibiotics and amino acids that penetrate through the
OprD channel.
7
1.4
Antibiotic uptake across the outer membrane
There exist three general pathways for antibiotic uptake in Gram negative bacteria: the
porin pathway, the hydrophobic pathway and the self promoted uptake pathway
(Hancock, 1997).
Small hydrophilic molecules enter the periplasm by diffusion through the porin channels
that are present in the outer membrane. Uptake of small hydrophilic antibiotics usually
occurs through the general porins. Antibiotic uptake through the specific porins is not
well documented, but it has been shown that the basic amino acid-specific OprD porin of
P.aeruginosa mediates the uptake of the B-lactam antibiotic imipenem (Sumita et al.,
1993; Yoneyama et al, 1993).
Lipophilic and amphipathic antibiotics enter the cell through the hydrophobic pathway by
passive permeation through the outer membrane bilayer (Hancock & Bell, 1988). The
mechanism of uptake of hydrophobic and amphipathic antibiotics is not well understood
but probably involves the partitioning into the hydrocarbon core of the outer membrane
and simple diffusion. It has become clear that the intrinsic and high level of resistance of
bacteria to many hydrophobic antimicrobials is mediated by active efflux mechanisms
rather than changes in outer-membrane permeability.
Cationic antimicrobials such as the aminoglycosides and cationic antimicrobial peptides
enter the cells by competing for divalent cation binding sites on the surface LPS in a
process that has been termed self promoted uptake (Hancock, 1997). It is believed that
the binding of these compounds to the outer membrane displaces the native divalent
8
cations and, due to their bulkiness, they destabilize (permeabilize) the outer membrane
and thereby facilitate further uptake of themselves and other molecules.
1.5
Imipenem
Imipenem, or N-formimidoyl thienamycin, is derived from thienamycin, a natural product
of the soil organism Streptomyces
cattleya
(Kahan et al., 1979). Thienamycin has unique
structural features that distinguish it from all natural and synthetic (3-lactam antibiotics
previously described (Albers-Schonberg et al., 1978). It is the first representative of a
new class of antibiotics, the carbepenems (Fig. 2). Imipenem is of particular interest
because of it's high potency, broad spectrum and lack of microbial cross resistance to
other (3-lactam antibiotics (Rohnson et al., 1986). Furthermore, imipenem had greater
bactericidal activity
in vivo
and greater protective efficacy in experimental infections
against pathogenic species than other antibiotics (Kropp et al., 1980). The high potency
and unusually broad spectrum of antimicrobial activity of imipenem is due to three
aspects. Firstly, it is able to relatively easily penetrate the outer membranes of many
Gram-negative bacteria. Imipenem has a compact structure with a molecular weight of
229 and is zwitterionic, and both these features facilitate its diffusion through the outer
membranes of Gram-negative bacteria by specific porin channels (Lipman and Nea,
1988). In P.aeruginosa, imipenem can overcome the poor outer membrane permeability
by penetrating through the specific porin OprD (Trias and Nikaido, 1990a). Secondly,
imipenem has high affinity for its target penicillin binding proteins (PBPs), namely PBP1 and PBP-2 (Hashizame et al., 1984). Thirdly, imipemen is a poor substrate for a range
9
of P-lactamases. Despite being a strong inducer of chromosomal cephalosporinases, a
class of P-lactamases induced in the presence of selected P-lactams and capable of
hydrolyzing many P-lactams, imipenem is only weakly hydrolyzed by these P-lactamases
(Livermore and Yang, 1987). These properties may account for the general lack of cross
resistance of microbes to imipenem with other P-lactams.
Resistance to imipenem can arise through three different mechanisms. Firstly, the
constituants of the outer membrane may be modified to prevent the uptake of imipenem.
Imipenem resistant mutants of P. aeruginosa have been isolated both in the laboratory and
from clinical sources. Their resistance usually stems from the reduced or lack of
expression of the outer membrane porin, OprD (Lynch et a l , 1987). Genetic analysis of
these mutants has shown that the loss of OprD expression is due to rearrangements in the
oprD coding region or the upstream promoter region (Yoneyana and Nakae, 1993).
Secondly, imipenems target, the PBPs, may be altered to reduce its effect on the cell wall
(Helinger and Brewer, 1991). Thirdly, P-lactamases may be expressed which are capable
of hydrolyzing the P-lactam ring of imipenem thereby inactivating it. Pseudomonas
maltophilia, which is resistant to imipenem, produces an imipenem-hydrolyzing Plactamase (Saino et al., 1982). This enzyme is part of a unique class of Zn -containing p2+
lactamases analogous to the metaloproteases. Interestingly, full resistance to imipenem in
P.aeruginosa seems to require both reduced permeability due to the loss of OprD and
slow hydrolysis due to production of the chromosomally encoded
(Livermore, 1992).
10
P-lactamase
1.6
Multiple-antibiotic Resistance in Pseudomonas aeruginosa
Infections of low-virulence bacteria in immunocompromised patients are of major
concern. Bacteria such as Staphylococcus aureus, Streptococcus spp., Enterococcus, and
Pseudomonas aeruginosa, found commonly in hospitals, often have low or no virulence
in healthy individuals but can cause potentially life threatening infections in people where
the immune system is not working properly. These types of infections can usually be
treated successfully with antibiotics. A major drawback of the use of antibiotics is the
selection of strains that exhibit antibiotic resistance. Antibiotic resistance, as stated
previously, can arise by different mechanisms. The most worrying of these mechanisms is
active efflux, as this mechanism gives resistance to a wide range of structurally unrelated
antibiotics (for a comprehensive review see Paulsen et al., 1996).
Multiple-antibiotic resistance in P.aeruginosa has been shown to correlate with resistance
to the quinolone class of antimicrobial agents (Hirai and Mitsuhashi, 1987). The
fluoroquinolones in particular seem to select strains demonstrating cross-resistance, not
only to other quinolones, but to structurally unrelated antibiotics (Nikaido, 1996). Three
of these mutants, nalB, nfxB, and nfxC, which display a multiple-drug resistance
phenotype (MDR) phenotype and overexpress outer membrane proteins in the range of
50kDa, have been described in P.aeruginosa (Rella and Hass, 1982; Hirai et al., 1987;
Fukuda etal., 1990).
NalB-type
mutants
chloramphenicol,
show increased
tetracycline,
resistance
|3-lactam
to meropenem, fluoroquinolones,
antibiotics
(including
penicillins
and
cephalosporins), as well as 2,2'-dipyridyl (Rella and Hass, 1982). These mutants also
11
overexpress an outer membrane protein with an apparent molecular weight of 50kDa.
Poole et al., cloned an operon that overcame a growth deficiency in a mutant that lacked
the ferri-pyoverdin receptor in the presence of the non-metabolizable iron-chelator, 2,2'dipyridyl. The operon contained three open reading frames, later designated mexA, mexB
and oprM (Fig. 3) (Poole et al., 1993). These genes coded for proteins of 41, 112 and
50kDa respectively. MexB had amino acid similarity to other members of the RestrictionNodulation-Division (RND) family of cytoplasmic pump proteins. OprM has been shown
to be in the outer membrane and to be the same protein seen in nalB-type mutants (Gotoh
et al., 1994). MexA was predicted to be anchored in the cytoplasmic membrane and to
form a bridge between, MexB in the cytoplasmic membrane, and OprM in the outer
membrane. It has been proposed that these proteins, as for their homologues in E.coli,
form a continuous channel from the cytoplasm to the external medium, and use the proton
motive force to actively efflux their substrates (Fig. 1). A small ORF designated mexR
was found upstream of the mexA/B-oprM operon and was shown to negatively regulate
the efflux operon (Fig. 3) (Poole et al., 1996). As yet, the nalB gene has not been cloned.
Mutants of the nfxB-type M D R phenotype show increased resistance to fluoroquinolones,
chloramphenicol, tetracycline, erythromycin and cefpirome, and increased susceptibility
to the classic P-lactams (Hirai et al., 1987). Outer membrane analysis of these mutants
demonstrated the overexpression of a 54kDa protein designated OprJ. Poole et al. cloned
the gene encoding OprJ and sequence analysis of the upstream region revealed the
presence of two additional coding regions (Poole et al., 1996). These genes were
designated mexC, which codes for a 112kDa membrane fusion protein, and mexD, which
12
encodes a 40kDa cytoplasmic membrane protein. These proteins showed significant
homology to the MexA and MexB proteins respectively. The nfxB gene was located
upstream of the mexCD-oprJ operon and coded for a putative D N A binding protein (Fig.
3). It was subsequently shown to negatively regulate itself and the efflux operon. The
nfxB mutants produce a defective repressor that results in derepression of the mexC/DoprJ operon and, therefore, overproduction of the MexC, MexD, and OprJ proteins
(Poole etal., 1996).
NfxC-type mutants show increased resistance to chloramphenicol, quinolones and the
carbepenem, imipenem (Fukuda et al., 1990). They show increased susceptibility towards
most p-lactam antibiotics (except imipemen) and aminoglycoside antibiotics (Fukuda et
al., 1990). Unlike the other types of mutations there are two changes in the outer
membrane profile. There is a reduction in expression of the imipenem specific porin,
OprD, and overexpression of a 51kDa protein designated OprN (Masuda et al., 1995).
Kohler et al. recently cloned the operon responsible for the nfxC-type phenotype (Kohler
et al., 1997). It consisted of three ORFs which coded for a cytoplasmic membrane protein
MexF (115kDa), a membrane fusion protein MexE (45kDa), and an outer membrane
protein OprN (51kDa). MexE MexF and OprN showed high similarity the MexA, MexB
and OprM, respectively. Upstream of this operon was an ORF that encoded a putative
LysR-type transcriptional activator (Fig. 3). Overexpression of this fragment resulted in
overexpression of OprN and reduction of a protein with an apparent molecular mass of
45kDa. It has been suggested that this efflux machinery is involved in the excretion of
intermediates for the biosynthesis of pyocyanain (Kohler et al., 1997).
13
mexA-mexB-oprM
nalB
mexR
mexA
• •'•
mexB
oprM
• • • • •
mexC-mexD-oprJ
nfxB
23K
21K
mexC
mexD
oprJ
AA
mexE-mexF-oprN
oprD
exT
m
mexE
mexF
oprN
Fig 3:
Schematic representation of the organization of the mex efflux operons in
P.aeruginosa.
Distances between genes and their sizes do not correspond to their physical sizes.
Arrows above operons correspond to the direction of transcription of the genes in the
operon. Arrows below correspond to regulatory proteins produced and their putative
binding positions.
14
1.7
OprD and its regulation
As stated previously, imipenem is highly active against P.aeruginosa. However, during
antibiotic therapy, imipenem resistant isolates arise at a significant rate (Quinn et al.,
1986), and usually the resistant strains are not cross resistant to other antibiotics, don't
show any changes in their PBPs and cannot hydrolyze or modify imipenem. Instead, they
lack the outer membrane protein, OprD (Quinn et al., 1986; Lynch et al., 1987).
OprD has an apparent molecular weight of 46,000 in an SDS-polyacrylamide gel
electrophoretogram when the protein is solubilized at 95°C (Yoshihara et al., 1991). This
corresponds to its monomer form. OprD exists as trimers in the native outer membrane
(Yoshihara et al., 1991). Native OprD shows a p-strand-rich conformation which is
typical of many other porins (Huang et al., 1995). OprD has been proposed to function as
a general porin, allowing the diffusion of monosaccharides, disacchrides and amino acids
at a significant rate . Of more importance, OprD has been shown to act as a specific porin
for the uptake of imipenem, basic amino acids and their structural analogues (Fig. 2)
(Trias and Nikaido, 1990).
While a lot is known about the structure and function of OprD, little is known about the
regulation of its expression . Huang et al. described the isolation of a Tn501 mutant of
P.aeruginosa that was resistant to imipenem and lacking OprD (Huang et al., 1992). This
mutant could be complemented both with relation to imipenem susceptibility and OprD
expression with a 6.0-kb fragment of D N A from the region of the chromosome of the
wild-type strain surrounding the site of Tn501 insertion. When sequenced this region
contained three open reading frames (ORF), one of which was found to be interrupted by
15
the Tn501 transposon. The latter ORF was designated opdE (for putative regulator of
oprD expression). It coded for a putative hydrophobic protein of molecular mass (Mr)
41,592. They hypothesized that the opdE gene, or one or two of the other ORFs, encodes
a protein that influences the expression of OprD. Recent work done in this lab has cast
doubt on the presumption that the insertion in the opdE gene is responsible for the
reduction in OprD expression (Ochs, unpublished data). Work to elucidate this is in
progress.
More recently, Park et al. described the cloning and characterisation of a gene, argR,
involved in the regulation of arginine biosynthesis and catabolism (Park et al., 1997).
This gene was found to be the sixth and terminal gene of an operon for the transport of
arginine. The argR gene was inactivated by gene replacement, using a gentamicin
cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes
encoded by the car and argF operons. Both the car operon, which encodes
carbamoyltransferase (CPS), and the argF operon, which encodes the anabolic ornithine
carbamoyltransferase (aOCT), are negatively regulated by arginine. The argR gene
product, ArgR was also shown to regulate the aru operon. This operon is involved in the
arginine succinyltransferase (AST) pathway, which converts arginine to glutamate, and is
considered the major route for catabolism in P.aeruginosa under aerobic conditions (Hass
et al., 1990). Enzymes of the A S T pathway catalyze the conversion of arginine to
glutamate and are strongly induced by exogenous arginine. Arginine also induces the
catabolic NAD-dependent glutamate dehydrogenase (NAD-GDH), which is required for
16
the utilization of glutamate and regeneration of succinyl coenzyme A (succinyl-CoA),
through the citric acid cycle (Hass et al., 1990).
ArgR was purified to homogeneity. Using DNase footprinting, it was shown that ArgR
bound directly to the promoter regions of the car, argF, and aru operons (Park et al.,
1997). Alignment of the ArgR binding sites revealed that the ArgR binding sites
contained a consensus sequence. The consensus sequence is T G T C G C N A A N . They
8
5
also looked at the promoter sequence of oprD which, as previously stated, is involved in
the uptake of basic amino acids. A n ArgR consensus sequence was found upstream of the
oprD coding region. DNase footprinting experiments showed direct binding of ArgR to
the oprD promoter region (Park et al., unpublished data). This is the first report of a
regulatory protein directly binding to the oprD promoter region and thereby affecting
OprD expression.
Work done in this lab looked at the effect of different carbon and nitrogen sources on
OprD expression. A l l amino acids tested (His, Arg, Ala, Glu), strongly induced OprD
when used as sole carbon sources (Ochs, unpublished data). This induction could be
abolished by succinate. Non-inducing carbon sources included succinate, glucose,
gluconate and glycerol. The effect of different nitrogen sources was also looked at.
Histidine and arginine slightly induced OprD expression, while alanine and glutamate
resulted in strong induction when used as sole nitrogen sources (Ochs, unpublished data).
Addition of ammonium sulphate abolished induction. Induction of OprD by using
arginine as the sole nitrogen source was abolished in an argR mutant background.
Interestingly in an argR mutant background, glutamate still induced OprD expression
17
(Ochs, unpublished data). This suggests that there is a separate mechanism for the
regulation of OprD by glutamate.
Recent analysis of the promoter region of oprD has found potential sigma70 and sigma54
consensus sequences. Investigations to see i f indeed oprD is regulated by these sigma
factors is underway in the lab. Taken together, the above data suggest that oprD is a
highly regulated gene with a complex network of regulatory signals and factors involved.
This is not unexpected due to the observation that oprD is poorly expressed from its own
promoter in E.coli (Huang et al., 1992). This observation is consistent with a gene that
has a weak promoter and that is highly regulated.
1.8
Aims of this Study
OprD, a specific porin for imipenem and basic amino acids has been studied extensively
as a model for the mechanisms of antibiotic and nutrient uptake through the specific
porins. Despite all this research, little is known about the regulation of expression of the
oprD gene itself. Recent work and other work performed during the investigations
described here, suggest that oprD expression is highly regulated by multiple factors. One
of the regulatory factors described was a putative transcriptional regulator belonging to
the LysR-type family of transcriptional regulators. This regulatory ORF was found
upstream of the mexE-mexF-oprN and was shown to positively regulate the operon and
potentially negatively regulate oprD.
The aims of this study were, 1) to clone, sequence and analyze this putative
transcriptional regulator; 2) show it directly regulates transcription of oprD; 3) attempt to
18
identify possible cofactors and 4) try to find conditions under which this gene regulates
oprD expression.
19
2
2.1
METHODS AND MATERIALS
Strains, plasmids and growth conditions
Strains used in these studies are listed in Table I and all plasmids used are listed in Table
II. E. coli strains were routinely grown in Luria Bertani (LB) broth (1.0% Tryptone,
0.5% yeast extract,0.5% NaCl) containing 0.5 % glucose; for solid media, agar was added
to 1.5% (wt/vol). P.aeruginosa strains were routinely grown in Mueller Hinton broth; for
solid media, agar was added to 1.5% (wt/vol). B M 2 minimal media was made as
previously described (Poole et al., 1995). A l l media components were purchased from
Difco Laboratories (Detroit, MI). Antibiotics were used for E. coli when required at the
following concentrations:
ampicillin,
100
(J.g/ml; for P.aeruginosa: carbenicillin,
400|ag/ml; norfloxacin, l|j,g/ml; gentamycin, 10(ag/ml.
2.2
Reagents
Chemical reagents used in this study were purchased from Sigma Chemical (St. Louis,
MO).
Restriction enzymes, polymerases and other molecular biology reagents were
purchased from Gibco B R L (Burlington, ON). A l l enzymes were used as described in the
manufacturer's instructions Rabbit anti-OprD monoclonal antibody (YD11), mouse antiOprM monoclonal antibody (TM01), mouse anti-OprJ (HJ004) and mouse anti-OprN
(TN009) monoclonal antibodies were all provided by Dr. Gotoh. Alkaline phosphatase
conjugated anti-goat and anti-mouse antibodies were purchased from BioRad (Richmond,
CA).
20
Table I:
Strain:
Bacterial strains
Relevant characteristics:
Source/reference:
supE44, /act/169 (<j) 80/acZM15), hsdRU, recAl, endAJ,
gyrA96, thi-1, relAX
BRL
P A O l prototroph: wild type reference strain
HI 03 containing a chromosomal oprD-xylE fusion
Clinical isolate
nfxC-type mutant of 1008
Hancock, et al., 1979
M . Ochs
Masuda, et al., 1995
Masuda, et al., 1995
E. coli:
DH5ct
P. aeruginosa
H103
H103-1
1008
1008OIR01
2.3
Genetic manipulations
General molecular biology protocols were performed according to standard methods
described in Sambrook et al. (1989). D N A fragments were isolated using the Gene Clean
kit (BiolOl Inc., Vista, CA). Sticky end ligations were performed at RT for 30-60 min;
blunt end ligations were done overnight in a thermocycler programmed to continuously
cycle between 10°C and 30°C for 30 sec at each temperature.
2.4
D N A sequencing
D N A sequencing was done on an A B I 373 automated D N A sequencer (Applied
Biosystems, Foster City, C A ) using the A B I Prism dye terminator sequencing kit
according to the manufacturer's directions. Plasmid D N A was prepared for sequencing
using Qiagen columns (Qiagen, Inc., Chatsworth, CA) and plasmid D N A was quantified
using a Hoefer T K O 100 mini spectrofluorimeter. Oligonucleotide sequencing primers
were synthesized as needed on a A B I 392 D N A / R N A oligonucleotide synthesizer
according to the manufacturer's directions.
21
Table II:
Plasmids used in study
Plasmid:
Relevant characteristics:
pUCP18/19
pUC18/19-derived broad host range plasmid which can be maintained
in both E.coli and P.aeruginosa
pUCPl 8 containing a 1.2kb BamHIIEcoRl fragment encoding mexT
cloned in the reverse orientation to the promoterless lacZgene
pUCPl 9 containing a 1.2kb BamHIIEcoRl fragment encoding mexT
cloned in the same orientation as the promoterless lacZ gene
pMCl with a 500bp BamHIIBgUI de letion in the 5' end of the mexT
gene
pMCl with a 700bp Sail deletion in the 5' end of the wex^gene
pMCl
pMC2
pMC3
pMC4
Source/re ferenee:
Schweizer, 1991
This study
This study
This study
This study
Oligonucleotide primers were purified as follows: following deprotection for 8 h at 55°C,
the oligonucleotides were lyophilized and redissolved in 500 ul deionized water. The
resuspended oligonucleotide was centrifuged for 5 min at maximum speed (-14,000 rpm)
in a microcentrifuge to remove insoluble material and the supernatant was retained. The
concentration of oligonucleotide was determined by measuring the absorbance at 260 nm.
To completely sequence the cloned mexT D N A fragment two strategies were used. One
was the building of custom oligonucleotide primers (Table III). The second was the
construction of two plasmids containing deletions in the mexTUNA fragment (Table II).
The first deletion was from the BamHI site of the pUCP18 M C S to the BgUI site oimexT
(Fig. 6). The second deletion was from the Sail site of the pUCP18 M C S to the Sail site
of mexT (Fig. 6). Nucleotide and deduced amino acid sequences were analyzed with the
PC Gene software package (Intelligenetics Inc., Mountain View, CA). The D N A
sequence was used for homology searches of the National Center for Biotechnology
Investigation
(NCBI)
non-redundant
database
22
with
the
BLASTX
program
(vvrv^.ncbi.nlm.nih.gov/cgi-bin/BLAST/). The default settings for the B L A S T X program
was used for all the searches. The D N A sequence was also used for homology searches of
the contents of the Pseudomonas aeruginosa genome sequencing project with the
B L A S T N program (www.ncbi.nlm.nih.gov/BLAST/pseudoabl.html).
2.5
Transfer of D N A into P. aeruginosa
Plasmids were transferred in P.aeruginosa by transformation (Olsen et al., 1982). Briefly,
cells to be transformed were grown to an O D
600
of 0.6-0.8 in M H . Cells were pelleted and
resuspended in 150 m M M g C l , placed on ice for 5 min. This step was repeated except
2
for the cells were put on ice for 20 min. The cells were then pelleted and resuspended in
1/10 volume of ice cold 150 m M M g C l , and kept at 4°C for 16 hrs. D N A (0.1 to ljig)
2
was added to 200ul of competent cells and left on ice for 1 hr. Cells were heat shocked by
placing them at 42°C for 5 min and then were returned to ice for a further 5 min. 1ml of
M H broth was added and the mixture was incubated at 37°C for 2-3 hrs to allow
expression of the plasmid antibiotic resistance gene. Aliquots (50-100 ul) of cells were
plated on selective medium and grown for 24 to 48 hrs.
2.6
Cloning strategy for the mexT gene
According to Kohler et al., the mexT gene was directly upstream and in the same
orientation as the mexE gene (Kohler et al., 1997). The sequence of mexE and its
upstream region was retrieved from the EBI databank under accession number X99514.
The 5' end of the sequence and all the available upstream sequence was used to search the
contents of the P.aeruginosa genome sequencing project for a Contig containing the 5'
end of mexE and as much upstream sequence as possible. Contig 1523 was retrieved and
23
when analysed was found to contain the 5' end of mexE and one other ORF upstream.
The sequence of this ORF was used for homology searches of the National Center for
Biotechnology Investigation (NCBI) non-redundant database with the B L A S T X program
(www.ncbi.nlm.nih.gov/cgi-bin/BLAST/V It showed high homology to LysR-type
regulators which was consistent with it being mexT. Using this sequence primers were
designed to allow amplification of the mexT gene using P C R (Table. III). To allow easier
cloning the primers contained convenient restriction sites. Primer mexTF89 contained a
BamHI site while mexTRext contained an EcoRI site.
2.7
Cloning and analysis of the mexT gene
Amplification of the mexT gene from P.aeruginosa P A O l (HI03) was achieved using
PCR. The reaction mixture (total volume lOOul) included: 2 units of Vent polymerase
(New
England Biolab, Beverly, M A ) , l x Vent reaction buffer, 0.3
m M each
deoxynucleoside triphosphate, 0.5 u M of each primer (mexTF89 and mexTRext), and
lOOng of HI03 genomic D N A . The mixture was treated for 5 min at 95°C, followed by
one cycle of 5 min at 95°C, 2 min at 55-60°C and 1 min 45 sec at 72°C. This was
followed with 30 more cycles of 2 min at 95°C, 2 min at 55-60°C and 1 min 45 sec at
72°C, before finishing with 10 min at 72°C. P C R products were examined on a 0.8%
(wt/vol) agarose gel and purified with the QIAquick-spin PCR purification kit (Qiagen
Inc., Chatsworth, CA).
2.8
Overexpression of the mexT gene in P. aeruginosa
To overexpress MexT, the 1.2 kb BamHIIEcoRl fragment from p M C l containing the
mexT gene was cloned into pUCP19 to form the plasmid pMC2, so that the direction of
24
expression of the mexT gene was in the same orientation as the lac promoter (Fig. 5). It
was then transformed into P.aeruginosa HI03 and HI03-1.
2.9
Membrane isolation and Protein analysis
Outer membranes were prepared by sucrose gradient density centrifugation (Hancock and
Nikaido, 1978) or by differential Sarcosyl solubilisation (Poxton et al., 1985). A l l
samples were taken from cultures at an O D
600
of 1.0. Analysis of protein profiles was
done by electrophoresis through SDS 12%- polyacrylamide gels (PAGE) (Mutharia &
Hancock, 1983). Proteins were solubilized in sample buffer (0.5 M Tris-HCl buffer, pH
6.8, containing 2% SDS, 10% glycerol, 5% P-mercaptoethanol and bromophenol blue)
for 10 min at 98°C before being applied to the gel. Following electrophoresis proteins
were visualized by staining with Coomassie Brilliant Blue R250 (Bio-Rad, Richmond,
CA). Immunoblotting procedures were performed as described previously (Mutharia &
Hancock, 1983). Antibodies for Western immunoblotting were used at the following
dilutions: OprD, 1:5000; OprM, 1:3000; OprJ, 1:714; OprN, 1:1250.
25
F i g 4:
Diagram of plasmid pMCl utilized for sequencing of the mexT gene.
The coding region of the mexT gene is shown. The orientations of the ampicillin
resistance gene, origin of replication and the lac promoter are indicated. The 1.8 kb
stabilizing fragment is for maintenance of the plasmid in P. aeruginosa.
26
Oligonucleotide primers used in study
Table III:
Primer:
D N A Sequence from 5' to 3':
mexTF89
TTACATG<JATCCTGGCTATGCCTGTCAGTG
mexTRext
TTCTACGAATTCCGTTTTCTGTAACAGTCG
UNI
TGTAAAACGACGGCCAGT
REV
CAGGAAACAGCTATGACC
mexT17AFwd
CTGCTGATCGTGTTCGAGACC
mexT17BRev
CATGGCGGTGGAGATGGAACT
mexT13ARev
GAGGATCTTCGGCTTGCTGCG
mexT12ARev
CAAGCCATCGACAGTTCGAAG
mexT21AFwd
CTTCGAACTGTCGATGGCTTG
2.10
Determination of Minimal Inhibitory Concentration (MIC) Assay
The MIC of various antimicrobial agents for P. aeruginosa strains was determined by the
broth dilution method (Amsterdam., 1991). Briefly, cells were grown overnight at 37°C
in Mueller-Hinton and diluted 1 in 10 000 in the same medium to give concentrations of
about 10 -10 colony-forming units/ml. Serial dilutions of each antibiotic were made in
4
5
Mueller-Hinton medium in 96-well microtitre plates (Costar).
Each well was then
inoculated with 10u.l of the test organism. Samples of the bacterial inoculum were plated
to ensure they were within the proper range. The MIC was determined after overnight
incubation of the plates at 37°C. The microtitre plates were scored for growth in the wells
and the M I C was taken as the lowest antibiotic concentration at which growth was
inhibited. The influence of iron and zinc on the antibiotic susceptibilities of P. aeruginosa
strains was determined using B M 2 minimal media with succinate as a carbon source and
varying concentrations of each ion.
27
Fig 5:
Diagram of plasmid pMC2 utilized for the overexpression of the mexT gene.
The coding region of the mexT gene is shown. The orientations of the ampicillin
resistance gene, origin of replication and the lac promoter are indicated. The 1.8 kb
stabilizing fragment is for maintenance of the plasmid in P. aeruginosa.
28
2.11
X y l E assays
P.aeruginosa strains containing a chromosomal oprD::xylE transcriptional fusion and
either no plasmid, pUCP19, or pMC2, were assayed for X y l E activity as described
previously (Deretic and Konyecsni, 1988). Briefly, cells were grown with appropriate
antibiotic selection to and O D
600
of -0.5
and then harvested. Cell pellets were
resuspended in 750ul of 50mM K phosphate buffer, pH 7.5, and 10% acetone. Cells were
broken by 1 min sonication on ice. Unbroken cells and cell debris were removed by
centrifugation for 20 min at 5000 rpm, at 4°C. Supernatant was removed to a clean tube.
Protein concentration was determined as described previously. Two volumes of
supernatant were assayed from each sample in a total volume of 1 ml containing 50mM K
phosphate buffer, p H 7.5 and 0.3 m M catechol. Reaction rates were recorded in a PerkinElmer (Lambda3) dual-beam spectrophotometer coupled to a Perkin-Elmer 561 chart
recorder, by following the change of absorbance at 375 nm. Results are given in pmol of
product produced per min per \xg of extract. The molar extinction coefficient of the
product, 2-hydroxymuconic semialdehyde, is 4.4 x 10 . A l l experiments were done in
4
triplicate.
2.12
Growth phase experiments
For Growth phase experiments, P.aeruginosa P A O l (HI03) was grown in M H broth with
shaking at 37°C. Samples for analysis of the outer membrane profiles were taken in midlog, late-log and stationary phases of growth. Stage of growth was determined by
monitoring O D
600
and plotting the growth curve.
29
3
RESULTS
Chapter One: Analysis of a Gene Involved in the Regulation of Expression of OprD
3.1.1
Molecular cloning of the mexT Gene
Using the primers designed on the basis of the data from the P.aeruginosa genome
sequencing project, a P C R reaction was done. A fragment of approximately 1.2 kb was
obtained. This was consistent with the size predicted from analysis of the sequence in the
P.aeruginosa genome sequencing project. The 1.2 kb fragment was cut with BamHI and
EcoRI and cloned into pUCP18 to yield plasmid p M C l (reverse orientation compared to
the lac promoter) (Fig. 4), and into pUCP19 to yield plasmid pMC2 (forward orientation
compared to the lac promoter) (Fig. 5).
BgH(34)
BamHI(l)
BgUI(564)
,
/
AraI(5S6)
tadl(38)
B«I
/
BgJI(869)
s»Hf7«l
P™n(673)
t !
S ! d I
7 4 3
C M (1076)
\EnII(904)
\
B M I\
Piral(1080)
,
EcoRI(1151)
1151
mexT
lOObp
Fig 6:
Physical map of the mexT gene.
Some of the endonuclease restriction sites used for the cloning and subcloning of mexT
are shown. The BamHI and EcoRI restriction sites were engineered into the primers used
to P C R up the mexT gene and are not part of the sequence.
30
3.1.2
Nucleotide sequence of the mexT gene
Both strands of the 1.2 kb BamHI/'EcoRI P C R fragment were sequenced. When this
sequence was compared to the sequence from the P.aeruginosa genome sequencing
project, a number of discrepancies were noticed. There was an 8bp duplication, a lbp
deletion and 4bps that were different to the sequence obtained. From analysis of the
chromatograms obtained during the sequencing of the P C R product, it was determined
that the sequence shown in Fig. 7 is the correct sequence. The errors in the genome
project sequence probably resulted from the fact that this sequence from the sequencing
project has not been fully checked and edited yet.
The fragment contains a single open reading frame (ORF) of 912 nucleotides (Fig. 7). It
encodes a 304 amino acid protein with a calculated molecular weight of 33,422.
Consistent with this, overexpression of the gene, designated mexT, using the pET-based
expression system (Invitrogen, Ca.), yielded a major protein band with an M r of
approximately 33kDa (data not shown). The GC content of the whole gene is 68%, and at
the third codon position it reaches 82.2%. Both of these sequence features are typical of a
P.aeruginosa gene. Putative -35 and -10 promoter regions were identified upstream of
mexT (Fig. 7). The 6 nucleotides, A A G G A G , which constitute a consensus bacterial
ribosome binding site (SD) are not present upstream of the mexT start site. However a
putative SD sequence, A A C G A G , does exist (Fig. 7). There are at least two potential
A T G start codons at the predicted 5' end of the mexT gene, although the largest ORF is
likely to be correct in the light of the similarity between the largest deduced MexT
product and other bacterial proteins (see below).
31
CCTGTCAGTGATCCTATGCCCCTCCGGCACCTCGCCAGGCCCCGCCCCGTCTCGCACGCA
-35
-10
SD
60
+l->
AGGCTTGACGGCGAGCCCCCGCGGTTGCAGCCTCTAGCCCCTGGAAACGAGGAACGCCAT
M
120
1
GAACCGAAACGACCTGCGCCGCGTCGATCTGAACCTGCTGATCGTGTTCGAGACCCTGAT
N
R
N
D
L
R
R
V
D
L
N
L
L
I
V
F
E
T
L
M
2
1
180
GCACGAACGCAGCGTGACCCGCGCCGCAGAGAAACTGTTCCTCGGCCAGCCGGCCATCAG
H
E
R
S
V
T
R
A
A
E
K
L
F
L
G
Q
P
A
I
S
4
1
CGCCGCGCTGTCGCGCCTGCGCACGCTGTTCGACGACCCGCTGTTCGTCCGTACCGGACG
A
A
L
S
R
L
R
T
L
F
D
D
P
L
F
V
R
T
G
R
6
1
CAGCATGGAGCCCACCGCGCGAGCCCAGGAAATCTTCGCCCACCTGTCGCCGGCGCTGGA
S
M
E
P
T
A
R
A
Q
E
I
F
A
H
L
S
P
A
L
D
8
1
240
300
3 60
TTCCATCTCCACCGCCATGAGTCGCGCCAGCGAGTTCGATCCGGCGACCAGCACCGCGGT
S
I
S
T
A
M
S
R
A
S
E
F
D
P
A
T
S
T
A
V
4 20
101
GTTCCGCATCGGCCTTTCCGACGACGTCGAGTTCGGCCTGTTGCCGCCCATGCTCCGCCG
F
R
I
G
L
S
D
D
V
E
F
G
L
L
P
P
M
L
R
R
480
121
CCTGCGCGCGGAGGCGCCGGGGATCGTCCTCGTCGTGCGCCGCGCCAACTATCTATTGAT
L
R
A
E
A
P
G
I
V
L
V
V
R
R
A
N
Y
L
L
M
540
141
GCCGAACCTGCTGGCCTCGGGGGAGATCTCGGTGGGCGTCAGCTACACCGACGAACTGCC
P
N
L
L
A
S
G
E
I
S
V
G
V
S
Y
T
D
E
L
P
600
161
GGCCAACGCCAAGCGCAAGACCGTGCGCCGCAGCAAGCCGAAGATCCTCCGCGCCGACTC
A
N
A
K
R
K
T
V
R
R
S
K
P
K
I
L
R
A
D
S
660
181
CGCGCCCGGCCAGCTGACCCTCGACGACTACTGCGCGCGACCGCACGCGCTGGTGTCCTT
A
P
G
Q
L
T
L
D
D
Y
C
A
R
P
H
A
L
V
S
F
7 20
201
CGCCGGCGACCTCAGCGGCTTCGTCGACGAGGAGCTGGAAAAATTCGGCCGCAAACGCAA
A
G
D
L
S
G
F
V
D
E
E
L
E
K
F
G
R
K
R
K
7 80
221
GGTGGTCCTGGCGGTGCCGCAGTTCAACGGCCTCGGCACCCTCCTGGCCGGCACCGACAT
V
V
L
A
V
P
Q
F
N
G
L
G
T
L
L
A
G
T
D
I
840
241
CATCGCCACCGTGCCCGACTACGCCGCCCAGGCGCTGATCGCCGCCGGCGGCCTACGCGC
I
A
T
V
P
D
Y
A
A
Q
A
L
I
A
A
G
G
L
R
A
900
261
CGAGGACCCACCGTTCGAGACCCGCGCCTTCGAACTGTCGATGGCTTGGCGCGGCGCCCA
E
D
P
P
F
E
T
R
A
F
E
L
S
M
A
W
R
G
A
Q
960
281
GGACAACGATCCGGCCGAACGCTGGCTGCGCTCGCGGATCAGCATGTTCATCGGCGATCC
D
N
D
P
A
E
R
W
L
R
S
R
I
S
M
F
I
G
D
P
1020
301
GGACAGTCTCTGAGCCCTCCGGCAGCTACCCGCACGAGGCGTCGCAACGGGAAAATCGAT
D S L *
1080
304
CGCGCGCCGCGGGTGTGCGGCTTATTCCATCCGAAAGCACTGTCCATAACCATCGACTGT
1040
TACAGAAAACG
1051
Fig 7:
Nucleotide and deduced amino acid sequence o f the mexTgene.
A potential Shine-Dalgarno (SD) sequence is shown (dashed line). The start site o f the
mexTgene is indicted along with its putative - 1 0 and - 3 5 promoter regions.
32
3.1.3
MexT shows similarity to NahR and other LysR-type transcriptional regulators
The deduced MexT protein sequence was compared with sequences in the Genbank
database using the B L A S T algorithm. The search for amino acid sequences similar to
MexT identified 14 proteins with similariity scores greater than 115. Thirteen out of the
first 14 were NodD proteins from various species of Rhizobium. The protein with the
highest similarity was NahR. NahR is a LysR-type transcriptional regulator (LTTR) that
regulates production of enzymes for the metabolism of naphthalene and salicylate as sole
carbon sources in Pseudomonas putida (You., et al. 1988).
Alignment of the two sequences (MexT, 304 aa; NahR, 300 aa) revealed that there were
33% identical residues and 14.5% conserved residue changes (Fig. 8). The regions of
similarity were concentrated somewhat in the N-terminal portion of the proteins. This
region of NahR contains the D N A binding helix-turn-helix - (HTH) domain (Fig. 8.
residues 26-56) of the protein. A number of very highly conserved residues in NahR
essential for D N A binding (Ala 27, Pro 35, Thr 56) were also conserved in MexT (Fig. 8.
bold). It is likely therefore that the N-terminal region of MexT contained a D N A binding
H T H domain. Analysis of the region of NahR which binds salicylate as a coninducer
(Fig. 8. aa 95-170), and the residues important for binding, showed little similarity and no
conservation of residues in the same region of MexT. This was consistent with other
LTTRs which show little similarity in this region. The C-terminal portion of the two
proteins also showed limited similarity.
33
MEXT
MNRNDLRRVDLNLLIVFETLMHERSVTRAAEKLFLGQPAISAALSRLRTL
*
_**
_*****_**
*
* *
**
* * ***
* **
****
NAHR
M
MEXT
FDDPLFVRTGRSMEPTARAQEIFAHLSPALDSISTAMSRASEFDPATSTA
*******
****
*
_
..
*.
.
*
***
**
100
NAHR
LQDPLFVRTHQGMEPTPYAAHLAEPVTSAMHALRNALQHHESFDPLTSER
97
MEXT
VFRIGLSDDVEFGLLPPMLRRLRAEAPGIVLVVRRANYLLMPNLLASGEI
150
ELRDLDLNLLVVFNQLLVDRRVSITAENLGLTQPAVSNALKRLRTS
*
*
*
*
*
**
*
*
*
*
NAHR
TFTLAMTDIGEIYFMPRLMDVLAHQAPNCVISTVRDSSMSLMQALQNGTV
MEXT
SVGVSYTDELPANAKRKTVRRSKPKILRADSAPGQ
*
* _
*
LTLDDYCARPHA
*
* * * * *
50
4 7
14 7
197
NAHR
DLAVGLLPNLQTGFFQRRLLQNHYVCLCRKDHPVTREPLTLERFCSYGHV
197
MEXT
LVSFAGDLSGFVDEELEKFGRKRKVVLAVPQFNGLGTLLAGTDIIATVPD
2 47
*
**
***
^ ^ *
_*
_ * * * *
_ *
_ *
**__****
NAHR
RVIAAGTGHGEVDTYMTRVGIRRDIRLEVPHFAAVGHILQRTDLLATVPI
2 47
MEXT
YAAQALIAAGGLRAEDPPFETRAFELSMAWRGAQDNDPAERWLRSRISMF
*
_
**
*
*
* *
* *
***
2 97
NAHR
RLADCCVEPFGLSALPHPVVLPEIAINMFWHAKYHKDLANIWLRQLMFDL
2 97
MEXT
IGDPDSL
*
304
NAHR
FTD
300
Fig 8.
Similarity of MexT to NahR.
Amino acid sequences of MexT and NahR were aligned with the P A L I G N program of the
PCGene software package (Intelligenetics Inc.). The character to show that two aligned
residues are identical was '*'. The character to show that two aligned residues are similar
was '.' Amino acids considered to be 'similar' are: A,S,T; D,E; N,Q; R,K; I,L,M,V;
F,Y,W
34
3.1.4
Overexpression of the mexT gene in P.aeruginosa
To determine the effect of overexpressing mexT in P.aeruginosa, plasmid pMC2 was
transformed into a P.aeruginosa wild-type strain, HI03. mexT is in the forward
orientation compared to the lac promoter, allowing overexpression of mexT, as the lac
promoter is constitutive in P.aeruginosa. The antimicrobial susceptibility and outer
membrane profiles of this strain were examined.
The MICs for the various strains are shown in Table IV. Overexpression of mexT resulted
in a 32 fold increase in resistance to chloramphenicol, an 8-fold increase in resistance to
norfloxacin and a 2-4-fold increase in resistance to imipenem. There was a 2-fold
increase in susceptibility to gentamicin while there was no change in the M I C of
tetracycline. This was in comparison to the wild-type strain with and without the vector.
The high M I C for carbenicillin in the strains containing pUCP19 and pMC2 was due to
the plasmid encoded antibiotic marker. The antibiotic susceptibility profile when mexT
was overexpressed was consistent with that of the nfxC-type mutant 1008OIR01 (Table
IV). The characteristic increase in susceptibility to carbenicillin seen in nfxC-type
mutants could not be confirmed due the use of carbenicillin to maintain the plasmids.
Analysis by SDS-PAGE of the outer membrane proteins of the strains in Table IV
showed reduced expression of a 45kDa protein (Fig. 9, lane 4) in the mexT
overexpressing strain compared to the control strains (Fig. 9, lanes 2 and 3). This was
confirmed to be OprD by Western blot analysis using an OprD specific mAb (Fig. 10A,
lanes 2, 3 and 4).
35
Table IV:
MICs for P. a e r u g i n o s a strains.
Strains
H103
H103 + p U C P 1 9
H103 + pMC2
1008
1008OIR01
MICs ( ucj/ml)
CARB
CEFP
IM
GENT
NOR
CM
TET
25
>400
>400
25
6.25
0.5
1
1
0.5
0.5
1
0.5
2
1
4
0.39
0.39
.0.195
0.39
0.195
0.195
0.195
1.56
0.195
3.13
12.5
12.5
400
25
>800
3.13
1.56
3.13
3.13
3.13
•Abbreviations: CARB, carbenicillin; CEFP, cefpirome; IM, imipenem; GENT, gentamicin; NOR, norfloxacin; CM,
chloamphenicol; TET, tetracycline.
This reduction of OprD in the outer membrane was consistent with the nficC mutant (Fig.
9, lane 6 and Fig. 10A, lane 6). Outer membrane preparations were further analysed by
Western blotting using anti OprM, OprJ and OprN mAbs. In the mexT
overexpressing
and nfxC mutant, a protein of 50kDa hybridised with the OprN mAb (Fig. 10B, lanes 4
and 6). No OprN expression was detected in the parent and control strains (Fig. 10B,
lanes 2, 3 and 5 ). No cross hybridization bands were seen with the OprJ antibody (data
not shown), while the OprM mAb revealed only wild-type levels of OprM in all strains
(Fig. 10C, lanes 2-6). No other changes in the outer membrane profiles were observed.
These results suggest that overexpression of mexT
n/xC-type phenotype.
36
is itself sufficient to reproduce an
OprD (45kDa)
1
Fig 9:
gene.
2
3
4
5
6
SDS-PAGE demonstrating repression of OprD by overexpression of the mexT
The banding position of OprD is indicated by the arrow on the right. Lanes: 1, molecular
markers; 2, H103; 3, H103(pUCP19); 4, H103(pMC2); 5, 1008; 6, 1008OIR01. For each
lane, 20pg of outer membrane protein was applied.
37
.
4fc
—«
4
OprD(46kDa)
4
OprN (50kDa)
B
1
Fig 10:
2
3
4
5
6
Western immunoblots of P. aeruginosa strains.
Lanes: 1, molecular markers; 2, H103; 3, H103(pUCP19); 4, H103(pMC2); 5, 1008; 6,
1008OIR01. For each lane, 20ug of outer membrane protein was applied. A , B, C were
blotted with anti-OprD m A B , anti-OprN mAb and anti-OprM mAB respectively.
38
3.1.5
MexT reduces transcription of oprD
Given the fact that MexT had high homology to the LysR-type transcriptional regulator
NahR, and that overexpression of mexT resulted in reduction in the level of OprD in the
outer membrane, it was believed likely that MexT exerted its effect at the transcriptional
level. To test this directly, plasmid pMC2 containing the mexT gene in the forward
orientation relative to the lac promoter, was transformed into the P. aeruginosa strain,
HI03-1,
which had an oprDr.xylE
transcriptional
fusion in the chromosome.
Overexpression of mexT in this strain resulted in a 2.7-fold reduction in X y l E activity
(Table V). To rule out the possible involvement of other genes present on the pM2
plasmid in the observed repression, vector alone (pUCP19) was transformed in HI03-1.
In this instance only a 10% reduction in X y l E activity was detected (Table V). The effect
of overexpressing mexT'm E.coli on an oprDr.xylE plasmid encoded fusion could not be
determined due to the instability of the mexT overexpressing construct in E.coli. This
would have allowed us to test whether mexT regulates oprD via a third gene.
39
Table V: Effect of MexT on an oprDr.xylE chromosomal transcriptional fusion in
P.aeruginosa.
i
!
Strain
H103-1
Vector
(
none
|
XylE activity (values given in
I pmol of product per min per ug extract)*
|
61 +/- 5
pUCP19
I
55+/-12
pMC2
22 +/- 5
*Data are reported as the means of three separate experiments. Data from each
experiment was in turn the mean of two different measurements using different
protein concentrations
3.1.6
Summary
The mexT structural gene was obtained by using sequence data from the P. aeruginosa
genome sequencing project to design primers that allowed amplification of the gene from
the genome of P.aeruginosa P A O l using PCR. D N A sequencing of the obtained PCR
product revealed an ORF of 912 bp, that encoded a 304 aa mature MexT protein. The
sequence showed homology to the LysR-type transcriptional regulator (LTTR) family of
proteins. It showed highest homology to the LTTR, NahR from P.putida. Overexpression
of the mexT gene in P.aeruginosa increased resistance to norfloxacin, chloramphenicol
and imipenem. Outer membrane profiles were also changed by mexT overexpression,
with reduction of OprD and expression of the outer membrane component of the mexE/FoprN efflux operon, OprN. MexT was shown to reduce transcription of the oprD gene
using a chromosomal oprDr.xylE transcriptional fusion.
40
Chapter Two: The search for factors affecting mexT expression in P.aeruginosa.
3.1.7
Introduction
In E.coli, salicylate, a plant-derived compound, has been shown to reduce the expression
of outer membrane porins as well as induce low-level resistance to some antibiotics.
Salicylate does this by antagonizing the repressor activity of the MarR protein which
negatively regulates another regulatory locus, marAB. By reducing binding of MarR to
the promoter region of the marAB genes, salicylate relieves the repression of MarA which
in turn results in changes in the expression of several unlinked target genes. Among these,
two have clear relevance to antibiotic resistance. The levels of the major outer membrane
porin OmpF are substantially reduced, and expression of the multi-drug efflux pump,
AcrAB is induced (for review see Miller and Sulavik, 1996).
In P.aeruginosa, it has been shown that salicylate reduces expression of an outer
membrane protein with a molecular mass of 45 kDa (Sumita and Fukasawa, 1993).
Salicylate has also been suggested to induce expression of a 50-kDa protein thought to be
OprN (Masuda et al., 1995). With these results in mind, as well as the fact that MexT was
found to be similar to NahR, we were interested in investigating the possibility that
salicylate or other structurally related compounds may be an inducer of the mexE/F-oprN
operon by acting as a cofactor of the MexT protein. This was done by examining the
effects of these compounds on the antimicrobial susceptibilities and outer membrane
profiles of P. aeruginosa.
41
A number of other factors suspected to play a role in the expression of the known efflux
operons as well as expression of the imipenem specific porin, OprD, in P.aeruginosa
were investigated. These included iron and zinc concentrations as well as the growth
phase of the bacterium. This was done by looking at either MICs, outer membrane
profiles or a combination of both.
3.1.8
Salicylate and sodium benzoate; effects on antibiotic susceptibilities and outer
membrane protein production in P.aeruginosa.
Shown in Table VI are the antibiotic susceptibilities of P.aeruginosa P A O l in the
presence of salicylate and a number of other compounds known to induce multiple
antibiotic resistance in E.coli. Salicylate induced an 8 fold increase in resistance to
imipenem, a 2 fold increase in resistance to chloramphenicol, norfloxacin and cefpirome
and a 4 fold increase in susceptibility to carbenicillin (Table VI). The only significant
change in M I C with sodium benzoate was a 4 fold increase in resistance to imipenem
(Table VI). Dinitrophenol, naphthalene, and catechol induced no significant changes in
susceptibility to any of the antibiotics tested, while naphthaquinone showed only a 2 fold
increase in resistance to carbenicillin, cefpirome, norfloxacin and tetracycline (Table VI).
Interestingly, the antimicrobial susceptibility profile of P.aeruginosa in the presence of
salicylate was similar to that of an nfxC-type mutant;
i.e. increased resistance to
norfloxacin, chloramphenico, imipenem and increased sensitivity to carbenicillin (Table
IV). To determine whether these changes were due to expression of one of the efflux
operons, we examined the outer membrane profiles of P.aeruginosa when grown in the
presence of 32 m M salicylate. We also looked at the outer membrane profiles of
42
P.aeruginosa when grown in the presence of 16 m M sodium benzoate to see whether the
increased resistance to imipenem might be due to reduced OprD.
43
Table V I :
Antibiotic susceptibilities of P.aeruginosa P A O l in Mueller-Hinton in the
presence of salicylate and other structurally related compounds.
I'" '
Compound
none
|
Salicylate I
Sodium benzoate
Dinitrophenol
Naphthalene
Naphlhaquinone
Catechol
" "~
|MIC (ug/ml)*
CARB
25
'
25
12.5
2 5
50
50
'
—
CEFP
0.5
1 "~
1
0.5
"1
1
1
_
1
IM
|
1
!
8
l~
4
I
1
!
1 " '
1
|
1
_j_
GENT
0.39
1.56
0.78
0.39
0.39
0.39
o : 3 g
NOR
CM
IET
0.195
12.5
3.13
" 0.39
25
3.13
0.195
12.5 " " 3 . 1 3
0.195 | 12.5
0.195 " 1 2 . 5
3.13
0.39
" 6.25
" 0.195 ^ 6.25 ~
3.13
3
1 3
"
[
"Abbreviations: CARB , carbenicil in; CEFP, cefpirome; IM, imipenem; GENT, gentamicin;
NOR, norfloxacin; CM, chloramphenicol; TET, tetracycline.
|
Salicylate and sodium benzoate both reduced expression of a 45 kDa protein when
included in the media at the appropriate concentrations (Sal, 32 m M ; SB, 16 mM) (Fig.
11, lanes 3 and 4), compared to media alone (Fig. 11, lane 2). This was confirmed to be
OprD using Western blotting with anti-OprD mAb (Fig 12A, lanes 2-4). Wild type levels
of OprM were detected in all samples using anti-OprM mAb (Fig. 12B, lanes 2-4). No
cross-reacting bands were revealed with the OprJ antibody (data not shown), or the OprN
antibody (Fig. 12C, lanes 4-6).
44
OprD(46kDa)
1
Fig 11:
2
3
4
Effect of salicylate and sodium benzoate on the outer membrane proteins of
P.aeruginosa P A O l .
Lanes: 1, molecular markers; 2, H103 grown in M H ; 3, H103 grown in M H plus
salicylate (32mM); 4, H103 grown in M H plus sodium benzoate (16mM). For each lane,
20u.g of outer membrane protein was applied.
45
•sin
«•*
Hjjjj
'
g
<—
<«— O p r M
(50kDa)
OprD
(46kDa)
1
2
3
4
OprN(50kDa)
F i g 12:
Western immunoblots o f outer membranes o f P. aeruginosa grown in the
presence o f 32 m M salicylate and 16 m M sodium benzoate.
A and B Lanes: 1, molecular markers; 2, H I 0 3 grown in M H ; 3, H I 0 3 grown in M H plus
salicylate (32mM); 4, H103 grown in M H plus sodium benzoate (16mM). C Lanes: 1,
molecular markers; 2, 1008; 3, 1008OIR01; 4, H103 grown in M H ; 5, H103 grown in
M H plus salicylate (32mM); 6, H I 0 3 grown in M H plus sodium benzoate (16mM). For
each lane, 20ug o f outer membrane protein was applied. A , B, C were blotted with antiOprD m A B , anti-OprM m A b and anti-OprN m A b respectively.
46
3.1.9
Salicylate reduces transcription of the oprD gene in P.aeruginosa.
To further examine the mechanism through which salicylate and sodium benzoate reduce
OprD levels in the outer membrane, we looked at the effect the compounds had on the
oprDr.xylE chromosomal transcriptional fusion in P.aeruginosa P A O l . When grown in
M H supplemented with 32 m M salicylate, X y l E activity was reduced by 3.3 fold
compared to M H alone (Table VII). This indicated that salicylate reduced OprD
expression by reducing transcription of the oprD gene. When grown in M H with 16 m M
sodium benzoate X y l E activity increased by 1.3 fold compared to M H alone. This
suggests that
sodium benzoate
exerts its
transcriptionally.
47
effects
on OprD
expression
post-
Table VII:
Effect of salicylate and sodium benzoate on an oprDr.xylE chromosomal
transcriptional fusion in P.aeruginosa.
pmol of product per min per ug extract)*
H103-1
none
[ 82 +/-11
\
Ii
32mM Sal
j 25+/-9
i
M i l +/-14"
TBrnWI S&
Data are reported as the means of three separate experiments. Data from
each experiment is in turn the mean of two different measurements using
fwo different "protein concentrations
I
|
I
" S a l , salicylate (32m M); SB, sodium benzoate (TBmM).
3.1.10 Effect of iron and zinc concentrations on the antibiotic susceptibilities of
P.aeruginosa.
Table VIII shows the antimicrobial susceptibilities of P.aeruginosa P A O l to various
antibiotics grown in M H and B M 2 minimal medium with varying concentrations of iron
and zinc.
The addition of excess iron to M H resulted in no changes in the M I C of the antibiotics
tested (Table VIII). Addition of supplementary zinc to the M H media resulted in a four
fold increase in resistance to imipenem and a two fold increase in resistance to cefpirome
(Table VIII). When B M 2 minimal medium supplemented with 20mM iron was used
instead of M H , there was a two fold increase in susceptibility to tetracycline,
chloramphenicol, imipenem, and cefpirome, no change for carbenicillin and norfloxacin,
and a two fold increase in resistance to gentamicin (Table VIII). When no iron was added
to the B M 2 minimal media making it iron deficient, there was a consistent two to four
48
fold increase in the susceptibility to all the antibiotics tested compared to iron sufficient
medium, except for gentamicin which showed a two fold increase in its MIC (Table
VIII). Interestingly, when iron deficient B M 2 was supplemented with 0.1 mM zinc, the
MIC of any of the antibiotics could not be determined due to no growth in any of the
wells.
49
Table VIII:
Effect of iron and zinc concentrations on the antibiotic susceptibilities of
P.aeruginosa
Media
MH
MH + 20mM Fe**
MH + 0.1 mM Zn**
BM2 + 20mM Fe
BM2 - Fe
BM2 - Fe + 0.1 mM Zn
CARB
25
25
25
25
12.5
NG
CEFP
1
1
2
0.5
0.25
NG
IM
1
1
4
0.5
0.25
NG
MIC (ug/ml)*
GENT
0.39
0.39
0.39
0.78
0.78
NG
NOR
0.195
0.195
0.195
0.195
0.097
NG
CM
12.5
12.5
12.5
6.25
3.13
NG
TET
3.13
3.13
3.13
1.56
0.78
NG
"Abbreviations: CARB, carbenicillin; CEFP, cefpirome; IM, imipenem; GENT, gentamicin; NOR, norfloxacin;
CM, chloramphenicol; TET, tetracycline. NG, no growth
**lron and Zinc added as Fe and Zn sulphate.
The increase in resistance of P.aeruginosa to imipenem in the presence of zinc was
investigated further to try to identify the mechanism of resistance. Expression of the
imipenem specific porin, OprD, was examined in the presence of varying concentrations
of zinc (Fig. 13). There was no change in OprD expression as observed by using SDSP A G E (Fig. 13A), and Western blotting with an OprD mAb (Fig. 13 B). Expression of
OprM, OprJ and OprN were also checked but no expression of OprJ or OprN was
detected by Western blotting with the respective mAbs, while only wild type levels of
OprM were found (data not shown).
50
OprD
(46kDa)
OprD(46kDa)
1
2
3
4
Fig 13:
Outer membrane profiles o f P.aeruginosa P A O l grown i n Mueller-Hinton
with different concentrations of zinc.
(A) S D S - P A G E and (B) Western-immunoblot with anti-OprD m A b . Lanes: 1, molecular
standards; 2 , M H only; 3, M H with zinc (6ug/ml); 4, M H with zinc (12ug/ml). For each
lane, 20ug o f outer membrane protein was applied.
51
3.1.11 Effect of growth phase on expression of the mex efflux operons of P.aeruginosa.
Outer membrane profiles of P.aeruginosa P A O l during the various growth phases were
examined using SDS-PAGE and Western blotting. Samples were taken during mid-log
phase (Fig. 14 A , Lane 2), late log phase (Fig. 14 A , Lane 3), and late stationary phase
(Fig. 14, Lane 4). No visible changes in OprD, OprE or OprF levels were observed during
the growth. The level of OprG seemed slightly elevated during log phase compared to
late log and stationary phase (Fig 14 A , lane2). OprH was highly produced during log
phase (Fig. 14 A , lane 2), while, OprL showed increased levels in stationary phase (Fig.
14 A , lane 4).
Analysis by Western blotting using monoclonal antibodies against OprM, OprJ and
OprN, was also done. The OprM antibody detected only wild-type levels of OprM
throughout growth (Fig. 14 B, lanes 2-4). No cross reacting bands were detected with
either the OprJ or OprN antibodies in any of the samples (data not shown).
52
+
<
OprD(46kDa)
' OprE(44kDa)
OprF(35kDa)
OprG(25kDa)
OprH(21kDa)
OprL(20kDa)
B
Fig 14:
OprM(50kDa)
Outer membrane profiles of P.aeruginosa P A O l during growth phases.
(A) SDS-PAGE and (B) Western-immunoblot with anti-OprM mAb. Lanes: 1, molecular
standards; 2, HI03 mid-log phase; 3, HI03 late-log phase; 4, late stationary phase. For
each lane, 20ug of outer membrane protein was applied.
53
3.1.12 Summary
When grown in the presence of 32mM salicylate, P.aeruginosa had increased resistance
to imipenem, chloramphenicol, norfloxacin and gentamicin while becoming more
sensitive to carbenicillin. Sodium benzoate gave increased resistance to imipenem while
none of the other compounds tested, naphthalene, naphthaquinone, dinitrophenol or
catechol, resulted in any significant increase in resistance to any of the antibiotics tested.
Salicylate and sodium benzoate reduced the amount of the outer membrane porin, OprD.
No other changes in the outer membrane profiles were noticed.
Iron concentrations had no effect on the antibiotic susceptibilities oi P. aeruginosa while
zinc increased its resistance to imipenem. This increase was not due to the loss of the
imipenem specific porin OprD as seen by the normal levels of OprD seen in the outer
membrane when grown in media containing high levels of zinc.
The outer membrane profiles of P.aeruginosa were examined during the various growth
phases for the presence of the 3 efflux operons known in P.aeruginosa, mexA/B-oprM,
mexCD-oprJ and mexEF-oprN. There were no changes in the amounts, with only wildtype levels of OprM and no presence of either OprJ or OprN detected in the outer
membrane during any of the growth phases.
54
4
DISCUSSION
The P.aeruginosa outer membrane porin, OprD, is an interesting protein as it facilitates
the diffusion of basic amino acids and imipenem, a potent antibiotic that has been used
for the treatment of P.aeruginosa. While much has been discovered about OprD's
structure function relationships little has been uncovered about its regulation until
recently. In this study the cloning, sequencing and characterization of a gene, mexT,
involved not only in the regulation of oprD, but also in the regulation of an efflux operon,
mexEF-oprN, is described. These and other tests set the ground for the detailed analysis
of the mechanism of regulation of these genes by the MexT protein.
MexT; a new member of the LysR-type transcriptional regulator family.
D N A sequencing of the mexT gene detected a 33kDa ORF which probably represents the
amino acid sequence of the MexT protein (Fig. 7). This amino acid sequence has
extensive similarity with the derived sequence of the NahR protein of Pseudomonas
putida (Fig. 8). It also has lower but significant similarity with the derived sequences of
the NodD proteins of Rhizobium species (data not shown). A l l three of these proteins are
nearly identically sized transcriptional activators and it is likely that that the regions of
greatest similarity between them represent protein domains involved in common
functions (e.g., D N A binding or transcriptional activation or both). The amino acid
sequence of the domain of MexT containing the region of greatest similarity between the
three (common domain; residues 24-60) (Fig. 8), had a protein sequence which was
55
similar to the amino acid sequence of the helix-turn-helix (HTH) DNA-binding motif of
the lambda Cro protein (Dodd and Egan, 1987).
Henikoff et al. reported the discovery of a family of prokaryotic transcriptional activators
evolutionary related to LysR (Henikoff et a l , 1988). This family included the NodD
proteins and a number of other proteins, all of which were approximately 300 residues in
length and most, if not all, of which are transcriptional activators. More recently, NahR
has also been shown to belong to this family of transcriptional regulators (Schell and
Sukordhaman, 1989). Comparison of the MexT protein sequence with the Henikoff
consensus sequence for this protein family clearly showed that MexT is a member of this
family (41% similarity over the first 150 residues; 33% similarity overall) (Henikoff et
al., 1988).
Schell and Sukordhaman (Schell and Sukordhaman, 1989) noticed that NahR and the
NodD proteins had much greater similarity to each other than any other members of the
family. They suggested that these two proteins might represent a subfamily within the
LysR-type family of transcriptional activators. MexT may represent another member of
this proposed subfamily based on its much greater similarity to NahR and the NodD
proteins than any other members of the LysR-type family of transcriptional activators.
Additional evidence that the more conserved N-terminal region of MexT is indeed as
proposed here, a H T H domain, comes from the comparison of the LysR-type regulators
consensus H T H sequence (Henikoff et a l , 1988), with the same region from MexT. This
region showed 50% identity and 20% conserved residues compared to the consensus
sequence.
56
MexT; an atypical LysR-type transcriptional regulator.
Most members of the LysR-type transcriptional regulator (LTTR) family share four
common characteristics: (a) They encode coinducer-responsive transcriptional activator
proteins, (b) Independent of the presence of coinducer they bind at regulated targets to
D N A sequences, (c) Each is divergently transcribed from a promoter that is very close to
and often overlaps a promoter of a regulated target gene, (d) Because the overlapping
divergent promoter structure
allows for simultaneous bi-directional control
of
transcription, most LTTRs repress their own transcription.
The mexT gene and the protein which it encodes, MexT, show some interesting
differences to the 'typical' members of the L T T R family. As judged from analysis of the
sequence from the P.aeruginosa genome sequencing project, the mexT gene lacks the
divergent promoter structure so characteristic of members of this family of proteins.
Transcription occurs in the same direction as that of the mexEF-oprN operon (sequence
data not shown) (Fig. 3 shows a schematic representation). Another exception to this rule
is the phcA L T T R of Pseudomonas solanacearum which is transcribed in the same
direction as its target genes (Brumbley et al., 1993)
When overexpressed from a multi-copy plasmid, MexT not only resulted in expression of
the mexEF-oprN operon, as judged by the presence of OprN in the outer membranes (Fig.
10B, Lane 4), but also resulted in reduced levels of the outer membrane porin, OprD (Fig.
10A, Lane 4). These result would suggest that MexT can not only act as an activator of
57
target genes, as in the case of the mexEF-oprN operon, but also as a repressor, as in the
case of oprD.
Most LTTRs described to date positively regulate their target genes while negatively
regulating their own expression. They do this by binding to the same target D N A
sequence in both cases, but the positioning of this sequence is very different. In activated
genes the target sequence is upstream of the -35 region of the promoter, while in cases
where the gene is repressed the target sequence overlaps the transcriptional start site. It
seems likely that MexT would act in a similar manner with relation to the activation of
the mexEF-oprN operon, but as discussed below, the mechanism of repression is unclear.
While unusual, this would not be the first example of an L T T R acting both as a repressor
and an activator. The TfdS protein of Agrobacterium eutrophus is reportedly a repressoractivator (Kaphammer and Olsen, 1990). Interestingly, this protein's repressor activity is
low for a repressor, with a 40%
maximal repression of transcription. MexT
overproduction resulted in a 64%
repression of an oprDr.xylE chromosomal
transcriptional fusion in P. aeruginosa (Table V). This result is consistent with the level of
OprD in the outer membrane in both the mexT overexpressing strain and the nfxC mutant
(Fig. 10A, Lanes 4 and 6). This would suggest that the nfxC mutation results in
overexpression of the mexT gene rather than by making the protein independent of any
coinducer. It is be possible that MexT does not need a coinducer to function, as is the of
AmpR protein of Enterobacter cloacae (Honore, et al., 1986) but it is more than likely
that MexT has at least one coinducer that it responds to.
58
The mechanism by which MexT represses oprD expression is an interesting question.
Normally, L T T R proteins repress transcription by occluding the promoter region by
binding to and overlapping the transcription start site. However, in the case described
here, one still gets some expression of the oprD gene. Once the exact binding site of the
MexT protein in the promoter region is identified, it will allow us examine possible
repression mechanisms more closely.
Recent unpublished data (Kohler et al., 1998) has suggested that there is no mutation in
the coding region of the mexT gene in the nfxC-type mutants. This would tend to rule out
the possibility of a change in the binding properties and hence the normal function of the
MexT protein. So how might the mexT gene get overexpressed in nfxC-type mutants as
suggested here? It is likely that the mexT gene negatively autoregulates. A n explanation
for this suspected mexT overexpression in nfxC-type mutants, would be that there is a
mutation in the MexT target D N A sequence in the promoter region of the mexT gene
itself. A mutation in the promoter region of the mexT gene that would eliminate this
negative autoregulation would allow for overexpression of the mexT gene resulting in
expression of the mexEF-oprN operon and repression of the oprD gene. Once the
sequence to which MexT binds has been determined, the sequence in the promoter region
oimexT in nfxC-type mutants can be examined for potential mutations.
Work is ongoing in the lab to purify the mature MexT protein from the wild-type
P. aeruginosa P A O l strain, and to test the binding of this protein to the promoter regions
of mexT, mexE, and oprD. To assist this I have cloned the mexT gene into a His-Tag
vector. Possible cofactors of MexT, as discussed below, will also be examined for their
59
affect on the binding of this protein to its target D N A sequences. These experiments will
help reveal the mechanism by which MexT exerts its effects on its target genes.
Salicylate; a repressor of oprD transcription and possible cofactor of the LysR-type
transcriptional regulator protein, MexT.
Given the observed apparent homology between the transcriptional regulators, MexT and
NahR, and the previous reports that salicylate, one of NahR's natural cofactors, reduced
expression of a 45kDa protein in P.aeruginosa, we were intrigued with the possibility
that salicylate reduced oprD expression via the MexT protein.
Salicylate is a membrane-permeable weak acid and is known to suppress the synthesis of
some outer membrane porins in a number of Gram negative bacteria (Burns et al., 1992;
Sawai, et al., 1987). The mechanism by which this occurs has not been elucidated thus
far. In the presence of 32 m M salicylate, the levels of a 45kDa protein in the outer
membrane and the susceptibility of P.aeruginosa to the carbepenem, imipenem, were
reduced. This was consistent with previous reports (Sumita and Fukasawa, 1993 and
Masuda et al., 1995). However, this thesis is the first report confirming that the 45kDa
protein with reduced expression was indeed OprD (Fig. 12A). Using an oprDr.xylE
chromosomal fusion, I determined that the mechanism by which salicylate reduces oprD
expression was at the level of transcription. When grown in 32 m M salicylate, oprD
expression was reduced by 3.3 fold compared to normal (Table VII). This level is similar
to the level of repression seen when mexT was overexpressed (Table V). While the
60
involvement of another regulator cannot be ruled out, I hypothesize here that salicylate
does indeed repress oprD expression via the MexT protein. Unfortunately, a mexT knock
out, which would have allowed the direct testing of this hypothesis, could not be obtained
during the course of this study. Its construction is ongoing in the lab and will hopefully
allow us to conclusively show whether or not MexT represses oprD expression in
response to salicylate.
Masuda et al. (1995), reported that increased resistance to ofloxacin and the induction of
a protein of approximately 50kDa occurred in the presence of 32 m M salicylate. This
protein was not heat modifiable and was therefore thought to be OprN and not OprM. In
the paper by Fukasawa and Sumita (Fukasawa and Sumita, 1993) however, no change in
the M I C to quinolones was seen and no expression of a heat unmodifiable protein was
noted in the presence of 32 m M salicylate. Our results showed an M I C profile consistent
with that of an nfxC-type mutant but, as stated in the results, there was only a two fold
difference in any of the M I C values (Table VI). These changes are not considered
significant enough to suspect meaningful induction of an efflux operon. The outer
membrane profiles showed that only OprD expression was changed and that there were
wild-type levels of expression of OprM and the usual lack of expression of either OprJ or
OprN (Fig. 12). These data seem to suggest that salicylate alone does not induce
expression of the mexEF-oprN operon. This does not contradict the hypothesis that MexT
down-regulates oprD in response to salicylate. LysR-type regulators often regulate
different genes in response to different signals. MexT may repress oprD in response to
salicylate while not affecting expression of the mexEF-oprN operon. A s stated
61
previously, work to purify the MexT protein is underway in the lab. Promoter binding
studies in the presence and absence of salicylate along with the knockout of the mexT
gene, will hopefully shed some light on whether salicylate indeed affects MexT
interactions with its target D N A promoter sequences.
Sodium benzoate reduced the levels of OprD in the outer membranes of P.aeruginosa
almost to the same level as seen with salicylate (Fig . 12 and 13, lane 4). Similarly, no
change in production of any of the efflux operon was observed (Fig. 13 B and C, lanes 4
and 6). Unlike salicylate though, sodium benzoate was found not to reduce transcription
of the oprD gene. No conclusions can be drawn from this except to say that the
mechanism of repression of oprD expression would be presumed to be posttranscriptional.
Iron and zinc as possible signals for efflux operon expression.
While the role of the three mex operons of P.aeruginosa in antibiotic efflux has been
widely documented (for a review see Nakae, 1998), their real function and natural
substrates, i f any, await identification. It was originally proposed that the mexAB-oprM
operon is involved in the excretion of the siderophore pyoverdine (Poole et al., 1993).
More recently this theory has come into question (Poole, 1994). To try and elucidate
whether or not iron concentrations in the growth medium were actually involved in the
expression of any of the efflux operons, I looked at the effect of varying concentrations of
iron on the MICs of P.aeruginosa to various antibiotics. Apart from a general increase in
62
susceptibility when the iron concentrations were limited (Table VIII), there were no
noticeable changes in the pattern of susceptibility to any of the antibiotics tested. These
increases in susceptibility can be simply explained by the reduced growth rate in minimal
media, like B M 2 , compared to rich media, like M H .
Another ion suspected in the induction of the efflux operons in P. aeruginosa is zinc. Two
independent reports have shown that the high concentrations of zinc in the media can give
rise to levels of imipenem resistance that are considered clinically significant (Cooper et
al., 1993; Daly et al., 1997). Concentrations of 6 p.g/ml zinc were shown to significantly
increase resistance to imipenem. This was consistent with the results obtained here, in
which the M I C for imipenem rose from 1 u,g/ml to 4 ug/ml in the presence of
supplementary zinc (Table VIII). This did still not represent a rise that would be
considered 'resistant' but is a significant increase in M I C for imipenem.
Previous studies have shown that cations such as zinc, calcium and magnesium affect
aminoglycoside activity against P.aeruginosa (Riller et al., 1974; Casillas et a l , 1981).
However, in general, cation influence on P-lactam activity has not been frequently
described. Zinc-dependent P-lactamases have been isolated from only a few bacteria
including from one P.aeruginosa isolate (Watanabe et al., 1991). Nonetheless, it is highly
unlikely that zinc induction of P-lactamase production is a possible explanation for the
increased resistance to imipenem in the presence of high zinc concentrations. Such a Plactamase would have to have limited activity against antipseudomonal cephalosporins,
as seen from the only two fold increase in resistance to cefpirome (TableVIII), and would
be able to specifically inactivate imipenem. Only one P-lactamase is present in the
63
P.aeruginosa P A O genome and it has been well described as having properties that could
not be used to explain this zinc effect. A n enzyme which inactivates carbepenems but not
cephalosporins has been found in Serratia marcescens (Yang and Livermore, 1990), but
not in P.aeruginosa. Interestingly, it is activated by zinc. A n alternative explanation to
the increased resistance to imipenem in the presence of high zinc concentrations, is that
zinc may effect the binding of imipenem to their target PBPs. This has been shown in
other bacteria but as yet not in P.aeruginosa (Sabath et al., 1990).
Could zinc be acting by altering the uptake of imipenem into the cell? A n obvious
candidate for this was the imipenem specific porin, OprD. Even when grown in high
levels of zinc (12u.g/ml), there was no change in OprD expression (Fig. 13). Additionally,
there was no change in expression of any of the outer membrane components of the mex
efflux operon, namely, OprM, OprJ and OprN (data not shown). This was consistent with
the MIC data obtained (Table VIII), where no increase in MIC was observed in any of the
antibiotics known to be substrates of these efflux pumps. A final alternative for the
observed increase in resistance to imipenem in the presence of zinc, is that zinc may be
altering the chemical properties of imipenem prior to uptake into the cell reducing its
activity.
Poole et al. isolated a mutant that was deficient in the production of the siderophore,
pyochelin, and the ferripyochelin receptor (Poole et al., 1993). This mutant when grown
in iron-deficient B M 2 media supplemented with 0.1 m M ZnS0 , induced expression of a
4
protein of -50 kDa that was not heat modifiable, later thought to be OprN. The outer
membrane profile also showed reduction in a protein of approximately 45 kDa, probably
x
64
OprD. Another independent report also suggested that zinc induced OprN expression in
media containing high zinc concentrations (Hofte et al., 1991). As shown in Table VIII,
MICs done under these very low iron and high zinc conditions resulted in no growth,
although growth was observed in the preculture before the start of the assay.
Interestingly, i f the preculture was grown under the same conditions except for the
presence of zinc, an M I C could be determined. This would indicate that in the presence of
zinc, iron limitation is so severe that the susceptibility of P. aeruginosa to the antibiotics
tested was high enough to result in killing even at low antibiotic concentrations.
Unfortunately, the question of whether or not the mexEF-oprN operon is indeed
expressed under these conditions is unresolved. If expression of this efflux operon can be
shown under these conditions, it would shed a lot of light onto possible natural roles of
these systems.
Expression and role of the mex efflux operons in P.aeruginosa.
The prevalence of PMF-dependent multidrug systems in a diversity of organisms raises
several questions. What is the normal physiological role of such efflux systems? Is their
primary role to protect the cell from toxic compounds in the environment? Or do they
play roles other than detoxification, such as the transport of a particular substrate? In
which cases are their abilities to efflux a wide range of structurally unrelated compounds
only fortuitous rather than by design?
65
As yet it is not possible to answer these questions definitively, although, light is being
shed slowly on the natural roles of these systems. The broad substrate specificity of these
systems suggests that their natural role is to 'detoxify' the cells by excreting either toxic
compounds from the environment that enter the cell, or getting rid of toxic by products
produced by the cells metabolic processes. As stated previously the P.aeruginosa mexABoprM is highly expressed under conditions of iron starvation but is also expressed
constitutively at low levels even in the presence of excess iron (Poole et al., 1993). On the
other hand, nothing is know about the expression of the mexCD-oprJ and mexEF-oprN
efflux operons. The most likely stage for the expression of these efflux operons is in the
conversion stage from late log to stationary phase, when there is a build up of metabolic
byproducts. Our experiments show that there is no change in expression of any of the
three known efflux operons in P.aeruginosa under the growth conditions tested (Fig. 14).
This seems to suggest that these efflux operons, especially the mexCD-oprJ and mexEFoprN efflux operons are tightly regulated and are not expressed under 'normal' lab
growth conditions. A possible role for the mexEF-oprN efflux operon might be to excrete
toxic byproducts from the metabolism of basic amino acids. Since OprD is known to be
involved in the uptake of basic amino acids in P.aeruginosa and that it is linked via a
common transcriptional regulator, MexT, it is conceivable that when the cellular levels of
toxic compounds reach a certain level, it turns on the efflux pump to 'detoxify' the cell
while turning off any further OprD production. Recent data from the lab using the
P.aeruginosa strain harboring a oprDr.xylE chromosomal fusion and grown in medium
where the basic amino acid arginine was used as the sole carbon source, suggest that in
stationary phase, oprD expression is reduced (Ochs, unpublished data).
66
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