Download A Strategy to Identify Novel Antimicrobial Compounds

A Strategy to Identify Novel Antimicrobial Compounds
– A Bioinformatics and HTS Approach
Sara Garbom
Department of Molecular Biology
Umeå University
Umeå, Sweden, 2006
Copyright © 2006 by Sara Garbom
ISBN 91-7264-190-8
PRINTED BY ARKITEKTKOPIA AB, UMEÅ, SWEDEN, 2006
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Ju mer man tänker, ju mer inser man att det inte finns
något enkelt svar
/Nalle Puh
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Table of Contents
List of Papers ................................................................ 6
Abstract ....................................................................... 7
Abbreviations ................................................................ 8
Introduction.................................................................. 9
THE THREAT OF BACTERIAL INFECTIONS................................................. 9
ANTIBIOTICS AND RESISTANCE..........................................................9
MECHANISMS OF ANTIBIOTICS AND THE DEVELOPMENT OF RESISTANCE .................. 9
THE RIBOSOME AND TRANSLATION..................................................... 12
IDENTIFICATION OF TARGETS AND ANTIMICROBIAL COMPOUNDS .................... 15
DISCOVERING ANTIMICROBIAL COMPOUNDS ............................................ 15
WHAT IS A GOOD TARGET?............................................................ 16
TARGETING VIRULENCE AND IN VIVO SURVIVAL GENES ................................. 16
EXPERIMENTAL APPROACHES TO IDENTIFY TARGETS ................................... 17
GENOME COMPARISONS TO IDENTIFY TARGETS ........................................ 18
EVOLUTION AND CONDENSATION OF MICROBIAL GENOMES .......................... 20
MINIMAL GENOME CONCEPT ........................................................... 20
GENOME CONDENSATION .............................................................. 20
YERSINIA—OUR MODEL SYSTEM ...................................................... 23
YERSINIAE ............................................................................. 23
INVASION AND THE BATTLE AGAINST THE IMMUNE SYSTEM ............................. 23
REGULATION OF THE T3SS ............................................................ 27
YERSINIA—AN INTRACELLULAR PATHOGEN?............................................ 28
General and Specific Aims .............................................. 30
Results and Discussion ................................................... 31
IDENTIFICATION OF NOVEL VIRULENCE-ASSOCIATED GENES (PAPER I).............. 31
EVALUATION OF THE VAGS IN A MOUSE MODEL OF INFECTION YIELDED 5 ATTENUATED
MUTANTS ............................................................................. 32
VAGA ................................................................................. 33
VAGH ................................................................................. 34
PHENOTYPIC CHARACTERIZATION OF THE VAGH MUTANT (PAPER II) ............... 34
VAGH IS A SAM-DEPENDENT METHYLTRANSFERASE ................................... 35
MICROARRAY AND 2D-GEL ANALYSIS AND A LINK TO THE T3SS ....................... 35
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THE VAGH MUTANT BEHAVES LIKE A T3SS STRAIN IN THE MOUSE MODEL OF INFECTION
AND IN THE INTERACTION WITH MACROPHAGES ........................................ 36
VAGH AND THE CONNECTION TO THE T3SS IN MORE DETAIL (PAPER III) ........... 37
SCREENING FOR VAGH INHIBITORS (PAPER IV) ...................................... 39
Conclusions................................................................. 41
Tack!!!....................................................................... 42
References ................................................................. 44
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List of Papers
This thesis is based on the following publications referred to in the text by their roman
numerals (I-IV).
I
Sara Garbom, Åke Forsberg, Hans Wolf-Watz, Britt-Marie Kihlberg
Identification of novel virulence-associated genes via genome analysis of
hypothetical genes
Infect Immun. 2004 Mar;72(3):1333-40. Reprinted with permission from the publisher.
II Sara Garbom, Martina Olofsson, Ann-Catrin Björnfot, Manoj Kumar Srivastava
Victoria L. Robinson, Petra C.F. Oyston, Richard W. Titbull, Hans Wolf-Watz
Phenotypic characterization of a virulence associated protein, VagH, of Yersinia
pseudotuberculosis reveals a tight link between VagH and the T3SS
Submitted manuscript
III Sara Garbom, Hans Wolf-Watz
The negative regulator YopD regulates LcrF expression on the translational level
as studied by a vagH mutant of Yersinia
Manuscript
IV Sara Garbom, Martina Olofsson, Hans Wolf-Watz, Mikael Elofsson
Small molecule inhibitors of the methyltransferase VagH rescue cells from T3SSdependent toxicity
Manuscript
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Abstract
Bacterial infections are again becoming difficult to treat because the microbes are growing
increasingly resistant to the antibiotics in use today. The need for novel antimicrobial
compounds is urgent and to achieve this new targets are crucial. In this thesis we present a
strategy for identification of such targets via a bioinformatics approach. In our first study
we compared proteins with unknown and hypothetical function of the spirochete
Treponema pallidum to five other pathogens also causing chronic or persistent infections in
humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia
burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for
the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at
identifying conserved proteins important for in vivo survival or virulence of the pathogens
we reasoned that T. pallidum would have deleted genes not important in the human host.
This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our
model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was
evaluated in a mouse model of infection. Five genes were found to be essential for
virulence and thus constitute possible antimicrobial drug targets.
We have studied one of these virulence associated genes (vags), vagH, in more detail.
Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine
dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis
also showed that very few genes and proteins were differentially expressed in the vagH
mutant compared to wild-type Yersinia. One major finding was that expression of the Type
III secretion system effectors, the Yops, were down regulated in a vagH mutant. We
dissected this phenotype further and found that the down regulation was due to lowered
amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD
or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that
YopD in addition to its role in translational regulation of the Yops also plays a part in the
regulation of LcrF translation. We suggest also that the translation of LcrF is particularly
sensitive to the amount of translation competent ribosomes and that one effect of a vagH
mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in
turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS.
This down regulation is likely the cause of the attenuated virulence of the vagH mutant.
Finally, we set up a high throughput screening assay to screen a library of small
molecules for compounds with inhibiting the VagH methyltransferase activity. Five such
compounds were identified and two were found to inhibit VagH also in bacterial culture.
Furthermore, analogues to one of the compounds showed improved inhibitory properties
and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on
HeLa cells.
We have successfully identified five novel targets for antimicrobial compounds and in
addition we have discovered a new class of molecules with antimicrobial properties.
Keywords: Antibiotic resistance, Yersinia, T3SS, virulence associated genes, VagH,
HemK/PrmC, Release factors, small molecular inhibitors, HTS.
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Abbreviations
•
A, P, E-site
Acceptor, Peptidyltransfer, Exit sites on the ribosome
•
A, U, T, G, C
Adenine, Uracil, Thymine, Guanine, Cytosine
•
Ca
Calcium
•
Da
Dalton
•
EF-G, EF-TU
Elongation factors
•
G, Q
Glycin, Glutamine
•
GI
Gastrointestinal
•
GTP, GDP
Guanosine-5'-triphosphate, Guanosine-5'-diphosphate
•
3
Tritium
•
HTS
High Throughput Screen
•
ID50
50% Infectious dose
•
IF1, 2, 3
Initiation factors
•
IVET
In Vivo Expression Technology
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Kb, Mb
Kilo-basepairs, Mega-basepairs
•
Lcr
Low Calcium Response protein
•
M-cells
Microfold cells
•
MDR
Multi Drug Resistance
•
MTase
Methyltransferase
•
PP
Peyer´s Patches
•
RF1, RF2
Release Factor 1, Release Factor 2
•
RNA
Ribonucleic acid, mRNA – messenger, tRNA – transport
H
rRNA – ribosomal
•
RRF
Ribosomal recycling factor
•
SAM
S-adenosyl-methionine
•
SD
Shine-Dalgarno
•
STM
Signature Tagged Mutagenesis
•
T3SS
Type III Secretion System
•
vag
Virulence Associated Gene
•
Yop
Yersinia Outer Protein
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Introduction
The Threat of Bacterial Infections
Bacterial infections were a big problem before the introduction of antibiotics (penicillin) in
the 1940s, and millions of people died annually from various bacterial infections. In the
1960s it was proclaimed that the threat of bacterial infections was a problem of the past and
that the war against microbes was won. This has, however, unfortunately been proven
wrong over the last decades with the appearance of microbes resistant to the antibiotics in
use. Resistance to antibiotics is an emerging problem worldwide, and novel antibiotics are
necessary to combat infections of resistant pathogens.
This thesis deals with the possibilities of identifying novel antibacterial drug targets
and compounds to meet the development of resistant pathogens.
Antibiotics and Resistance
Mechanisms of Antibiotics and the Development of Resistance
Clinically important pathogens such as Staphylococcus aureus, Streptococcus pneumoniae,
and Mycobacterium tuberculosis are becoming multiresistant to antibiotics and difficult to
treat [1]. In the past 30 years, only two new classes of antibiotics have reached the
market—oxazolidinone and lipopeptide daptomycin—and most new antibiotics are simply
modifications of or combinations of already present compounds [2]. Resistance toward
penicillin was found already in the mid-1940s, within two years of the introduction of the
compound on the market [3]. The reasons for the rapid development of resistance to
antibiotics by microorganisms are manifold and a net outcome of the fitness increase and
the cost of the alterations introduced to confer resistance. The alterations can be either
mutations in genes or acquisition of new gene elements; the first is exemplified in the
resistance toward rifampicin, which is conferred by a mutation in the gene encoding for the
RNA polymerase subunit ß, rpoB [4], and the latter by the resistance to ß-lactams (that is,
penicillin) by the introduction of a gene-expressing ß-lactamase [5]. Gene acquisition can
occur by three different processes: transformation, transduction, or conjugation [6].
Resistance to a particular antibiotic can develop in alternative ways in different situations: a
gene encoding ß-lactamase can be introduced conferring resistance; secondly, acquisition of
efflux pumps prevent accumulation of the antibiotic inside the bacteria; thirdly, the
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synthesis of the cell wall is altered so that the antibiotic no longer can bind; and finally,
down regulation of porins that allow the influx of the antibiotics may occur—all of these
alterations lead to resistance [7]. The antibiotics available today target many different
aspects of cell metabolism and growth; the mechanisms and resistance mutations are
summarized in Figure 3 and Table 1. A clinical case demonstrating the rapid spread of
resistance involves a four-year-old girl who was severely ill with an Escherichia coli
infection initially resistant to ampicillin and narrow-spectrum cephalosporins but not thirdgeneration cephalosporins. During the course of treatment (two months), the bacteria
became resistant to the third-generation compounds through the acquisition of an additional
ß-lactamase gene. A fraction of the bacteria had also lost the surface expression of the
OmpF porin involved in the influx of the antibiotics, giving rise to additional resistance.
The bacteria thus evolved different mechanisms of resistance in the blood of the infected
child in a short period of time. [7]
Some bacteria are naturally resistant to antibiotics, and the soil-living bacteria
belonging to the Streptomyces genus are natural antibiotic producers as well as resistant to
many of the antibiotics, both synthetic and natural compounds, in use today [8]. How, and
if, the resistance elements can be transferred directly from soil bacteria to human pathogens
is not known, but excessive use of vancomycin on farms is believed to have contributed to
S. aureus resistance toward this compound, and the resistance elements could be transferred
via soil bacteria [9].
Fig. 1. Antibiotics and targets. See Table 1 for references and more details.
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Table 1. Antibiotics, mechanisms, and resistance
Class/Antibiotic
ß-lactams &
cephalosporins
Function
Acetylates (and inhibits) active
sites of transpeptidases
involved in peptidoglycan
synthesis [10].
Resistance mechanism
Acquisition of a gene coding for
ß-lactamase that disrupts the ring
structure of the antibiotic [11] or
mutations in the transpeptidases
[12].
Glycopeptides
Binds to D-Ala-D-ala dipeptide
in the peptidoglycan layer of
the cell wall of G+ bacteria,
inhibiting cell growth.
Peptide antibiotics that disrupt
the outer membrane [14].
Modification of the peptidoglycan
recognition site for the antibiotic
[13].
Flouroquinolones
Inhibits DNA replication by the
targeting of DNA gyrase [16].
Mutations in DNA gyrase and
drug efflux [17].
Rifampicin
Inhibits mRNA synthesis by
binding to RNA polymerase
[18].
Binds to the ribosomal exit
tunnel, inhibiting translation
[19]. Inhibits the formation of
the 50S subunit by binding to
precursors [20].
Binds to the 30S subunit and
blocks tRNA binding to the A
site [24, 25].
Cationic inhibitor of
translation. Binds to 16S RNA
in the A site of the ribosome,
affecting ribosome accuracy
[27].
Mutations in the gene encoding
RNA polymerase, rpoB [18].
Oxazolidinones
Interacts with 23S RNA and
inhibits translation [31].
Mutations in 23S RNA [31].
Trimethoprim &
sulfonamides
Inhibits the production of
tetrahydrofolate, and thereby
the production of purines,
methionine, and thymidine
[32].
Mutations in the target enzymes
or introduction of an enzyme
resistant to the compounds [32].
Polymyxins
Macrolides
Tetracyclins
Aminoglycosides
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Changes in the lipopolysacharide
inhibit the binding of the
antibiotic [15].
Methylation of the 23S RNA
inhibits binding [21] or
production of peptides binding the
macrolides [22, 23].
Mainly drug efflux [26].
Modification of the antibiotic by
acetylation, phosphorylation, or
adenylation [8], or by mutations
[28] and methylation [29, 30] of
the 16S RNA.
High-level resistance often requires that several consecutive mutations occur. The
initial mutation is enough for a slight resistance toward the antimicrobial compound,
allowing for sequential mutations in this population, conferring high-level resistance [7,
33]. The alterations introduced that provide resistance often decrease the overall fitness of
the bacteria, and this can be compensated for by additional mutations, so-called
compensatory mutations [33, 34]. The bacteria can also revert back to the original
genotype, but this is less likely as there are many more possibilities for compensatory
mutations compared to reversion [34]. The compensatory mutations can be intragenic and
intergenic as well as result in increased amounts of a protein produced or duplication of the
target to allow for multiple functions [34]. The introduction of compensatory mutations
often stabilizes the original mutation as a strain with only the compensatory mutation
having decreased fitness, but in combination with the original mutation, it leads to an
increase of fitness [34]. It has also been shown that spread of resistance is delayed if the
mutations necessary for resistance inflict a high fitness cost for the microbe; such mutations
are unlikely to spread as the decrease in fitness would be too disadvantageous [35]. There is
also data that combinations of different antibiotics and a multitude of targets delay
development of resistance and make the drugs more efficient, as for trimethoprim and
sulfamethoxazole [36, 37]. Both antibiotics target tetrahydrofolate synthesis but at different
stages. The combination of the two drugs is more efficient, and the bacteria have to develop
two resistance mechanisms to survive, thereby delaying resistance.
The Ribosome and Translation
The ribosome is a very large, complex structure, and its importance for bacterial survival
has made it an excellent target for antibiotics, as seen in Table 1. With the increased
knowledge about the structure of the ribosome, the process of translation has been
elucidated. In many instances translational inhibitors, such as antibiotics, have been used to
freeze the ribosome in a certain position before crystallization to clarify the different steps
of translation. These studies have also revealed that although the ribosome is a much
conserved structure, the mechanisms for inhibition of an antibiotic might differ between
organisms [38]. Translation can be divided into four steps: initiation, elongation,
termination and, finally, ribosome recycling. Each step requires the correct positioning of
the ribosome and the recruitment of the right factors. Initiation of translation occurs before
transcription has ended, making transcription and translation a coupled event in bacteria.
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This requires three initiation factors (IF1, IF2, IF3), initiator tRNA and, of course, the
ribosome and an mRNA with a Shine-Dalgarno (SD) sequence. The ribosome consists of
two subunits (30S and 50S) (see Figure 2 for a schematic overview of ribosome structure)
that dissociate prior to initiation. This dissociation is triggered by IF3 binding to the 30S
subunit, and the action of IF3 is enhanced by IF1 [39, 40]. IF2, initiator tRNA, and mRNA
thereafter associate with the small subunit. The SD sequence interacts with the anti-SD on
the ribosome, and the start codon is adjusted so that it enters the P site of the ribosome [41].
This complex is unstable and undergoes changes forming the initiator 30S complex;
thereafter IF1 and IF3 dissociate from the complex, and IF2 aids in the binding of the 30S
to the 50S subunit, forming the 70S ribosome [42, 43]. The initiator tRNA is now
positioned in the P site, IF2 is released, GTP is hydrolyzed to GDP in the process, and the
initiator 70S complex is now ready for elongation. The A site of the ribosome now has the
second codon of the mRNA in place; Ef-Tu:GTP bound to an elongator tRNA is recruited
and bound to the second codon on the mRNA. The decoding process is very complex, as
can be expected for such an important feature. Recent crystal structures have somewhat
elucidated this event; conformational changes of the ribosome are induced when a tRNAEf-Tu:GTP is bound to the mRNA, and this allows elongation to proceed if there is a match
between the tRNA and the mRNA in the A site [44-47]. Ef-Tu itself is also involved in this
process, and after correct positioning, Ef-Tu:GTP is hydrolyzed to Ef-Tu:GDP and
released. Thereafter the tRNA “swings” into the peptidyltransferase center, and a peptide
bond is spontaneously and rapidly formed [48]. Crystal structures of the ribosome bound to
a peptidyltransferase inhibitor (“Yarus inhibitor”) proved that it is the 23S rRNA that is
responsible for this bond formation [49, 50]. At this time, the P-site tRNA is deacetylated,
and the peptide chain has grown with one amino acid in the A site; for elongation to
proceed, a translocation of the tRNAs in the A and P sites must occur so that the A site
again is free to accept a new tRNA on the next codon. The translocation process is not fully
understood, but it requires EF-G and GTP hydrolysis for efficient movement, and it likely
includes a hybrid state with the tRNAs bound in both A/P and P/E sites simultaneously
[51]. The elongation now proceeds until the ribosome reaches a stop codon on the mRNA
(UAA; UGA or UAG). For termination of translation to occur, release factors are essential
as no tRNAs exist for these codons. There are two class I release factors in bacteria: RF1
recognizing UAA and UAG, and RF2 recognizing UAA and UGA [52]. Eukaryotes, on the
other hand, have only one class I release factor, eRF1, which recognizes all stop codons.
RF1/2 binds to the stop codon in the A site and thereby promotes the release of the newly
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synthesized peptide. A universally conserved essential motif, GGQ, the glutamine (Q) in
this motif if post-translationally modified to a N5-glutamine by the SAM-dependent
methyltransferase HemK [53-55]. This domain is believed to interact with the
peptidyltransferase center, the methylation increases the efficiency of translational
termination of the release factors although not essential for function [53]. Once the peptide
is released, a class II release factor, RF3, induces the release of RF1/2 [56]. The complex
ribosome machinery needs to be recycled for another round of translation; this is catalyzed
by the binding of ribosome recycling factor (RRF), IF-3, and EF-G [57-59]. This process is
not fully understood and it is not clear in what order the mRNA, peptidyl-tRNA, and
ribosome subunits dissociate.
During translation ribosomes can stall at for instance rare codons, if the amount of a
certain tRNA is low, or if the mRNA contains secondary structures difficult to translate for
some reason. Trans-translation is a process where stalled ribosomes are rescued by
recruitment of SsrA and SmbP. SsrA can act as both a tRNA and an mRNA and when a
ribosome has stalled the complex is recruited and SsrA, charged with an alanine, adds an
alanine to the growing peptide and thereafter acts as an mRNA for the ribosome adding a
specific tag to the peptide directing the faulty peptide to degradation. The ribosome is
thereafter released and recycled as after normal termination. [60]
Fig. 2. Structure of the ribosome subunits and the interaction with tRNA and mRNA..
(A) The individual subunits, 30S and 50S, with the active sites labeled A=entry,
P=peptidyltransfer, E=exit. (B) The associated subunits, 70S, in complex with mRNA, and
a tRNA seen from the side.
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Identification of Targets and Antimicrobial Compounds
Discovering Antimicrobial Compounds
Early in drug discovery, most compounds were discovered through whole-cell screening,
searching for compounds inhibiting growth; initially this method was very successful but
later proved limiting in that most screens resulted in the same molecules identified. This
lead to a shift in the strategies used, a shift toward target-based screening methods where
high throughput screening (HTS) is applied to screen large collections of random molecules
for inhibitory effects on, for instance, enzymatic activity. [61] To be able to perform HTS
for compounds inhibiting a certain enzymatic activity, a potential target first has to be
found; different ways to do this are discussed in the sections below. The process of
identifying novel antimicrobial compounds is outlined in Figure 3.
Fig. 3. Outline for finding potential antimicrobial drugs. First, a target is identified,
which is followed by a functional analysis of the target and development of an assay to
screen for compounds inhibiting the target protein function. When a lead compound has
been identified, this can be further improved with chemical genetics and modification of the
identified compounds. This is then followed by characterization of the compounds,
mechanisms, toxicity, and so on.
The advantages of a HTS strategy are that the targets of the compound found are known
and that the mechanism of action is thereby easier to deduce. One disadvantage is that high
throughput screens for inhibition of enzymatic activity often lead to compounds that are
unable to enter the bacterial cell or are efficiently pumped out of the cell by already present
efflux systems [62], whereas whole-cell screening systems avoid these molecules already at
the screening stage. There are, however, ways to modify the molecules so that they are
efficiently taken up by the bacteria, arguing that the whole-cell screening method has the
disadvantage of missing inhibitory molecules not entering the cell [63]. It is also possible to
modify an initial lead compound with weak inhibitory effects through structure-activity
relationship (SAR) analysis. FabI is an enzyme involved in the metabolism of fatty acids,
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and an initially identified lead compound inhibiting this enzyme exhibited low or no
antibacterial activity. However, through SAR analysis specific inhibitors with a 350-fold
higher activity were identified [64].
What Is a Good Target?
The first step in performing a HTS is to find a target to pursue; different strategies to find
targets are discussed in the following sections. First, one has to decide what a good target
is. The general criteria for a good target are that it should exhibit a function essential for
either growth or pathogenesis of the bacteria, be expressed during infection, and also have
an enzymatic activity that can be modified [2]. It is also preferred if the target does not have
a close human homologue to avoid toxicity problems further down the line. This does,
however, seem to be rather gene specific—as exemplified by trimethoprim, a specific
inhibitor of bacterial dihydrofolate reductase—even though the identity to the human
homologue is 28% [65, 66]. In some cases the human homologue is closer to a particular
bacterial drug target than the bacterial targets are to each other, and still the drug is
effective and nontoxic [63]. Metabolic enzymes are considered good targets; in a study of
the metabolic pathways essential for in vivo survival and pathogenesis of Salmonella, the
authors concluded that very few novel broad-spectrum antimicrobial drug targets could be
found in this group [67]. The study combined already published results with new in vivo
expression data, virulence data, and metabolic networking. Enzymes with homologues in
humans or no homologues in distantly related pathogens were discarded. The low amount
of in vivo essential genes was attributed to metabolic robustness due to functional
redundancy and to the host’s providing the microbe with nutrients, which renders the
bacteria nondependent on its own metabolic pathways in vivo [67]. The apparent limitation
in essential enzymes might be why so few novel antibiotics have been developed during the
past 30 years [67]. However, there is much we do not know about microbes and their
pathogenesis and in the group of genes with an unknown or hypothetical function,
additional good targets may exist. This group (and other genes) probably contains suitable
targets that are not metabolic enzymes but perhaps regulators, adhesins, virulence factors,
and so on.
Targeting Virulence and in vivo Survival Genes
Most antibiotics today target proteins or functions essential for bacterial survival per se;
another strategy is to target proteins directly involved in virulence, for example, type 3
secretion (T3S) components or proteins involved in in vivo survival of the pathogens but
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not essential for growth outside the host. The strategy not to directly target growth of the
microorganism is thought to delay the development of resistance [68]. All clinically used
antibiotics today inhibit growth or kill bacteria in vitro [2], so the idea to target virulence
genes or in vivo essential genes has not yet been evaluated. Nevertheless, compounds
inhibiting the type 3 secretion system (T3SS) have been identified in a number of studies
[69-72]. The targets for these small molecules are not known, but they have the potential to
be effective against all microbes that use the T3SS in infection, that is, Salmonella spp.,
Yersinia spp., Shigella spp., Pseudomonas aerigunosa, E. coli, and Chlamydia spp. [73].
Molecules that inhibit the enzymatic activity of the phosphatase YopH secreted by Yersinia
spp. have also been developed as a potential treatment for plague [74]. In another study,
mice could be protected from infection of Vibrio cholerae by virstatin, as it inhibits the
transcriptional regulator ToxT, which regulates virulence genes [75]. Quorum sensing (QS)
is important for many pathogens for proper regulation of virulence genes and also survival
inside the host; several studies have examined the potential of QS components as targets for
antimicrobial compounds [76-79]. Another important feature for many pathogens in vivo is
iron acquisition from the host during infection [80-82], and a study identified small
molecules inhibiting the iron siderophore biosynthesis in Yersinia pestis and M.
tuberculosis [83, 84]. These studies emphasize the potential of targeting either virulence
genes or genes important for survival in the host. The selection pressure for development of
resistance could be lower when the targets are not essential for growth per se and not
important or nonexisting in the bacteria of the normal gut flora, thereby perhaps delaying
the spread of resistance through horizontal gene transfer between microbes.
Experimental Approaches to Identify Targets
Experimental techniques to identify targets involved in virulence and not in vitro growth
include signature-tagged mutagenesis (STM) and in vivo expression technology (IVET).
STM is a technique introduced about 10 years ago making it possible to screen several
mutants simultaneously for their importance in virulence [85]. A pool of random transposon
mutants is used to infect an animal; the mutants not capable of colonizing the animal are
considered to carry mutations in genes important for virulence. Each mutant carries a
unique tag, the input and output pools of bacteria are compared, and the gene with an
insertion resulting in a strain killed by the host is identified with sequencing. This technique
has over the years been employed on 31 pathogens (until 2005) identifying 1,700 genes
important for in vivo survival of the pathogens (Reviewed in [86] and [87]). The majority of
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the genes identified are surface molecules (25%–30%), stressing their importance for
pathogens in vivo. Also interesting is that up to 50% of the genes identified have an
unknown function [87]. STM has also identified genes that are true virulence factors such
as the SPI-2, encoding a second T3SS of Salmonella in a screen by Shea et al. [88]. The
advantage of this technique is that it reduces the number of animals used and the identified
genes are all potentially good targets for antimicrobials. IVET and other techniques
evaluating the expression of genes in vivo have the limitation that after a gene is identified
as being differentially expressed, the gene has to be disrupted and the virulence of the
mutant evaluated. Also, genes that are equally expressed in vitro and in vivo can still be
interesting for evaluation, and genes expressed only in vivo are perhaps not essential for the
pathogen. Random transposon mutagenesis (or targeted mutagenesis) can also be used to
identify genes essential for growth if that category of targets is of interest.
Genome Comparisons to Identify Targets
Another way to limit the amount of genes screened, and also the number of animals used, is
to employ in silico methods. Today >350 microbial genomes are completely sequenced,
and almost 600 additional projects are underway (NCBI web site). With the techniques
available today, it is possible to sequence the genome of a microbe in a day or two. A lot of
the genomes sequenced are genomes of pathogens, and this has revolutionized the
possibilities to, via bioinformatics approaches, identify genes that constitute good
antimicrobial drug targets. Comparisons of genomes of closely related pathogens and the
subtraction of genes present in nonpathogenic bacteria or bacteria habituating a different
environmental niche yield targets that are specific for the related pathogens. This can be
used to, for example, identify targets important only for colonization of the lungs or the
intestine. Antibacterial compounds directed toward these targets are narrow in spectrum.
The identification of conserved genes in a wide variety of pathogens will instead yield
targets that are suitable for broad–spectrum antibiotics. In the comparisons, it is beneficial
to include microbes with condensed genomes to avoid identification of genes nonessential
for in vivo survival as these genomes have deleted genes that are not necessary for survival
in contact with a eukaryotic cell. The in silico analysis has to be followed by experiments
determining whether the genes identified are indeed essential for survival or virulence of a
pathogen. To do so, it is important to use an animal model where the expression of the
target genes can be investigated and where the virulence of a mutant can be assessed.
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With the increased amount of whole genome sequences of bacteria available, there was
a great anticipation that the information within the sequences would instantly yield many
new antimicrobial drug targets. This has not been the case. However, a few drug targets
have been found or validated with the aid of genomics, such as peptide deformylase
inhibitors, compounds targeting fatty-acid biosynthesis, and aminoacyl-tRNA synthetase
inhibitors [1, 89]. Genome comparisons have also been used to screen genomes for already
known targets to assess against which pathogens a certain antibiotic will be effective [1].
The publishing of the human genome presented the possibility to investigate whether the
targets are present in the host and potential toxicity problems can thereby be avoided [67,
90]. But, as stated before, this might exclude potential targets to which the antimicrobial
drug would not induce toxicity, even though a close homologue is present in humans [63].
Zheng et al. have created a database of essential genes (DEG), in which experimental data
from a number of different studies have been incorporated into a searchable database [91].
This database is continuously updated and freely available, and it was used in a study where
essential genes of P. aerigunosa were identified [92]. The clusters of orthologous groups
(COG) database is a database in which proteins from different organisms are categorized
into families based on function and homology, and it is here easy to analyze the potential
spectrum of an antimicrobial drug directed toward a known target; eukaryotic genomes are
also included, so the presence of eukaryotic homologues can be evaluated [93, 94].
19
Evolution and Condensation of Microbial Genomes
Minimal Genome Concept
The concept of defining the minimal genome to sustain life has intrigued researchers for a
long time, as it was believed that a core set of genes essential for life in all microorganisms
could be identified. These genes would then constitute good antimicrobial drug targets, as
they would be essential for growth of all bacteria. When the first two sequenced genomes
of microbes, Haemophilus influenzae and Mycoplasma pneumoniae, were compared, the
minimal genome was believed to consist of the 256 genes conserved between these two
distantly related bacteria [95]. As more complete genome sequences are available, it is
becoming evident that even fewer genes are conserved throughout evolution; only 74 genes
are conserved in all microbes sequenced so far [96, 97]. There are insufficient universally
conserved genes to maintain basic cellular processes such as transcription and translation,
which means that microbes have evolved in different ways to minimize the size of the
genome and that nonhomologous genes/proteins can have the same function [96, 98]. This
complicates any attempt to define a minimal genome by computational methods. Using a
more experimental approach, genes have been knocked out one by one in various
organisms to determine which genes are essential and which should thus be included in the
minimal genome [99-104]. This approach is limited in that a particular gene may not be
essential if other genes are present, but a combination of deletions of two nonessential
genes might lead to death. Because of this, some genes essential for sustaining life are
missed, and therefore these methods do not really define the minimal set of genes. In fact,
the minimal set of genes required for life will vary depending on the nutrients available and
other growth conditions, such as temperature, oxygen, and osmolarity. It is perhaps not
interesting to define the set of genes required to sustain life under the most favorable
conditions but instead to study the naturally occurring genome condensation of obligate
parasites in order to define the set of genes necessary to sustain life under different
conditions [96, 97].
Genome Condensation
The eukaryotic cell provides a very stable, nutrient-rich environment, which is taken
advantage of by parasites living inside the cells. The microbe can simply benefit from the
nutrient-rich environment, or the relationship can be beneficial for both partners as for the
endosymbiont Buchnera aphidocola and its host, the aphids. The diet of the insect is very
20
low in amino acids, but this is instead provided for by the bacteria, and in return the
bacteria receive other nutrients from the host [105-107]. Another example is
Wigglesworthia glossinidia that colonizes tsetse flies and provides the host with vitamin B
that the fly cannot acquire from its diet [108]. The genomes of the endosymbionts are very
small and have evolved from much larger genomes. B. aphidicola is believed to have
diverged from an ancestor of E. coli, from the time when the ancestor colonized the aphids
approximately 75% of the coding capacity of the microbe has been lost [109]. Now most of
the genomes of these species are around 600 kb, but recently the genome sequence of a
Buchnera (Buchnera aphidicola BCc) with a genome size of 422kb was reported [110].
Interestingly, this strain has lost the capacity to synthesize tryptophan, an amino acid that
the host cannot obtain from its diet and usually is provided for by a strain of B. aphidicola.
This amino acid is instead likely provided for by a second endosymbiont, providing both
the host and the B. aphidicola with this essential nutrient [110]. Another striking finding is
the sequencing of Carsonella ruddii, this organism has a genome size of 160 kb encoding
for only 182 genes [111]. This is by far the smallest genome sequenced to date and it is
hypothesized that genes previously encoded for by the microorganism now instead has been
integrated into the chromosome of the host as it has been for organelles such as the
mitochondria [111].
The frequency of deletions and rearrangements was higher in the beginning of the hostmicrobe relationship, probably with large deletions of genes of unrelated function [106,
109]. The rate has decelerated over time, and now the genomes of the endosymbionts are
more stable than their free-living relatives [96, 106, 109]. Genome reduction is driven by
the host-microbe interaction; depending on the host, the microbe will lose genes in
metabolic pathways no longer necessary, because the nutrients can be acquired from the
host. In some cases, as described above for B. aphidicola and W. glossinidia, the microbes
will retain genes important for the host to maintain the host-microbe interaction. These
endosymbiont genomes have also reduced their mutational capacity by the deletion of recA
and also the removal of repetitive sequences; this factor and also probably deletions of
other recombination pathways make these genomes very stable [96, 106, 109]. The
genomes of parasites living in contact only with eukaryotic cells have also undergone
reductions. The spirochete Treponema pallidum is believed to have lost approximately 75%
of its previous coding capacity; the size of the genome is now 1.1 Mb [112]. T. pallidum,
the causative agent of syphilis, is an obligate human parasite that cannot be cultured
continuously in vitro [113-115]. Through the interaction with the eukaryotic host cell, it has
21
deleted genes necessary for de novo nucleotide synthesis, cofactor synthesis, and fatty-acid
synthesis [112]. A clear indication that a genome is in the process of adaptation is a large
quantity of pseudogenes; the pseudogene is thereafter deleted resulting in a reduced
genome size [116]. Gene elimination usually starts with a single base change, the
insertion/deletion of a single base, or the introduction of insertion elements (IS) into a gene
inactivating its expression, creating a psuedogene [116]. Gene rearrangements and deletions
can also be large, deleting several genes with different functions in one event. The selective
advantage of a small genome is not clear, but the reduction of genome size is probably the
result of the pathogen living in a stable environment where many genes are useless and an
increased genetic drift due to population bottlenecks [98]. The deletion of genes is not
always beneficial for the microbe; even genes that are beneficial are inactivated by chance,
and due to the small population size, these mutations can stabilize, even though there is no
increase in fitness for this population but rather a decrease [117, 118]. Deletions are also
balanced by acquisition of novel genes through horizontal gene transfer; this is also less
frequent for microbes living in an environment where few other organisms exist, also
contributing to the stability of these genomes.
The microbes with small, condensed genomes are often difficult to study in the lab, and
therefore model organisms are often used instead. In our studies, we have used Yersinia
pseudotuberculosis as a model organism to study the virulence of a set of human pathogens,
of which some are difficult to cultivate and modify. Y. pseudotuberculosis is a well-studied
bacterium easy to cultivate in vitro with a number of genetic tools available for mutagenesis
and modification. For a pathogen to be a useful model organism, it is also important that an
animal model is available for the evaluation of virulence. Mice infected with Y.
pseudotuberculosis develop a plague-like disease [119]. Animals can be infected via the
oral, subcutaneous, intravenous, or intraperitoneal route, and the outcome is the same—
systemic infection and death. The mouse model is, of course, not equivalent to infections in
humans, but it is still an acceptable model for evaluating virulence attributes of pathogens.
22
Yersinia—Our Model System
Yersiniae
Y. pseudotuberculosis is a gram-negative, rod-shaped protobacteria belonging to the
Enterobacteriacea family. The genus Yersinia consists of 11 species; of these, Y.
enterocolitica, Y. pseudotuberculosis, and Y. pestis are known to cause disease in humans
[120, 121]. Y. pestis is, in contrast to the two other strains, a facultative pathogen with
limited ability to survive outside its hosts (humans and rats) and perhaps the most famous
of the three responsible for causing plague outbreaks in history. Y. enterocolitica and Y.
pseudotuberculosis, on the other hand, cause enteritis and mesenteric adenitis that usually
are self-limiting within two weeks in healthy individuals. In rare cases reactive arthritis is a
complication of Yersinia infection [122, 123]. Although very similar to Y.
pseudotuberculosis on the genetic level, Y. pestis causes a dramatically different disease.
Interestingly, the two other strains—Y. enterocolitica and Y. pseudotuberculosis—that
induce similar symptoms are evolutionary more distantly related [124]. Of the genes in Y.
pseudotuberculosis (IP32953), 75% are >97% identical to their counterparts in Y. pestis
(CO-92), and it is believed that Y. pestis evolved from Y. pseudotuberculosis very
recently—between 1,500 and 20,000 years ago [125]. Y. pestis has acquired two additional
plasmids by horizontal gene transfer since the divergence from Y. pseudotuberculosis:
pPCP1 and pMT1. The plasmids encode genes important for tissue invasion [126], capsule
formation [127], and infection of the flea vector [128, 129]. Y. pestis CO-92 also carries
147 unique pseudogenes compared to Y. pseudotuberculosis [130], indicating that the
genome is in the process of eliminating genes not necessary for survival in the new niche.
Genes in the process of being deleted encode for proteins involved in processes important
for an enteropathogen but not for the new transmission route of Y. pestis via the flea vector
such as genes important for adhesion and motility [131]. The increased virulence of Y.
pestis is not easy to understand from genomic analysis; the reason for the difference in
pathogenicity is likely a combination of gene inactivation and acquisition [130]. One
common plasmid, and virulence feature, of the three pathogenic strains is a 70 kb virulence
plasmid encoding a T3SS described in more detail below.
Invasion and the Battle against the Immune System
Y. pseudotuberculosis and Y. enterocolitica are transmitted via the oral-fecal route, and
infected humans have usually ingested contaminated food or water. If infected via the oral
23
route, Y. pseudotuberculosis first encounters the epithelial-cell layer in the gastrointestinal
(GI) tract. Microfold cells (M cells) are specialized cells in the GI tract that sample antigens
in the lumen and allow macrophages to pass through from the underlying lymphoid tissue
[132]. The M cells are positioned over the Peyer’s patches (PP) of the lymphoid tissue, and
the sampling ability of the M cells is used by many bacteria to reach the lymphoid
circulation [133]. Invasin is a 103 kDa adhesion molecule that via ß1-integrins binds to the
M cells and promotes uptake of the bacteria; it is present in Y. enterocolitica and Y.
pseudotuberculosis but not in Y. pestis [134]. Invasin is enough to promote uptake into
cultured epithelial cells and turn a noninvasive Escherichia coli into an invasive organism
[135]. If invasin is not present on the surface of Yersinia, the bacteria cannot efficiently
bind to M cells, but are still capable of causing systemic disease [136, 137]. The route of
entry in this case is not known, but it has recently been shown that Yersinia can multiply in
the intestine and thereafter spread to spleen and liver in mice lacking PP [138]. In the same
study, the authors also could show that the pool of bacteria first entering the PP of wildtype C57BL/6J mice was rapidly cleared from this site of infection, not reaching the liver
and spleen in contrast to the pool of bacteria first replicating in the intestine. The authors
could not conclude how the bacteria entered the deeper tissues, such as the liver and spleen
if not via the PP. YadA is another adhesion molecule present on the surface of Yersinia. It
is not required for virulence of Y. pseudotuberculosis. A yadAinv double mutant is at least
as virulent as the wild-type strain [139, 140], and by the deletion of yadA in an inv
background, the strain again can invade M cells by an unknown mechanism [141]. YadA
binds to collagen, laminin, and fibronectin; it confers serum resistance; and YadA also
causes auto-agglutination and fibril formations [142-144]. pH6 antigen is an additional
adhesion molecule involved in hemagglutination and attachment to eukaryotic cells; it is
essential for the virulence of Y. pestis but not the other Yersiniae likely due to the fact that
Y. pestis lacks functional invasin and YadA [130, 145-147]. Apart from the adhesion
molecules, other chromosomally encoded genes important for virulence have also been
identified. STM of Y. pseudotuberculosis identified many genes involved in polysaccharide
synthesis (LPS core and O antigen) and also genes encoding phospolipase A, RseA, a
negative regulator of sigma 24 transcription factor, and proteins with unknown functions
[148]. In addition, the ribosomal rescue system trans-translation has been shown to be
important for virulence [149].
When Yersiniae reach the PP, liver, spleen, or other deep tissues, it has to combat the
immune system of the host. The bacteria first encounter parts of the innate immune
24
system—for example, macrophages and neutrophils. Yersinia is viewed mainly as an extra
cellular pathogen, although it can replicate inside macrophages (discussed below). To stay
extracellular, it has a sophisticated system to avoid phagocytosis, T3SS. The T3SS is a
secretion system that is conserved among a wide variety of gram-negative pathogens,
ranging from the symbiotic microbe Rhizobium, which infects plants, to Y. pestis. It can
play diverse roles in the pathogenesis; for Salmonella, it is important for the invasion of
eukaryotic cells; for enteropathogenic E. coli (EPEC), it is involved in attachment to cells;
and for Yersinia, it is used to avoid uptake [134, 150, 151]. Common characteristics of
these secretion systems are: induction of secretion upon cell contact, delivery of toxins into
the cytoplasm of the eukaryotic target cell, and also a needle-like structure protruding from
the bacterial cell surface. The needle is attached to the basal body of the T3S machinery,
spanning the inner and outer membranes of the bacteria creating a hollow conduit through
which the toxins are believed to be secreted (see Figure 5). The T3S machinery has
extensive homology to the components of the flagella, and it is believed to have developed
from a flagella ancestor [73]. It is important to note that secretion through the needle-like
structure and translocation via a pore in the eukaryotic cell membrane induced by Yersinia
has not been shown conclusively. Therefore, other possibilities for the process of
translocation from the bacterial cytoplasm into the eukaryotic target cell are possible. The
process is, however, dependent on a functional T3S apparatus, and the toxins are
transported into the target cell in a T3SS-dependent manner. The functions of the various
toxins, the Yops and YpkA, have been studied extensively the past decades, and the cellular
targets for most are now elucidated; this is summarized in Figure 4. The results of the
effects of the toxins translocated are inhibition of phagocytosis, apoptosis, and
inflammation. This enables the bacteria to stay extracellular, and to avoid detection and
clearance by the immune system. No single Yop is essential for virulence in the mouse
model of infection, although it is important at different stages. But the absence of both
YopE and YopH in the same strain renders the bacteria unable to colonize mice [152]. The
effects of YopE on epithelial cells can be easily evaluated in a cellular model system. The
actin cytoskeletal rearrangements caused by the enzymatic activities of YopE can be scored
morphologically by studying the effects on epithelial cells such as HeLa cells; see Figure 7.
In vitro expression and secretion of the T3SS can be induced by the depletion of calcium
from the growth media (Figure 5). The relevance of this calcium induction is not clear, but
it provides a nice tool to study regulation and function of the T3SS in a test tube.
25
Fig. 4. The toxins and targets. YopH is a potent phosphatase (PTPase) [153]
dephosphorylating a number of eukaryotic target molecules [154-159] leading to inhibition
of phagocytosis [134], inactivation of T and B cells [160-162], and inhibition of Ca2+
signaling in response to Yersinia’s binding to ß1-integrins [163]. YopE is a GTPaseactivating protein (GAP) [164, 165] targeting RhoA and RacI in vivo [166]. The
downregulation of these small GTPases lead to actin cytoskeleton rearrangements [164,
165, 167, 168], inhibition of phagocytosis [134, 169], prevention of Yersinia-induced pore
formation in the eukaryotic cell membrane [170], and disruption of tight junctions [171].
YpkA is a Ser/Thr kinase [172] that binds to actin [173] and small GTPases [174, 175],
which is probably the cause of the actin disruption properties of YpkA [174-176], and it
also phosphorylates otubain 1 [177]. YopM is a scaffolding protein interacting with
antitrypsin 1 [178] and two kinases modulating their activity [179]. It has also been shown
to localize to the nucleus [180]. YopJ is an acetyltransferase (ACTase), and its activity
inhibits NFκB-dependent apoptosis [181]. LcrV is not translocated but situated at the tip of
the needle-like structure of Yersinia [182]. It interacts with toll-like receptor 2 (TLR2) and
inhibits the secretion of IL-10, inhibiting the inflammatory response [183]. It is also
essential for the translocation of the other toxins [184].
26
Fig. 5. HeLa cells infected with Yersinia, (A) yopE mutant (B) wild-type. The actin
cytoskeleton is disrupted by the action of YopE [185] and in infection with a yopE mutant
strain the HeLa cell remains flat. (C) Calcium response of Yersinia. In media lacking
calcium the expression of the Yops is induced. (D) Schematic picture of the T3Smachinery. (D) T3S-apparatuses purified from Salmonella and visualized by electron
microscopy.
Regulation of the T3SS
LcrF is the main positive regulator of the T3SS in Yersinia. LcrF belongs to the AraCfamily of transcriptional regulators [186-188]. The transcription of this activator is
regulated by changes in DNA topology and also on the level of translation by formation of
a secondary structure hiding the SD-sequence [189, 190]. When bacteria are grown at 37°C,
LcrF expression is high, which in turn activates the expression of effectors and machinery
genes. At lower temperatures Ymo, a small histone like protein encoded for on the
chromosome, inhibits transcription from the yop promoters [191, 192]. YmoA is degraded
by ClpXP and Lon proteases at elevated temperature thereby relieving the transcriptional
inhibition [192]. There is also an negative regulatory loop at work at 37°C before cell
27
contact and also when bacteria are grown in media containing calcium, this loop consists of
the virulence plasmid encoded proteins LcrQ, YopD, and LcrH [193-195]. YopD is a
multifunctional protein; in addition to its role in regulation, it is directly involved in the
translocation process, and it is also translocated itself [185, 195, 196]. LcrH is a chaperone
for YopD and also YopB in addition to its function in regulation [193, 197]. LcrQ does not
have any other known roles besides regulation, and it is not under the regulatory control of
LcrF [194]. It has been hypothesized that LcrQ, LcrH, and YopD together form a complex
capable of inhibiting translation of the Yops until cell contact is achived. LcrQ is thereafter
sequestered by SycH and secreted [198-201], the complex is dissolved and translation can
occur [202, 203]. An additional regulatory level is imposed by the secretion machinery
itself; if the machinery is not correctly assembled, the negative regulators are not secreted
and the system remains repressed. Also, LcrG and YopN are believed to form lids on the
inside and outside of the machinery, preventing premature secretion [204, 205].
Yersinia—an Intracellular Pathogen?
It has been debated over the years whether Y. pseudotuberculosis has an intracellular stage
in the infection process [206-209]. The discrepancies in the results obtained in cell culture
between these studies are likely due to the different serotypes examined. YPIII (serogroup
O3) is not able to replicate inside of J774.1 cells, while strains of serogroup O4 are. The
difference between serogroups is also evident for Y. enterocolitica with some strains
capable of intracellular replication and some not [210-213]. Y. pestis has for a long time
been known to be able to replicate inside macrophages and neutrophils, and this ability is
believed to be important early in infection before the T3SS has been turned on and the
bacteria are able to avoid uptake [214-216]. The situation is different for the
enteropathogens, because the orally ingested bacteria grow at the T3SS-inducing
temperature (37°C) for some time before encountering neutrophils and macrophages. But
even when the T3SS is induced, about 50% of the bacteria are internalized by macrophages
[134], and the ability to survive and replicate inside the macrophages might be important
for this fraction of bacteria [216]. There is also evidence of intracellular Yersinia when
tissue sections from animals infected orally were analyzed early in infection (4 hours); the
bacteria could then be found inside monocytic cells [217]. The relevance for this feature is
not clear, but hypotheses include protecting from neutrophils, avoiding antigen presentation
(thereby delaying the immune response), and using macrophages as a vehicle to reach
deeper tissues [216]. The intracellular replication is not dependent on the plasmid-encoded
28
T3SS [208], and in addition to this, Y. pestis and Y. pseudotuberculosis harbor one more
T3SS on the chromosome that is similar to the SPI-2 of Salmonella. In Salmonella this
system is essential for survival inside macrophages [218], but when deleted in Yersinia, no
effect on intracellular replication was observed [216]. The two component system
PhoP/PhoQ is required for the ability to replicate inside macrophages, and a strain without
phoP is attenuated in a mouse model of virulence, which suggests that intracellular
replication is important in vivo [219].
29
General and Specific Aims
The aim of this thesis has been to develop a strategy for the identification of antimicrobial
drug targets present in a wide spectrum of human pathogens. It also includes studying these
target proteins in the model pathogen Y. pseudotuberculosis and evaluating their potential
as drug targets. This process can be divided into the following steps:
1.
Identification of targets (Paper I).
2.
Target evaluation in a model system of virulence (Paper I).
3.
Functional studies of targets (Papers II & III).
4.
Development of an assay to perform a HTS for inhibitors (Papers II & IV).
30
Results and Discussion
In this section, the main findings of the papers included in the thesis are presented and
discussed.
Identification of Novel Virulence-Associated Genes (Paper I)
With the increasing threat of microbes’ becoming resistant to available antibiotics, it is
essential to identify new targets for antimicrobial compounds and also to develop new
strategies for finding such targets. Two different classes of targets exist: genes that are
essential for growth of the microbe and those that are only essential for growth in vivo or
for virulence of the pathogen. Bioinformatics can be of great help when selecting targets. In
our first paper, we took a bioinformatics approach to identify novel virulence-associated
genes (vags) that constitute possible antimicrobial drug targets. We compared the genomes
of distantly related human pathogens, and the target genes identified are thus suitable for
the development of broad-spectrum antibiotics. The pathogens we chose to compare were:
Treponema pallidum, Borrelia burgdorferi, Neisseria gonorrhoeae, Helicobacter pylori,
Streptococcus pneumoniae, and Yersinia pestis. T. pallidum, the causative agent of syphilis,
was the starting point for our comparisons; it has a small and condensed genome (1.1 Mb)
[112] that reflects its parasitic lifestyle. The condensed genome served as a good start for
identifying genes essential for survival within the human host, and as we wanted to identify
novel genes, we limited the starting gene pool to genes annotated as unknown or
hypothetical (215 genes). To make the search nonbiased for codon usage, we used protein
sequences and the BLASTP alignment tool in all of our comparisons. The sequences were
compared to five additional microbes with parasitic lifestyles; the features of these are
summarized in Table 2. We considered two sequences as homologous if the comparison
yielded an E-value of <10-7, which resulted in 17 open reading frames that were conserved
in all species (see Table 1, Paper I for a complete list). These proteins (Vags) are all
potential targets for antimicrobial compounds, but to be interesting, the protein has to
contribute to the in vivo survival of the pathogens. To evaluate this, we deleted the genes
encoding these proteins in our model organism Yersinia pseudotuberculosis. This
bacterium is a very close relative to Y. pestis; in fact, Y. pestis is believed to have evolved
from Y. pseudotuberculosis less than 20,000 years ago [130, 220].
31
Pathogen
N. gonorrhoeae
B. burgdorferi
Table 2. Pathogens included in the comparison
Disease
Characteristics
Gonorrhea
Gram-negative
bacteria, human
specific, and host
dependent.
Lyme disease
Spirochete, humanspecific pathogen, and
host dependent. Small
genome, 1.6 Mb.
S. pneumoniae
Pneumoniae,
meningitis
Gram positive. Part of
the normal
nasopharyngeal flora.
H. pylori
Stomach ulcers,
gastric cancer
T. pallidum
Syphilis
Y. pestis
Plague
Gram negative.
Colonizes 80% of the
population in the
Western world.
Spirochete, obligate
parasite. Condensed
genome, 1.1 Mb.
Facultative pathogen.
Recent clone from Y.
pseudotuberculosis.
Potential bio-weapon.
Resistant to
Penicillin,
cephalosporins,
quinolones [6].
Not a big problem.
Resistance to
erythromycin has
been reported
[221].
Penicillin,
macrolides,
cephalosporins,
tetracyclines, and
MDR strains [6].
Tetracycline,
macrolides,
quinolones [222].
Macrolides [223].
Rare cases of
multi-drug and
tetracycline
resistance [224].
Evaluation of the vags in a Mouse Model of Infection Yielded 5 Attenuated
Mutants
Unlike its relative Y. pestis, Y. pseudotuberculosis can grow freely in the environment, and
there is also a well-established animal model for evaluation of the virulence of this
pathogen. Strains with mutations in 14 out of the 17 genes identified were constructed, and
the virulence of these insertion mutants was evaluated in C57/BL6 mice infected via the
oral route. Out of the 14 mutants investigated, 9 displayed an attenuated phenotype when
compared to the wild-type strain. We could not create mutations in 3 of the genes, and these
are likely essential for growth of Y. pseudotuberculosis in vitro. Because we were interested
only in genes not essential in vitro but in vivo, these genes were not further analyzed. In this
first screen, we created insertion mutants, and as bacteria often have their genes organized
in operons, insertions in the first gene of an operon will probably silence all the
downstream genes. We now created in-frame deletions of the 9 genes identified in this first
32
screen. We again infected mice orally with the mutant strains, and out of the 9, 5 were still
attenuated (Paper I, Table 3). The mutants were also analyzed with respect to growth, Yop
secretion, and ability to induce a cytotoxic response on HeLa cells. Yop secretion and
cytotoxicity are important virulence traits for Y. pseudotuberculosis and dependent on a
functional T3SS. The deletion mutants grew as wild-type in rich media, but the vagH
mutant had a growth defect when grown in minimal defined media (MDM).
In an attempt to validate our approach, we searched the literature for genes implicated
in virulence by, for example, STM [85, 225-227] and selected capture of transcribed
sequences (SCOTS) [228]. These genes were then analyzed in the same way as the
hypothetical and unknown genes of T. pallidum. 99 proteins were compared; 5 were
conserved in all species (Paper I, Table 2). Out of the 5 mutants constructed, 3 were
attenuated in the mouse model. These results confirm that pathogens do rely on similar
genes and functions for in vivo survival and virulence, and they also confirm that our in
silico analysis resulted in genes important for the virulence of several microbes.
VagA
The vagA gene encodes for a universally conserved GTPase termed YchF in E. coli. The
function for this GTPase is unknown, but a study regarding the bacteria Brucella melitensis
16M implicated that the homologue is involved in iron metabolism [229]. It was, however,
not essential for virulence for this human pathogen causing chronic fever [229, 230]. In an
STM analysis of Neisseria meningitides, an important cause of meningitis especially in
children, the vagA homologue was found to be important for virulence [231]. Avian
pathogenic E. coli (APEC) is also dependent on a functional vagA gene for virulence [232],
and in Chlamydophila pneumoniae, the gene was upregulated in response to IFN-γ
treatment [233]. In a study where essential genes in E. coli were identified with transposon
mutagenesis, ychF was noted as essential [234] for growth in rich media. In our study the
vagA mutant has a growth phenotype indistinguishable from wild-type, both in rich media
and MDM (minimal defined media); this makes VagA a good antimicrobial drug target by
our definition, together with the findings of vagA as being important for virulence in other
organisms. In collaboration with the labs of M. Rehn and B. Henriques-Normark, the
homologues of vagA have been deleted in Salmonella typhi and S. pneumoniae, and the
resulting mutants are attenuated in animal models of virulence (unpublished results), further
validating the potential of vagA as a target for broad-spectrum antibiotics and also the
approach taken to identify genes important for virulence in a broad spectrum of pathogens.
33
VagH
The vagH mutant, in addition to a growth defect in MDM, showed lower Yop secretion as
well as a slightly delayed cytotoxicity towards HeLa cells. It also displayed the most severe
virulence attenuation. VagH is homologous to HemK in E. coli shown to be an S-adenosylmethionine (SAM)–dependent N5-methyltransferase transferring a methyl group to Release
Factors 1 and 2 (RF1 and RF2) [53, 55]. This protein and its function in Y.
pseudotuberculosis are discussed in more detail in Paper II. Homologues to vagH have
been implicated in several virulence studies since ours. In EPEC, the homologue is
upregulated in infected patients [235], and in Porphyromonas gingivalis, one of the
causative agents of adult periodontitis, the gene was also found to be induced in epithelial
cell cultures by Park and coworkers [236]. The expression of the vagH homologue in S.
pneumoniae has in two different studies been shown to be induced in vivo [237, 238], and
in one case the operon containing the vagH homologue was also shown to be important for
virulence [238], but in the other study a deletion of the gene did not affect virulence [237].
However, the implications of induced expression of vagH homologues in other pathogens
makes this gene interesting for further evaluation; see Papers II, III, and IV.
Phenotypic Characterization of the vagH Mutant (Paper II)
We decided to study the vagH mutant further and to evaluate the potential of VagH as an
antimicrobial drug target. HemK was first misannotated as protoporphyrinogen oxidase
[239] and later hypothesized to be a DNA methyltransferase [240] before its correct
function as a glutamine methyltransferase was established [53, 55]. Glutamine
transmethylation is very unusual; only one additional case is known in bacteria: the
methylation of the ribosomal protein L3 [241, 242]. The targets of the MTase activity of
HemK in E. coli are RF1 and RF2 [53, 55]. These proteins are class I release factors
involved in translational termination. When a translating ribosome encounters a stop codon
on an mRNA, the release factors are recruited to decode the stop codon and induce the
release of the synthesized peptide. The importance of the methylation is different in E. coli
K-12 compared to most other bacteria due to a threonine in position 246 of RF2 in K-12;
this amino acid is an alanine or a serine in other organisms. The threonine renders RF2
more dependent on the methylation for efficient function [243]; that is also why a hemK
mutant has a reduced growth rate that can be rescued by a mutation changing the threonine
to an alanine [53, 55]. In line with this, the vagH mutant grows as wild-type in rich media
(Paper I).
34
VagH Is a SAM-Dependent Methyltransferase
We first wanted to establish whether VagH possessed the same MTase activity as HemK.
We set up an assay developed by Colson et al. [244] using purified VagH or HemK
together with total protein lysates prepared from different strains and incubated this
together with radio-labeled SAM ([3H]-SAM). The incorporation of [3H]-CH3 was
measured by scintillation counting or by autoradiography. From these experiments (Figures
1–3 in Paper II), it was clear that VagH also is a SAM-dependent methyltransferase
transmethylating the same targets as HemK. The results also showed that the targets are
saturated in the wild-type strain and also that the RF2 of Y. pseudotuberculosis migrates
differently compared to the E. coli homologue in a SDS-PAGE gel (Figure 3, Paper II). The
difference in theoretical molecular weight between these two proteins is only 42 Da, and
the difference in apparent molecular weight is not easily explained due to differences in
sequence or methylation status. We have tried to solve this issue by careful
masspectrometry analysis (Åke Engström, IMBIM) but not been successful; another
strategy could be to create hybrids between the E. coli and Y. pseudotuberculosis proteins
to identify the domain responsible for this difference.
Microarray and 2D-Gel Analysis and a Link to the T3SS
Now that we had established the function of VagH, we wanted to study the effects of a
mutation in vagH on a large scale, genome, and proteome. To achieve this, we performed
microarray and 2D-gel proteome analysis comparing the vagH mutant and the wild-type
strain. The results from these analyses are presented in Tables 1 and 2 in Paper II. In both
assays, very few genes/proteins showed differential expression levels in the mutant
compared to the wild-type. In a microarray analysis of the hemK mutant in E. coli, 260
genes were differentially expressed [55], but in our case only 22 genes were found to have
different expression levels. This might reflect the differences in position 246 in RF2 of the
two organisms and the growth reduction of the hemK mutant. Of the 22 genes with different
expression levels, 6 ribosomal genes were downregulated in the vagH mutant, indicating
that the balance of ribosomal proteins is altered in the mutant. The proteome analysis
prompted us to analyze an iron-binding protein 2.8-fold downregulated in the mutant. Iron
acquisition is important for Yersinia in vivo [245], and therefore this was an interesting
finding. However, when this gene was deleted, no difference in virulence between the
mutant and wild-type was observed in the mouse model of infection. YopD, a part of the
T3SS, was upregulated twofold in the mutant; this was interesting as we, in Paper I, found
35
the Yop secretion of a vagH mutant to be downregulated. YopD is involved in both
negative regulation of the T3SS [195] and the process of translocation of the toxins into the
eukaryotic target cell [196]. As we here grew the bacteria under noninducing conditions
(37°C, 2.5 mM calcium), the data are not necessarily conflicting; an upregulation of a
negative regulator is in agreement with the results of the previous experiments. To establish
whether the effects on the T3SS of VagH were dependent on its MTase activity, we
overexpressed either wild-type or enzymatically inactive VagH in the vagH mutant and
analyzed Yop secretion. The enzymatically inactive variant of VagH could not transcomplement the vagH mutant with respect to Yop secretion, but the wild-type VagH could.
The effect of VagH on the T3SS is discussed in more detail in Paper III.
The vagH Mutant Behaves like a T3SS Strain in the Mouse Model of Infection
and in the Interaction with Macrophages
We were also interested in doing a more careful virulence study of the vagH mutant and
analyzing where the mutant was cleared from the mice and whether it was capable of
reaching deeper tissues (liver, spleen). We infected mice orally and followed the infection
over time by dissecting PP and spleens. The vagH mutant behaved like a Y.
pseudotuberculosis strain cured of the virulence plasmid (encoding the T3SS) incapable of
reaching deeper tissues and also unable to proliferate in PP (Figure 4, Paper II). When mice
were infected intraperitoneally, the vagH mutant was severely attenuated with an ID50 value
500-fold higher than wild-type (ID50 wild-type; 3.2x103 cfu; vagH; 1.6x106 cfu), again
demonstrating the importance of vagH in virulence. The effects on Yop secretion together
with the fact that the mutant behaves like a T3SS null strain in the animal model lead to the
conclusion that the virulence attenuation observed is due to the lack of a properly
functioning T3SS.
To rule out other possibilities contributing to the virulence attenuation, we also
investigated the vagH mutant and its response to eukaryotic cells such as J774.1 cells
(mouse macrophage cell line) and HeLa cells (epithelial cell line). Y. pseudotuberculosis is
capable of intracellular growth in macrophages. The survival in macrophages is not
dependent on the T3SS [208], but it is important for virulence [219]. To study a virulence
feature not dependent on the T3SS, we analyzed the ability of the vagH mutant to replicate
inside macrophages and found it to be similar to the wild-type strain (Figure 6a, Paper II).
In contrast to this, when the mutant is grown in presence of macrophages allowing for
T3SS induction and extracellular growth, the mutant did not proliferate as well as the wild-
36
type (Figure 6b, Paper II). If cultivated in the presence of HeLa cells, the mutant and wildtype again showed similar phenotypes. HeLa cells are nonprofessional phagocytes in
contrast to macrophages. The vagH mutant does possess a functional T3SS, because it is
able to translocate YopE into HeLa cells, causing cytotoxicity (Paper I); it can therefore
inhibit phagocytosis by the epithelial cells. In the interaction with macrophages, on the
other hand, the reduction of Yop secretion might be enough for these phagocytes to kill the
bacteria.
The results in this paper strongly suggest that the attenuated virulence phenotype of the
vagH mutant is mainly due to the effects the deletion of vagH has on the T3SS. We can also
conclude that VagH is a SAM-dependent MTase targeting RF1 and RF2.
VagH and the connection to the T3SS in more detail (Paper III)
As we had found a link between VagH and the T3SS we wanted to dissect this connection
further. The first step we took was to identify at what level the effects of vagH mutation
had on the T3SS. We did this by insertion of a transcriptional fusion of yopE to luxAB
under the native yopE promoter. This creates a fusion where we can estimate the
transcriptional activity of the yopE promoter by measuring the light activity of LuxAB.
Fig. 6. A schematic overview of the yopE:luxAB fusion mRNA. If an inhibitor binds to
the 5´-UTR of the yopE:luxAB transcript this will decrease the amount of YopE produced
compared to LuxAB if the inhibitor is not present.
37
We can also use this construct to measure the translational efficiency of YopE over LuxAB,
see Figure 6 for an overview, as the proteins have individual ribosomal binding sites and
thereby independent translation. When performing this analysis we found that the vagH
mutant had lower output from the yopE promoter, and also decreased efficiency of
translation of yopE over luxAB, compared to the wild-type strain (see Paper III, Figure 1).
The transcription could be restored to wild-type levels by an additional deletion of the
negative regulator yopD, this deletion also restores the translational efficiency of YopE. In
a single yopD mutant strain the efficiency of translation of YopE is increased indicating, in
line with previous reports [193, 202, 203], that YopD inhibits translation of yopE. A
deletion of lcrG, however, does not restore either transcription or translation in a vagH
mutant strain although a single lcrG mutant has a more efficient translation of yopE
compared to wild-type. LcrG and YopD belong to two different classes of regulators; it is
believed that LcrG exerts its regulatory function by inhibiting premature secretion of the
negative regulators [205]. The finding that a vagH mutant is affected differently by a
mutation in lcrG and yopD is interesting as it discriminates between these two classes of
regulatory mutants. Further studies of these two double mutants (vagHlcrG and vagHyopD)
will probably elucidate the regulatory functions of both LcrG and YopD.
LcrF is the central positive regulator of the T3SS in Yersinia, and in addition to its
previously described role as a transcriptional activator we have found evidence that it also
plays a role in translational regulation of YopE expression. Over expression of LcrF from
an inducible promoter increases the efficiency of translation of yopE in all strains tested
(vagH, lcrG, yopD, vagHlcrG, vagHyopD, and wild-type), see Figure 3, Paper III. This
could be due to a direct effect of LcrF or an indirect effect through the increased expression
of a protein under the control of LcrF. We reasoned if the latter was the case this would be
a protein encoded for on the virulence plasmid and we therefore over expressed all of the
proteins encoded for by the yscB-yscL operon. No positive effects of over expression of
these proteins on the translational efficiency of YopE were observed. However, over
expression of YscH and YscI reduced the induction of the T3SS. We also investigated
potential effects of LcrV, LcrG, YscU but found non of these proteins to influence the
YopE translation. Thus, to the best of our knowledge, it is a direct effect of increased
amounts of LcrF that improves the efficiency of translation of YopE.
We also found evidence of YopD acting as a negative regulator of LcrF translation. In
a vagH mutant, the level of LcrF is reduced although no effect is seen on the transcription
of lcrF (measured by a lcrF:luxAB fusion). This suggests that the translation of LcrF is
38
reduced in the vagH mutant, this reduction can be suppressed by a deletion of yopD arguing
for a role of YopD in translational regulation of lcrF similar to its role in regulation of yopE
translation. The finding that YopD regulates the expression of LcrF connects the negative
regulatory loop with the positive loop.
Another way to increase the amount of LcrF in a vagH mutant is to over express the
ribosomal recycling factor (RRF) (see Figure 4, Paper III). Over expression of RRF
probably increases the amount of translation competent ribosomes in the bacteria as it is
involved in ribosome recycling. It is plausible that a vagH mutant has less free ribosomes
due to lowered efficiency of translational termination. A hemK mutant of E. coli shows
increased amounts of tmRNA tagged proteins, an indication of stalled ribosomes [55].
These results suggest that translation of LcrF is particularly sensitive to the amount of
available free ribosomes perhaps due to a weak ribosomal binding site or blockage of this
site. Over expression of RRF also increases the expression of YopE, possibly both by
increasing transcription and translation and this is achieved by the increased amount of
LcrF.
Screening for VagH Inhibitors (Paper IV)
Now that we had elucidated the function of VagH, we set up a high throughput screen
searching for molecules inhibiting the VagH enzymatic activity. A small molecule library
containing 9,400 molecules was screened using the assay described in Paper II with the
exception that a potential inhibitor was added to each reaction mixture. In the screen, we
identified five compounds that inhibited the in vitro activity of VagH. Two molecules
(compounds 2 and 3, Figure 1, Paper IV) belong to the same chemical class. Compound 1
was also identified as its 2-(2-naphtyl)-acetic acid salt indicating that the screening assay is
robust and reproducible. Two additional molecules belonging to the same class as 2 and 3
were also present in the library. These were retested but again failed to inhibit the reaction;
this result, and the fact that our assay picked up similar molecules, validates the screening
method.
For these compounds to be interesting as possible antimicrobial agents, they have to be
able to enter the bacteria and inhibit the VagH enzymatic activity inside the bacteria. To
evaluate this, we took advantage of the effect that the MTase activity of VagH has on Yop
expression (see Papers I, II, and III). Bacteria were grown in the presence of compounds 1-
39
5 under conditions known to induce the T3SS (37°C and depletion of calcium). To evaluate
the induction of the T3SS we monitored the expression of one of the Yops, YopB (Figure 2,
Paper IV). Only compounds 2 and 3 had an effect on the expression of YopB. We do not
know the reason why the other compounds now failed to inhibit VagH; possible
explanations include that the compounds do not enter the bacteria, or that they are
hydrolyzed in the broth or inside the bacteria and thereby inactivated. The compounds that
failed to inhibit the reaction in vivo were discarded from further analysis, and we instead
continued to study the class of compounds represented by compounds 2 and 3 in more
detail. The antimicrobial compound should inhibit survival only in vivo and not in vitro,
because a deletion of vagH has this phenotype; therefore the compounds should not inhibit
growth of Y. pseudotuberculosis in rich media. To evaluate this, bacteria were grown in the
presence of compounds 2 and 3, and the OD600 was monitored over time. The compounds
did not inhibit or slow the growth of Y. pseudotuberculosis in rich media (Figure 3, Paper
III). Next we wanted to expand the number of molecules in our analysis from this class of
compounds; we therefore investigated the inhibitory properties of 13 analogues to
compound 3. The results from this are summarized in Figure 4, Paper IV. The most
promising, compounds 6 and 15, were subjected to a more careful analysis of their in vivo
inhibitory effects and were found to inhibit Yop expression at 20 μM and 40 μM, which is
lower compared to compound 3 (~80 μM). We also examined the effects of the compounds
in a cellular-based assay where HeLa cells were infected with an lcrG mutant strain of Y.
pseudotuberculosis. Before the addition to the cells, the bacteria were grown in the
presence of various concentrations of the compounds; thereafter changes in the morphology
of the HeLa cells were monitored. We infected the HeLa cells with an lcrG mutant strain,
as we have found that a double vagHlcrG mutant is incapable of inducing a cytotoxic
response in the HeLa cells, a phenotype mainly associated mainly with the deletion of
vagH. In this assay, the compounds inhibited the translocation of the Yops, delaying the
cytotoxic response (Figure 5, Paper III), which confirms their in vivo inhibitory effects. We
also tried to set up a more purified system based on His-tagged, purified RF2 to establish
MICs (Minimal inhibitory concentration) of the compounds. Unfortunately, most of the
compounds failed to inhibit this reaction; only compound 4 without in vivo inhibitory
effects could inhibit the VagH enzymatic activity in the purer system, and we were,
therefore, unable to further evaluate the inhibitory properties of the compounds.
40
Conclusions
We have here outlined a strategy to identify novel antimicrobial compounds by a
bioinformatics and HTS approach. The main findings in this thesis are:
•
We have by a bioinformatics approach identified five virulence associated genes
(vags) in Y. pseudotuberculosis. These vags are conserved in a wide spectrum of
human pathogens making them all possible antimicrobial drug targets.
•
One of the vags, vagH, has been studied in more detail for its potential as a drug
target.
•
VagH is an S-adenosyl-methionine dependent methyltransferase targeting RF1 and
RF2. In Y. pseudotuberculosis the strongest phenotypes of a vagH mutant are:
•
o
Virulence attenuation.
o
Down regulated T3SS.
The effect on the T3SS is likely the cause of the virulence attenuation. The down
regulation is due to lower production of the positive T3SS activator LcrF. This
can be increased by a deletion of yopD, the amount of LcrF can also be increased
both in the wild-type and the vagH mutant by over expression of RRF. The
analysis of the vagH mutant has elucidated some new regulatory aspects of the
T3SS.
•
o
YopD regulates translation of LcrF.
o
LcrF expression is sensitive to the amount of free ribosomes.
We set up an in vitro assay system where a library of small molecules was
screened in a high throughput manner for inhibitory effects on the VagH
methyltransferase activity
o
Five out of 9, 400 compounds screened were found to inhibit VagH.
o
Two of these five also had an effect in bacterial culture on VagH as
measured by the expression of T3S-effectors (YopB and YopE)
o
Evaluation of structural analogues to one of the compounds decreased the
concentration necessary for inhibition in the bacterial system.
o
•
The compounds could also inhibit T3SS dependent cytotoxicity.
We have identified a novel class of compounds with antimicrobial properties.
41
Tack!!!
Åren på molekylär biologen blev många och roliga, detta till stor del på grund alla som
jobbar eller har jobbat på avdelningen. Först ut i raden av personer som ska tackas är min
handledare, Hasse, du har varit en toppen handledare under dessa år. Tack för att du trott
på mig och låtit mig jobba på men funnits där med support när det gått trögt eller med
entusiasm när det gått bra. Ibland är jobbet mindre viktigt än annars, det har du förstått och
det är jag väldigt tacksam för. Tillslut så handledde du mig även praktiskt i labbet, bättre
sent än aldrig! Tack också till Rolle N som däremot tidigt fick visa runt i labbet och svara
på 1 miljon frågor.
Britt-Marie Kihlberg för att du fick den första artikeln skriven och publicerad, Åke
Forsberg för samarbete, Mikael Elofsson för detsamma!
Janne för delat skrivrum och även för många insikter i hur det fungerar här i huset,
och dessutom en hel radda cyklar. Det har varit tomt efter dig! Ann-Catrin och Elin, roligt
med nya tillskott (det har ju bara gått två år…). Ann-Catrin, tack också för det arbete du
gjort (nästan alltid med ett leende…) och all hjälp med smått och gott i labbet.
Kaffehörnan - alltid där och oftast med kaffe om inte Rolle R har kaffevecka ☺
Cathrin P, för att man känner sig pigg när man ser dig på morgonen, Maria F för att du
försöker få tiden att växa, jobba på. Tack också till alla glada deltagare i Journal Clubs och
gruppmöten: Margareta, Sara C, Lisandro (lycka till!), Linda, Barbara, Medisa,
Anthony (från Apan ☺), Moa (tack för samarbete), Jeanette, Matt, Vicky, Debbie och
alla andra från nu och förr. Katrin, syjuntor, VM-tips och torsdagsluncher, jag kan till och
med (nästan) förlåta att du håller på Färjestad. Myggflickorna Jenny och Maria, roligt
med parasiter, tack för musvaktandet. Christer – tack för hjälpen med mössen och för att
du alltid är så nice. Det är ni andra Borrelia/Chlamydia män också (Pär och Leslie). StorPia, Lill-Pia, Pia Charlotte jag får byta namn så att jag också kan få ett jobb… och alla ni
andra på Innate, speciellt Henrik för hjälp med molekyler och sånt som jag inte förstår
mig på.
Petra, du är så glad och söt, dessutom är den mest omtänksamma vän man kan tänka
sig, tack för all hjälp i och utanför labbet!
Martina, du har bidragit mycket till denna avhandling! Jag tänker inte bara på
typsnitt och slavarbete utan också för att du är så fin. Saknar dig mycket! Magnus, du är
också fin, engagerad och entusiastisk dessutom, det är alltid roligt att locka fram
42
blodådrorna med lite Ebba Grön… David E, med stort tålamod med mig i fiskeribranschen
och även som extra fru, jag ställer gärna upp igen! Miss J, du är starkare än somliga (de
flesta) tror, glöm inte det och kom även ihåg att du är lysande på många sätt (dock inte på
att passa tiden…) Sofia, snäll och godhjärtad som få, du ställer upp i alla väder vad det än
gäller. Johan, hård utanpå men mycket mjuk inuti, en av de argaste men trognaste MoDo
supportrarna. Christina, det hade absolut inte varit lika kul utan dig, skidåkning, Malaysia
osv. Fredrik, Maria och Viktor, det är sååå roligt att ni flyttat till Malmö! Goda Grannar:
Gunnarn, du tillför sarkasm (gillas!) och lite kultur i vårt lilla gäng, Charlotte, vårt
rättesnöre som är väldigt generös och har varit mycket bra att ha under skrivandet, Niklas
och Maria mina nyaste grannar och tappra medkämpar i pendlarbranschen även om jag
tycker ni gav upp lite lätt ☺. Anna, vi får aldrig glömma Riksgränsen, ja, vissa saker kan vi
glömma…Erik, kalas, Kicki du är också kalas. Petra, Tom, Peter S, Sven, Håkan,
Linda, Tobias tack för allt roligt.
Annica, Anna, och Mia för att ni gjorde det roligt att springa omkring i overall och
för alla mysiga träffar sedan dess, roligt att alla blir fler. Malin, du var också söt i overall
och dessutom en mycket rolig lägenhetsdelare.. Katta, du är verkligen en frisk fläkt och
råbra ☺.
Carro, du är en toppenvän, jag lovar att komma till Genéve snart och hälsa på dig och
Matte även om jag är lite besviken på att ni lagt ner mitt hotell i Solna…
Och så familjen. Det har minst sagt hänt mycket de senaste åren och med en del saker är det
svårt att se meningen. Jag är så glad för er; Alexander det lilla solskenet, Mamma du
finns alltid där, Eva syster-yster och min estetiska konsult, du är fabulös!
Henrik, längtar tills vi ska bo ihop för alltid! Du är min bäste vän och kärleken (L).
43
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