Download Genes required for Lactococcus garvieae survival in a fish host

Microbiology (2007), 153, 3286–3294
DOI 10.1099/mic.0.2007/007609-0
Genes required for Lactococcus garvieae survival
in a fish host
Aurora Menéndez, Lucia Fernández, Pilar Reimundo and José A. Guijarro
Correspondence
José A. Guijarro
Área de Microbiologı́a, Departamento de Biologı́a Funcional, Facultad de Medicina, IUBA,
Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
[email protected]
Received 2 March 2007
Revised
7 June 2007
Accepted 12 June 2007
Lactococcus garvieae is considered an emergent pathogen in aquaculture and it is also
associated with mastitis in domestic animals as well as human endocarditis and septicaemia. In
spite of this, the pathogenic mechanisms of this bacterium are poorly understood.
Signature-tagged mutagenesis was used to identify virulence factors and to establish the basis of
pathogen–host interactions. A library of 1250 L. garvieae UNIUD074-tagged Tn917 mutants in
25 pools was screened for the ability to grow in fish. Among them, 29 mutants (approx. 2.4 %)
were identified which could not be recovered from rainbow trout following infection. Sequence
analysis of the tagged Tn917-interrupted genes in these mutants indicated the participation in
pathogenesis of the transcriptional regulatory proteins homologous to GidA and MerR; the
metabolic enzymes asparagine synthetase A and a-acetolactate synthase; the ABC transport
system of glutamine and a calcium-transporting ATPase; the dltA locus involved in alanylation of
teichoic acids; and hypothetical proteins containing EAL and Eis domains, among others.
Competence index experiments in several of the selected mutants confirmed the relevance of the
Tn917-interrupted genes in the development of the infection process. The results suggested
some of the metabolic routes and enzymic systems necessary for the complete virulence of this
bacterium. This work is believed to represent the first report of a genome-wide scan for virulence
factors in L. garvieae. The identified genes will further our understanding of the pathogenesis
of L. garvieae infections and may provide targets for intervention or lead to the development of
novel therapies.
INTRODUCTION
Lactococcus garvieae is the aetiological agent of lactococcosis, an emergent disease, which affects cultured freshwater and marine fish with special incidence in rainbow
trout (Onchorhynchus mykiss) (Eldar et al., 1999a) and
yellowtail (Seriola quinqueradiata) (Kusuda & Kawai,
1998), particularly during the summer given its association
with high water temperatures (for a review see Vendrell et
al., 2006). In addition, L. garvieae has been isolated from
buffalos with mastitis (Teixeira et al., 1996), from clinical
specimens of human blood and urine (Elliott et al., 1991)
and from patients with bacterial endocarditis and different
tissue infections (Aguirre & Collins, 1993; Fefer et al., 1998;
James et al., 2000; Mofredj et al., 2000; Fihman et al., 2006;
Vinh et al., 2006; Wang et al., 2006; Yiu et al., 2007).
In fish farming, outbreaks are treated with antibiotics,
although they are often ineffective and do not prevent
Abbreviations: CI, competitive index; STM, signature-tagged mutagenesis.
The GenBank/EMBL/DDBJ accession numbers for the DNA
sequences corresponding to mutants I–XXVI are EF450028–
EF450053, respectively.
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reinfection. On the other hand, vaccination with inactivated whole cells by intraperitoneal injection is only
protective for a limited period of time (Ravelo et al.,
2005). In recent years, progress has been made in
diagnostic techniques (Endo et al., 1998; Zlotkin et al.,
1998; Goh et al., 2000; Wilson & Carson, 2003), strain
typing (Eldar et al., 1999b; Vela et al., 2000; Wilson et al.,
2002; Ravelo et al., 2003; Barnes & Ellis, 2004; Eyngor et al.,
2004; Kawanishi et al., 2006) and the knowledge about the
immune response to infection (Ooyama et al., 1999; Barnes
et al., 2002b; Schmidtke & Carson, 2003; Shin et al., 2007).
Despite the importance of this syndrome, there is little
information about the precise pathogenic mechanisms by
which this bacterium is able to defeat the host defences and
cause disease. Up to now, it has only been established that
virulence of this bacterium is, in part, dependent upon its
ability to form a capsule (Yoshida et al., 1997; Barnes et al.,
2002a).
Laboratory media appear to be a limited tool when trying
to study the molecular mechanisms of disease, since it is
very difficult to mimic the complex and changing
environment of the host. In an attempt to overcome these
limitations, Hensel et al. (1995) developed a method for
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Pathogenesis of Lactoccous garvieae
the identification of bacterial virulence genes by screening
in living animals, called signature-tagged mutagenesis
(STM). STM relies on two key elements. First, a negative
selection will select against replication of mutant strains
where a transposon-mediated disruption of genes related
to virulence has occurred. Second, as each mutagenizing
transposon carries ‘signature tags’ (DNA fragments consisting of a central variable region that is flanked by two
invariant arms to which oligoprimers may bind for PCR
amplification), STM is capable of distinguishing between
the different mutants. A limitation of the application of
STM is that the screen will be unlikely to detect mutations
in virulence determinants whose function can be transcomplemented by the presence of other strains, i.e. toxins,
adhesins and binding proteins. Conversely, it may favour
the identification of genes having a longer-term impact on
in vivo growth and persistence. The STM methodology has
been adapted and used to identify virulence genes in several
microbial pathogens (Saenz & Dehio, 2005). Modifications
of this method include the use of pre-selected tagged
transposons to construct the mutant library, which
simplifies the screening significantly (Mei et al., 1997).
This modification was used in the present work.
In this study, STM was used to identify genes required for
growth and survival in a fish model of infection. A library
of 1250 L. garvieae UNIUD074-tagged Tn917 mutants in
25 pools was screened for attenuation. This led to the
identification of 29 mutants defective for survival in the
animal host, as judged by their inability to compete with
the wild-type strain in mixed infections. Thus, this work
establishes a first approach to the study of the genes that
are required for survival of L. garvieae in rainbow trout.
METHODS
Bacterial strains, plasmids and culture conditions. L. garvieae
instructions (Roche Applied Science). For dot-blot hybridizations,
plasmid DNA (1 mg) was transferred onto a Hybond-N+ membrane
(Amersham) using a Bio-Dot Microfiltration Apparatus (Bio-Rad).
DNA on the membranes was denatured by alkali treatment and fixed
by UV cross-linking according to the manufacturer’s instructions.
Southern hybridization analysis was performed by standard methods
on EcoRI-digested genomic DNA using an 819 bp fragment from the
bla gene present in the transposon Tn917 as a probe (Menendez et al.,
2006)
Cloning and selection of tags. pTV408 is a thermo-sensitive
plasmid able to replicate at temperatures below 37 uC but not above
this temperature (Slater et al., 2003). It harbours the Tn917
transposon conferring erythromycin resistance. A single EcoRI site
is present in the plasmid and it is located within the Tn917
transposon. This EcoRI site was used to introduce the PCR-generated
tags. Double-stranded 89 bp DNA signature tags were obtained by
PCR using the variable oligonucleotide pool RT (59CTAGAATTCTACAACCTCAAGCTT-[NK]20-AAGCTTGGTTAGAATGGAATTCATG-39) as template DNA, and primers P3 (59CTAGAATTCTACAACCTC-39) and P5 (59-CATGAATTCCATTCTAAC-39), which are the same as primers P5 and P3 (Hensel et al.,
1995) except that the 59 ends have a site for EcoRI instead of KpnI.
The PCR-amplified tags were digested with EcoRI and gel purified,
then ligated with EcoRI-digested, dephosphorylated pTV408 to
generate pTV408tag. The ligated DNA was transformed into L.
garvieae UNIUD074 by electroporation (Menendez et al., 2006).
Transformed bacteria were plated onto BHI containing erythromycin
and grown at 28 uC overnight. The total pool of transformants was
then screened by colony blot hybridization with their corresponding
DIG-labelled tags to identify 50 tags that amplified and labelled
efficiently. These tags were then tested for cross-hybridization (Hensel
et al., 1995) and 50 transformants were chosen for library
construction.
Generation of the L. garvieae mutant bank. A single colony of
each of the transformants chosen was transferred into a microtitre
dish well containing BHI broth and erythromycin. The microtitre
dish was incubated at 28 uC overnight. Glycerol was added to each
well to give a final concentration of 50 % (v/v), and the plate
(designated the master plate) was stored at 280 uC.
strain UNIUD074 was obtained from Dr L. Gusmani (University of
Udine, Italy). Plasmid pTV408 (Slater et al., 2003) was obtained from
Dr J. P. May (Department of Clinical Veterinary Medicine, University
of Cambridge, UK). L. garvieae strains were routinely cultured in
brain heart infusion medium (BHI) (Difco) at 20, 28 or 40 uC.
Escherichia coli strain DH5lpir was grown in 26 TY (tryptone/yeast)
medium at 37 uC. Two per cent agar was added to obtain solid media.
The following antibiotics were added to the media as needed: 1 mg
erythromycin ml21 for L. garvieae and 100 mg ampicillin ml21 for E.
coli.
To generate 50 different Tn917 mutants, bacteria from the master
plate were replicated using a microtitre dish replicator (Sigma) into
the wells of a second microtitre dish containing 200 ml BHI broth and
erythromycin. This dish was incubated at 40 uC overnight, and then
bacteria from each well were streaked onto BHI agar containing
erythromycin and incubated at 40 uC to obtain single colonies. One
different colony from each well was then transferred into the
corresponding well of a microtitre dish containing BHI broth and
erythromycin. Mutant pools were also stored at 280 uC in 50 % (v/v)
glycerol.
DNA manipulations, PCR, digoxigenin labelling and hybridizations. Extraction of chromosomal and plasmid DNA from L. garvieae
A series of identical membranes for dot-blot hybridizations was
prepared by transferring 1 mg of each of the selected 50 plasmids onto
Hybond-N+ membranes using the Bio-dot Microfiltration apparatus.
was perfomed as described by Leenhouts et al. (1989). Plasmid DNA
from E. coli was prepared by alkaline lysis (Birnboim & Doly, 1979).
Routine DNA manipulation was conducted as described by Sambrook
& Russell (2001). Phage T4 DNA ligase and calf intestinal alkaline
phosphatase were from Amersham, and oligonucleotides were from
Sigma.
The tag region was amplified by PCR using the primers P2
(59-ATTCTACAACCTCAAGC-39) and P4 (59-ATTCCATTCTAACCAAGC-39) (Hensel et al., 1995). Synthesis of digoxigenin (DIG)labelled tags, hybridization and development were perfomed with a
DIG DNA labelling and detection kit according to the manufacturer’s
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In vivo mutant selection. Each pool of 50 mutants was grown in
BHI supplemented with erythromycin in a microtitre dish at 20 uC
overnight. The bacteria were pooled, washed twice with PBS and
resuspended in PBS. According to previous results obtained by LD50
experiments using the parental strain, conditions for selection of
mutants in vivo were defined (A. Menéndez & J. A. Guijarro,
unpublished). Rainbow trout (O. mykiss) weighing from 10 to 15 g
were infected by intraperitoneal injection (for screening the library)
with doses of 105 mutant cells in 100 ml PBS. Fish were kept in 60 l
tanks at 20 uC in continually flowing dechlorinated water. At 72 h
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A. Menéndez and others
post-infection, the animals were sacrificed and dissected, and the liver
and spleen were homogenized in BHI with a stomacher. Afterwards,
the pool of bacteria was grown in BHI supplemented with
erythromycin, and chromosomal DNA was isolated for the generation
of the output PCR probe (Hensel et al., 1995). The tags present in the
recovered pools were compared with the tags present in the inoculum
pools by PCR amplification from DNA samples, DIG labelling using
primers P2 and P4 and hybridizations to the 50 plasmids on HybondN+ membranes.
Characterization of transposon insertion sites. For plasmid
rescue of mutants with attenuated virulence, genomic DNA
previously digested with EcoRI was religated and the ligation mixture
was used for transforming E. coli DH5lpir electrocompetent cells.
Transformants were selected on 26 TY agar plates containing 100 mg
ampicillin ml21. Plasmid DNA was obtained from the transformants
and sequencing analysis was performed, as described by Slater et al.
(2003), using the Tn917 seq (59-AGAGAGATCTCACCGTCAAGT39) designed to read out from the transposon. The dideoxy chaintermination method was used for DNA sequencing using a DR
terminator taq FS sequencing kit (Applied Biosystems). The sequence
was obtained in an ABI Prism 310A automated DNA sequencer from
Perkin-Elmer, according to the manufacturer’s instructions, at the
Universidad de Oviedo facility. Sequence analysis was performed
using the BLASTX computer program.
Competition experiments. For in vivo competition assays, mutant
strains and the wild-type strain UNIUD074 were grown separately in
BHI at 20 uC for approximately 18 h. Bacteria were washed in PBS as
described above, and each mutant was mixed with the wild-type at a
concentration of 105 c.f.u. ml21 each (26105 c.f.u. total bacteria
ml21). Dilutions of this suspension were plated onto BHI (to measure
total c.f.u.) and BHI with erythromycin (to determine mutant c.f.u.).
From this, the exact input ratio of mutant to wild-type was calculated.
A sample of 0.1 ml of the mixed suspension was used to infect
rainbow trout weighing from 10 to 15 g by intraperitoneal injection.
After 72 h, spleens and livers were recovered as described above, and
homogenates were plated onto selective media to determine the
output ratio of mutant to wild-type. The competitive index (CI) is
defined as the output ratio (mutant/wild-type) divided by the input
ratio (mutant/wild-type).
For in vitro competition assays, 5 ml BHI in a test tube was inoculated
with approximately 105 c.f.u. ml21 of the mutant and the wild-type.
The cultures were grown at 20 uC for 18 h (final OD600 was 1.5–2).
The input and output ratios of mutant to wild-type were determined
by selective plating as described above.
RESULTS AND DISCUSSION
Construction of signature-tagged L. garvieae
mutant library
From the original pool of signature-tagged transposons, 50
uniquely tagged transposons were selected and used to
construct the mutant library. The criteria of selection were
efficient amplification and labelling, and lack of crosshybridization to other tags. The plate containing this array
of 50 transformants was designated the master plate and
was used for all subsequent mutagenesis of L. garvieae. To
generate 50 different Tn917 mutants, bacteria from the
master plate were replicated into the wells of a second
microtitre dish, grown at the non-permissive temperature
(40 uC) and then mutant bacteria from each well were
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purified and transferred into a third microtitre dish, as
described in Methods. Thus, a total of 1250 mutants was
arrayed in 25 pools of 50 mutants each.
Screening for essential genes for infection in fish
For the in vivo selection of attenuated strains, a total of
1250 different tagged mutants were assembled into 25
pools of 50 different mutants. The presence of attenuated
strains in each pool was analysed by comparison between
the strains that were inoculated (input) to those that were
recovered from the livers and spleens of the animals
(output). Failure of recovery from the output was
considered as possible attenuation. Output pools were
recovered 72 h post-infection because intraperitoneal
experimental infection in rainbow trout caused the first
symptoms at that time after inoculation. Bacterial doses
were defined by previous LD50 experiments using the
parental strain (A. Menéndez & J. A. Guijarro, unpublished). Mutants were identified as attenuated if they showed
a reproducible decrease in the hybridization signal between
input and output pools in two animals. An example of the
results of a hybridization analysis is shown in Fig. 1.
Hybridization signals at positions B1, B3, C6 and E1 are
weaker on the blots probed with tags from the recovered
pools (output pools) than on the blot probed with tags from
the inoculum pool (input pool). Twenty-nine putative
attenuated mutants were identified in the STM screening of
1250 mutants (approx. 2.4 %). To verify that selected
mutants carried a single chromosomal insertion of Tn917,
chromosomal DNA samples from the individual mutant
strains were digested with EcoRI and subjected to Southern
analysis using part of the bla gene as a probe. For each of the
analysed mutants, a single hybridizing fragment of different
size was observed in each lane. This indicates that Tn917
insertions occurred singly in the chromosome of the L.
garvieae mutants (data not shown).
Identification of disrupted genes
The DNA regions flanking the pTV408tag side of the
transposon insertion points of 29 mutants were cloned as
described in Methods. The nucleotide sequences of the
flanking regions were determined and subsequently analysed
by searching the DNA and protein databases for identical or
similar genes (Table 1). Twenty mutants had Tn917 flanking
DNA encoding proteins with identities to known proteins.
Transcriptional products of flanking DNA cloned from four
mutants did not display any similarity to sequences in the
public databases. Finally, another five mutants showed
identities with proteins of unknown function.
Unexpectedly, four out of the 20 mutants in genes
encoding known proteins had transposon insertions in
different locations within a 1.7 kb DNA genomic region
containing two genes (asnA and merR). Random Tn917
transposon insertion throughout the chromosome of
Streptococcus mutans (Gutierrez et al., 1996), group A
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Pathogenesis of Lactoccous garvieae
Fig. 1. Dot-blot hybridization of input and two
output pools of STM-generated L. garvieae
mutants. (a) Tags were amplified from a mixed
mutant inoculum and probed against a DNA
dot-blot of 50 different tags on filters. (b)
Surviving cells were obtained from the infected
fish three days after the infection and tags
were amplified from the recovered colonies
and probed again. Hybridization signals of the
mutants XVII, XXII, XXII and VIII, corresponding
to wells B1, B3, C6 and E1, respectively, are
shown.
Table 1. Characteristics of the genes identified by STM screens with fish
Mutant
Homology
Hypothesized function
I
II
AsnA/Clostridium perfringens
GidC/Lactococcus lactis subsp. lactis
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
ChiA/L. lactis subsp. cremoris
Als/L. lactis subsp. lactis
Hypothetical protein/Pediococcus pentosaceus
MerR/Enterococcus faecium
Xre/L. lactis subsp. cremoris
AsnA/Lactobacillus salivarius subsp. salivarius
Hypothetical protein/Staphylococcus aureus
subsp. aureus
YrbI/L. lactis subsp. lactis Il1403
Unknown protein/Enterococcus faecalis
No homology in public databases
Calcium-transporting ATPase/Ent. faecium
No homology in public databases
No homology in public databases
Hypothetical protein/Bacillus cereus
XerC/Streptococcus pneumoniae
DltA/L. lactis subsp. lactis IL1403
XIX
XX
XXI
XXII
GlnP/L. lactis subsp. lactis IL1403
No homology in public databases
DprA/L. lactis subsp. lactis
Hypothetical protein/Lactobacillus gasseri
XXIII
XXIV
XXV
Hypothetical protein/Ent. faecium
Hypothetical protein/L. lactis
Hypothetical protein/L. lactis subsp. cremoris
SK11
Intergenic region between asnA and merR
XXVI
% Amino acid
CI in vitro CI in vivo
identity (homology)
Asparagine synthetase
Enzyme possibly involved in
translation
Chitinase
a-Acetolactate synthase
Unknown
Transcriptional regulator
Transcriptional regulator
Asparagine synthetase
Unknown
57 (74)
91 (95)
52
82
51
59
33
28
100
(65)
(92)
(78)
(83)
(59)
(38)
(100)
–
0.8
–
0.3
0.75
0.45
–
0.93
0.91
0.87
0.4
0.09
0.14
–
0.03
0.95
0.2
,0.0006
Transcriptional regulator
Unknown
34 (59)
46 (67)
–
–
–
–
Inorganic ion transport
49 (66)
–
–
50 (66)
62 (78)
65 (74)
0.85
–
–
–
0.79
–
0.11
–
–
–
0.34
–
78 (90)
–
58 (85)
37 (69)
0.42
–
–
1.5
,0.01
–
–
0.46
32 (46)
37 (58)
52 (69)
0.64
–
1.25
0.4
–
0.01
–
–
–
Unknown
Integrase/recombinase
D-Alanine-D-alanyl carrier protein
ligase
ABC transporter glutamine
DNA processing SMF protein
RNA polymerase binding protein
(family Spx)
Unknown
Protein with an EAL domain
Acetyltransferase involved in
intracellular survival
–, No data.
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A. Menéndez and others
streptococci (Eichenbaum & Scott, 1997) and Streptococcus
equi (Slater et al., 2003) has been assumed based on the
appearance of different-sized hybridizing fragments on
Southern blots. However, caution must be used when
interpreting these results because, as Slater et al. (2003)
demonstrated, when the insertions were analysed using the
Strep. equi genome sequence database, 60 % of the
transposition insertions in this bacterium were found
within a 15 kb region of its genome, whereas the remaining
40 % appeared to be random (Slater et al., 2003). Previous
studies by Southern blot analysis of Tn917 insertion into
the L. garvieae genome suggested that this occurred
apparently randomly (Menendez et al., 2006). Nevertheless, the selection in the present work of seven mutants
that had transposon insertions in four different locations
within a DNA region of 1.7 kb suggests that a hot-spot
for Tn917 insertion exists in the chromosome of L.
garvieae. This is consistent with the idea that all
transposons exhibit some sequence bias for transposition
(Coulter et al., 1998).
Competitive assays with selected mutants
In vivo competition assays were performed to validate the
results of our STM screen and to quantify the degree of
virulence attenuation of individual mutants. In the assay,
mixed infections with mutant and wild-type strains are
used to provide an in vivo measure of virulence attenuation
referred to as the in vivo CI (see Methods). A total of 13 of
the 24 different mutants were selected. The mutants
displayed a range of attenuation in the in vivo competitive
assays (Table 1), ranging from subtle attenuation (XXII,
CI50.46) to severe attenuation (IX, CI,0.0006). Mutant
VII was not attenuated in the in vivo competition assay
(Table 1). This mutant represents a false-positive mutant, a
result that has also been seen in other screens (Camacho
et al., 1999; Ruley et al., 2004). In addition, comparable
general growth defects under optimal laboratory growth
conditions were ruled out by in vitro competition assays
(see Methods). Thus, three mutants (IV, IX and XIX)
showed a growth defect, whereas the rest of the analysed
mutants showed essentially wild-type growth (Table 1).
Inferred function of STM selected genes
Approximately 70 % of the mutants identified had
transposon insertions within genes encoding proteins
similar to known proteins in the public databases.
The ability to adapt to the host environment is a key
component of pathogenicity. The nutritional environment
of the host’s cells imposes a requirement for de novo
biosynthesis of various amino acids, cofactors and nucleotides in many pathogens. The identification of mutants with
transposon insertions that induce auxotrophy demonstrated
that auxotrophic mutants are cleared from the host. Two
mutants (I, VIII) were found with transposon insertions in
different positions of an asparagine synthetase (AsnA) gene
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homologue that seems to be necessary for the survival of L.
garvieae in the host.
Mutant IV had a transposon insertion in an a-acetolactate
synthase gene homologue. This enzyme is involved in the
biosynthesis of acetoin and 2,3-butanediol. The metabolic
function of this process appears to be to counteract lethal
acidification as cells approach stationary phase by redirecting pyruvate into neutral rather than acidic end products.
The 2,3-butanediol pathway may also participate in the
regulation of the NAD/NADH ratio in bacteria (Johansen
et al., 1975). Recently, Yoon & Mekalanos (2006) obtained
evidence that 2,3-butanediol synthesis gives Vibrio cholerae
El Tor biotypes a survival advantage during infection,
which is important for colonization. The results obtained
in L. garvieae indicate the importance of the 2,3-butanediol
pathway for in vivo survival.
A number of loci encoding putative regulatory genes were
identified in the screening, suggesting the importance of
regulation of bacterial gene expression for in vivo survival
of L. garvieae. Mutant II had a transposon insertion in a
homologue of the gidA gene. gidA is widely distributed and
highly conserved in both prokaryotes and eukaryotes,
having a translational regulatory function. (Kinscherf &
Willis, 2002; Sha et al., 2004).
The DNA flanking the transposon insertion in mutant VI
had homology to the regulatory protein MerR of
Enterococcus faecium. The MerR family is a group of
transcriptional activators that have been found in a wide
range of bacterial genera. The majority of regulators in this
family respond to environmental stimuli, such as oxidative
stress, heavy metals or antibiotics (Amabile-Cuevas &
Demple, 1991; Brown et al., 2001; Kim et al., 2002).
Mutant XXIV had a transposon insertion in a gene
encoding a putative protein that contains an EAL domain.
Proteins with this domain are predicted to regulate cell
surface adhesiveness, biofilm formation and virulence in
response to extracellular cues, by controlling the level of
the newly recognized bacterial second messenger 39,59cyclic diguanylic acid (c-diGMP) (Römling & Amikam,
2006). Genetic and biochemical evidence suggests that the
EAL protein domains act as a phosphodiesterase for c-diGMP degradation (D’Argenio & Miller, 2004). Recently,
STM of Salmonella spp. found that the cdgR gene is
required in order to resist the host phagocyte oxidase in
vivo. CdgR consists solely of an EAL domain. Thus, besides
its known role in regulating biofilm formation, bacterial cdiGMP also regulates host–pathogen interactions involving
antioxidant defence and cytotoxicity (Hisert et al., 2005).
Other regulatory functions affecting in vivo survival were
also identified, including a transcriptional regulator (Yrb1)
from Lactococcus lactis (mutant X), and a putative RNA
polymerase binding protein (mutant XXII). The assortment of independent mutations recovered in regulatory
genes emphasizes the biological significance of these loci
for in vivo survival of L. garvieae.
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Pathogenesis of Lactoccous garvieae
Two loci identified in the screening included homologues
of transporter genes. DNA flanking the transposon
insertion in mutant XIX showed homology to glnP of L.
lactis. The in vivo attenuated growth seen in this mutant is
probably caused by the polar effects of transposon
insertion, as glnP and glnQ are usually organized in an
operon. glnP encodes a glutamine ABC transporter
permease and glnQ encodes a glutamine ABC transporter
ATP-binding protein. It seems that the concentration of
glutamine in the host is a critical parameter for in vivo
survival as occurred in group B streptococci (Tamura et al.,
2002) and Streptococcus pneumoniae (Polissi et al., 1998).
Mutant XIII had a transposon insertion in a gene coding
for a cation-transporting ATPase. A mutant in a hypothetical cation-transporting ATPase was also identified by
STM in Strep. pneumoniae (Polissi et al., 1998).
Other genes identified were associated with several cellular
processes. Mutant XVII contained an insertion in a
homologue to the tyrosine recombinase (XerC) of Strep.
pneumoniae. XerC has a role in the segregation of
replicated chromosomes during cell division. The xerC
null mutants of Strep. pneumoniae and Staphylococcus
aureus were found to be attenuated in a murine infection
model (Chalker et al., 2000). A site-specific recombinase
was also found to be as important for survival of Erwinia
amylovora in plants (Wang & Beer, 2006). This result,
together with that obtained in L. garvieae, suggests that this
gene may control processes affecting virulence.
Mutant XVIII had a transposon insertion in a gene
encoding a protein homologous to DltA. This gene encodes
a D-alanine-D-alanyl carrier protein ligase involved in Dalanylation of teichoic acids in Gram-positive pathogens
(Abachin et al., 2002; Weidenmaier et al., 2005; Kovács
et al., 2006; Wartha et al., 2007). Interestingly, dltA
mutants of Staph. aureus (Weidenmaier et al., 2005),
Strep. pneumoniae (Wartha et al., 2007) and Listeria
monocytogenes (Abachin et al., 2002) showed attenuated
virulence. In Strep. pneumoniae the alanylation of teichoic
acids is essential for protection against neutrophils during
the infection process (Wartha et al., 2007). It seems that the
dltA gene is important for growth of L. garvieae in the host,
but further studies are needed to elucidate the role of this
gene in the L. garvieae infection process.
Mutant XXI carried an insertion in the dprA gene, which
has been described as a competence gene. The DprA
protein has been suggested to be involved in the protection
of incoming DNA. However, members of the dprA gene
family (also called smf) can be detected in virtually all
bacterial species, which suggests that their gene products
have a more general function. The basic function of dprA/
smf remains unclear (Smeets et al., 2006).
Mutant XXV had a transposon insertion in a gene coding
for a putative protein that contains an Eis domain. The
proteins with this domain are hypothetical and have
unknown function. However, the Eis domain seems to be
http://mic.sgmjournals.org
involved in intracellular survival. Although its function
remains unknown, it was found to enhance intracellular
survival of Mycobacterium smegmatis in a human macrophage-like cell line when eis was introduced into M.
smegmatis on a multicopy vector (Wei et al., 2000).
Intracellular survival may be essential for the progress of
the infection by L. garvieae. Additional studies are planned
to further characterize this mutant and to determine the
exact role of this protein.
The DNA flanking the transposon insertion in mutant III
had homology to a chitinase of L. lactis. The exact role
of this protein during the infection is unknown. This
mutant was severely attenuated in the CI assay in vivo
(Table 1). Further studies are under way to characterize
this mutant.
Analysis of DNA sequences from nine mutants did not
reveal any significant similarities to entries in the DNA and
protein databases (XX, XII, XIV, XV) or reveal significant
similarities to genes with unknown function (V, IX, XI,
XVI, XXIII). CI assay analysis of one of the strains with
mutations in genes with unknown function (XXIII)
showed that it was significantly attenuated compared with
the wild-type strain (Table 1).
CONCLUSIONS
The STM technique is a powerful method that allows large
numbers of mutants to be screened for attenuation
(competitive defects) in an animal model of infection.
STM has been applied successfully to other Gram-positive
bacterial pathogens including Strep. pneumoniae, Staph.
aureus and Streptococcus agalactiae (Polissi et al., 1998; Mei
et al., 1997; Jones et al., 2000). The purpose of this study
was to assess the usefulness of the technique for identifying
virulence genes of L. garvieae.
STM analyses of L. garvieae identified a wide variety of
functional gene classes underscoring the diversity of
bacterial processes required for the infection process.
Currently, the function of the genes identified in the
screen can only be inferred by homology. Multiple mutants
were obtained in genes homologous to transport systems,
regulatory proteins and metabolism enzymes, suggesting
the importance of their respective functions for infection.
Mutants lacking a-acetolactate synthase, teichoic acids
alanylation protein (DltA), EAL and Eis proteins domains,
and MerR and GidA regulatory proteins, seem to be
interesting candidates for future studies. As in other STM
studies, a relatively high percentage of the attenuated
mutants were disrupted in genes of unknown function.
These genes could potentially encode novel factors that
may play important roles in the bacterial infection process.
The identification of the entire ORF and search for specific
motifs or domains may give clues about the putative
function of these proteins. In addition, virulence studies of
some of the mutants could give information about their
potential for use as attenuated vaccines.
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3291
A. Menéndez and others
These results are believed to represent the first report of a
genome-wide scan for virulence factors in L. garvieae, and a
number of important putative virulence factor genes
worthy of further study have been identified.
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Biodiversity of Lactococcus garvieae strains isolated from fish in
Europe, Asia, and Australia. Appl Environ Microbiol 65, 1005–1008.
Eldar, A., Hurvitz, H., Bercovier, H. & Ghittino, C. (1999b). Lactococcus
ACKNOWLEDGEMENTS
This study was supported in part by the Spanish MEC (grant
AGL2003-229). A. M. and L. F. were recipients of predoctoral
fellowships from the MEC. We thank Dr B. Mayo for his advice in
Lactococcus genetics and Dr A. F. Braña for critical reading of the
manuscript. We also thank Dr L. Gusmani and Dr J. P. May for
sending us the L. garvieae strain UNIUD074 and the plasmid pTV408,
respectively.
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