INFECTION AND IMMUNITY, Dec. 2008, p. 5826–5833
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 12
Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of
the LigB Gene Does Not Affect Virulence in Animal Models
Julio Croda,1,2Claudio Pereira Figueira,1Elsio A. Wunder, Jr.,1Cleiton S. Santos,1
Mitermayer G. Reis,1Albert I. Ko,1,3* and Mathieu Picardeau4
Gonc ¸alo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Brazil1; Laboratory of Pathology of
Transmissible Diseases, Department of Pathology, Medicine School, Sa ˜o Paulo University, Sa ˜o Paulo, Brazil2; Division of
International Medicine and Infectious Disease, Weill Medical College of Cornell University, New York,
New York3; and Unite ´ de Biologie des Spiroche `tes, Institut Pasteur, Paris, France4
Received 7 August 2008/Returned for modification 27 August 2008/Accepted 16 September 2008
The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely
unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species.
Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has
never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were
previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by
allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spcr) gene replaced a portion of
the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluores-
cence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster
model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of
the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together,
our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species.
Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the
host and in the development of acute disease manifestations or persistent renal colonization.
Leptospirosis is a widespread zoonosis that has emerged as
a major public health problem in developing countries in
Southeast Asia and South America (6, 22, 29). This increas-
ingly common disease occurs in poor urban centers subject to
frequent flooding (20). Rodents are the main reservoir of the
disease, excreting the bacteria in their urine (14, 22). Humans
are usually infected through contaminated water. More than
500,000 cases of severe leptospirosis are estimated to occur
worldwide each year (46), and the fatality rate is 5 to 20% (29).
The control methods for leptospirosis implemented to date
have been ineffective (29). A significant barrier to control and
prevention of leptospirosis has been our limited understanding
of the pathogenesis of the disease, due in part to the lack of
genome sequences and tools to genetically manipulate the
pathogens. Most of the barriers have now been overcome. The
genomes of two pathogenic species and one saprophytic spe-
cies have been sequenced (8, 32, 39, 40). Furthermore, we
developed a transposon-mediated mutagenesis system for
pathogenic Leptospira species (7). This advance allowed char-
acterization of the first genetically defined virulence factor in
pathogenic Leptospira spp. (41). However, the generation of
targeted mutants of pathogenic species was not feasible until
High-molecular-weight leptospiral immunoglobulin-like re-
peat (Lig) proteins were previously identified as putative vir-
ulence factors in pathogenic Leptospira spp. (21, 26, 34). This
family of three proteins, LigA, LigB, and LigC, belongs to the
superfamily of bacterial immunoglobulin-like (Big) repeat do-
main proteins, which includes virulence determinants such as
intimin from enteropathogenic Escherichia coli, invasin from
Yersinia pseudotuberculosis, and BipA from Bordetella spp.
(26). This superfamily appears to mediate pathogen-host cell
interactions, such as invasion and host cell attachment, during
infection. Choy et al. and Lin and Chang recently showed that
recombinant Lig proteins can mediate in vitro interactions with
host extracellular matrix proteins, including fibronectin, fibrin-
ogen, collagen, and laminin (9, 23). In addition, lig genes are
upregulated at physiological osmolarity (27) and encode sur-
face-exposed proteins that are strongly recognized by sera
from human leptospirosis patients (10, 26, 43). Finally, several
studies have shown that Lig proteins are protective antigens in
animal models of leptospirosis (21, 35, 42).
In this study, we produced a ligB mutant of L. interrogans by
allelic exchange and evaluated the effect of the deletion in this
mutant using both cell adhesion assays and animal models. The
results provided the first demonstration of targeted mutagen-
esis of Leptospira pathogenic strains.
(This research was conducted by J. Croda in partial fulfill-
ment of the requirements for a Ph.D. from the Department of
Pathology, Medicine School, Sa ˜o Paulo University, and by
C. P. Figueira and E. Wunder in partial fulfillment of the
requirements for Ph.D. from Goncalo Moniz Research Center,
Oswaldo Cruz Foundation, Brazil.)
* Corresponding author. Mailing address: Centro de Pesquisas Gon-
c ¸alo Moniz, Fundac ¸a ˜o Oswaldo Cruz/MS, Rua Waldemar Falca ˜o, 121,
Salvador, Bahia 40296-710, Brazil. Phone: 55 71 3176 2302. Fax: 55 71
3176 2281. E-mail: firstname.lastname@example.org.
?Published ahead of print on 22 September 2008.
MATERIALS AND METHODS
Bacterial strains and growth conditions. Leptospires were cultivated in liquid
Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (13, 19) or on 1%
agar plates at 30°C and were counted in a Petroff-Hausser counting chamber
(Fisher Scientific). L. interrogans serovar Copenhageni strain Fiocruz L1-130, a
virulent clinical isolate from Brazil (20, 32), was used in all experiments. E. coli
was grown in Luria-Bertani medium. When necessary, spectinomycin or kana-
mycin was added to culture media at a concentration of 50 ?g/ml.
Polyclonal and monoclonal antibodies. We prepared immune sera against
previously described recombinant fragments of LigA (LigANI; amino acid po-
sitions 625 to 1225) and LigB (LigBNI; amino acid positions 625 to 1257) (42).
These fragments contain the 6th to 13th and 6th to 12th Big repeat domains of
LigA and LigB, respectively, and do not include the portions of these molecules
which have identical amino acid sequences (26). New Zealand White rabbits
were immunized intravenously on days 0, 7, and 14 with three doses of 80 ?g of
recombinant protein fragments, using aluminum hydroxide as an adjuvant. Rab-
bits were bled on day 28 to obtain immune sera. For quality control, the reac-
tivities of immune sera with recombinant and native Lig proteins were evaluated
using enzyme-linked immunosorbent and immunoblot assays as previously de-
scribed. Prebleed sera, as well as sera from rabbits immunized with phosphate-
buffered saline (PBS) and alhydrogel, were used as control samples. Ascites fluid
containing monoclonal antibodies (MAb) against a recombinant LigB protein
fragment, LigBrep (26, 42), were provided by Jose ´ Aleixo, Federal University of
Pelotas. The LigBrep fragment corresponds to the six N-terminal Big repeat
domains of the LigB molecule (amino acid positions 131 to 649).
Targeted mutagenesis. For allelic exchange, the gene fragment which encodes
the LigB nonidentical region (nucleotides 1891 to 5450), also called the B2
region, was amplified from the genomic DNA of L. interrogans serovar Copen-
hageni strain Fiocruz L1-130 using primers LigBU1F-SmaI (5? TCCCCCGGG
GCTGAAATTAAAAATACCAGTGGAAG 3?) and P2R-SmaI (5? TCCCCCG
GGCCTATGTAGAGATAAGATCCACTTGC 3?). The PCR product was
digested with SmaI and cloned into a PvuII-digested pGKm plasmid vector. The
resulting plasmid was then digested with EcoRI, which removed a 699-bp se-
quence from the ligB B2 domain. The Spcrcassette was amplified from plasmid
pGSpc DNA using primers Spc-EcorI3? (5? ACGGAATTCAACGTAAAG
TAAG 3?) and Spc-EcorI5? (5? TCGGAATTCAACGCGTCCCGAGC 3?). The
PCR product was digested with EcoRI and subsequently inserted into the plas-
mid from which the 699-bp ligB sequence had been removed to obtain plasmid
pB2SK (Fig. 1B). For gene inactivation by plasmid insertion, an internal DNA
fragment of ligB (nucleotides 1891 to 3771), also called the B1 region, was
amplified from the genomic DNA of L. interrogans Copenhageni strain Fiocruz
L1-130 using primers LigBU1F-SmaI (5? TCCCCCGGGGCTGAAATTAAAA
ATACCAGTGGAAG 3?) and LigBU2R-SmaI (5? TCCCCCGGGCACTTGG
TTTAAGGAATTACAAACT 3?). The PCR product was digested with SmaI
and cloned into a PvuII-digested pGSpc plasmid vector, resulting in plasmid
pB1S. Plasmids pGKm and pGSpc (complete sequences are available on re-
quest) were derived from the cloning vector pGEM-7Zf(?) (Promega Corpo-
ration, Madison, WI) as previously described (15). We used the Enterococcus
faecalis kanamycin and Staphylococcus aureus Spcrcassettes as previously de-
The plasmid constructs, which are not replicative in Leptospira spp., were used
to deliver the inactivated allele into L. interrogans by electroporation. Cells were
grown to exponential phase (optical density at 420 nm, 0.10 to 0.20) and then
centrifuged at 4,000 ? g and concentrated to obtain 1010bacteria/ml in sterile
water. Suicide plasmids containing the inactivated allele were subjected to 5 to
30 s of UV treatment (254 nm, 400 ?W/cm2) using a UV chamber (GS Gene
linker; Bio-Rad). Two hundred microliters of cells was electroporated (1.8-kV,
200-?, 25-?F electric pulse in a prechilled 0.2-cm-diameter cuvette) in the
presence of 100 to 500 ng of plasmid DNA and then transferred to 1 ml of EMJH
liquid medium, in which the cells were incubated for 24 h at 30°C. The bacteria
were then plated on EMJH medium supplemented with spectinomycin (50 ?g/
ml). After 4 to 6 weeks of incubation, Spcrcolonies were picked and examined
for allelic exchange in the target gene by PCR, Western blotting, and immuno-
Genomic DNA analysis. Genomic DNA was prepared from liquid cultures by
use of a cell DNA purification kit (Maxwell, Promega, Madison, WI). To check
for double homologous recombination, primers B2EF (5? CATACACACTTGT
AGTCAACAAACAAG 3?) and B2ER (5? CGTAACGTAATTCGGAACCG
3?) and primers LBN (5? GGGAATTCCATATGAAGAAAATATTTTGTAT
TTCG 3?) and P2R (5? TATGTAGAGATAAGATCCACTTGC 3?) were used
for amplification of the ligB locus (Fig. 1A). Gene inactivation by plasmid
insertion was confirmed by using primers B1F (5? ACCTGGAATTCCTCTAA
TACGGATATT 3?) and B1R (5? GAATATAAAGGTTTGGAAAAAGA
AACG 3?) for PCR amplification.
Immunoblotting. Mutant and wild-type L. interrogans Fiocruz L1-130 strains
were grown in EMJH medium until the optical density at 420 nm was 0.2.
Leptospira biflexa was also used as a control in these experiments. Bacteria were
washed in PBS. After the concentration was adjusted to 2 ? 108bacteria/per well
(20 ?l), the cells were solubilized in 62.5 mM Tris hydrochloride (pH 6.8)–10%
glycerol–5% 2-mercaptoethanol–2% sodium dodecyl sulfate. Crude protein ex-
tracts were resolved by 8% sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis using a discontinuous buffer system. After transfer to nitrocellulose
membranes, immunoblots were blocked in 0.05 M Tris-buffered saline (pH
7.4)–0.05% (vol/vol) Tween 20 with 5% (wt/vol) nonfat dry milk. The blots were
washed, incubated for 1 h at room temperature with a 1,000-fold dilution of
mouse ascites containing MAb to the LigB identical repeat region (LigA/B) or
with a 10,000-fold dilution of hyperimmune rabbit antisera to LipL41, and
probed with goat anti-mouse and anti-rabbit immunoglobulin G (IgG) antibodies
conjugated to alkaline phosphatase (Sigma). Immunoblots were developed in a
nitroblue tetrazolium—5-bromo-4-chloro-3-indolylphosphate (BCIP) solution
Immunofluorescence assays. Immunofluorescence labeling was performed us-
ing a modified protocol of Cullen et al. (12). Suspensions containing 107live
leptospires in 10 ?l of PBS were placed on poly-L-lysine-coated (Sigma) slides
and incubated for 1 h in a humidified chamber. The slides were washed twice
with PBS, blocked with PBS containing 1% bovine serum albumin (BSA)
(Sigma) (PBS-BSA), and incubated for 1 h with hyperimmune rabbit antisera to
the LigB nonidentical region (LigBNI) and the LigA nonidentical region
(LigANI) (diluted 1:100 in PBS-BSA) and with control rabbit antisera to a
leishmanial antigen. The slides were washed gently with PBS-BSA and incubated
with donkey anti-mouse IgG antibodies conjugated to Alexa dye (Molecular
Probes) or with goat anti-rat IgG antibodies conjugated to fluorescein isothio-
cyanate (Jackson ImmunoResearch Laboratories) for 1 h at 37°C. The slides
were washed twice with PBS-BSA and incubated with 1 ?g/ml 4?,6?-diamidino-
2-phenylindole (DAPI) (Molecular Probes) for 1 h at room temperature. The
slides were washed and then mounted in antifading solution (Prolong; Molecular
Probes) and visualized by fluorescence microscopy (Olympus BX51).
Hamster model of acute infection. Groups of four Golden Syrian male ham-
sters that were 5 to 8 weeks old were inoculated intraperitoneally with 10, 102,
104, and 106cells of the wild-type and ligB mutant L. interrogans Fiocruz L1-130
strains. Negative control animals were inoculated intraperitoneally with 1 ml of
EMJH medium. Animals were monitored daily for clinical signs of leptospirosis
(prostration and jaundice) and survival. Surviving animals were killed after a
21-day postchallenge follow-up period. The 50% lethal dose (LD50) of L. inter-
rogans strain Fiocruz L1-130 in 5- to 8-week-old hamsters was approximately 101
leptospires. Culture isolation and immunofluorescence studies were performed
using kidney and liver samples (42) to determine whether surviving animals had
a persistent infection. The protocols used for animal experiments followed the
guidelines of the Animal Care and Use Committee of Fundac ¸a ˜o Oswaldo Cruz.
Rat model of chronic infection. Groups of four or eight Wistar rats (Fiocruz,
Rio de Janeiro, Brazil) that were 4 to 5 weeks old were inoculated intraperito-
neally with 108cells of the wild-type and ligB mutant strains in 1 ml EMJH
medium. Control animals were inoculated intraperitoneally with 1 ml of sterile
EMJH medium. Animals were sacrificed 15 days after infection. Necropsies were
performed immediately after sacrifice. Kidney and liver samples were fixed in 4%
formalin, embedded in paraffin, and cut into 4- to 5-?m sections for conventional
histology analysis. Renal tissue samples were homogenized in 5 ml of EMJH
liquid medium for 10 min. After separation of the supernatant from the tissues,
500 ?l of the supernatant was used to inoculate 5 ml EMJH liquid medium,
which was subsequently incubated at 29°C. The cultures were examined weekly
for growth by dark-field microscopy for up to 6 weeks.
Histopathology studies. Groups of three hamsters were inoculated with 106
cells of the wild-type and ligB mutant L. interrogans Fiocruz L1-130 strains and
euthanized on day 9 postchallenge. Tissues (liver, kidneys, and lungs) were fixed
in 10% buffered formaldehyde, embedded in paraffin, and sectioned using rou-
tine histological procedures to obtain 4- to 5-?m sections that were then stained
with hematoxylin and eosin. For immunohistochemistry analysis, the paraffin was
removed from the sections with xylene and ethanol. The tissues were blocked by
incubation of sections with 1.0% BSA at room temperature for 20 min. The
tissues were incubated with a 1,000-fold dilution of antiserum to LipL32 (17) at
room temperature for 1 h. Samples were treated with 0.3% hydrogen peroxide
for 15 min at room temperature and then incubated at room temperature for 30
min with goat anti-mouse or anti-rabbit antibodies conjugated to peroxidase
(Histostain-Plus kit; Invitrogen). Enzyme reactions were developed using 3,3?-
VOL. 76, 2008ligB IS NOT ESSENTIAL FOR VIRULENCE OF L. INTERROGANS5827
Host cell adhesion assay. Madin-Darby canine kidney (MDCK) cells were
harvested by treating cell cultures with 0.05% trypsin and 0.02% EDTA in PBS
and then plated on 24-well plates in Dulbecco’s modified Eagle’s medium (Cul-
tilab) without antibiotics. Cell viability was determined by trypan blue exclusion,
and 500-?l portions of cell suspensions containing 2 ? 105cells per ml were
layered on round glass coverslips in 24-well tissue culture plates. The plates were
incubated for 24 h and washed twice with PBS to remove nonadherent cells.
Bacteria were suspended in warm (37°C) cell culture medium at a concentration
of 2 ? 107cells per ml. Wild-type and ligB mutant strains were used at the same
time in each experiment. A 500-?l aliquot of each bacterial suspension was then
added to the wells at a bacterium/cell ratio of 100:1. The plates were incubated
under static conditions for 1 h at 37°C (30). Experiments were performed in
triplicate. Coverslips were washed three times in PBS to remove nonadherent
bacteria. An immunofluorescence analysis was performed as described above.
The first antibody was anti-LipL32 MAb or anti-Omp Salmonella MAb, and the
second antibody was anti-mouse antibody conjugated with Alexa 488 (Molecular
Probes). DAPI and Alcian Blue were used to stain the nucleus and the cyto-
plasm, respectively. The numbers of leptospires and MDCK cells were deter-
FIG. 1. Disruption of ligB in L. interrogans strain Fiocruz L1-130. (A) Schematic representation of the genotype of the parental (Fiocruz wt)
and ligB (Fiocruz KO2) mutant strains. The LigB protein has a tripartite structure which includes an N-terminal identical repeat region, a
nonidentical repeat region, and the C-terminal region. The vertical bars indicate EcoRI restriction sites. The locations of primers used to check
for allelic exchange, as well as the expected sizes of amplified products, are indicated. (B) Map of the pB2SK plasmid, in which the Spcrcassette
was inserted between EcoRI sites in the B2 region of ligB. (C) PCR amplification of chromosomal DNA from the L. interrogans wild-type strain
(lane 1), L. biflexa strain Patoc1 (lane 2), the L. interrogans ligB KO2 mutant (lane 3), and the L. interrogans ligB KO2 mutant reisolated from
hamsters after infection (lane 4) with primers B2EF and B2ER, as shown in panel A. (D) Western blot of LigA and LigB expression in the L.
interrogans wild-type strain (lane 1), L. biflexa strain Patoc1 (lane 2), the L. interrogans ligB mutant (lane 3), and the L. interrogans ligB mutant which
was reisolated from hamsters (lane 4). Blots were also probed with LipL41 antiserum as a reference. (E) Immunofluorescence assays were
performed with L. interrogans wild-type (wt) and ligB mutant (KO2) strains. Strains were labeled with antibodies against LigANI (?-LigA0),
LigBNI (?-LigB), and a control. Alexa- and fluorescein isothiocyanate-conjugated secondary antibodies were used to detect surface-bound
antibodies to LigANI and LigBNI, respectively. A DAPI counterstain was used to document the presence of leptospires. The photomicrograph
show the results of one of three representative experiments.
5828CRODA ET AL.INFECT. IMMUN.
mined by examining 10 high-power fields during fluorescence microscopy. Stu-
dent’s t test was used to evaluate the significance of differences between the
numbers of associated leptospires per host cell in incubations with wild-type and
ligB mutant strains.
Allelic exchange mutagenesis of L. interrogans ligB. Patho-
genic Leptospira species possess between one and three lig
genes. L. interrogans serovar Copenhageni strain Fiocruz L1-
130 contains two lig genes, ligA (3,675 bp) and ligB (5,673 bp),
which encode polypeptides with molecular masses of 128 and
201 kDa, respectively. The third gene, ligC, was identified as a
pseudogene in this L. interrogans strain (26). The ligB locus was
used as a target for mutagenesis by allelic exchange in L.
interrogans strain Fiocruz L1-130. A gene replacement con-
struct was generated by cloning ligB into a suicide vector, which
deleted a portion of the ligB open reading frame, which was
replaced by an Spcrcassette. The amounts of homologous L.
interrogans DNA present on the two sides of the spectinomycin
marker were 1.8 and 1 kb (Fig. 1A).
The origin of replication used in the plasmid construct was
that from pGEM7Zf, which is nonfunctional in Leptospira spp.
Thus, any Spcrcolonies arising after electroporation of this
plasmid into L. interrogans should have resulted from recom-
bination of the plasmid with the host genome. Strain Fiocruz
L1-130 was electroporated with the UV-irradiated plasmid
construct as previously described (38) and plated on solid me-
dium containing spectinomycin. A total of six transformation
experiments were performed, one of which yielded two trans-
formants. The two mutant clones were obtained on the same
plate and may have been mutants of siblings.
To confirm that homologous recombination events occurred
at the origin of the Spcrphenotype, the ligB locus was analyzed
by PCR mapping of genomic DNA obtained from L. interro-
gans transformants. PCR amplification with primers B2EF and
B2ER, which normally produce a 0.7-kb product with wild-type
L. interrogans, generated a 1.3-kb DNA fragment with the two
Spcrrecombinants analyzed (Fig. 1B). The amplified product
was the expected size if a ligB mutant resulted from integration
of the Spcrcassette by double-crossover recombination in the
ligB chromosomal locus (Fig. 1A and B). Furthermore, PCR
amplification with primers B2N and P2R yielded a product
which had a size that was consistent with a gene replacement
event in ligB loci. Immunoblotting with an anti-LigA/B MAb
confirmed that LigB-reactive polypeptides were not present in
the mutant (Fig. 1C). Moreover, by evaluating the reactivity
with antibodies raised against LigA, we were able to detect
LigA, which is encoded by the lig gene located upstream of ligB
(26). This finding indicates that ligB inactivation does not mod-
ify ligA expression. Immunofluorescence studies also demon-
strated that the ligB mutant did not express LigB, whereas the
wild-type strain did express this protein (Fig. 1D). In contrast,
antisera to LigA labeled live ligB mutant and wild-type lepto-
spiral strains similarly.
By using another approach, we cloned an internal fragment
of ligB lacking the 5? and 3? ends of the open reading frame in
a suicide vector. Five transformants were recovered after elec-
troporation in L. interrogans strain Fiocruz L1-130 in one of
four transformation experiments. With all clones tested we
obtained integration of the plasmid via a single crossover
event, which generated two copies of the targeted gene, one
with a deletion at the 5? end of the gene and the other with a
deletion at the 3? end, thereby rendering it inactive, as con-
firmed by immunoblotting (data not shown).
In addition to the differences in genotype mentioned above,
the transformants resulting from allelic exchange and plasmid
insertion did not produce LigB; hence, the term ligB mutant
refers to the double-crossover recombinant KO2 mutant below
unless indicated otherwise.
Loss of the ligB gene does not affect virulence and persis-
tence in animal models. The ligB mutant and wild-type strains
had similar cell growth kinetics in liquid EMJH medium (the
generation time for the parent and mutant strains was approx-
imately 20 h). Inactivation of ligB did not affect cell morphol-
ogy and motility.
In order to determine whether LigB may have a role in
virulence in vivo, we evaluated ligB mutants and the parental
wild-type strain using the standard hamster model for acute
leptospirosis. Different numbers of organisms (log increases in
the challenge dose) were inoculated intraperitoneally to pro-
duce infection. The proportion of hamsters which died and the
proportion which survived for each bacterial concentration
were used to calculate the LD50. Three independent experi-
ments in which groups of four animals were infected with each
challenge dose were performed (Table 1). The LD50was less
than 100 bacteria for both the wild-type and mutant strains.
Thus, the lack of LigB expression did not result in loss of
virulence as measured by the LD50. In addition, no significant
differences in the time to death were observed with the ligB and
wild-type strains (data not shown). The general health status of
the hamsters infected with the ligB and wild-type strains was
also assessed. Infections with the ligB and wild-type strains
produced similar pathological findings (jaundice, pulmonary
hemorrhage, dissociation of hepatic trabecula, and acute dam-
age of renal tubular epithelia with cell swelling in proximal
segments). The immunohistochemistry results showed the same
distribution of Leptospira in the renal parenchyma (Fig. 2).
The virulence of the ligB mutant was evaluated using the
rat model for renal colonization. In three separate experi-
ments, groups of four or eight rats were infected intraperi-
TABLE 1. Virulence of the wild-type and ligB mutant L. interrogans
Fiocruz L1-130 strains in the hamster model of leptospirosis
No. deaths (% of total) with a challenge dose ofa:
ligB KO2 mutant
ligB KO2 mutant
ligB KO2 mutant
aGroups of four hamsters were inoculated with each challenge dose.
bND, not determined.
VOL. 76, 2008ligB IS NOT ESSENTIAL FOR VIRULENCE OF L. INTERROGANS 5829
toneally with 108cells of either the ligB mutant or wild-type
strain as previously described (1). Rats were sacrificed 15
days after infection. The ligB mutant behaved like the wild
type. High levels of both strains were recovered in rat kid-
neys (Table 2). In hamster and rat experiments, the double-
crossover disruptant KO2 was recovered from animals at the
time of sacrifice and 2 weeks postchallenge, respectively,
and the genotype was confirmed by PCR and immunoblot
analysis (Fig. 1). This suggests that the ligB disruption was
stable in the absence of selection.
In vitro adherence of the ligB mutant to MDCK cells. Inter-
actions of L. interrogans wild-type and mutant strains with
cultured epithelial cells were assayed by examining the adher-
ence of leptospires to epithelial monolayers of MDCK cells.
Cell monolayers were incubated using a multiplicity of infec-
tion of 100 bacteria per MDCK cell, and subsequent binding
was quantified by microscopic analysis. There was not any
statistically significant difference between the L. interrogans
ligB mutant and the wild-type strain in the number of bacteria
associated with MDCK cells (Fig. 3). These findings suggest
that the ligB genotype does not influence in vitro host cell
Compared to other bacterial species, the genetic data for
leptospires and determination of the molecular basis of the
pathogenesis of these organisms are in their infancy. Analysis
of the complete genome sequences of pathogenic Leptospira
species revealed that more than 50% of the predicted open
reading frames did not exhibit similarity to genes encoding
proteins with known functions (8, 32, 39, 40). Until a method
for constructing pathogenic leptospire mutants is developed,
any function of leptospire proteins, including virulence factors,
remains speculative. Previous attempts to inactivate genes in
pathogenic Leptospira species have been unsuccessful. The pu-
tative role of LigB in virulence (21, 26, 34) prompted us to
generate an L. interrogans ligB mutant.
We used approaches used previously for saprophytic Lepto-
spira species (5, 16, 24, 25, 28, 37, 38, 45) to carry out gene
targeting by homologous recombination in the pathogen L.
interrogans. Although the efficiency of the transformations was
low, our results show the feasibility of performing allelic ex-
change in pathogenic Leptospira spp. by homologous recombi-
nation. Our previous attempts to generate homologous recom-
bination in L. interrogans were not successful (unpublished
data), presumably due to the target gene chosen. The use of a
large region of homologous DNA (more than 1 kb) may have
increased the probability of homologous recombination. The
ligAB locus appears to be the target of fragment rearrange-
ments and recombination events. It has been suggested that
ligA was created from ligB by gene duplication, since the frag-
ments which encode the first six Big domains are identical in
the two genes (26). Furthermore, sequence analysis of lig genes
from collections of pathogenic Leptospira species resulted in
evidence of recombination between Leptospira species at this
locus (unpublished data). Finally, in addition to allelic ex-
change derived from a double homologous recombination
event, we showed that targeted integration of a suicide plasmid
which contains a 5?- and 3?-truncated fragment of the gene of
interest can facilitate targeted mutagenesis in L. interrogans.
For members of the superfamily containing the Big proteins,
previous studies have demonstrated that compared to the wild-
FIG. 2. Pathology in hamsters infected with the ligB mutant.
(A) Gross appearance of hamsters infected with the wild-type (wt) and
mutant ligB mutant (KO2) strains and a representative uninfected
control hamster. (B) Livers and kidneys from hamsters infected with
the wild-type and ligB mutant strains of L. interrogans. Tissues were
stained with hematoxylin and eosin (HE) (magnification, ?400), and
the immunohistochemistry analysis was performed with antiserum spe-
cific for LipL32 (IHC) (magnification, ?1,000).
TABLE 2. Renal colonization of Rattus norvegicus with the wild-
type and ligB mutant strains of L. interrogans Fiocruz
L1-130 after experimental challenge
No. animals with evidence of Leptospira renal
colonization (% of total) based ona:
ligB KO2 mutant
ligB KO2 mutant
ligB KO2 mutant
aGroups of eight rats and groups of four rats were inoculated with 108
leptospires in experiments 1 and 3 and in experiment 2, respectively.
5830CRODA ET AL.INFECT. IMMUN.
type strain, an intimin-deficient enteropathogenic E. coli strain
is defective for adherence to cultured cells and for intestinal
colonization (33). Similarly, analysis of a Y. enterocolitica inv
mutant suggested that invasin is necessary for efficient trans-
location of the bacteria across the intestinal epithelium (36).
ligB is conserved among all pathogenic Leptospira species and
is upregulated when bacteria confront the host environment
(26, 34). Furthermore, expression of ligB is correlated with the
virulence status of Leptospira strains (26). Therefore, the work-
ing hypothesis has been that LigB is essential for the bacteria
to survive, disseminate, and/or colonize in the host.
Yet we found that loss of ligB was not associated with a loss
of virulence phenotypes. Inoculation of ligB mutants produced
the same acute disease manifestations and lethal outcomes
that were observed in hamsters infected with wild-type strains.
In addition, inoculation of ligB mutants resulted in efficient
renal colonization in experimental rats similar to that observed
with wild-type strains.
Moreover, we found that the L. interrogans ligB mutant was
able to adhere to epithelial cells in vitro. The interaction of L.
interrogans with host cells is critical for dissemination in the
host (4). Although the LigB protein has been shown to bind in
vitro to host extracellular matrix moieties (9, 23), our findings
suggest that there may be other modes of leptospiral attach-
ment to host epithelial cells. As a caveat, we did not examine
whether ligB mutants bind to extracellular matrix components,
including fibronectin, and further studies are required to eval-
uate this possibility. Furthermore, the mechanism of associa-
tion of the pathogen with the host cell in vivo may be quite
different than what is observed in vitro; therefore, we cannot
exclude the possibility that Lig proteins mediate host cell in-
teractions based on observations made with in vitro assays
Because of the location of the Spcrcassette in the 3? end of
ligB, a truncated LigB protein could have been expressed.
However, immunoblot analysis using polyclonal and monoclo-
nal antibodies against recombinant fragments located up-
stream of the disruption site did not allow identification of any
fragments in the mutant strains. Furthermore, whereas these
antibodies stained strongly with the wild-type strain in immu-
nofluorescence studies, no signal was associated with the mu-
tant strains. It is therefore unlikely that a truncated ligB frag-
ment was expressed in the mutants.
In the present study, the data obtained with the ligB mutant
suggest that an absence of LigB does not lead to a loss of
virulence and a loss of colonization in the acutely and chron-
ically infected animal models, respectively. In our challenge
experiments, hamsters and rats were infected by intraperito-
neal inoculation of leptospires. We cannot exclude the possi-
bility that LigB may play a role in penetration of the host or
other early events during infection. Alternatively, the fact that
the ligB mutant remained virulent may have been due to func-
FIG. 3. Adherence of the L. interrogans ligB mutant to MDCK epithelial cells. The adherence of the L. interrogans wild-type (A) and ligB mutant
KO2 (B) strains to MDCK epithelial cells was examined. Anti-Salmonella OmpA MAb was used as a control (C). Representative micrographs
obtained by fluorescence microscopy are shown. (D) Attachment ratios (means ? standard deviations) determined using 10 random fields. wt, wild
VOL. 76, 2008ligB IS NOT ESSENTIAL FOR VIRULENCE OF L. INTERROGANS5831
tional redundancy in the bacteria. The numerous lipoproteins
which are present in leptospires (11) in addition to the LigB
protein may compensate for the loss of LigB expression. Sev-
eral surface-associated Leptospira proteins, including LigA,
have been shown to interact in vitro with extracellular matrix
components (2, 3, 18, 31, 44). Thus, the function of LigB may
be replaced to various extents by other lipoproteins which may
play a role in host-cell interactions. LigA and LigB proteins
contain Big domains that may have redundant functions (9, 27,
34). Choy et al. demonstrated that domains within LigA and
LigB proteins bind specifically to fibronectin in vitro (9). A
phenotype distinct from that of the parental strain may occur
only when both genes are disrupted. Therefore, further studies
should include generation of ligA and ligAB mutants.
In conclusion, we demonstrated for the first time that site-
directed homologous recombination can be successfully
achieved in pathogenic Leptospira. The approaches used in this
study, therefore, make it feasible to produce knockout muta-
tions in putative virulence-associated genes in Leptospira and
evaluate the roles that these genes may play in leptospiral
This work was supported by a Fiocruz-Pasteur Scientific Coopera-
tion Agreement, by the Brazilian National Research Council (Instituto
Mile ˆnio 420067/2005), by the French Ministry of Research ANR
Jeunes Chercheurs (no. 05-JCJC-0105-01), and by the National Insti-
tutes of Health (grants 5 R01 AI052473, 2 R01 AI034431, and 2 D43
TW00919). J. Croda received scholarship 2007/00083-2 from the Re-
search Support Foundation of the State of Sa ˜o Paulo.
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Editor: A. Camilli
VOL. 76, 2008ligB IS NOT ESSENTIAL FOR VIRULENCE OF L. INTERROGANS 5833