The OmpA-Like Protein Loa22 Is Essential
for Leptospiral Virulence
Paula Ristow1,2, Pascale Bourhy1, Fla ´via Weykamp da Cruz McBride3, Claudio Pereira Figueira3, Michel Huerre4,
Patrick Ave4, Isabelle Saint Girons1, Albert I. Ko3,5, Mathieu Picardeau1*
1 Unite ´ de Biologie des Spiroche `tes, Institut Pasteur, Paris, France, 2 Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil, 3 Centro de Pesquisas Gonc ¸alo Moniz, Fundac ¸a ˜o Oswaldo Cruz, Salvador, Brazil, 4 Unite ´ de Recherche et d’Expertise Histotechnologie et Pathologie, Institut
Pasteur, Paris, France, 5 Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University, New York, New York, United States of America
Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is
mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a
mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has
a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was
attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented
strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host
infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal
model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.
Citation: Ristow P, Bourhy P, Weykamp da Cruz McBride F, Pereira Figueira C, Huerre M, et al. (2007) The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS
Pathog 3(7): e97. doi:10.1371/journal.ppat.0030097
Leptospira interrogans is a spirochete responsible for lep-
tospirosis. This disease, which is considered the most geo-
graphically widespread zoonosis, has emerged as a major
public health problem in developing countries [1–3]. Numer-
ous mammalian species, including rodents, excrete the
pathogen in their urine and serve as reservoirs for trans-
mission. Humans are usually infected through contact with
contaminated water or soil. Leptospirosis imparts its greatest
burden on poor rural farming and urban slum populations in
developing countries [1–3]. More than 500,000 cases of severe
leptospirosis occur each year, with a mortality rate of 5% to
20% . Little is understood of Leptospira pathogenesis, which
in turn has hampered the identification of new intervention
Leptospires are highly motile bacteria that are able to
penetrate skin and mucous membranes and rapidly dissem-
inate to other tissues shortly after infection. In susceptible
hosts such as humans, systemic infection produces severe
multi-organ manifestations, including jaundice, acute renal
failure, and severe hemorrhage in the lungs and other organs.
However, in animal reservoirs such as the domestic rat,
infection produces chronic and persistent asymptomatic
carriage in the renal tubules [1–3].
The virulence mechanisms, and more generally the
fundamental understanding of the biology of the causative
agents of leptospirosis, remain largely unknown. To date,
only a few proteins have been identified as putative virulence
factors. Pathogenic leptospires have been shown to express
adhesins [5,6], hemolysins , and many lipoproteins prom-
inent in leptospires and other spirochetes that could play a
role in host–cell interactions . The recent completion of
the genome sequence of pathogenic Leptospira strains [9–11]
has provided a basis for understanding the pathogenesis of
leptospirosis. However, to date, the role of putative virulence
factors that were identified in the genome sequence remains
speculative. The lack of genetic tools to manipulate patho-
genic Leptospira spp. has prevented testing of Koch’s
molecular postulates  and researchers have been unable
to elucidate the role of these determinants in virulence.
We recently provided evidence of gene transfer in L.
interrogans, which involved the transposition of a transposon
of eukaryotic origin . This advance has now made it
possible to apply genetic approaches to the identification of
virulence determinants and vaccine candidates in pathogenic
Leptospira spp. In this study, we characterized a mutant of the
pathogen L. interrogans, which we obtained by random
transposon mutagenesis. This mutant exhibited transposon
insertion in a gene, loa22, which was described by Koizumi et
al.  as encoding for a lipoprotein (Loa22) of 22 kDa with a
C-terminal OmpA domain. Previous studies suggested that
this protein may play an important role in infection [14–17].
Herein, we show that the mutant loa22?strain is avirulent in
animal models, therefore demonstrating that Loa22 is
essential for in vivo infection of pathogenic leptospires.
Disruption and Complementation of loa22 in L.
interrogans Serovar Lai
Plasmid pMSL  was used to deliver the spectinomycin-
resistant Himar1 transposon into L. interrogans serovar Lai
strain Lai. One of the transposon mutants exhibited an
insertion in a putative gene, LA0222, encoding a protein (195
Editor: Michael R. Wessels, Children’s Hospital Boston, United States of America
Received April 9, 2007; Accepted May 17, 2007; Published July 13, 2007
Copyright: ? 2007 Ristow et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviation: ELISA, enzyme-linked immunosorbent assay
* To whom correspondence should be addressed. E-mail: email@example.com
PLoS Pathogens | www.plospathogens.orgJuly 2007 | Volume 3 | Issue 7 | e97 0894
amino acids in length) that was reported by Koizumi et al. 
to be Loa22, a 22-kDa Leptospira lipoprotein with an OmpA
domain; we will therefore refer to this protein henceforth as
Loa22. The L. interrogans serovar Lai protein (LA0222)
exhibits 99% and 96% similarity with orthologs in the
pathogens L. interrogans serovar Copenhageni (LIC_10191)
and L. borgpetersenii serovar Hardjobovis (LBL_2925 /
The protein Loa22 exhibits a bipartite structure, which
includes an N-terminal domain (residues 1–77) that is
unrelated to other eukaryotic or prokaryotic protein do-
mains, followed by an OmpA domain (residues 78–186).
According to SpLip , an algorithm for the prediction of
spirochetal lipoproteins, Loa22 is a possible lipoprotein with
an atypical Leu residue prior to Cys or a probable lipoprotein
with a cleavage site between residues 20 and 21, as indicated
by the LipoP algorithm for lipoprotein prediction in Gram-
negative eubacteria . C-terminal amino acid sequence
analysis of Loa22 revealed that other proteins of L. interrogans
(LA4337, LA3685, LA0056, LA3615, and LB328) have
sequence homology with members of the OmpA family.
These L. interrogans putative proteins, including Loa22, share
between 46% and 59% sequence similarity in their C-
terminal domain, but they have significant amino acid
sequence heterogeneity in their N-terminal domains.
Because there is no replicative plasmid vector available for
pathogenic Leptospira, we reintroduced the wild-type copy of
the gene encoding Loa22 into the spectinomycin-resistant
mutant strain by using a kanamycin-resistant transposon
carrying loa22 (Figure 1C). Transposition within the chromo-
some is random, so we identified the transposon insertion
sites in several transformants and selected one strain, TK2,
for further studies (Figure 1A and 1B). Enzyme-linked
immunosorbent assay (ELISA) (Figure 1D) and immunoblot
analysis (unpublished data) confirmed the absence of detect-
able Loa22 in the mutant loa22?strain, whereas the protein
was expressed in the wild-type and complemented strains
(Figure 1D). Inactivation of L. interrogans loa22 did not affect
cell morphology and motility. The wild-type, loa22?, and TK2
strains had similar cell growth kinetics in liquid Ellinghausen-
McCullough-Johnson-Harris (EMJH) medium, indicating that
genetic manipulation did not alter growth in vitro.
Loa22 Is a Surface-Exposed Protein
Immunofluorescence studies found that Loa22 is a surface-
exposed moiety (Figure 2). Antiserum to Loa22 labeled the
surface of live wild-type and complemented TK2 strains but
did not label the surface of the mutant loa22?strain. In
control experiments, antisera to LipL32 (Figure 2) and
LipL41 (unpublished data) labeled the surface of wild-type,
mutant loa22?, and TK2 strains, indicating that the labeling
method was able to detect previously described surface-
exposed LipL32 and LipL41 , but not Loa22, in the
mutant loa22?strain. The procedure specifically detected
antibodies bound to the leptospiral surface: antisera to
LipL31, a previously described lipoprotein associated with
the inner membrane  (Figure 2), and cytoplasmic heat-
shock protein GroEL (unpublished data) did not label live
leptospires in this procedure, although the antisera strongly
labeled fixed, permeabilized leptospires (unpublished data).
These results indicate that Loa22 is a surface-exposed
component of the leptospiral outer membrane as previously
Virulence of the Mutant loa22?Strain Is Attenuated in
Experimental Animal Models
The guinea pig and hamster, the standard experimental
models for leptospirosis [1,2], were used to evaluate the
virulence of the wild-type, mutant loa22?, and complemented
strains (Table 1). In two experiments, ten of fourteen and
eight of eight of the guinea pigs died when inoculated with
intraperitoneal challenges of 2 3 108and 4 3 108wild-type
bacteria, respectively. Infected guinea pigs developed lepto-
spirosis with characteristic signs such as prostration and
jaundice (Figure 3), and died within 4 to 6 d after the infection
In contrast, the mutant loa22?strain demonstrated loss of
virulence, as reflected by the inability of challenge doses of 23
108and 4 3 108bacteria to produce death in guinea pigs (14
and eight animals, respectively) (Table 1). The difference in
mortality was significantly lower for animals challenged with
the loa22?than those challenged with the wild-type strains
(0% versus 71% and 0% versus 100% in experiments 1 and 2,
respectively, p , 0.05). Guinea pigs infected with the loa22?
strain did not demonstrate clinical signs of leptospirosis
during the 21-d follow-up period. The mutant loa22?strain
was isolated from blood at post-challenge day 3 in four of four
infected guinea pigs that were infected with 2 3 108bacteria
in a separate experiment. In addition, the loa22?strain was
isolated from the kidneys of five of seven guinea pigs killed at
post-challenge day 21 (experiment 1, Table 1). However,
cultures of kidneys from animals infected with the loa22?
mutant required an incubation period of more than 2 wk to
test positive for the bacteria, suggesting that the number of
viable leptospires in these tissues was low. In addition, when
cultures of livers of guinea pigs infected with the wild-type
strain were positive for infection, we were not able to isolate
the loa22?strain by culture of liver tissues from seven guinea
pigs killed at post-challenge day 21. These findings indicated
that although the mutant did not induce disease, it was able to
cause bacteremia and colonization following infection.
Sequential in vivo passaging and re-isolation of the loa22?
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Virulence Attenuation in Leptospira interrogans
The spirochetes, which include medically important pathogens such
as the causative agents of Lyme disease, syphilis, and leptospirosis,
constitute an evolutionarily unique group of bacteria. Leptospirosis
is a zoonotic disease that causes a high rate of mortality and
morbidity in humans and animals throughout the world each year.
The year 2007 marks the centenary of the discovery of the causative
agent of leptospirosis, Leptospira interrogans. Until now, the genetic
obstacles posed by leptospires (principally, the difficulties in
generating targeted mutants) have hampered the identification of
virulence genes. In this study, we describe an avirulent mutant in a
pathogenic Leptospira that was obtained via disruption of loa22, a
gene that encodes an outer membrane protein containing an OmpA
domain. This mutation resulted in an avirulent mutant in the guinea
pig model, and reintroduction of loa22 into the mutant restored
Leptospira’s ability to kill guinea pigs. Our results therefore indicate
that loa22 is a virulence determinant that is, to our knowledge, the
first identified for this pathogen.
strain from blood or tissues of infected guinea pigs (seven
cycles in total) failed to recover a virulent isolate that could
induce clinical disease or death in guinea pigs.
Complementation of loa22?restored the virulence pheno-
type of the mutant loa22?strain in the guinea pig infection
model. Challenge doses of 23108and 43108of TK2 bacteria
caused death in 43% and 75%, respectively, of the inoculated
animals (Table 1). Deaths occurred 5 to 9 d after challenge.
There were no significant differences between the death rates
among guinea pig groups challenged with the wild-type and
TK2 strains. DNA was extracted from TK2 strains that were
used to challenge guinea pigs and TK2 strains that were re-
isolated from guinea pigs during autopsy. Southern blot and
PCR analyses demonstrated that these isolates had the
complemented loa22 genotype and the spectinomycin and
kanamycin cassettes (unpublished data), indicating that the
observed restoration in virulence was not due to contami-
nation of inoculating cultures with the wild-type strain.
Figure 1. Disruption and Complementation of loa22 in L. interrogans
(A and B) Analysis of chromosomal DNA from the parental (lane 1), mutant loa22?(lane 2), and complemented TK2 strains (lane 3) by PCR with primers
S1a and S1b (A) and Southern blot of EcoRI-digested DNA probed for hybridization with the spectinomycin (SpcR)- and kanamycin (KmR)-resistant
cassettes (B). Primers S1a and S1b are located in the flanking sequences of the insertion site of the spectinomycin-resistant transposon into loa22. This
analysis revealed that there was an insertion of 1.3 kb in the mutant loa22?strain and that an additional copy of loa22 was present in the
(C) Schematic representation of the genotype of the parental (wt), mutant, and complemented strains. Arrowheads in white indicate the position of
(D) ELISA of plates with total bacterial antigens and Loa22 antiserum (serum dilution 1:800).
PLoS Pathogens | www.plospathogens.orgJuly 2007 | Volume 3 | Issue 7 | e97 0896
Virulence Attenuation in Leptospira interrogans
Hamsters were challenged with wild-type, mutant loa22?,
and TK2 strains to confirm the findings observed in the
guinea pig model. Inoculation with 108and 53107wild-type
bacteria induced death in 100% and 90%, respectively, of the
animals (Table 1, experiments 3 and 4, respectively). In
contrast to what was observed in the guinea pig model,
challenge with mutant loa22?bacteria caused death in one of
ten hamsters in the two experiments. Autopsy evaluation
performed in experiment 4 found that the hamster died from
manifestations of leptospirosis. However, death rates were
significantly lower (10% versus 100%, p ¼ 0.00011; and 10%
versus 90%, p ¼ 0.001 for experiments 3 and 4, respectively)
for hamsters challenged with loa22?than those challenged
with wild-type strains. Challenge with the TK2 strain
produced death in 60% (six of ten) and 80% (eight of ten)
of the hamsters in experiments 3 and 4, respectively,
indicating that as in the guinea pig model, complementation
of loa22 in the mutant strain partially restored virulence.
Figure 2. Surface Localization of Loa22
Surface immunofluorescence assay was performed with L. interrogans wild-type strain (wt), mutant loa22?(loa22?), and mutant loa22?complemented
with wild-type loa22 (TK2). Strains were labeled with antibodies against Loa22 and the following lipoproteins: LipL32, a surface-exposed lipoprotein
, and LipL31, a lipoprotein that is associated with the inner membrane and not surface-exposed . Alexa and fluorescein isothiocyanate (FITC)–
conjugated secondary antibodies were used to detect surface-bound antibodies to Loa22 and LipL32 and LipL31, respectively. A DAPI counterstain was
used to document the presence of leptospires. A photomicrograph is shown from one of three representative experiments.
PLoS Pathogens | www.plospathogens.org July 2007 | Volume 3 | Issue 7 | e970897
Virulence Attenuation in Leptospira interrogans
Mutant loa22?Strain Does Not Produce Tissue Pathology
in the Guinea Pig Model
Necropsy evaluation of guinea pigs infected with wild-type
strain at post-challenge days 5 and 6 found macroscopic
lesions associated with leptospirosis (Figure 3A). Diffuse
hemorrhage was observed in kidneys, and multi-focal
hemorrhage was seen in lungs, stomachs, and intestines
(unpublished data). Splenomegaly was observed, as well as
jaundice of the liver and subcutaneous, ascites, and hemo-
thorax. None of these findings was observed, except for
Table 1. Virulence of L. interrogans Serovar Lai Strain Lai and Its Derivatives in the Guinea Pig and Hamster Infection Models
Number of Animals
Time to Death
Experiment 1: Guinea pigs; challenge dose, 2 3 108
Experiment 2: Guinea pigs; challenge dose, 4 3 108
Experiment 3: Hamsters; challenge dose, 108
Experiment 4: Hamsters; challenge dose, 5 3 107
awt, wild-type strain; loa22?, mutant loa22?; TK2, complemented strain.
bFisher test was performed to determine if there was a significant difference in mortality between the wt and other strains.
Figure 3. Gross Examination of Infected Guinea Pigs
Left panel: Guinea pigs infected with the wild-type (A) and complemented strains (C) with clinical findings of jaundice and hemorrhages that are absent
in guinea pigs infected with the mutant loa22?strain (B).
Right panel: Lungs of a guinea pig infected with mutant loa22?did not exhibit macroscopic hemorrhage (B), in contrast with lungs of guinea pigs
infected with the wild-type (A) and complemented strains (C). Tissues were observed 6 d post-inoculation.
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Virulence Attenuation in Leptospira interrogans
splenomegaly, in necropsies of guinea pigs infected with the
mutant loa22?strain (Figure 3B). Infection with the TK2
strain, in which loa22?was complemented, produced the
complete spectrum of gross lesions observed in infections
with wild-type strain (Figure 3C).
Hematoxilin and eosin staining of sectioned lung, kidney,
spleen, and liver from guinea pigs infected with the wild-type
strain demonstrated characteristic histopathologic findings
for leptospirosis (Figure 4). Spleens were hemorrhagic, with
focal necrosis in the red pulp (unpublished data). Intra-
alveolar hemorrhage associated with interstitial infiltration
with polymorphonuclear and mononuclear cells was a
prominent finding in lung sections (unpublished data).
However, infection with mutant loa22?strain produced
markedly reduced or absent inflammatory responses and
tissue pathology in guinea pigs on post-challenge day 6
(Figure 4). Liver tissues demonstrated mild parenchymal
dystrabeculaton and periportal infiltrates without focal
necrosis or hemorrhage (Figure 4A, middle panel). Kidneys,
spleens, and lungs from mutant-infected animals exhibited
sparse or absent inflammatory infiltrates. Infection with the
TK2 strain, in which loa22?was complemented, produced
Figure 4. Livers and Kidneys from Guinea Pigs Infected with the Wild-Type Strain and Mutant loa22?Strain
All images are from guinea pigs 6 d post-inoculation. The right panels show normal livers (A–C) and kidneys (D and E). Tissues were stained by
hematoxylin and eosin (3200, [A and D]), Warthin–Starry (31000, [B and E]) and immunohistochemistry with antiserum specific to LipL32 (3200, [C]). Left
panel, wild-type strain (wt); middle panel, mutant loa22?strain.
(A–C) Livers of wt-infected animals exhibit important periportal lymphoplasmocitary inflammatory infiltration, loss of parenchymal architecture, and
increase of biliary canalicules in comparison with a normal aspect of mutant loa22?. Distortion of liver cords is related to numerous leptospires along
cell membranes of hepatocytes (arrow, [B]) in wt-infected animals.
(D and E) Kidneys of wt-infected animals present hemorrhages in Bowman’s spaces, lumen of renal tubules, and interstitium (D). A large number of
leptospires is seen in Bowman’s space (arrow), sometimes forming a cap (E). Histology of kidneys infected with mutant loa22?was considered as normal
(D and E).
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Virulence Attenuation in Leptospira interrogans
similar pathological findings as observed for the wild-type
strain (Figure 5).
Silver staining and immunohistochemistry demonstrated
the abundant numbers of leptospires in the livers (Figure 4B
and 4C, left panel) and kidneys (Figure 4E, left panel) of
guinea pigs infected with the wild-type strain at post-
challenge day 6. Sparse numbers of leptospires were found
in the interstitial and alveolar spaces of the lungs. In contrast,
leptospires were not detected in tissues of guinea pigs
infected with the loa22?strain at post-challenge days 6
(Figure 4B, 4C, and 4E, middle panel) and 21. In sectioned
kidneys and livers from guinea pigs infected with the
complemented TK2 strain (Figure 5), immunohistochemical
analyses identified leptospires in numbers similar to those
observed for wild-type infections. Antiserum to Loa22 stained
all wild-type (Figure 6) and TK2 (unpublished data) lepto-
spires found in kidney and liver sections, demonstrating that
this protein is expressed during acute leptospirosis.
The recent completion of the genome sequences of
pathogenic Leptospira strains has led to the identification of
putative determinants that may play a role in virulence [9–
11]. One such determinant, loa22, is up-regulated during host
infection  and encodes a lipoprotein with an OmpA
domain  that is strongly recognized by sera from human
leptospirosis patients . Furthermore, Loa22 is conserved
among pathogenic Leptospira [14–16], suggesting that it may
play a specific role in disease pathogenesis. However, its role
has not been elucidated until now, because targeted muta-
genesis was not previously feasible in pathogenic Leptospira.
Recently, we showed that the Himar1 mariner transposon
permits random mutagenesis in the pathogen L. interrogans
. In search of mutants that might be affected in virulence,
we identified an L. interrogans mutant exhibiting Himar1
insertion into loa22. By analysis of the loa22?strain, we now
show that Loa22 is required for virulence of the pathogen
within animal models and fulfills the molecular Koch’s
postulates  as a virulence factor.
Complementation of the virulence phenotype of the loa22?
strain by chromosome insertion of loa22 demonstrated that
the virulence defect was due to the inactivation of loa22 and
not to a second-site mutation. Transcriptional data and
sequence analysis of the transposon insertion sites in the
mutant and complemented strains further confirm that
Himar1 insertion did not affect another gene that could be
involved in virulence (unpublished data). The parental and
Figure 5. Histopathologic sections on Liver, Kidney, and Lung of Guinea
Pigs Infected by Complemented Strain TK2 6 d Post-Inoculation
Left panel: Hematoxylin and eosin staining (3200) of infected guinea
pigs. Right panel: Immunochemistry with antiserum specific for LipL32
(3200; except [C], 31,000). Pictures of histopathology were similar
between animals infected with wild-type and complemented strains.
(A) The liver has a great loss of architecture and areas of necrosis and
inflammatory infiltration, which are both associated with the presence of
(B) Kidneys present hemorrhages, tubular necrosis, and inflammatory
infiltration, with leptospires mainly located in Bowman’s spaces and
(C) Lungs have marked intra-alveolar hemorrhages with inflammatory
infiltrates, and few leptospires are present within septal membranes and,
sometimes, in macrophages ([C], right panel).
Figure 6. In Vivo Expression of Loa22 in Liver and Kidney of Guinea Pigs Infected with L. interrogans Serovar Lai
(A) Liver, (B) kidney. Histopathologic sections were stained by immunochemistry using Loa22 antiserum (31,000). Intact organisms were found in biliary
ducts (A) and in large number in Bowman’s spaces and proximal tubules (B). Scale bar ¼ 20 lm.
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Virulence Attenuation in Leptospira interrogans
mutant strains of L. interrogans showed similar cell morphol-
ogy and growth characteristics in vitro, which demonstrates
that loa22 is not essential for in vitro growth. Although there
was no statistical difference in death rates among animals
challenged with wild-type and complemented strains, infec-
tions with the complemented strain did not cause death in all
animals. The inability to restore complete virulence (100%
lethality) in the complemented strain did not appear to be
due to instability of the construct, because the complemented
strain resulted from chromosome insertion. The comple-
mented strain that was re-isolated from animals expressed
Loa22. In addition, infection with another strain, TK1, for
which the loa22 gene was complemented at another chromo-
somal site, caused death rates in guinea pigs (six of eight
guinea pigs; challenge dose: 4 3 108bacteria/ml ), which was
not significantly different from results obtained for infections
with the TK2 strain (Table 1). The complemented strains were
subjected to more in vitro passages than the wild-type strain
due to electroporation-mediated transformation followed by
plating onto solid medium. Leptospires are known to lose
their virulence phenotype with prolonged in vitro culture
passages , although the mechanism for this loss is not well
understood. It is also possible that the complemented loa22
gene did not attain the optimal level of expression required
for the virulence phenotype.
Infection with L. interrogans produces a lethal infection in
the standard hamster and guinea pig model and mimics the
clinical presentation of severe leptospirosis in humans (i.e.,
jaundice and pulmonary hemorrhage) [1–3]. Loss of loa22
attenuated the ability of leptospires to cause clinical disease
in addition to death in guinea pigs and hamsters. Consistent
with the lack of disease manifestations, mildly abnormal or no
pathologic changes were observed in tissues of guinea pigs
infected with the loa22?strain. Although the loa22?strain was
recovered by culture isolation from blood on post-challenge
day 3 and in kidneys of guinea pigs during the 21-d post-
inoculation period, immunohistochemical analysis did not
detect leptospires in these tissues, suggesting that loss of the
loa22 genotype reduced the pathogen burden in tissues
during infection. The lack of tissue pathology observed in
loa22?-infected animals presumably reflects decreased inflam-
mation elicited by lower numbers of leptospires. Loa22 may
influence one of several virulence steps during infection, such
as the ability to disseminate throughout the host after
inoculation, adhere to host cells, and establish persistent
infection, which may, in turn, explain the finding that mutant
loa22?strain did not achieve sufficient numbers in tissues to
produce disease manifestations and pathology. The standard
inoculation method used in animal models of leptospirosis—
intraperitoneal injection—may not reflect conditions en-
countered during natural infection, because leptospires enter
the host by penetrating breaks in the skin or traversing the
mucosal membranes. Further studies that use subconjunctival
or subcutaneous challenge routes will need to be performed
to determine whether Loa22 plays a role in the initial steps of
The process of host infection by pathogens is usually
complex and multifactorial. We observed that loss of loa22
genotype was associated with complete loss of virulence in the
guinea pig model, as indicated by the inability to induce
death in these animals. In hamsters, infection with the mutant
loa22?strain was associated with significantly reduced death
rates (10% versus 100%, mutant versus wild-type strain,
respectively) but did not lead to complete loss in the ability to
produce a lethal infection. The observed differences may
reflect intrinsic differences in susceptibility of the two animal
models to infection with the strain L. interrogans serovar Lai,
which was used in this study.
Our results demonstrate that Loa22 is exposed on the
surface of L. interrogans, confirming the localization of the
protein to the outer membrane . The structure of Loa22 is
composed of a C-terminal OmpA domain of approximately
110 amino acids. This OmpA domain refers to the C terminus
of Escherichia coli OmpA, a major outer membrane protein of
E. coli. Orthologs of the OmpA domain are found in proteins
from a wide range of bacterial species. Predictions for the
structure of this C-terminal OmpA domain have ranged from
that of a globular domain containing a predicted peptido-
glycan-associating motif that is located in the periplasm
[22,23], to a domain containing a significant proportion of
anti-parallel b sheets that are associated with the outer
membrane [24,25]. The N-terminal domain of E. coli OmpA
was crystallized as a b-barrel–structured porin, which is
believed to be inserted into the outer membrane .
However, this N-terminal region has no significant sequence
similarity to Loa22. Because there is no sequence similarity
between Loa22 and other OmpA-like proteins in this N-
terminal region, these proteins may be structurally distinct.
The role of Loa22 during pathogenesis remains to be
determined. The OmpA protein of E. coli and other Gram-
negative bacteria are believed to play a multifunctional role
in bacterial physiology and pathogenesis. In Gram-negative
bacteria, OmpA has been shown to be an adhesin [27–29] and
to induce cytokine production by dendritic cells [30,31]. In a
recent study, recombinant Loa22 was shown to bind in vitro
to a limited extent with components of the extracellular
matrix such as plasma fibronectin and collagen types I and IV
, suggesting that the surface-exposed domain of Loa22
may, in fact, act as an adhesin. Furthermore, the lipopeptide
moieties of spirochetes are potent mediators of the inflam-
matory response . Loa22, which has a lipobox sequence,
may therefore induce severe disease manifestations by
eliciting the host immunopathogenic responses.
Proteins of the OmpA family have been proposed to play a
role in the stabilization of the envelope structure . Loa22 ,
which includes a predicted peptidoglycan-associating motif
in its C-terminal domain , may form a bridge linking the
protoplasmic cylinder, including the peptidoglycan layer, and
the outer membrane. Although the loa22?strain was
recovered from the animal, we cannot rule out that the
absence of this protein in the membrane may affect several
steps in host infection, such as the stability of the outer
membrane, survival of the leptospiral pathogen in vivo, and
the ability to penetrate tissues during dissemination or
adhere during colonization.
In conclusion, this study identified the first virulence
factor, to our knowledge, in pathogenic Leptospira and will
form the basis for further investigation of the role that Loa22
plays in leptospiral pathogenesis. Furthermore, Loa22 is
expressed on the leptospiral surface, suggesting that immu-
nization with this protein may elicit bacteriocidal or patho-
genesis-blocking immune responses. Bacterin-based vaccines
have been used in some countries but they present a number
of disadvantages, including adverse reactions, short duration
PLoS Pathogens | www.plospathogens.orgJuly 2007 | Volume 3 | Issue 7 | e970901
Virulence Attenuation in Leptospira interrogans
of efficacy, and lack of protection against serovars not
included in the vaccine preparations [1–3]. A better under-
standing of the role of Loa22 may facilitate identification of
defined and more effective subunit vaccine candidates for
Materials and Methods
Bacterial strains and growth conditions. L. interrogans serovar Lai
strain Lai 56601 (gift from the National Institute for Communicable
Disease Control and Prevention, ICDC China CDC) and other
Leptospira strains were grown at 30 8C in EMJH [32,33] liquid medium
or on 1% agar plates. E. coli was grown in Luria-Bertani (LB) medium.
When necessary, spectinomycin or kanamycin was added to culture
media at 50 lg/ml.
Construction of mutant and complemented strains. Random
insertion mutagenesis was carried out in low-passage L. interrogans
serovar Lai strain Lai 56601 with plasmid pMSL as previously
described . After 4 to 6 wk of incubation, spectinomycin-resistant
transformants were inoculated in liquid medium. Genomic DNA was
then extracted, and the transposon insertion site of each trans-
formant was identified by ligation-mediated PCR as previously
described . Among the transformants, we selected a mutant with
an insertion into loa22, also called LA0222, at position 220548 in the
large chromosome (CI) of L. interrogans for further characterization.
For complementation, loa22 was amplified with primers OMIA (59-
AGTCGACGGTTTTGGTGGGATGGATAG-39) and OMIB (59-AGTC-
GACAGACGTTGAGTTGCCACAGC-39) and cloned into the SalI
restriction site of the kanamycin-resistant transposon carried by
plasmid pMKL, resulting in plasmid pMKLoa22. The mutant loa22?
strain was then electrotransformed by pMKLoa22, and the transposon
insertion site of some transformants was identified as described
above. Two transformants, strains TK1 and TK2, were further studied;
one, strain TK1, exhibited the kanamycin-resistant transposon at
position 1079614 (between LA1074 and LA1075), and the other, strain
TK2, at position 84051 (into LA0071) of the large chromosome of L.
interrogans. Confirmation of genotypes was performed by using PCR
with primers S1a (59-TTGTTGTGGTGCGGAAGTCG-39) and S1b (59-
GGTCCCGAACAAGCAGAAGG-39), which are located in the flanking
sequences of the transposon inserted into loa22, and Southern blots.
Enzyme-linked immmunosorbent assay (ELISA). L. interrogans
strains were grown in EMJH until the culture reaches an optical
density at 420 nm (OD420) of 0.4. L. biflexa was also used a control.
Concentrations were adjusted to 109bacteria/ml in a volume of 20 ml
of EMJH, and 40 ll of 37% formaldehyde was added, then incubated 2
h at room temperature and boiled for 30 min. After adjusting pH at
9.6, cultures were centrifuged at 8,000g for 20 min and pellets were
resuspended in 10 ml of 0.05 M bicarbonate buffer. Ninety-six–well
flat-bottom polystyrene assay plates (Immulon, VWR, http://www.vwr.
com/) were coated overnight at 4 8C with 50 ll of total bacterial
Plates were washed three times with phosphate buffered saline
(PBS) (pH 7.2) and wells were blocked with 50 ll of 5% nonfat milk
PBS for 45 min at 37 8C. Plates were incubated 45 min at 37 8C with 50
ll of an 800-fold dilution of mouse polyclonal antiserum to Loa22
 diluted in milk PBS, washed, and incubated for 1 h at 37 8C with
50 ll of a 2,500-fold dilution of horseradish peroxidase–conjugated
sheep affinity–purified antibody specific to mouse immunoglobulin G
(IgG) (Promega, http://www.promega.com/). After washing of the
plates, 50 lL of ABTS peroxidase substrate (Roche, http://www.
roche.com/) was added, and the plates were incubated in the dark at
room temperature for 25 min. Optical density was measured using an
ELISA reader (Labsystems Multiskan MS; Thermo Scientific, http://
www.thermo.com/) at 405 nm.
Localization of Loa22 by immunofluorescence. Surface immuno-
fluorescence labeling was performed according to a modified
protocol of Cullen et al. . Suspensions of 107live leptospires in
10 ll of PBS were placed onto poly-L-lysine–coated (Sigma, http://
www.sigmaaldrich.com/) slides for 1 h in a humidified chamber. The
slides were washed twice with PBS with 2% bovine serum albumin
(PBS-BSA) and were incubated for 1 h with antisera (diluted 1:100 in
PBS-BSA) to recombinant Leptospira proteins. After incubation with
mouse antiserum to Loa22  and rat antisera to LipL32, LipL41,
LipL31, and GroEL, the slides were washed gently with PBS-BSA.
Leptospires were fixed by applying cold methanol and incubating the
slides for 10 min at ?20 8C. The slides were then washed and
incubated with donkey anti-mouse IgG antibodies conjugated to
Alexa dye (Molecular Probes, http://probes.invitrogen.com/) or goat
anti-rat IgG antibodies conjugated to fluorescein isothiocyanate
(Jackson ImmunoResearch Laboratories, http://www.jacksonimmuno.
com/) for 1 h at 37 8C. The slides were washed twice with PBS-BSA and
incubated with 1 lg/ml DAPI (Molecular Probes) for 1 h at room
temperature. The slides were mounted in anti-fading solution after
washing and before visualization of stained organisms with fluo-
Animal infections. Golden Syrian male hamsters, 5 to 8 wk old, and
Hartley male guinea pigs (Charles River Laboratories, http://www.
criver.com/), 2 to 3wk old, were used for this study. Animals were
maintained under standard conditions according to institutional
guidelines. Water and food were given ad libitum. All animal
infections were performed with intraperitoneal injection of low-
passage strains in 1 ml of EMJH medium. Negative control animals
were injected intraperitoneally with 1 ml of EMJH medium. Animals
were monitored daily for characteristic signs of leptospirosis (i.e.,
prostation and jaundice) and survival. Surviving animals were killed
after a 21-d post-challenge follow-up period. The 50% lethal dose
(LD50) for L. interrogans serovar Lai in 2- to 3-wk-old guinea pigs and
5- to 8-wk-old hamsters was approximately 108and 107leptospires,
respectively. Protocols for animal experiments were prepared
according to the guidelines of the Animal Care and Use Committees
of the Institut Pasteur and Fundac ¸a ˜o Oswaldo Cruz.
Histopathology. Guinea pigs were inoculated with 23108bacteria
of wild-type, mutant loa22?, and complemented strains of L.
interrogans serovar Lai strain Lai or EMJH alone. For mutant loa22?
strain and EMJH control group infections, three guinea pigs were
killed 6 and 21 d post-inoculation. For wild-type and TK2 strain
group infections, tissues were collected at the day of death (5 or 6 d
post-inoculation). Tissues (liver, kidneys, spleens, and lungs) were
fixed in 10% buffered formaldehyde, embedded in paraffin, and
sectioned according to routine histological procedures to produce 5-
lm sections that were then stained with hematoxylin and eosin and
Warthin–Starry silver impregnation . For immunohistochemistry,
paraffin was removed from the sections with xylene and ethanol.
Tissues were then treated in citrate buffer (pH 6) at 98 8C for 1 h and
nonspecific sites were blocked by incubation of sections with 1.5%
BSA at room temperature for 20 min. Tissues were incubated with
6,000- and 1,000-fold dilution of LipL32  and Loa22  antisera,
respectively, overnight at 4 8C. Samples were treated with 0.3%
hydrogen peroxide for 30 min at room temperature, then incubated
at room temperature for 30 min with goat anti-mouse or anti-rabbit
antibodies conjugated to peroxidase (Dako Cytomation, http://www.
dako.com/). Enzyme reactions were developed using AEC (3-Amino-9-
ethylcarbazole) staining kit (Sigma). The pathologist viewed the
histopathological preparations without knowing the infection status
of the animals.
The Entrez Genome (http://www.ncbi.nlm.nih.gov/sites/entrez?
db¼Genome) accession numbers for the genes and gene products
discussed in this paper are L. borgpetersenii serovar Hardjobovis
(NC_008508 and CP000348), L. interrogans serovar Copenhageni
strain Fiocruz L1–130 (AE016823), and L. interrogans serovar Lai strain
Lai 56601 (NC_004342).
We are thankful to N. Koizumi for the generous gifts of Loa22
antiserum and plasmid construct for production of Loa22 recombi-
nant and to G. P. Zhao for providing the Lai sequenced strain. We are
thankful to L. S. Fonseca and W. Lilembaum for their support and
encouragement. This work is part of the doctoral thesis of P. Ristow
at the Universidade Federal do Rio de Janeiro, Brazil.
Author contributions. PR, PB, MH, ISG, AIK, and MP conceived
and designed the experiments. PR, PB, FWdCM, CPF, PA, and MP
performed the experiments. PR, AIK, and MP wrote the paper.
Funding. This work was supported by the French Ministry of
Research ‘‘ANR Jeunes Chercheurs’’ (n805-JCJC-0105–01), Fiocruz–
Pasteur scientific cooperation program, Brazilian National Research
Council (300861/1996, 420067/2005, 554788/2006), and the National
Institutes of Health/US (TW00919). PR was supported by a fellowship
from the CAPES, Brazil, and the Fiocruz–Pasteur scientific cooper-
Competing interests. The authors have declared that no competing
PLoS Pathogens | www.plospathogens.org July 2007 | Volume 3 | Issue 7 | e970902
Virulence Attenuation in Leptospira interrogans
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PLoS Pathogens | www.plospathogens.orgJuly 2007 | Volume 3 | Issue 7 | e970903
Virulence Attenuation in Leptospira interrogans