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Leptospirosis is a zoonotic disease which has emerged as a major cause of morbidity and mortality among impoverished populations. One centenary after the discovery of the causative spirochaetal agent, little is understood of Leptospira pathogenesis, which in turn has hampered the identification of new intervention strategies to address this neglected disease. However the recent availability of complete genome sequences for Leptospira and discovery of genetic tools to transform the pathogen has led to major insights into the biology and pathogenesis of this pathogen. We discuss the life cycle of the bacterium and the new advances that have been made and their implications for the future prevention of this disease.
Descriptions of leptospirosis-like syndromes were reported in the scripts of ancient civilisations 1, but the first modern clinical description of the leptospirosis was that of Weil in 1886 2. Inada et al., in their landmark study from 1916, isolated leptospires, identified the organism as the causal agent of leptospirosis, and determined that rats are a reservoir for transmission to humans 3. Leptospires were subsequently isolated from a wide range of animal reservoir species and classified into serogroups and serovars as a function of their antigenic determinants (Box 1).
The genus Leptospira belongs to the phylum of spirochaetes 12. The subgroup of saprophytes (L. biflexa, L. wolbachii, L. kmetyi, L. meyeri, L. vanthielii, L. terpstrae, and L. yanagawae) form the deepest branch within the genus, while another subgroup includes the pathogenic species with 8 species (L. interrogans, L. kirschneri, L. borgpetersenii, L. santarosai, L. noguchii, L. weilii, L. alexanderi, and L. alstonii). Another evolutionary branch comprises the so-called “intermediate” group (L. inadai, L. broomii, L. fainei, L. wolffii, L. licerasiae), which contains species of unclear pathogenicity 4, 5. Leptospira spp. are also serologically classified into serovars, of which there are more than two hundred pathogenic serovars, on the basis of structural heterogeneity in the carbohydrate component of the lipopolysaccharide (LPS) 4, 5. Serotyping of leptospires is important for clinical or epidemiological investigations, since identification of serovars and serogroups provides clues on the host reservoirs involved in transmission. However, serotyping is performed in few reference laboratories worldwide. Furthermore, several studies have shown that the system of serogroups was not related to molecular classifications 4, suggesting that genes determining serotypes may be laterally transferred into different species. Consequently, the classification system based on genetic similarities is being used in conjunction with classical antigenic classification. Recently, the releases of genome sequences allowed the introduction of several approaches to genotype Leptospira spp, which include multilocus variable-number tandem-repeat (VNTR) analysis 145 and multilocus sequence typing (MLST) 44, 146, a typing method that is based on the partial sequences of housekeeping genes and may evolve as a standard genotyping method as it has for other bacterial species.
Leptospirosis, a zoonotic disease [AU: GT] with a worldwide distribution, is now recognised as an emerging infectious disease 4. Over the last decade, outbreaks during sporting events, adventure tourism and disasters underscore the ability of the disease to become a public health problem in non-traditional settings 4-6. Yet leptospirosis is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries and tropical regions 6. Leptospirosis, in addition to being an endemic disease of subsistence farmers 1, 4, 5, has emerged as a widespread problem in urban slum populations where inadequate sanitation has produced the conditions for rat-borne transmission of the disease 7-9. More than 500,000 cases of severe leptospirosis are reported each year, with case fatality rates exceeding 10% 10.
Previous reviews summarized our knowledge of the epidemiology, diagnosis, and clinical features of leptospirosis 1, 4-6 as well as the genomics of Leptospira spp. 11. This review will focus on the pathogenesis of leptospirosis and highlight the recent advances with respect to genetic approaches taken and the virulence factors discovered.
The genus Leptospira comprises of saprophytic and pathogenic species and belongs to the phylum of spirochaetes (Box 1) 12. Saprophytic leptospires, such as L. biflexa, are free-living organisms found in water and soil and unlike pathogenic Leptospira spp., do not infect animal hosts 1. Leptospires are thin, highly motile, slow-growing obligate aerobes with an optimal growth temperature of 30°C and can be distinguished morphologically from other spirochaetes on the basis of their unique hook or question mark-shaped ends 13 (Figure 1A).
The genomes for two pathogenic species, L. interrogans and L. borgpetersenii, and one saprophytic species, L. biflexa, have been sequenced 14-17. The majority (77-81%) of the genes in the Leptospira genome do not have orthologues found in the genomes of other spirochaetes, confirming that large degree by which leptospires have genetically diverged from other members of the phylum 12. Furthermore, comparative analysis of genomes of pathogens and saprophytes 11, 17 has provided insights on the genetic determinants that may be involved in pathogenesis (Box 2).
A major advance in the understanding of Leptospira and its pathogenesis has been the recent sequencing of the genomes of two pathogenic species, L. interrogans and L. borgpetersenii, and the saprophytic species L. biflexa 14-17. Overall, the genomes have a G + C content of between 35% and 41% and possess two circular chromosomes of approximately 4 Mb and 300 kb in size. The presence of a 74-kb replicon has also been identified in L. biflexa 17, which can possess a fourth circular replicon, the 74-kb leptospiral bacteriophage LE1 74, 147. A comparative analysis of Leptospira genomes provides clues with respect to the genetic determinants responsible for the different lifestyles of the spirochaetes 11. Comparison of the proteins across the genomes has revealed a common backbone of 2052 proteins for this genus 17. The L. interrogans and L. borgpetersenii genomes contain approximately 3400 and 2800 predicted coding regions (excluding transposases and pseudogenes), of which 656 of which are pathogen-specific and not found in the saprophyte L. biflexa. The functions of most (59%) of these genes are unknown, suggesting the presence of pathogenic mechanisms unique to Leptospira. The saprophyte L. biflexa, which survives exclusively in the external environment, has many more genes encoding environmental sensing and metabolic proteins than pathogenic leptospires 17. Although L. interrogans and L. borgpetersenii share 2708 genes between them, there are 627 and 265 genes from L. interrogans and L. borgpetersenii, respectively which are not shared with the other pathogenic species. L. interrogans have retained more genes from its free-living ancestor, most of which relate to survival in the external environment 17. L. borgpetersenii has a smaller genome (3.9 vs 4.6 Mb) and a much larger proportion of transposase genes or pseudogenes (20 vs 2%) than L. interrogans. Together these findings indicate that L. borgspetersenii is undergoing a process of genome reduction and specialization in the bacterium 16. Gene loss appears to have impaired the ability of L. borgpetersenii to survive in the external environment, and therefore rely on direct contact between host animals (i.e., cows), rather than indirect environmental exposures, as its principle mode of transmission.
Transmission requires continuous enzootic circulation [AU: GT] of the pathogen among animal reservoir, or as commonly referred, maintenance hosts (Figure 2). Leptospira serovars demonstrate specific host preferences with respect to their ability to produce high-grade carriage. For example, rats (genus Rattus) serve as reservoirs for the Icterohaemorragiae serogroup, whereas house mice (Mus musculus) are the reservoir for the Ballum serogroup 4, 5, 18. Furthermore, serovars often do not cause significant disease in reservoir hosts to which they are highly adapted (Box 3).
Leptospirosis is considered the most geographically widespread zoonotic disease 4 because of the wide range of animals, mainly mammalian species, for which it infects. Rodents are the primary reservoir for maintaining enzootic transmission in most settings (Figure 2) 5. This group includes not only rats and mice, but also voles, shrews, hedgehogs, and marsupials, all of which may serve as reservoirs of leptospirosis 1. However, some rodents, including hamsters and guinea pigs in particular, are nevertheless highly susceptible to leptospirosis and can be used as animal models of human leptospirosis. Amphibians, snakes and freshwater fish have also been shown to have the potential to harbour pathogenic Leptospira 148. Finally, although leptospires do not survive in seawater, leptospirosis has been reported in sea lions and seals, which were presumably infected in coastal rookeries 149. Direct modes of transmission, including venereal, congenital and suckling exposures, play a more important role in animals than in humans (Figure 2A) 1. Leptospirosis causes a broad spectrum of pathogenic processes in animals, for which acute disease and chronic colonization represent opposite poles. Humans are susceptible hosts in which infection causes severe acute manifestations but does not produce carriage. Infection in maintenance hosts such as rats causes an asymptomatic infection with persistent carriage 18. Leptospirosis in other animals is a mixture of the two processes: infection causes a range of acute-to-chronic manifestations and produces a carrier state for which duration varies considerably between species 1. In addition to being a human health problem, leptospirosis is a major veterinary disease associated with large economic costs 1. In animals such as dogs, deer and pigs, leptospirosis causes acute manifestations, such as jaundice, renal failure and bleeding, as are observed in human disease 1, 150, 151. Furthermore, leptospirosis causes a range of chronic manifestations in livestock, particularly cattle, pigs, sheep and goats, which are associated with reproductive losses, decreased mild production, stillbirths and abortions 152-154. Recurrent uveitis due to leptospirosis is a major problem among horses 155.
The pathogen colonises and is shed from the renal tubules of a broad spectrum of animals (Box 3). Leptospires survive for weeks to months in moist soil and water after excretion in the urine 19. Cell aggregation 19 and biofilm formation 20 (Figure 1B) may contribute to the survival of leptospires outside their hosts.
Pathogenic Leptospira spp. produce a systemic infection after an environmental exposure, establish persistent renal carriage and urinary shedding in reservoir animals, and cause tissue damage in multiple organs of susceptible hosts. Acute disease and chronic colonisation represent opposite poles of a wide spectrum of disease presentations (Box 3). Humans are incidental hosts in which leptospirosis causes acute disease manifestations and yet does not induce a carrier state required for transmission of the pathogen.
Leptospires penetrate abraded skin and mucous membranes and quickly establish a systemic infection by crossing tissue barriers and haematogenous [AU:GT] dissemination 1. It was believed that leptospires, like other spirochaetes, spread by transiting though intercellular junctions 21. However, leptospires have been shown to efficiently enter host cells in vitro 22, 23 and rapidly translocate across polarized cell monolayers without altering the trans-epithelial electrical resistance 24, 25. Leptospires are not facultative intracellular organisms for they are rarely observed intracellulary in infected tissues and appear to reside transiently within host cells as they cross cell monolayers in vitro 25. The process by which leptospires enter host cells is not understood: internalized leptospires have been observed in cytoplasmic 24, 25 and phagosome compartments 23 of normally non-phagocytic host cells. Nonetheless, these findings suggest that leptospires use host cell entry and rapid translocation as a mechanism to spread to target organs and evade immune killing.
Infection causes a prolonged leptospiraemia until the host mounts an effective acquired immune response which occurs one to two weeks after exposure (Figure 3A) 26. Leptospires are isolated from the bloodstream within minutes after inoculation 1 and are detected in multiple organs by the 3rd day after infection 26-29. Leptospires, whose burden in blood and tissues may reach up to 106-107 organisms/ml or g in patients 30, 31 and infected animals 29, are able to evade the host innate immune response during the early-phase of infection. The organism is resistant to the alternative pathway of complement activation 32, 33 and acquire Factor H and related fluid-phase regulators 34, 35, through ligands such as Leptospiral endostatin-like (LEN) proteins 36, 37. Host C4BP binds to the surface of leptospires 38, suggesting that a similar process may confer some protection against the classical pathway of complement activation.
The essential component of the pathogen’s life cycle is its ability to produce persistent renal carriage in reservoir animals. In rats, leptospires cause a systemic infection but are cleared from all organs except the renal tubules 28, 39. Colonised tubules are densely populated with leptospires, which aggregate together with an amorphous biofilm-like structure (Figure 1D). Rats have been shown to excrete leptospires in high concentrations (107 organisms/ml 39) for periods of 9 months after experimental infection 40.
Leptospires isolated from chronically-infected rat kidneys have significantly higher amounts of lipopolysaccharide (LPS) O-antigen than those isolated from livers of hamsters with acute disease, suggesting that expression of O-antigen content may facilitate induction of carriage 39. The renal tubule is an immunoprivileged site, a feature which may contribute to high-grade persistence of the pathogen. Moreover leptospires which are shed in the urine down-regulate the expression of proteins recognised by the humoral immune response in rats 41.
Infection does not produce disease until 5-14 days (incubation period, 2-30 days) after environmental exposure (Figure 3A) 1. In humans, leptospirosis causes a febrile illness which in its early-phase, often cannot be differentiated from other causes of acute fever. In most patients, illness resolves after the 1st week of symptoms. Yet a subset (5-15%) of patients progress to develop severe late-phase manifestations 6. Unlike bacterial infections such as gram-negative sepsis, leptospirosis does not cause a fulminating disease manifestations shortly after the onset of illness, which may relate to the low endotoxic potency of Leptospira LPS 1. Severe late-phase manifestations occurs four to six days after onset of illness (Figure 3A) but may vary depending on the infecting inoculum dose and other disease determinants. Weil’s disease is the classic presentation of severe leptospirosis and characterized by jaundice, acute renal failure and bleeding. In addition, there is increasing awareness of a new emerging severe disease form, leptospirosis-associated pulmonary haemorrhage syndrome (LPHS) (Box 4) for which the case fatality rate is >50% 6.
Leptospirosis-associated pulmonary haemorrhage syndrome (LPHS), first described in Korea and China 156, was brought to world attention by a large outbreak of this severe disease form in Nicaragua in 1995 157. Subsequently LPHS has emerged as a major cause of haemorrhagic fever in developing countries 30, 158-160. LPHS is striking for its fulminant presentation of massive pulmonary bleeding and acute lung injury and is associated with poorer clinical outcomes 6 indicating that the pathogenesis of LPHS may be different from that of Weil’s disease. LPHS patients have high amounts of leptospiral DNA (≥106 organisms/g) in lung tissues 30. However, scant numbers of intact leptospires are found in lung 49 The major lesion associated with LPHS is damage of the vascular endothelium 49, 50. More recently several reports have observed linear deposition of immunoglobulin and complement along the alveolar basement membrane and in the intra-alveolar space of lung tissues 69, 161, 162, suggesting a possible underlying autoimmune process. The sudden appearance of LPHS in certain settings 160, suggests that introduction of clones with enhanced virulence may also contribute to the recent emergence of this syndrome.
Development of leptospirosis and disease progression are influenced by the virulence characteristics of the strain, host susceptibility factors and infecting inoculum size during environmental exposure. Specific Leptospira species and serovars are more frequently found to cause severe disease in humans 42, 43. Thaipadungpanit et al. found that a single circulating clone caused a large and sustained nationwide epidemic in Thailand 44. Clonal transmission of strains has been described in other outbreaks and settings of endemic transmission 45, 46 and may reflect localized clusters of transmission 45. However the magnitude and duration of the epidemic in Thailand suggests that predominant clones may indeed possess specific factors which contribute to their overall biological success. The advent of high-throughput whole-genome sequencing provides an opportunity to determine whether such factors exist by screening isolate genomes for genetic polymorphisms associated with clinical and transmission-related phenotypes.
Our understanding remains limited on the acquired and innate host factors which influence infection and disease progression. An investigation of a triathlon-related outbreak identified HLA-DQ6 genotype as the first and to date only genetic susceptibility factor reported for leptospirosis 47. The authors found a synergistic risk interaction between HLA-DQ6 and swallowing water while swimming during the triathlon event. This environmental exposure was a likely proxy for an inoculum size effect. It is well known that increasing inoculum size shortens the incubation period and decreases survival in a dose-dependent manner in experimental animals (Figure 3B) 26, 48. The synergism between HLA-DQ6 and environmental exposures found during the triathlon outbreak constitutes the first gene-environment interaction identified for an infectious disease.
The onset of disease correlates with the appearance of agglutinating antibodies and clearance of leptospires by antibody-mediated opsonisation and lysis (Figure 3A) 1. Vascular endothelial damage is a hallmark feature of severe leptospirosis 49, 50 and causes capillary leakage, haemorrhage, and in a subset of cases, vasculitis. Leptospirosis activates the coagulation cascade 51, 52 and has been reported to cause disseminated intravascular coagulation in up to 50% of patients with severe disease manifestations 51.
Leptospiral components released after immune killing stimulate production of pro-inflammatory cytokines 53-56 and mediate inflammation and damage of end-organ tissues. The Jarisch-Herxheimer reaction, caused by the sudden release of these cytokines, is a complication of antimicrobial therapy for leptospirosis. Moreover, TNF-alpha may play a key role in disease progression since levels of this cytokine are a predictor of poor clinical outcomes 57.
The Leptospira LPS has been shown to activate Toll-like receptor 2 (TLR2) rather than the TLR4 pathway in human cells 58, an unusual finding that may relate to a 1-methylphosphate moiety which is not found in other bacterial lipid A 59. In addition, leptospiral lipoproteins induce innate responses by activating the TLR2 pathway 58, 60. As a caveat, Leptospira LPS activates both TLR2 and TLR4 pathways in mouse cells, indicating that there are species-specific differences with respect to TLR activation 61. Leptospires stimulate expansion of gamma-delta T cell populations in naïve peripheral blood mononuclear cells and leptospirosis patients have increased numbers of this specific subset 54, suggesting that acquired cell-mediated responses, in addition to innate and acquired humoral responses, may promote inflammation.
Infection causes pronounced physiological disturbances in the kidney and liver, which has led to the speculation that leptospires liberate a toxin. Leptospirosis produces a peculiar hypokalaemic non-oliguric form of acute renal failure characterized by impaired tubular sodium reabsorption 62. Leptospira-derived non-esterified unsaturated fatty acids have been found to inhibit kidney Na+, K+ ATPase 63. However, it seems more plausible that the renal manifestations are the direct result of a focal tubulointerstitial nephritis. Leptospiral outer membrane proteins, such as LipL32, activates TLR-dependent pathways which leads to induction of nuclear transcription factor kappa B, mitogen-activated kinases and cytokines and subsequently, tubular damage 60. Furthermore, activation of these pathways may provide a possible explanation for the dysregulation of sodium transporters in infected kidneys, a finding which has shown to be associated with impaired sodium reabsorption 64, 65.
Leptospires have been reported to induce apoptosis in macrophages and hepatocytes 22, 66, 67, yet the overall contribution of apoptosis in disease pathogenesis has not been delineated. Leptospirosis elicits production of autoantibodies, such as anti-cardiolipin antibodies 68. Several reports suggest that autoimmune mechanisms may play a role in the development of uveitis 37 and LPHS 69 during infection.
The virulence mechanisms, and more generally the fundamental understanding of the biology of the causative agents of leptospirosis, remain largely unknown. Before 2000, the lack of genetic tools available for use in leptospires, in either pathogenic or saprophytic species, precluded the full characterisation of genes of interest. In the first genetic studies carried out in the 1990s, several Leptospira genes were isolated by the functional complementation of E. coli mutants. This method led to the identification of the L. biflexa recA gene 70, the L. interrogans rfb genes 71, and a number of amino acid biosynthesis genes, such as asd and trpE 72, 73.
The origins of replication from the LE1 temperate leptophage 74, a 74-kb extrachromosomal element of L. biflexa 17, and a genomic island that can excise from the L. interrogans chromosome 75 were used to generate a plasmid vector able to replicate autonomously in both L. biflexa and E. coli 76. DNA can be introduced into Leptospira by electroporation 76, 77 and conjugation 78. However, to date, there is no replicative plasmid vector available for pathogenic Leptospira.
Deletion of chromosomal genes, including flaB, trpE, metY, metX, metW, hemH, and recA by targeted mutagenesis was achieved in the saprophyte L. biflexa with a suicide plasmid 79. Recently the first gene, ligB, was disrupted in the pathogenic L. interrogans 80 by site-directed homologous recombination.
A system for random mutagenesis using the Himar1 mariner transposon has been developed in both saprophytic and pathogenic Leptospira strains 77, 81, 82. In L. biflexa an extensive library of mutants can be generated that can be screened for phenotypes affecting diverse aspects of metabolism and physiology, such as amino-acid biosynthesis and iron acquisition systems 82, 83. However pathogenic leptospires remain much less easily transformable with Himar177. At the end of three years of transformation experiments performed simultaneously in two different laboratories, we obtained about 1000 random mutants with characterised transposon insertion points in L. interrogans (Table 1) 81. In total, 721 of the mutations identified affected the protein coding regions of 551 different genes. The challenge at the moment is to improve existing methods and to identify more readily transformable pathogenic strains for further genetic studies in L. interrogans. If successful this approach should make it possible to generate a library for the high-throughput screening of mutants for specific processes known to be involved in pathogenesis.
Guinea pigs and hamsters are the standard experimental model for acute leptospirosis 1. Infection with low inocula (<100 leptospires) produces similar disease kinetics (Figure 3B) and severe manifestations as observed in humans (Figure 3B) 48. Mice and gerbils have been used to study the genetics of the immune response to leptospirosis 61, 84, 85 and as models for vaccine-mediated immunity (Table 2) 86. However mice are relatively resistant to infection and require high inocula (up to 108 organisms) to produce disease, a situation which may not parallel what occurs during naturally-occurring exposures. Furthermore mice, when administered with high inoculum doses required to induce a lethal infection, develop a more fulminant clinical course and tend to die within significantly shorter intervals (five days) than that observed in patients or hamsters infected with low-inoculum lethal challenges (Figure 3B). This finding raises concerns that this experimental animal model may not reproduce the disease dynamics and pathogenic processes observed in natural infections. Rats have been used as a model to study persistent colonization but also require high inocula 28, 39. Like mice, it is not understood why this common reservoir in nature is relatively difficult to infect experimentally. Natural infection with leptospirosis occurs in non-human primates, which in turn have been used as models to study the disease 87, and more recently, the development of pulmonary haemorrhage syndrome 88.
The virulence factor determined to date are primarily surface proteins, which are thought to mediate the interaction between the bacterium and the host tissues. Although several proteins are secreted by Leptospira spp., including degradative enzymes, there is no evidence for any dedicated protein secretion pathway for injection of proteins into host cells, such as the Type III and Type IV secretion machinery of Gram negative bacteria. Other virulence factors promote motility and iron acquisition, but many other factors, including proteins that mediate host-cell interactions or cause tissue damage are likely to be discovered.
The development of genetic tools and the availability of complete genome sequences of pathogenic Leptospira have made it possible to apply state-of-the-art approaches to determine the virulence and survival mechanisms used by these bacteria to ensure their persistence in different ecological niches.
Previous microarray studies have shown that exposure of L. interrogans to the osmolarity conditions found in host tissues induces a profound shift in global transcription profiles. Thus, osmolarity and temperature 89, 90 are important factors regulating the expression of proteins mediating the infection of mammalian hosts. Nineteen of the 25 most strongly salt-induced L. interrogans genes encode hypothetical proteins 90. These genes may encode response regulators and environment-sensing proteins involved in survival or persistence in the environment or in the infected host.
Moieties expressed on the surface of leptospires, are believed to be determinants in the pathogen’s interaction with the host and ability to cause virulence. Leptospires adhere and enter in vitro host mammalian cells (Figure 1C), a phenotype which is observed in virulent leptospires and not in culture-attenuated or saprophytic organisms 22, 24, 25, 91. The attachment of pathogenic leptospires to eukaryotic cells (Figure 1C) is a key step in the process of infection that may involve molecules secreted by the bacterium or present on its surface (Figure 4). Several leptospiral proteins have been shown to bind in vitro to several components of the extracellular matrix 36, 92-96. Furthermore, virulent leptospires had significantly lower numbers of protein particles on the outer membrane surface as determined by freeze-fracture electron microscopy, and expressed different protein and LPS profiles than culture-attenuated strains 97.
Like other spirochaetes, the genomes of Leptospira spp. possess a much higher number of lipoprotein genes than that of other bacterial genomes. Analysis of the genome sequences of L. interrogans led to the detection of approximately 145 putative lipoproteins 98 and several putative extracellular and outer membrane proteins 99, 100.
Consistent with the predicted ability of Leptospira to migrate through host tissues, its genome encodes a wide range of putative hemolysins and proteases that may facilitate this process. An analysis of the L. interrogans genome identified nine genes which encode putative hemolysins, including sphingomyelinase genes that are not found in the saprophyte L. biflexa 17 and a pore-forming protein gene 101. Sphingomyelinase C was found to be up-regulated by increases in osmolarity to the levels found in mammalian host tissues 90. The L. interrogans genome also contains a microbial collagenase, which is hypothesised to be involved in the destruction of host tissues.
Few proteins have been experimentally shown to be present on the leptospiral surface 102. Together, about twelve proteins have been identified as outer membrane proteins and include OmpL1 103, LipL32 104, LigB 105, LenA 36, LenD 36, and Loa22 106. Our knowledge of the surface of leptospires thus remains limited and the further development and improvement of tools for accurate localisation of surface-associated determinants are required.
The only gene to date that fulfils Koch’s molecular postulates for a virulence factor gene is loa22. Disruption of loa22 by Himar1 insertion in L. interrogans led to a complete loss of virulence in the guinea pig model (Table 1) 106. Loa22 is exposed on the bacterial surface 106 and recognised by sera from human leptospirosis patients 107 and its expression is up-regulated in an acute model of infection 108. The observed Loa22 in vitro binding with components of the extracellular matrix is relatively weak 109. The C-terminal of Loa22 consists of an OmpA domain, which contains a predicted peptidoglycan-binding motif. Although the non-pathogenic L. biflexa genome contains an orthologue of loa22 17, differential expression of this gene in pathogenic and non-pathogenic leptospires or pathogen-specific sialylation of Loa22 dependent on pathogen-specific sialic acid modification pathways (J. Ricaldi and J. Vinetz, personal communication) may explain why this protein, post-translationally modified, is a critical determinant of L. interrogans virulence.
LipL32 104, also designated Hap-1 for haemolysis-associated protein 110, is surface-exposed 104 and accounts for 75% of the outer membrane proteome 111. The lipoprotein is highly conserved among pathogenic Leptospira 112; there are no orthologues of lipL32 in the saprophyte L. biflexa 17. LipL32 was long believed to be a putative virulence factor. Higher levels of LipL32 are expressed in leptospires during acute lethal infections than in leptospires cultured in vitro 108. The C-terminus of LipL32 binds in vitro to laminin, collagen I, collagen IV collagen V and plasma fibronectin 94, 95. The crystal structure of LipL32 was elucidated recently and it was shown to present structural homologies with proteins such as collagenase that bind to components of the extracellular matrix 113. Yet a LipL32 mutant, obtained by Himar1 insertion mutagenesis, was found to be as efficient as the wild-type strain in causing an acute disease and chronic colonisation in experimental animals (Table 1) 114. The role of this major outer membrane protein in pathogenesis remains unclear and is a matter for debate.
A family of three high-molecular weight Leptospira proteins — LigA, LigB and LigC — was identified as a novel member of the bacterial immunoglobulin (Ig)-like (Big) protein superfamily 86, 105, 115. Lig proteins are anchored to the outer membrane and have 12 to 13 tandem Big repeats domains. Like lipL32, lig genes are exclusively present in pathogenic Leptospira. Recombinant Lig proteins bind in vitro to host extracellular matrix proteins, including fibronectin, fibrinogen, collagen, and laminin 96, 116. Furthermore, the repeat domain portion of the LigB molecule binds Ca2+ which in turn, appears to enhance its ability to adhere to fibronectin 117. The lig genes are up-regulated at physiological osmolarity 90 and encode surface-exposed proteins strongly recognised by sera from human patients with leptospirosis 105, 118, 119. Lig proteins are considered a putative virulence factor 105 since members of the bacterial Ig-like superfamily mediate pathogen-host cell interactions, such as invasion and host cell attachment, in other bacteria. However, a ligB mutation in L. interrogans, which also contains a ligA gene 80, does not affect the ability of the bacterium to cause acute leptospirosis or persistent renal colonisation in hamsters and rats, respectively. The presence of several other putative adhesins with potentially redundant functions, including LigA, may have obscured the detection of clear phenotypes for the ligB mutant.
The motility of the bacterium may be of relevance to its basic biology and, despite also being common to saprophytes, may be considered a virulence factor. Freshly-isolated pathogenic leptospires have higher translational and helical motility in comparison to strains passaged in vitro 120. The corkscrew motility allows these organisms to swim through gel-like medium, such as connective tissues 13. However, it has not been determined whether loss of motility directly results in attenuation of virulence for pathogenic leptospires. L. biflexa flaB mutants cannot form functional endoflagella, but their cell bodies remain intact and helical 121. The endoflagella are therefore not responsible for dictating the helical shape of the cell body in Leptospira spp as they do in Borrelia burgdorferi 122. Proteins known to be involved in the morphogenetic system of rod-shaped bacteria, such as MreBCD and penicillin-binding proteins, are encoded by genes present in the leptospire genome. Leptospiral cell morphology may thus be determined by the cytoskeleton and maintained by the rigid murein layer.
Multiple methyl-accepting chemotaxis proteins have been identified in Leptospira, suggesting that chemotactic responses to various chemoattractants/repellents may occur. Unlike avirulent or saprophytic strains, L. interrogans displays positive chemotaxis towards haemoglobin 123.
Iron acquisition is important for virulence in many bacterial pathogens, and Leptospira species have been found to contain several iron uptake systems, including TonB-dependent outer membrane receptors 83. Leptospira spp. possess a haem oxygenase, encoded by hemO, which degrades the tetrapyrrole ring of the haem molecule, releasing ferrous iron. Disruption of the hemO gene in L. interrogans decreases virulence in the hamster model of leptospirosis (Table 1) 124, suggesting that Leptospira uses haem as its principal source of iron during infection.
Mutations in the genes encoding the surface-associated proteins LenB and LenE, which were considered putative virulence factors 36 did not have an effect on virulence (Table 1) 80, 81. Two attenuated mutants with disruptions in hypothetical genes may correspond to novel virulence factors in L. interrogans 81, but these findings need to be confirmed with complementation studies.
The humoral response is believed to be the primary mechanism of immunity to leptospirosis 125. LPS appears to be the major target for the protective antibody response, since passive transfer of immunity correlates with levels of agglutinating anti-LPS antibodies in patient sera 126 and anti-LPS monoclonal antibodies passively protect naïve animals from leptospirosis 127. However, it is not known whether antibody responses against leptospiral antigens in addition to LPS also confer protection.
Recent work has contributed to the understanding that immunity to leptospirosis is not limited to the humoral response. Mice require intact TLR2 (Chassin et al., submitted) and TLR4 85 activation pathways of innate immunity in order to control a lethal infection. In contrast to immunity in hosts susceptible to acute leptospirosis, protective immunity against L. borgpetersenii serovar Hardjo in bovine maintenance hosts is cell-mediated. Immunisation trials in cattle found that protection against this serovar, conferred by whole Leptospira-based vaccines, correlated with TH1 responses and not with agglutinating antibody titres 128-130
Ido et al. provided the first demonstration in 1916 that immunisation with killed leptospires protects against experimental infection 131. Since then, whole Leptospira-based vaccines have been routinely administered to livestock and domestic animals and used for immunization of human populations 6. However there are major concerns with respect to their use 132. Whole Leptospira-based vaccines are associated with high rates of adverse reactions and confer only short-term serovar-specific immunity 1. Polyvalent vaccines are used to provide coverage for circulating serovar agents and need to be reformulated at significant cost when new serovars emerge 133. Furthermore whole-Leptospira vaccines are not universally effective in preventing carriage, which limits their use as a transmission-blocking intervention.
Due to these limitations, efforts have focussed on developing sub-unit vaccine candidates (Table 2) and more specifically, identifying surface-associated proteins which are conserved among serovars and targets for bacteriocidal immune responses. Haake et al. provided the first evidence for the feasibility of this approach by demonstrating that immunisation with E. coli outer membrane vesicles containing recombinant LipL41 and OmpL1 partially protected against a lethal challenge of leptospires in hamsters 134. Subsequently, LipL32 has been shown to elicit immunoprotection when administered in naked DNA 135, BCG 136, and adenovirus 137 delivery systems. Yet, overall efficacy of these formulations is low (40-75%) in experimental animals. The most promising sub-unit vaccine candidate is the Lig proteins, which have been shown to confer high-level protection (Table 2), approaching 100% in mice 86 and hamsters 138-140. The ability of Lig proteins to elicit cross-protective immunity against the spectrum of serovar agents needs to be determined since amino acid sequence identity for this protein is 70-100% among Leptospira spp 141.
The availability of multiple genome sequences provides an opportunity to apply high-throughput strategies for identifying novel vaccine candidates 107. The ultimate goal for vaccine development will be to identify a candidate which protects against the spectrum of Leptospira agents. The L. interrogans and borgpetersenii genomes share 2708 ORFs, of which 656 are not present in the L. biflexa genome 16, 17 (Box 2). Strategies to refine the number of target candidates include sequencing of a wider representation of pathogenic Leptospira genomes and bioinformatic analysis and selection of ORFs which are highly conserved among these genomes and encode outer membrane proteins 100. Yet the major barrier in pursuing this strategy is the lack of in vitro correlates for immunity against leptospirosis. High throughput screening in experimental animals may not be feasible given the expected number of candidate antigens. A priority for vaccine development will be to prospectively determine whether infection with leptospirosis protects against subsequent re-infection in high-risk populations and identify the mechanisms of immunity which may be involved. Until epidemiologically-validated immune correlates are identified, discovery of vaccine candidates will likely continue to rely on the search for new virulence factors and outer membrane proteins.
There has been impressive recent progress in our knowledge of the basic aspects of the biology and pathogenesis of Leptospira spp., although modern molecular genetics was not applied to pathogenic leptospires until 2005, with the generation of the first mutants in L. interrogans 77. Further studies need to explain why it is so difficult to introduce DNA into pathogenic leptospires by methods commonly used for other bacteria. More efficient methods are needed to test the role of putative virulence factors. The presence of prophage-like loci in the genome of pathogenic Leptospira 75, 142 suggest that transduction may occur and phages could be used as tools for gene transfer. Despite the large evolutionary distance between the pathogenic and non-pathogenic species, Leptospira spp. share a core of approximately 2000 genes 17. L. biflexa could be used as a model bacterium to identify the precise functions of these common genes to gain an insight into the general biology of Leptospira spp.
Nevertheless, the discovery of genetic tools to transform leptospires has circumvented a major barrier to elucidating pathogen-related determinants of virulence and has led to the identification of Loa22 as the first virulence factor in Leptospira 106. LipL32 and Lig proteins were long-standing hypothesized virulence factors. Yet knockout mutagenesis of the genes which encode these factors did not result in attenuation of virulence, suggesting that there may be a high degree of redundancy in function among virulence factors and that classical knockout approaches may not be useful in identifying such factors. There is therefore a real need to use convergent genomic, proteomics and metabolomic approaches to systematically identify molecular phenotypes and link these phenotypes with the pathogen’s ability to cause disease in humans and animals. Our next hurdle is also to learn more about leptospiral gene regulation and the interactions among proteins. Microarrays represent a valuable tool to identify regulatory networks or pleiotropic effects of a mutation. The use of genetically distinct (or engineered) laboratory rodents together with micro-arrays or proteomic studies should permit to better delineate the mechanisms leading to chronic renal shedding. Ecological and metagenomics studies of soils will possibly provide information on the environmental persistence of leptospires which remains poorly understood.
Both host and microbiological factors probably contribute to the severity of leptospiral infection. Further studies should, for example, determine if the increasingly recognized syndrome of pulmonary hemorrhage is rather due to the emergence of a Leptospira clone with strain-specific factors or to innate or acquired host susceptibility factors. Elucidation of the molecular mechanisms of pathogenesis will contribute to the development of novel strategies for the treatment and prevention of leptospirosis which are urgently needed to address the large disease burden attributable to this emerging infectious disease in impoverished populations.
The authors would like to thank Claudio Figueira, Elsio Wunder, Evelyne Couture, Marie-Christine Prevost, and Paula Ristow from the Oswaldo Cruz Foundation and the Institut Pasteur for their help in providing the figures on leptospires and the pathology of leptospirosis and Lee Riley from University of California at Berkeley and Guy Baranton and Isabelle Saint Girons from Institut Pasteur and Mitermayer Reis and Guilherme Ribeiro from the Oswaldo Cruz Foundation for their critical advice during the preparation of the manuscript. Some of the work described was supported by a cooperative agreement between Institut Pasteur and the Oswaldo Cruz Foundation, Brazilian National Research Council (grants 01.06.0298.00 3773/2005, 554788/2006, INCTV), Research Support Foundation for the State of Bahia (54663), and the National Institutes of Health (grants 2R01 AI052473, 2D43 TW00919), the Institut Pasteur, and Agence Nationale de la Recherche (n°05-JCJC-0105 01).
The authors declare no competing financial interests.