Leptospirosis and leptospires
The spirochetes form a phylum with a deep branching lineage, and many studies have shown that they differ considerably from well-studied gram-negative and gram-positive bacteria. Leptospira are thin bacteria (0.1 micrometre of diameter) with a helically shaped cell cylinder and a periplasmic flagella, called endoflagella.
Movement of leptospires (A, from Goldstein and Charon, 1988) and electronic microscopy of Leptospira biflexa (B)
Leptospirosis is a widespread zoonosis that has emerged as a major public health problem in developing countries in South-East Asia and South America. This increasingly common disease occurs in poor urban centers subject to frequent flooding. Rodents are the main reservoir of the disease, excreting the bacteria in their urine. Humans are usually infected through contaminated water.
Cycle of leptospirosis (from Faine et al. 1999)
Over 500,000 cases of severe leptospirosis are thought to occur each year, with a mortality rate of more than 10 %. In France, about 500 cases are diagnosed each year. About half of these cases occur in oversea territories, where the incidence rate is more than one hundred times that for mainland France.
the Leptospira server of the Pasteur Institute with epidemiological reports in France
Antibiotic therapy is beneficial but must be administered in the early stage of the disease. However, leptospirosis is often diagnosed late, due to its wide spectrum of symptoms, ranging from a flu-like syndrome to renal failure. The symptoms mimic the clinical presentations of many other diseases, including dengue fever and malaria. In addition, most cases of leptospirosis are currently detected by means of a complex and fastidious serological test, but antibodies are generally not detectable in the blood until about one week after the onset of symptoms. Human vaccines are available in some countries, but usually consist of killed bacteria of the most prevalent serovar. They present a number of disadvantages, including side effects, short duration of efficacy and incomplete protection against other serovars. Leptospirosis also affects animals worldwide, including livestock, and is therefore of economic importance. The main role of NRC for Leptospira is to carry out active surveillance and to determine the actual incidence of leptospirosis in France.
the WHO Collaborating Center for the epidemiology of leptospirosis
the National Reference Center for Leptospira
Whole-genome sequencing of the saprophyte Leptospira biflexa and comparative genomics of Leptospira species
The genus Leptospira has both saprophytic and pathogenic members, such as L. biflexa and L. interrogans, respectively. The recent completion of the L. interrogans and L. borgpetersenii genome sequences has provided a basis for understanding these organisms. A major challenge remains the assignment of a biological role to the predicted L. interrogans genes, more than 50 % of which show no similarity to proteins of known function. However, the slow growth of these bacteria, their virulence in humans and a lack of genetic tools make it difficult to work with these pathogens. We therefore sequenced the genome of the saprophyte L. biflexa serovar Patoc strain Patoc1, which is genetically manipulable. We identified putative Coding Sequences (CDSs) likely to encode proteins, using the web interface MaGe (Magnifying Genomes, Genoscope). The complete genome sequence of L. biflexa consists of a 3,600-kb large chromosome, a 278-kb small chromosome, and a 74-kb circular plasmid with a total of 3790 predicted coding genes, 60% of which are conserved in L. interrogans.
Link of Spiroscope, including the complete genome sequence of L. biflexa
We have begun the comparison of the genetic content of the saprophytic and pathogenic species. This comparative genomic analysis should provide clues to the lifestyle of Leptospira in the environment and in the infected host, increasing our understanding of the transition from environmental bacterium to major human and animal pathogen.
Random and targeted mutagenesis in pathogenic Leptospira
We recently provided the first evidence of gene transfer in a pathogenic Leptospira strain, involving the transposition of a transposon of eukaryotic origin. We used this methodology in L. interrogans serovar Lai strain Lai, a pathogenic strain which is entirely sequenced and relatively transformable, to construct a library of random mutants. The L. interrogans genome contains approximately 3500 predicted coding regions (excluding transposases and pseudogenes), of which half were assigned no biologic role, whereas the remaining half were assigned roles that await experimental validation. The analysis of a subset of random already allowed the characterization of mutants carrying insertions in genes encoding putative virulence factors, such as TonB-dependent receptors, sphingomyelinase, peptidases, outer membrane proteins, etc. We also identified an avirulent mutant that was obtained via disruption of loa22, a gene that encodes an outer membrane protein containing an OmpA domain, by insertion of the transposon Himar1. 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. This collection of insertional mutants will constitute a useful resource for functional studies of pathogenic strains and the library will be particularly useful for discovering new genes and studying protein functions.
Although targeted mutagenesis by allelic exchange has been used with success in Leptospira saprophytes, double recombination is a rare event and single recombination is the general rule. Previous attempts to transform pathogens for homolougs recombination resulted in no or very few transformants, all resulting from a single recombination. Recently, we were able to inactivate the ligB gene in L. interrogans serovar Copenhageni strain Fiocruz. Althought the tranformation efficiency was very low, this result represents the first demonstration of targeted mutagenesis of pathogenic strains. The use of large region of homologous DNA, together with the fact that the ligAB locus appears to be the target of fragment rearrangements and recombination events, may have increased the probability of homologous recombination By using a similar approach, we plan to inactivate other genes that have been described as putative virulence factors.
Further increase in transformation efficiency, through the identification of a hypertransformable strain or development of new genetic tools, will provide with opportunities to generate mutants. Untill recently, the successful introduction of DNA in Leptospira has only proved possible following electroporation. We now reported the first demonstration of conjugation between Escherichia coli and Leptospira spp. using RP4 derivative conjugative plasmids. This study, providing an alternative to electroporation for delivery of DNA to Leptospira, broadens the repertoire of genetic methods available to manipulate pathogenic strains.
Electronic microscopy of a conjugative transfer between Escherichia coli and Leptospira biflexa.
Development of new genetic tools
In comparison to other bacterial species, genetics and molecular basis of the pathogenesis of leptospires are in their infancy. It has to be noted that pathogenic Leptospira grow very slowly and may take up to a month to form colonies on solid media in culture. Our group has developed a number of tools for genetic manipulation of the saprophyte L. biflexa in recent years, including random and targeted mutagenesis. However, the further development and improvement of genetic tools are required to manipulate pathogenic strains. For example, we recenlty generated plasmid constructs containing a resistance marker, the LE1 replication origin, which allows autonomous plasmid replication in the saprophyte L. biflexa and leptospiral promoters fused with genes encoding the green fluorescent protein (GFP) or the red fluorescent protein mRFP1. Plasmids were then introduced into the saprophyte L. biflexa and expression of fluorescent proteins in transformants was evaluated by epifluorescence microscopy. All plasmid constructs proved to be functional in L. biflexa, making it feasible to visualize transformants by fluorescence. We now have to transfer these fluorescent alleles in virulent strains by using the Himar1 transposon (there is no replicative vector available in the pathogens). The use of fluorescent bacteria should be useful for the follow-up of the infectious process. Since the long term colonization of proximal renal tubules of mammalian maintenance host species by pathogenic leptospires is believed to proceed via the formation of cell aggregates biofilm-like structures, visualization of fluorescent bacteria in such biofilms may improve our understanding of the chronic carriage of L. interrogans in animal reservoirs. Transcriptional fusions placing GFP expression under the control of a bacterial promoter can also be a valuable tool to examine gene expression patterns during host infection or different in vitro conditions.
Fluorescent microscopy of Leptospira biflexa expressing the gfp and mRFP1 genes
Identification of virulence factors
A significant barrier to control and prevent leptospirosis has been the limited understanding of Leptospira pathogenesis, due in part to the lack of genome sequences and tools to genetically manipulate this pathogen. These barriers have now been overcome. The hallmark of Leptospira pathogenesis is the ability of these bacteria to penetrate skin and mucous membranes and to disseminate rapidly to other tissues shortly after infection. In susceptible hosts, such as humans, systemic infection produces severe manifestations, such as sepsis, hepatic and renal insufficiency and hemorrhage. However, in animal reservoirs such as the domestic rat, infection produces chronic asymptomatic disease, with persistent carriage in the renal tubules. We have developed experimental models with guinea pigs for acute severe disease. Our colleagues at Fiocruz have also developed an experimental model in Wister Rattus norvegicus for persistent carriage. We will evaluate the mutants obtained by random or targeted mutagenesis (see above) in these animal models. This will allow us to identify factors that play a role in acute disease pathogenesis and persistent colonization.
In the recent years, we studied the iron metabolism of leptospires as well as their ability to form biofilms. Thus, we have demonstrated that most of the Leptospira strains can form biofilms in vitro. The formation of biofilms is consistent with the life of saprophytic strains in water and may facilitate the pathogenic strains to survive in environmental niches and colonize the host. Biofilm-like structures of L. interrogans have been observed in rat kidney tubules. The presence of biofilms in proximal kidney tubules may play an important role in maintaining chronic carriage in rats. The genome sequences of Leptospira suggest that these bacteria possess several iron acquisition systems. An analysis of L. biflexa mutants obtained by random and targeted mutagenesis allowed the characterization of genes involved in iron uptake systems, such as TonB-dependent receptors, ABC transporters, and a two-component system. Further studies will include characterization of the mechanisms involved in metabolic responses to iron availability, through regulators such as the ferric uptake regulator protein Fur. This project should make it possible to evaluate the role of iron in the virulence of pathogenic Leptospira in infected hosts.
Electronic microscopy of biofilms of leptospires
The precise involvement of putative virulence factors in pathogenesis will be determined by reintroducing the wild type copy of the gene into the mutant strain, using a Himar1 transposon containing the wild-type copy By using this strategy, we were able to restore the expression of the ompA-like gene in the corresponding mutant by introducing the wild-type copy via a transposon containing a second selectable marker.
Molecular typing of pathogenic leptospires
The main function of the NRC for Leptospira is surveillance of this zoonosis, based on the collection of diagnosis data from laboratories around the country. The NRC is one of the few laboratories that can confirm leptospirosis diagnosis by means of the MAT test in France. This testing makes it possible to detect outbreaks rapidly, and to alert Health authorities accordingly. It enables the NRC to update knowledge of the epidemiological traits of leptospirosis and the situation of the disease regularly, both in mainland France and in French overseas territories (New Caledonia, French West Indies, La Réunion, Mayotte, French Guyana, and French Polynesia). The NRC has a duty to improve diagnostic techniques. We have developed a rapid and simple typing method based on analysis of the polymorphism of variable-number tandem repeats (VNTR) loci in the genome of pathogenic Leptospira species. VNTR typing could provide an accessible mean of testing for research and public health laboratories, particularly in developing countries. This method is now into routine used at the NRC. Further improvements to this method are required, so that MLVA (Multiple Locus VNTR analysis) can be applied directly to biological (serum or urine of patients and samples from animals) and environmental samples without the need to culture the pathogen. This typing method would help to improve the surveillance of this underestimated disease. This method should also facilitate the sharing of results and the generation of databases.
the MLVA database