REVIEWS Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen Albert I. Ko*‡, Cyrille Goarant § and Mathieu Picardeau|| Abstract | Leptospirosis is a zoonotic disease that has emerged as an important cause of morbidity and mortality among impoverished populations. One hundred years after the discovery of the causative spirochaetal agent, little is understood about Leptospira spp. pathogenesis, which in turn has hampered the development of new intervention strategies to address this neglected disease. However, the recent availability of complete genome sequences for Leptospira spp. and the discovery of genetic tools for their transformation have led to important insights into the biology of these pathogens and their pathogenesis. We discuss the life cycle of the bacterium, the recent advances in our understanding and the implications for the future prevention of leptospirosis. The question mark-shaped bacterium Zoonotic disease Descriptions of leptospirosis-like syndromes were 1 An infectious disease of animals reported in the scripts of ancient civilizations , but The genus Leptospira belongs to the phylum that can be transmitted to the first modern clinical description of leptospirosis Spirochaetes and comprises saprophytic and patho- humans, whatever its was published by Weil in 1886 (REF. 2). In a landmark genic species11 (BOX 1). Saprophytic leptospires, such as transmission mode (vector study in 1916, Inada et al. isolated leptospires, identi- Leptospira biflexa, are free-living organisms found in borne, directly or indirectly through a product derived from fied the organism as the causal agent of leptospirosis and water and soil and, unlike pathogenic Leptospira spp., 1 the host animal), and that determined that rats are a reservoir for transmission to do not infect animal hosts . Leptospires are thin, highly causes a disease syndrome in humans3. Leptospires were subsequently isolated from a motile, slow-growing obligate aerobes with an optimal susceptible individuals. wide range of animal reservoir species and classified into growth temperature of 30 °C and can be distinguished serogroups and serovars as a function of their antigenic from other spirochaetes on the basis of their unique determinants (BOX 1). hook or question mark-shaped ends12 (FIG. 1a). *Division of Infectious Leptospirosis, a zoonotic disease with a worldwide In addition to L. biflexa, the genomes of two patho- Disease, Weill Medical College distribution, is now recognized as an emerging infec- genic species, Leptospira interrogans and Leptospira of Cornell University, New York, 4 13–16 New York 10021, USA. tious disease . Over the past decade, outbreaks during borgpetersenii, have been sequenced . Most (77–81%) ‡Gonçalo Moniz Research sporting events, adventure tourism and disasters have of the genes in leptospiral genomes do not have ortho- Centre, Oswaldo Cruz underscored its ability to become a public health prob- logues in the genomes of other spirochaetes, indicat- Foundation, Brazilian lem in non-traditional settings4–6. However, leptospirosis ing the large degree by which leptospires have diverged Ministry of Health, Salvador, is a neglected disease that places its greatest burden on from other members of the phylum11. Furthermore, 40296-170, Brazil. §Institut Pasteur de Nouvelle- impoverished populations from developing countries comparative analysis of the genomes of the pathogenic 6 16,17 Calédonie, Laboratoire de and tropical regions . In addition to being an endemic and saprophytic species has provided insights into Recherche en Bactériologie, disease of subsistence farmers1,4,5, leptospirosis has the genetic determinants that may be involved in Nouméa, 98845, emerged as a widespread problem in urban slums, where pathogenesis (BOX 2). New Caledonia. ||Unité de Biologie des inadequate sanitation has produced the conditions for 7–9 Spirochètes, Institut Pasteur, rat-borne transmission . More than 500,000 cases of The transmission cycle 28 Rue du Docteur Roux, severe leptospirosis are reported each year, with case Transmission of leptospirosis requires continuous 75724 Paris Cedex 15, fatality rates exceeding 10%10. This Review focuses on enzootic circulation of the pathogen among animal res- France. the pathogenesis of leptospirosis and then highlights the ervoirs or, as they are commonly called, maintenance Correspondence to M.P. (FIG. 2) e-mail: mathieu.picardeau@ recent advances in the field with respect to the genetic hosts . Leptospira serovars demonstrate spe- pasteur.fr approaches that have been recently developed and the cific, although not entirely exclusive, host prefer- doi:10.1038/nrmicro2208 virulence factors that have been discovered. ences; for example, rats serve as reservoirs for the 736 | OCTOBER 2009 | VOLUME 7 www.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 24,25 Box 1 | Classification and molecular typing of Leptospira spp. resistance . Leptospires are not facultative intracellu- lar organisms; they are rarely observed within host cells 11 The genus Leptospira belongs to the phylum Spirochaetes . The subgroup of but instead seem to reside only transiently within these saprophytic species (Leptospira biflexa, Leptospira wolbachii, Leptospira kmetyi, cells as they cross cell monolayers in vitro25. The proc- Leptospira meyeri, Leptospira vanthielii, Leptospira terpstrae and Leptospira yanagawae) ess by which leptospires enter host cells is not under- forms the deepest branch in the genus and another subgroup includes the pathogenic species (Leptospira interrogans, Leptospira kirschneri, Leptospira borgpetersenii, stood. Internalized leptospires have been observed in 24,25 23 Leptospira santarosai, Leptospira noguchii, Leptospira weilii, Leptospira alexanderi cytoplasmic and phagosomal compartments of and Leptospira alstoni). Another evolutionary branch comprises the so-called normally non-phagocytic host cells. These findings ‘intermediate group’ (Leptospira inadai, Leptospira broomii, Leptospira fainei, Leptospira suggest that leptospires use host cell entry and rapid wolffii and Leptospira licerasiae), which contains species of unclear pathogenicity4,5. translocation as mechanisms to spread to target organs Pathogenic Leptospira spp. are classified into more than 200 serovars on the basis of and evade immune killing. structural heterogeneity in the carbohydrate component of the lipopolysaccharide4,5. Infection causes prolonged leptospiraemia until the Serotyping of leptospires is important for clinical or epidemiological investigations, as host mounts an effective acquired immune response, identification of serovars and serogroups provides clues about the host reservoirs that which occurs one to two weeks after exposure26 (FIG. 3a). are involved in pathogen transmission. However, serotyping is performed in only a few Leptospires can be isolated from the bloodstream within reference laboratories worldwide. Several studies have shown that the serogroups are 1 not related to the groups that are formed by molecular classifications4, suggesting that minutes after inoculation and are detected in multiple 26–29 the genes that determine the serotypes may be laterally transferred into different organs by the third day after infection ; they may reach 6 7 species. Consequently, a classification system based on genetic similarities is being used 10 –10 organisms per ml or per g in the blood and tis- in conjunction with antigenic classification. Recently, genome sequence information sues of patients30,31 and infected animals29. Leptospires allowed the introduction of several approaches to genotyping Leptospira spp., including evade the host innate immune response during the ini- multilocus variable-number tandem-repeat analysis144 and multilocus sequence tial stages of infection. They are resistant to the alternative typing44,145, a typing method that is based on the partial sequences of housekeeping pathway of complement activation32,33 and acquire genes and may evolve as a standard genotyping method for Leptospira spp. as it has complement factor H and related fluid-phase regula- for other bacterial species. tors34,35 through ligands such as the leptospiral endostatin- like (Len) proteins36,37. The host complement fragment C4b-binding protein alpha chain (C4BPA) binds to the Icterohaemorragiae serogroup, whereas house mice surface of leptospires38, suggesting that a similar process (Mus musculus) are the reservoir for the Ballum sero- may confer some protection against the classical pathway group4,5,18. Furthermore, serovars often do not cause of complement activation. serious disease in reservoir hosts to which they are highly adapted. Persistent colonization. The essential component of the Leptospires colonize and are shed from the renal life cycle of pathogenic Leptospira spp. is their ability to tubules of a wide range of animals (see Supplementary give rise to persistent renal carriage in reservoir animals. information S1 (box)). The bacteria survive for weeks or In rats, leptospires cause a systemic infection but are even months in moist soil and water after excretion in subsequently cleared from all organs except the renal the urine19. Cell aggregation19 and biofilm formation20 tubules28,39. Colonized tubules are densely populated with (FIG. 1b) may contribute to the survival of leptospires leptospires, which aggregate into an amorphous, biofilm- outside their hosts. like structure (FIG. 1d). Rats have been shown to excrete leptospires in high