Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen

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.

FIGURE 1. Leptospires in the environment and host

A. Leptospires are thin (cell diameter of 0.15 μm) and helical bacteria ranging from 10 to 20 μm long. Motility of leptospires is dependent on the presence of two endoflagella (or periplasmic flagella), one arising at each end of the spirochete, and extending along the cell body without overlapping in the central part of the cell. B. Scanning electron micrograph of L. interrogans biofilm on a glass surface. C. Scanning electron micrograph of L. interrogans adhering to polarized Mardin-Darby canine kidney cell monolayers. D. Photomicrograph of a Warthin-Starry stained section of kidney tissue from a captured sewer rat (Rattus norvegicus). Leptospires are seen as silver-impregnated filamentous structures within the proximal renal tubule lumen (400x magnification).

FIGURE 2. Cycle of infection

Mammalian species excrete the pathogen in their urine and serve as reservoirs for transmission. The pathogen is maintained in sylvatic and domestic environments by transmission among rodent species (-A-). In these reservoirs, infection produces chronic and persistent asymptomatic carriage in the renal tubules where L. interrogans forms aggregates (Figure 1D). Leptospires infect livestock and domestic animals and causes a range of disease manifestations and carrier states (Box 3). Maintenance of leptospirosis in these populations is due to continued exposure to rodent reservoirs or transmission within animal herds (-B-). Leptospirosis is transmitted to humans by direct contact with reservoir animals (-C-) or exposure to environmental surface water or soil contaminated with their urine (-D-). Leptospires penetrate abraded skin or mucous membranes (-1-), infect the bloodstream and disseminate throughout all the body tissue. Infection causes an acute febrile illness during the early “leptospiraemic” phase, which progresses during late “immune” phase to cause severe multi-system manifestations such as hepatic dysfunction and jaundice (-2-), acute renal failure (-3-), pulmonary haemorrhage syndrome (-4-), myocardidtis and meningoencephilitis (-5-). Although the immune response eventually eliminates the pathogen, leptospires may persist for prolonged periods in immunoprivileged sites, such as the anterior chamber and vitreous of the eye and the renal tubules, where they can produce respectively, uveitis (-7-) months after exposure and urinary shedding weeks after resolution of the illness (-8-). Humans are an accidental host and do not efficiently shed sufficient numbers of leptospires to serve as reservoirs for transmission.

FIGURE 3. Disease kinetics of leptospirosis

A. Schematic diagram of the kinetics of leptospiral infection and disease. Infection produces a leptospiraemia (black line) within the first days after exposure, which is followed by detection of leptospires in tissues of multiple organs (red line) by the 3rd day of infection. In humans, illness (fever, brown line) develops with the appearance of agglutinating antibodies 5-14 days after exposure (blue line). Leptospires are cleared from the bloodstream and organs as serum agglutinating antibodies titres increase. Although early-phase illness (yellow arrow) is mild and resolves in the majority of infected individuals, a subset of patients progress four to six days after the onset of illness to develop severe late-phase manifestations (red arrow) during the period of immune-mediated destruction and clearance of leptospires (black line). B. Survival curves for hamsters during experimental leptospirosis. Inoculation with increasing numbers of L. interrogans strain Fiocruz L1-130 (10⁷ organisms, red line; 10⁵, brown; 10³, green; 10¹, blue) is associated with shortening of the incubation period and increased mortality among Golden Syrian hamsters. Infection of hamsters with low inocula produces disease manifestations which are found in patients with severe leptospirosis.

FIGURE 4. Schematic diagram of the cell wall of leptospires

Leptospira spp. possess a double-membrane structure. The peptidoglycan cell wall is associated with the inner membrane . The leptospiral outer membrane is known to contain the transmembrane porin OmpL1, and lipoproteins LipL32, LipL36 (at the inner leaflet of the outer membrane), LipL41, and LigB (the surface-exposed Loa22, Len, LenD, LigA, and LigC proteins are not indicated in the schematic diagram). Leptospira spp. possess a lipopolysaccharide (LPS) which is composed of lipid A, a non-repeating oligosaccharide core and a distal polysaccharide (or O-antigen). Several TonB-dependent receptors (TBDR) were identified by genome analysis. Three of these TBDR were found to be involved in the transport of iron citrate (FecA-like transporter), the siderophore desferrioxamine, and hemin ^, . Both transport and induction functions require energy transduction from the TonB–ExbB–ExbD complex in the inner membrane (for simplicity, only one ExbB-ExbD-TonB-TBDR system is indicated). As in other spirochaetes, the endoflagella is located in the periplasm. The inner membrane contains the FeoAB-type iron , penicillin-binding proteins (PBP), and the lipoprotein LipL31. Homologues of the E. coli export systems of outer membrane proteins (OMPs) and lipoproteins were found in Leptospira; this includes inner membrane signal peptidases SP1 and SP2. Lipoproteins are first transported via the Sec system and bind to the ABC-transporter LolCDE. In E. coli, lipoproteins interact with LolA and the outer membrane receptor LolB to be inserted into the outer membrane. However, no LolA and LolB homologues are found in the Leptospira genomes. For OMPs, after transport via the Sec translocon, they are bound by the periplasmic chaperone Skp, then by the outer membrane protein Omp85 to be integrated into the lipid bilayer. An incomplete set of type II secretion-like genes is also present in the Leptospira genomes.