Leptospiral membrane proteins - variations on a theme?

Leptospirosis is considered to be the most widespread zoonosis in the world¹, reflecting the ability of the causative spirochetes to adapt to the renal tubules of a wide variety of mammalian reservoir hosts. Transmission to humans occurs either through direct contact with an infected animal or through indirect contact via soil or water contaminated with urine from an infected animal. Leptospiral infection in humans frequently results in a fulminant, life-threatening illness characterized by liver dysfunction, kidney failure, and pulmonary haemorrhage. Early diagnosis and treatment would almost certainly reduce the severity of complications and save lives. However, there is currently no reliable and convenient test available for the diagnosis of acute leptospirosis. The microscopic agglutination test, which is the standard serodiagnostic approach, relies on the immune response to leptospiral lipopolysaccharide (LPS). LPS is also thought to be the basis for the protective efficacy of whole cell leptospiral vaccines being marketed at this time for use in humans and domestic animals. Unfortunately, LPS antigens vary greatly among the many leptospiral serovariants. In contrast to LPS, leptospiral membrane proteins are thought to be highly conserved. For this reason, there is intense interest in leptospiral membrane proteins for development of accurate serodiagnostic tests and effective vaccines for protection of at risk individuals.

As in the Salmonellae, the variation in LPS is thought to reflect the diversity of mammalian hosts to which pathogenic Leptospira species have adapted. Leptospires have been isolated from hundreds of mammalian species and each mammalian reservoir host species tends to harbour organisms belonging to distinct leptospiral serovars. Rats are typically colonized by serovars belonging to the Icterohaemorrhagiae serogroup, while mice are reservoir hosts for serogroup Ballum. Associations also exist for domestic animals. For example, serovar Hardjo is associated with cattle, serovars Pomona and Bratislava are generally associated with pigs, while dogs tend to harbour Canicola. These associations presumably reflect antigenic adaptation of leptospiral serovars to their mammalian host. The most impressive example of this adaptation is the convergent evolution of the genetically unrelated, but antigenically indistinguishable Hardjo and Hardjoprajitno serovars to adapt to cattle in different parts of the world². The host-specificity of leptospiral LPS could be an immune evasion strategy allowing the spirochete to persist in the animal’s kidney for the life of the animal without stimulating an inflammatory response. The ability of leptospires to persist in mammalian renal tubules reflects a highly evolved form of parasitism and suggests an ancient relationship between mammals and their resident spirochetes. The host range and antiquity of the Genus Leptospira is reflected not only in the antigenic diversity of its LPS, but also in the genetic diversity of its DNA. Currently, 16 genomospecies have been defined by DNA-DNA hybridization techniques¹.

Despite their genetic and antigenic diversity, all pathogenic Leptospira species share the distinctive double-membrane architecture of spirochetes. Spirochetal structure has similarities with both Gram-positive and Gram-negative bacteria. As in Gram-positive bacteria, the inner (cytoplasmic) membrane of spirochetes is closely associated with the peptidoglycan cell wall. Spirochetes also have an outer membrane that provides a barrier shielding underlying antigens, such as the endoflagella, from the outside environment. However, the spirochetal outer membrane appears to be fluid and labile, which contrasts it with the outer membrane of Gram-negative bacteria. Several laboratories have studied the protein composition of the leptospiral outer membrane. The most straightforward method to isolate the leptospiral outer membrane involves selective solubilization using the nonionic detergents Triton X-100 or Triton X-114^3,4. Because of its low cloud-point, Triton X-114 has the added advantage of allowing separation of outer membrane proteins from contaminating periplasmic proteins. Studies involving isolation of the outer membrane in the form of membrane vesicles by sucrose density gradient ultracentrifugation have validated the Triton X-114 approach^5,6.

The study by Biswas and colleagues published in this issue⁷ indicates that sarcosine extraction may be an additional valid method to selectively solubilize the leptospiral outer membrane. Validation of the sarcosine approach awaits immunoblot analysis of the fractions using antisera to endoflagella⁴ or inner membrane markers such as LipL31 and ImpL63⁵. An alternative approach to understanding the leptospiral outer membrane is freeze-fracture electron microscopy, which indicates that, as in other spirochetes, the outer membrane of pathogenic Leptospira species has a relatively low density of transmembrane proteins⁴. The first transmembrane outer membrane protein (OMP) to be identified was OmpL1, a trimeric porin. In contrast to transmembrane OMPs, the leptospiral outer membrane contains large amounts of lipoproteins, such as LipL32 and LipL41. LipL32 has been referred to as the leptospiral major outer membrane protein because it is the most abundant protein in the entire organism³. ELISA studies involving recombinant OmpL1 and LipL32 indicate that a high percentage of human patients with leptospirosis form antibodies to these antigens⁸. Studies in animal models of leptospirosis indicate that OmpL1, LipL41, and LipL32 may also be vaccine candidates for prevention of leptospirosis^9,10. However, it is uncertain whether these leptospiral membrane proteins contain cross-reactive epitopes or to what extent immunization with these proteins is cross-protective.

Research by Biswas et al⁷ and others¹¹ indicates that, in contrast to LPS, the protein profiles and antigenicity of membrane proteins from diverse leptospiral isolates are highly conserved. The detailed studies by Biswas and colleagues provide important new information regarding the conservation of OMPs among leptospiral isolates from the Andaman Islands and other parts of India. Their results greatly strengthen the conclusion that of all the proteins in the leptospiral outer membrane, LipL32 is the major immunoreactive OMP shared by diverse serogroups. Cross-reactivity of leptospiral membrane proteins confirms the results of comparative sequence analysis of the genes encoding OmpL1, LipL41, and LipL32¹². The sequences of genes were analyzed from 38 strains belonging to the core group of pathogenic Leptospira species: L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii. The LipL32 amino acid sequences across these diverse species exhibited a mean pairwise sequence identity of 99.1 per cent. The LipL41 and OmpL1 amino acids sequences were more variable than those of LipL32, but were relatively conserved compared to OMP genes of other pathogenic bacteria. The amino acid sequence differences among OmpL1 variants clustered in four variable regions encoding surface loops. Interestingly, while phylogenetic trees for the 16S, lipL32, and lipL41 genes were relatively stable, 8 of 38 (20%) ompL1 sequences had mosaic compositions consistent with horizontal transfer of DNA between related leptospiral species. A Bayesian multiple change-point model was used to identify the most likely sites of recombination, revealing that segments of the mosaic ompL1 genes originated from a new branch of the leptospiral phylogenetic tree not represented by any known species. We wondered whether this new lineage of ompL1 gene fragments may have been donated by a leptospiral species that is fastidious and cannot be cultivated or became extinct at some point in leptospiral evolutionary history. In either case, these migratory ompL1 gene fragments appear to confer a selective advantage on the recipients, presumably related in some way to interactions between leptospires and their mammalian hosts.

More research is needed to determine how amino acid sequence variation in leptospiral outer membrane proteins affects their potential serodiagnostic utility or their ability to function as vaccines for prevention of leptospirosis in at risk populations. It is also possible that additional leptospiral proteins selectively expressed during infection may prove to be even better serodiagnostic or immunoprotective antigens than proteins expressed by cultivated organisms. Examples of leptospiral membrane proteins upregulated during mammalian infection belong to the family of Lig proteins containing immunoglobulin-like repeats, which were discovered by screening expression libraries with antisera from leptospirosis patients^13,14. Hundreds of additional genes encoding lipoproteins, transmembrane OMPs, and other potential leptospiral membrane proteins revealed through sequencing and analysis of leptospiral genomes^15,16 await further characterization by comparative sequence analysis, serodiagnostic studies, and vaccine trials.