Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

Abstract

Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B 12 . When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.

Use of Luminescent Leptospira interrogans for Enumeration in Biological Assays

“…An extracellular matrix-binding assay was conducted as described previously (7,15). In brief, quadruplicate wells of a microtiter tray were incubated at 4°C overnight with 100 ml of a 500-g/ml solution of the extracellular matrix-like compound Matrigel (BD Biosciences) in phosphate-buffered saline and washed, and then a known quantity of bacteria was added.…”

“…This is currently the only efficient means for constructing defined leptospiral mutants. This method has been applied for widespread random mutagenesis of the bacterial chromosome (13), facilitating the identification of two virulence factors (14,17) and the unexpected finding that the major outer membrane protein (LipL32) is not required for pathogenesis (15).…”

Simultaneous Detection of Pathogenic and Saprophyte Leptospira in Human Plasma by Multiplex Taqman Real Time PCR

“…LipL32 genes encode a lipoprotein that can be used as an adhesion molecule to laminin-collagens and fibronectin bands (15)(16)(17). LipL32 lipoprotein is a major outer membrane protein of Leptospira, which can only be found in pathogenic strains and absent in saprophytic bacteria (3,18,19).…”

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

“…Genome sequencing analyses have shown that saprophytic strains of Leptospira do not harbor genes orthologous to LipL32 (4), suggesting an important role of this protein in leptospiral pathogenesis. Although a knock-out of the lipL32 gene in pathogenic Leptospira did not prevent colonization of the renal tubules in a rat model of chronic infection (5), it is important to keep in mind that animal models may not reproduce all relevant conditions of infection and colonization in the host. Therefore, the precise role of LipL32 in Leptospira biology remains unclear.…”