The OmpA-like protein Loa22 is essential for leptospiral virulence

Abstract

Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. 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.

Figure 1. Disruption and Complementation of loa22 in L. interrogans

(A and B) Analysis of chromosomal DNA from the parental (lane 1), mutant loa22⁻ (lane 2), and complemented TK2 strains (lane 3) by PCR with primers S1a and S1b (A) and Southern blot of EcoRI-digested DNA probed for hybridization with the spectinomycin (Spc^R)- and kanamycin (Km^R)-resistant cassettes (B). Primers S1a and S1b are located in the flanking sequences of the insertion site of the spectinomycin-resistant transposon into loa22. This analysis revealed that there was an insertion of 1.3 kb in the mutant loa22⁻ strain and that an additional copy of loa22 was present in the complemented strain. (C) Schematic representation of the genotype of the parental (wt), mutant, and complemented strains. Arrowheads in white indicate the position of EcoRI sites. (D) ELISA of plates with total bacterial antigens and Loa22 antiserum (serum dilution 1:800).

Figure 2. Surface Localization of Loa22

Surface immunofluorescence assay was performed with L. interrogans wild-type strain (wt), mutant loa22⁻ (loa22⁻), and mutant loa22⁻ complemented with wild-type loa22 (TK2). Strains were labeled with antibodies against Loa22 and the following lipoproteins: LipL32, a surface-exposed lipoprotein [20], and LipL31, a lipoprotein that is associated with the inner membrane and not surface-exposed [20]. Alexa and fluorescein isothiocyanate (FITC)–conjugated secondary antibodies were used to detect surface-bound antibodies to Loa22 and LipL32 and LipL31, respectively. A DAPI counterstain was used to document the presence of leptospires. A photomicrograph is shown from one of three representative experiments.

Figure 3. Gross Examination of Infected Guinea Pigs

Left panel: Guinea pigs infected with the wild-type (A) and complemented strains (C) with clinical findings of jaundice and hemorrhages that are absent in guinea pigs infected with the mutant loa22⁻ strain (B). Right panel: Lungs of a guinea pig infected with mutant loa22⁻ did not exhibit macroscopic hemorrhage (B), in contrast with lungs of guinea pigs infected with the wild-type (A) and complemented strains (C). Tissues were observed 6 d post-inoculation.

Figure 4. Livers and Kidneys from Guinea Pigs Infected with the Wild-Type Strain and Mutant loa22⁻ Strain

All images are from guinea pigs 6 d post-inoculation. The right panels show normal livers (A–C) and kidneys (D and E). Tissues were stained by hematoxylin and eosin (×200, [A and D]), Warthin–Starry (×1000, [B and E]) and immunohistochemistry with antiserum specific to LipL32 (×200, [C]). Left panel, wild-type strain (wt); middle panel, mutant loa22⁻ strain. (A–C) Livers of wt-infected animals exhibit important periportal lymphoplasmocitary inflammatory infiltration, loss of parenchymal architecture, and increase of biliary canalicules in comparison with a normal aspect of mutant loa22⁻. Distortion of liver cords is related to numerous leptospires along cell membranes of hepatocytes (arrow, [B]) in wt-infected animals. (D and E) Kidneys of wt-infected animals present hemorrhages in Bowman's spaces, lumen of renal tubules, and interstitium (D). A large number of leptospires is seen in Bowman's space (arrow), sometimes forming a cap (E). Histology of kidneys infected with mutant loa22⁻ was considered as normal (D and E).

Figure 5. Histopathologic sections on Liver, Kidney, and Lung of Guinea Pigs Infected by Complemented Strain TK2 6 d Post-Inoculation

Left panel: Hematoxylin and eosin staining (×200) of infected guinea pigs. Right panel: Immunochemistry with antiserum specific for LipL32 (×200; except [C], ×1,000). Pictures of histopathology were similar between animals infected with wild-type and complemented strains. (A) The liver has a great loss of architecture and areas of necrosis and inflammatory infiltration, which are both associated with the presence of numerous leptospires. (B) Kidneys present hemorrhages, tubular necrosis, and inflammatory infiltration, with leptospires mainly located in Bowman's spaces and proximal tubules. (C) Lungs have marked intra-alveolar hemorrhages with inflammatory infiltrates, and few leptospires are present within septal membranes and, sometimes, in macrophages ([C], right panel).

Figure 6. In Vivo Expression of Loa22 in Liver and Kidney of Guinea Pigs Infected with L. interrogans Serovar Lai

(A) Liver, (B) kidney. Histopathologic sections were stained by immunochemistry using Loa22 antiserum (×1,000). Intact organisms were found in biliary ducts (A) and in large number in Bowman's spaces and proximal tubules (B). Scale bar = 20 μm.