The OmpA-like protein Loa22 is essential for leptospiral virulence.

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.

A methylated phosphate group and four amide-linked acyl chains in leptospira interrogans lipid A. The membrane anchor of an unusual lipopolysaccharide that activates TLR2.

Leptospira interrogans differs from other spirochetes in that it contains homologs of all the Escherichia coli lpx genes required for the biosynthesis of the lipid A anchor of lipopolysaccharide (LPS). LPS from L. interrogans cells is unusual in that it activates TLR2 rather than TLR4. The structure of L. interrogans lipid A has now been determined by a combination of matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR spectroscopy, and biochemical studies. Lipid A was released from LPS of L. interrogans serovar Pomona by 100 degrees C hydrolysis at pH 4.5 in the presence of SDS. Following purification by anion exchange and thin layer chromatography, the major component was shown to have a molecular weight of 1727. Mild hydrolysis with dilute NaOH reduced this to 1338, consistent with the presence of four N-linked and two O-linked acyl chains. The lipid A molecules of both the virulent and nonvirulent forms of L. interrogans serovar Icterohaemorrhagiae (strain Verdun) were identical to those of L. interrogans Pomona by the above criteria. Given the selectivity of L. interrogans LpxA for 3-hydroxylaurate, we propose that L. interrogans lipid A is acylated with R-3-hydroxylaurate at positions 3 and 3' and with R-3-hydroxypalmitate at positions 2 and 2'. The hydroxyacyl chain composition was validated by gas chromatography and mass spectrometry of fatty acid methyl esters. Intact hexa-acylated lipid A of L. interrogans Pomona was also analyzed by NMR, confirming the presence a beta-1',6-linked disaccharide of 2,3-diamino-2,3-dideoxy-d-glucopyranose units. Two secondary unsaturated acyl chains are attached to the distal residue. The 1-position of the disaccharide is derivatized with an axial phosphate moiety, but the 4'-OH is unsubstituted. (1)H and (31)P NMR analyses revealed that the 1-phosphate group is methylated. Purified L. interrogans lipid A is inactive against human THP-1 cells but does stimulate tumor necrosis factor production by mouse RAW264.7 cells.