Leptospiral Outer Membrane Protein LipL41 Is Not Essential for Acute Leptospirosis but Requires a Small Chaperone Protein, Lep, for Stable Expression

Abstract

f Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

A Putative Regulatory Genetic Locus Modulates Virulence in the Pathogen Leptospira interrogans

“…These include genes involved in motility (30,36), heme utilization (18), lipopolysaccharide biosynthesis (40), stress resistance (25,41,42), and the host-pathogen interaction (43) and genes that are involved in a manner that has not yet been determined (44,45). However, previous research has also negated the requirement of putative virulence factors LipL32 (46), LipL41 (47), and LigB (48) in the animal infection model. Taken together these studies suggest plasticity and redundancy in leptospiral genomes, where gene products can mimic the function of other genes in the event of a loss of function, thereby safeguarding survival of the bacteria.…”

The Leptospiral Outer Membrane

“…For this reason, the smaller gene has been designated lep for l ipL41 e xpression p artner (King et al, 2013). Even though lipL41 transcript levels were unaffected in a lep transposon mutant, LipL41 levels were greatly reduced.…”

“…Because Lep expression appeared to be required for stable expression of LipL41, perhaps by acting as a chaperone, researchers examined whether Lep bound to LipL41. Lep molecules were found to bind to LipL41 molecules at a molar ratio of 2:1 (King et al, 2013). Neither a lipL41 nor a lep mutant was attenuated for virulence in hamsters.…”

Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

“…For the representative membrane shown, 3.5 g of protein per sample was used for SDS-PAGE and polyvinylidene difluoride (PVDF) membrane transfer according to the manufacturer's instructions (Bio-Rad). Immunoblot analyses were performed as previously described (20), with the following primary antibodies: anti-LigA/B (1:1,000 dilution) and anti-FlaA2 (1:2,000 dilution in blocking buffer). Membranes were probed with goat anti-rabbit IgGhorseradish peroxidase (HRP)-conjugated antibody (1:100,000 dilution) according to the manufacturer's instructions (Sigma-Aldrich Chimie).…”