Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection

Abstract

We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322–2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundant L. kirschneri proteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adaptedL. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenic Leptospira species adapt to the host environment.

A novel leptospiral protein increases ICAM-1 and E-selectin expression in human umbilical vein endothelial cells

“…This is relevant because some proteins found in organisms grown under culture conditions are not expressed during infection. For example, LipL36 is expressed at high levels by cultivated Leptospira kirschneri but is not detectable either within infected hamster kidneys by immunohistochemistry or by sera from hamsters infected with a hostderived organism (Haake et al, 1998;Barnett et al, 1999). In contrast, LipL32 is detectable both by immunohistochemistry and by immunoblot (Haake et al, 2000).…”

Outer membrane proteins of pathogenic spirochetes

“…As well as being accessible to antibody on the spirochetal surface, any putative OMP-based vaccinogen must be expressed during either initial colonization of the host or during subsequent stages of infection. For example, the leptospiral outer membrane lipoprotein LipL36, which is exclusively located on the periplasmic face of the outer membrane [187] and down-regulated during infection [188] would be an unlikely protective immunogen. Similarly, the surface-exposed brachyspiral outer membrane lipoprotein SmpA [189] that is also down-regulated during infection [190] is unlikely to elicit immunoprotection.…”

Omp52 is a growth-phase-regulated outer membrane protein ofLeptospira santarosaiserovar Shermani

“…Some of leptospiral outer membrane proteins (OMPs) have been identified. The most abundant OMPs comprises the outer membrane lipoprotein, including the major OMP and immunodominant protein antigen LipL32 [3], the in vivo‐down‐regulated protein LipL36 [4], LipL48 [5] and the surface‐exposed protein LipL41 [6] and LipL21 [7]. OmpL1 is thought to function as a heat‐modifiable porin [8].…”