Global Analysis of Outer Membrane Proteins from Leptospira interrogans Serovar Lai

Abstract

Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37°C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30°C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30°C, but not at 30°C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature-and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.

High-coverage proteome analysis reveals the first insight of protein modification systems in the pathogenic spirochete Leptospira interrogans

“…The genome sequences of the pathogenic L. interrogans serovar Lai and serovar Copenhageni, the pathogenic L. borgpetersenii serovar Hardjo and the saprophytic L. biflexa serovar Patoc have been reported [3][4][5][6]. The accomplishment of these sequencing projects greatly facilitated the studies of Leptospira physiology and pathology at the genomic [4][5][6], transcriptomic [7,8] and proteomic levels [9][10][11][12][13][14]. However, there are discrepancies in the annotations for the same genome sequences by different institutions or by using diverse prediction methodologies, which hinders further studies [8].…”

Determinação do perfil protéico da membrana externa da Leptospira interrogans sorovariedade Hardjoprajitno

“…Uma PME de 42,75KDa foi encontrada nas linhas de análise 2 e 3 (r3), cuja massa molecular aproxima-se à LipL41, descrita por Shang et al (1996) e Cullen et al (2002). Nas linhas de análise L2 e L3, apareceram duas bandas de massas moleculares calculadas em 58,59KDa e em 57,06KDa (r1), respectivamente.…”

“…A eletroforese 2D foi realizada em tiras de gel imobilizado (Amersham Biosciences), com gradiente de pH de 4 a 7, medindo 13cm de comprimento, segundo técnica descrita por Cullen et al (2002). O gel foi corado com nitrato de prata ( Arrell et al, 2001).…”

“…A lipoproteína de 22KDa, denominada Loa22, considerada como o segundo maior constituinte das membranas externas das L. interrogans sorovariedades Lai (Cullen et al, 2002) e Manilae (Koizumi e Watanabe, 2003), foi associada à virulência das amostras de leptospiras, sendo expressa, principalmente, durante os processos de infecção, relacionandose aos casos agudos da doença (Ristow et al, 2007).…”

Avaliação e caracterização de candidatos vacinais voltados para o controle da leptospirose.

“…Devido à localização, essas proteínas podem participar das interações patógeno-hospedeiro e, ainda, servir de alvo para o sistema imune. Nesse sentido, algumas proteínas de membrana, analisadas por programas de bioinformática têm sido identificadas como possíveis candidatos vacinais em modelo animal (ATZINGEN et al, 2010;ATZINGEN et al, 2012;CULLEN et al, 2002;GAMBERINI et al, 2005;HAAKE et al, 1999;NASCIMENTO et al, 2004a;NASCIMENTO et al, 2004b;PALANIAPPAN et al, 2006;REN et al, 2003;YAN et al, 2009).…”