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. 2013 Oct;17(10):527-35.
doi: 10.1089/omi.2013.0043. Epub 2013 Jul 29.

Extracellular proteome analysis of Leptospira interrogans serovar Lai

Lingbing Zeng  1 , Yunyi Zhang, Yongzhang Zhu, Haidi Yin, Xuran Zhuang, Weinan Zhu, Xiaokui Guo, Jinhong Qin
Affiliations

Extracellular proteome analysis of Leptospira interrogans serovar Lai

Lingbing Zeng et al. OMICS. 2013 Oct.

Abstract

Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

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Figures

FIG. 1.
FIG. 1.
The growth curve of L. interrogans serovar Lai cultivated in protein-free medium C-70 and EMJH. Leptospira were diluted to a density of 5×107cells/mL and grown at 28°C in medium C-70 and EMJH. Triplicate samples were counted under a dark-field microscope with a Petroff-Hausser cell counter. Differences among the three groups were nonsignificant at all time points (p>0.05).
FIG. 2.
FIG. 2.
The profile of L. interrogans lysates and extracellular proteins. (A) The lysates of L. interrogans. (B) The extracellular proteins of L. interrogans. The protein were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and stained with Coomassie blue. (C) Equal amounts of protein from whole lysates and extracellular protein of L. interrogans were separated by SDS-PAGE and immunoblotted with antiserum against LA_2512. Migration of protein standards is shown to the left in kilodaltons. Abbreviations: C, whole cell of L. interrogans; M, protein marker; S, supernatant of L. interrogans.
FIG. 3.
FIG. 3.
The transcriptional differences of L. interrogans serovar Lai extracellular proteins encoding genes between in vivo and in vitro. Hamsters inoculated with L. interrogans serovar Lai were used as in vivo models. Real-time PCR was used for comparing the transcriptional level between in vivo and in vitro. + means these genes were upregulated twice in vivo compared to in vitro. – means these genes were downregulated twice in vivo compared to in vitro.
FIG. 4.
FIG. 4.
Schematic of the predicted secretion systems of L. interrogans serovar Lai. L. interrogans serovar Lai is a gram-negative bacteria that possesses an inner membrane and outer membrane. Based on the genomic and proteomic data available for L. interrogans (Cao et al., ; Zhong et al., 2011), BLASTP was used to search for homologs of components of the secretion system of T1SS, T2SS, T3SS, T4SS, T5SS, and T6SS in the NCBI in the L. interrogans genome and further to confirm the expression of the components by whole cell proteomics data search. This figure shows the schematic of a relatively complete T1SS and T2SS and incomplete T3SS, T4SS, T5SS, and T6SS in L. interrogans serovar Lai.
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