Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

Abstract

cThe saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Members of the genus Leptospira are spirochetal bacteria that share a conserved spiral morphology and motility. Leptospires can be broadly divided into host-associated species (including pathogens and the closely related intermediate species [1]) and free-living saprophytes, and Picardeau et al. proposed that the host-associated species may have evolved from a free-living progenitor (2). Therefore, comparisons between species with different ecological niches may elucidate the mechanisms that allow host-associated species to infect, persist in, and sometimes cause fatal disease in their hosts.Leptospira biflexa, a free-living saprophyte, was discovered over 100 years ago (3) and is often used as a model organism for studies of the genus as a whole (2, 4-6). Although host-associated species, such as Leptospira interrogans, are more intensively studied, they exhibit a low transformation efficiency that approaches the limits of detection. The higher in vitro growth rate and more efficient genetic tools available to manipulate L. biflexa make this organism better suited for laboratory studies. Techniques such as targeted allelic exchange, transposon mutagenesis, conjugative transfer, the use of fluorescent markers, and the use of an inducible promoter system were first developed in L. biflexa before use in L. interrogans (5, 7-9). However, despite its being a model organism, there are significant gaps in our knowledge of L. biflexa. No large-scale microarray data o...

Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

“…More recently, it was shown that protein expression, in response to changing environmental conditions, can be modified further by specific post-translational modifications (PTM; Eshghi et al, 2012 ). Indeed, both saprophytic and pathogenic leptospires have comprehensive bio-systems to modify proteins (Cao et al, 2010 ; Schmidt et al, 2011 ; Stewart et al, 2016 ). Recently, 32 phosphorylated, 46 acetylated, and 155 methylated proteins were identified that not only confirmed multiple modifications in prokaryotes, but also suggests that L. interrogans shares significant similarities with protein modification systems in eukaryotes (Cao et al, 2010 ).…”

A widely conserved bacterial cytoskeletal component influences unique helical shape and motility of the spirochete Leptospira biflexa

“…We termed the bactofilins of L. biflexa LbbA‐E, for Leptospira biflexa bactofilins A‐E, ranked by increasing number of amino acids (Table ). LbbD was the only bactofilin identified from a previous proteomic mapping experiment (Stewart et al ., ) and we selected this protein for further characterization of its contribution to L. biflexa morphology and physiology.…”

“…Furthermore, qRT‐PCR analysis of wild type and ΔLBBD strains for flaB transcript normalized to putative outer membrane protein B0SQ62 (Uniprot) demonstrated similar levels between strains (data not shown). This outer membrane protein was previously identified as an abundant membrane‐associated protein with Uniprot accession B0SGK2 (Stewart et al ., ). Finally, mutant cell morphology during translation in viscous media was similar to that of wild type (Supporting Information Figs S2 and S3).…”

“…This conservation across all leptospires indicates that these five protein families predate the speciation of the genera, and each bactofilin is important enough to the fitness of the cell that they remain highly conserved. As LbbD was the only bactofilin observed in the 2‐dimensional gel proteome mapping project, with the caveat that the mapping was not exhaustive (Stewart et al ., ), we deleted the gene (Fig. A and B) and characterized the phenotype of the mutant during in vitro cultivation.…”

“…Relative to many spirochetes, L. biflexa cells are fast growing and comparatively easy to genetically manipulate, thus making this organism a good model for structural studies. Previously, we generated a proteomic map of L. biflexa (Stewart et al ., ) and identified a bactofilin homolog, a family of proteins demonstrated to contribute to cell shape in other bacteria (Kuhn et al ., ). Bactofilins are cytoskeletal proteins, defined as containing the conserved DUF583 domain (synonymous with DUF342) (Kuhn et al ., ).…”