Comprehensive label-free quantitative proteomic analysis of the Corynebacterium pseudotuberculosis exoproteome and the implications on pathogenicity and resistance to stress

摘要

A three-phase partitioning methodology combined with a nanoLC-MS~(E) quantitative proteomic strategy provided confident and comprehensive coverage of the extracytoplasmic proteins from Corynebacterium pseudotuberculosis. Comparative exoproteome analysis of two wild type strains of different virulence status allowed us to detect considerable variations of the C. pseudotuberculosis extracellular proteome. In our study we did not observe phospholipase D in the cultured wild type 1002 exoproteome and this may contribute as one of the main factors responsible for the lowered virulence of this strain. Conversely we did observe the FagD and Cp40 proteins in the C231 strain. Several novel targets for future work on molecular determinants of virulence can be identified. Such proteins may represent promising new candidates for composing a vaccine more effective than the ones currently available. A mutant deficient in the alternative extracytoplasmic sigma factor sigE from strain 1002 produced a more diverse exoproteome both under normal and nitric oxide stress than its wild type counterpart. The exoproteome of the 1002 wild type strain showed little variation under NO stress indicating the reproducibility of the TPP and nanoLC-MS~(E) approach for the study of the exoprotein complement. The observation of a dioxygenase protein in this strain under NO stress suggests that additional studies are needed to confirm whether the expression of this species is really dependent on the alternative sigma factor sigE and, most importantly, whether this protein plays any role in detoxification of extracellular NO in C. pseudotuberculosis. It was not possible to identify known conserved domains in the putative secreted protein upregulated over 100-fold in the (DELTA)sigE mutant under nitrosative stress, thus its function remains elusive. The fact that it is highly expressed in the mutant strain in response to NO ensures that it warrants further investigation. This study is the first to demonstrate that the alternative sigma factor sigE may participate in the specific bacterial response triggered by NO and its contribution to resistance to nitrosative stress in vitro and in vivo and we propose a model for its participation in the alteration of C. pseudotuberculosis exoproteome in response to nitric oxide. After entering a host cell C. pseudotuberculosis is exposed to nitrosative stress generated by nitric oxide synthase (iNOS) within the phagosomal environment. Then, the wild-type strain of this bacterium secretes a few additional proteins that include a putative dioxygenase, which might aid in detoxification of intraphagosomal nitric oxide. The (DELTA)sigE mutant strain in turn, secretes more proteins normally involved in general stress responses, indicating a compensatory response. A highly up-regulated protein (indicated in red) is secreted only in the mutant strain following NO exposure, but its function is still unknown.