Altered Proteome in Burkholderia pseudomallei rpoE Operon Knockout Mutant: Insights into Mechanisms of rpoE Operon in Stress Tolerance, Survival, and Virulence

Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Journal of Proteome Research (Impact Factor: 4.25). 05/2007; 6(4):1334-41. DOI: 10.1021/pr060457t
Source: PubMed


We have previously shown that the alternative sigma factor sigmaE (RpoE), encoded by rpoE, is involved in stress tolerance and survival of Burkholderia pseudomallei. However, its molecular and pathogenic mechanisms remain unclear. In the present study, we applied gel-based, differential proteomics to compare the cellular proteome of an rpoE operon knockout mutant (RpoE Mut) to that of wild-type (K96243 WT) B. pseudomallei. Quantitative intensity analysis (n = 5 gels from 5 individual culture flasks in each group) revealed significantly differential expression of 52 proteins, which were subsequently identified by Q-TOF MS/MS. These included oxidative, osmotic, and other stress response proteins; chaperones; transcriptional/translational regulators; metabolic enzymes; proteins involved in cell wall synthesis, fatty synthesis, glycogen synthesis, and storage; exported proteins; secreted proteins; adhesion molecule; protease/peptidase; protease inhibitor; signaling proteins; and other miscellaneous proteins. The down-regulation of several stress response proteins, chaperones, transcriptional/translational regulators, and proteins involved in cell wall synthesis in RpoE Mut provided some new insights into the mechanisms of the rpoE operon for the stress tolerance and survival of B. pseudomallei. In addition, the proteomic data and in vivo study indicated that the rpoE operon is also involved in the virulence of B. pseudomallei. Our findings underscore the usefulness of proteomics for unraveling pathogenic mechanisms of diseases at the molecular level.

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Available from: Napat Songtawee, Jun 03, 2014
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    • "A B. pseudomallei rpoE insertional inactivation mutant has previously been constructed and showed increased susceptibility to hydrogen peroxide (H2O2), suggesting a role for σE in the oxidative stress response [13]. Furthermore, inactivation of B. pseudomallei σE resulted in reduced survival in J774A.1 macrophages and the mutant was attenuated in a murine model of infection [13, 14]. A proteomic comparison of B. pseudomallei wild type and the σE insertional mutant revealed the differential levels of proteins that may contribute to the stress tolerance and survival of B. pseudomallei [14] but this study was unable to identify all the proteins involved in this response, because of the limitations of the proteomic platform. "
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    ABSTRACT: Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative bacterium widely distributed in soil and water in endemic areas. This soil saprophyte can survive harsh environmental conditions, even in soils where herbicides (containing superoxide generators) are abundant. Sigma factor E (σE) is a key regulator of extra-cytoplasmic stress response in Gram-negative bacteria. In this study, we identified the B. pseudomallei σE regulon and characterized the indirect role that σE plays in the regulation of spermidine, contributing to the successful survival of B. pseudomallei in stressful environments. Changes in the global transcriptional profiles of B. pseudomallei wild type and σE mutant under physiological and oxidative stress (hydrogen peroxide) conditions were determined. We identified 307 up-regulated genes under oxidative stress condition. Comparison of the transcriptional profiles of B. pseudomallei wild type and σE mutant under control or oxidative stress conditions identified 85 oxidative-responsive genes regulated by σE, including genes involved in cell membrane repair, maintenance of protein folding and oxidative stress response and potential virulence factors such as a type VI secretion system (T6SS). Importantly, we identified that the speG gene, encoding spermidine-acetyltransferase, is a novel member of the B. pseudomallei σE regulon. The expression of speG was regulated by σE, implying that σE plays an indirect role in the regulation of physiological level of spermidine to protect the bacteria during oxidative stress. This study identified B. pseudomallei genes directly regulated by σE in response to oxidative stress and revealed the indirect role of σE in the regulation of the polyamine spermidine (via regulation of speG) for bacterial cell protection during oxidative stress. This study provides new insights into the regulatory mechanisms by which σE contributes to the survival of B. pseudomallei under stressful conditions.
    BMC Genomics 09/2014; 15(1):787. DOI:10.1186/1471-2164-15-787 · 3.99 Impact Factor
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    • "Therefore, an establishment of a 2DE dataset proteome reference map from one of these three species will be a valuable resource for comparing protein expression profile in order to study their pathogenesis differentially caused by B. pseudomallei and B. mallei comparing with B. thailandensis. To this end, given the fact that B. pseudomallei has the most biomedical impact between these three bacterial species, the proteome reference map constructed from B. pseudomallei will eventually lead to the most effective effort, not only for interspecies comparative proteomic analysis [7] but also for studying gene and environmental regulation in this bacterium [8] [9] [10]. We previously reported an initiation of a partial proteome reference map of B. pseudomallei grown at prolonged stationary phase to apply for a proteomic comparison between this virulent species and the nonvirulent species B. thailandensis [7]. "
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    ABSTRACT: Burkholderia pseudomallei is the etiologic agent of melioidosis. Using 2DE and MALDI-TOF MS, we report here a proteome reference map constructed from early stationary phase, a bacterial adaptation process. We identified 282 protein spots representing 220 ORFs; many of them have been implicated in bacterial pathogenesis. Up to 20% of identified ORFs belong to post-translational modification and stress responses. The proteome reference map will support future analysis of the bacterial gene and environmental regulation and facilitate comparative proteomics with its sibling species.
    BioMed Research International 09/2011; 2011(6699):530926. DOI:10.1155/2011/530926 · 2.71 Impact Factor
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    • "In addition, RpoS controls the acid resistance in E. coli by modulating gadC, a gene involved in the glutamate-dependent low pH-resistance, hdeAB, encoding pH-regulated periplasmic chaperons, and cfa, a gene for cycloporpane fatty acid synthesis [20]. As another stress-response sigma factor, RpoE regulates extracytoplasmic functions related to sensing and responding to bacterial periplasmic and extracellular environmental changes, which contributes to heat- and oxidative stress resistance in many Gram-negative bacteria, including E. coli, Pseudomonas and Salmonella [21,22]. The RpoE mutation in Salmonella reduces bacterial survival and growth in macrophages by the loss of RpoE-dependent gene expression such as htrA, a gene required for oxidative stress resistance [23,24]. "
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    ABSTRACT: Campylobacter jejuni is a leading foodborne pathogen worldwide. Despite the fastidious nature of C. jejuni growth, increasing numbers of human campylobacteriosis suggest that C. jejuni may possess unique mechanisms to survive under various stress conditions. C. jejuni possesses only three sigma factors (FliA, RpoD, and RpoN) and lacks stress-defense sigma factors. Since FliA and RpoD are dedicated to flagella synthesis and housekeeping, respectively, in this study, we investigated the role of RpoN in C. jejuni's defense against various stresses. Survivability of an rpoN mutant was compared with the wild-type C. jejuni under various stress conditions. While the growth of the rpoN mutant was as comparably as that of the wild type in shaking cultures, the rpoN mutant exhibited significant survival defects when cultured statically. The rpoN mutant was more sensitive to osmotic stress (0.8% NaCl) with abnormally-elongated cell morphology. Compared to the wile type, the rpoN mutant was more susceptible to acid stress (pH 5) and more resistant to hydrogen peroxide. However, the rpoN mutation had little effect on the resistance of C. jejuni to alkaline pH, heat, cold and antimicrobials. The results demonstrate that RpoN plays an important role in C. jejuni's defense against various stresses which this bacterial pathogen may encounter during transmission to and infection of humans.
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