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: 5). 05/2007; 6(4):1334-41. DOI: 10.1021/pr060457t
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Napat Songtawee, Jun 03, 2014
1 Follower
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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; 2011:530926. DOI:10.1155/2011/530926 · 2.71 Impact Factor
  • Source
    • "Proteomics studies demonstrated that the rpoE mutant displayed reduced expression of a number of stress response proteins and chaperones, as well as transcriptional regulators and proteins involved in cell wall synthesis (Thongboonkerd et al., 2007). The inability of the rpoE mutant to repair cell wall damage is probably responsible for the decreased intracellular survival of this mutant in macrophages and the observed attenuation in BALB/c mice (Korbsrisate et al., 2005; Thongboonkerd et al., 2007). However, the direct regulation of unknown virulence factors by RpoE cannot be completely excluded. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore & Krishnaswami (1912). The causative agent, Burkholderia pseudomallei, was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia (Currie et al., 2000). This health burden, plus the classification of B. pseudomallei as a category B biological agent (Rotz et al., 2002), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.
    FEMS microbiology reviews 08/2009; 33(6):1079-99. DOI:10.1111/j.1574-6976.2009.00189.x · 13.81 Impact Factor
  • Source
    • "This induction was not seen in the AL30 mutant. Recently, on the basis of proteomic analysis, we have reported that the expressions of GroEL and HtpG are downregulated in the rpoE operon mutant (Thongboonkerd et al., 2007). These data supported our hypothesis that the B. pseudomallei AL30 mutant has a defect in the regulation of heat shock protein, resulting in a heat-sensitive phenotype. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei rpoE locus in the response to heat stress. The rpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter-lacZ fusion revealed that heat stress induction of rpoH, which encodes heat shock sigma factor (sigma(H)), was abolished in the B. pseudomallei rpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of sigma(E)-dependent promoters. Moreover, the putative heat-induced sigma(H)-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the rpoE operon mutant. Altogether, our data suggest that the rpoE operon regulates B. pseudomallei heat stress response through the function of rpoH.
    FEMS Microbiology Letters 08/2008; 284(2):191-6. DOI:10.1111/j.1574-6968.2008.01216.x · 2.72 Impact Factor
Show more