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Regulation of the pspA virulence factor and essential pcsB murein biosynthetic genes by the phosphorylated VicR (YycF) response regulator in Streptococcus pneumoniae.

ABSTRACT: The VicRK (YycFG) two-component regulatory system (TCS) is required for virulence of the human respiratory pathogen Streptococcus pneumoniae (pneumococcus). The VicR (YycF) response regulator (RR) is essential through its positive regulation of pcsB, which encodes an extracellular protein that mediates murein biosynthesis. To determine other genes that are regulated by VicR, we performed microarray analyses on a unique DeltavicR deletion mutant, which was constructed by uncoupling regulation of pcsB. Results from these microarray experiments support the idea that the VicR RR exerts strong positive regulation on the transcription of a set of genes encoding important surface proteins, including the PspA virulence factor, two proteins (Spr0096 and Spr1875) containing LysM peptidoglycan-binding domains, and a putative membrane protein (Spr0709) of unknown function. To demonstrate direct regulation, we performed band shift and footprinting experiments using purified unphosphorylated VicR and phosphorylated VicR-P, which was prepared by reaction with acetyl phosphate. VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, spr1875, and spr0709. Phosphorylation of VicR to VicR-P increased the apparent strength and changed the nature of binding to these regions. DNase I footprinting of VicR and VicR-P bound to regions upstream of pcsB, pspA, spr0096, and spr1875 showed protection of extended regions containing a degenerate sequence related to a previously proposed consensus. These combined approaches did not support autoregulation of the vicRKX operon or substantive direct regulation of fatty acid biosynthesis by VicR or VicR-P. However, the DeltavicR mutant required fatty acids in some conditions, which supports the notion that the VicRK TCS may mediate membrane integrity as well as murein biosynthesis and virulence factor expression in S. pneumoniae.

SUBMITTER: Ng WL

PROVIDER: S-EPMC1272996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 from Streptococcus pneumoniae belongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD from Thermotoga maritima, PhoB from Escherichia coli, and PhoP from Bacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound alpha(5)beta(5); however, they differ remarkably in the fine detail of the beta4-alpha4 and alpha4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacterium S. pneumoniae R. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4 S. pneumoniae (as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 A, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 A and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.

Project description:BackgroundStreptococcus pneumoniae (SPN) is the agent responsible for causing respiratory diseases, including pneumonia, which causes severe health hazards and child deaths globally. Antibiotics are used to treat SPN as a first-line treatment, but nowadays, SPN is showing resistance to several antibiotics. A vaccine can overcome this global problem by preventing this deadly pathogen. The conventional methods of wet-laboratory vaccine design and development are an intense, lengthy, and costly procedure. In contrast, epitope-based in silico vaccine designing can save time, money, and energy. In this study, pneumococcal surface protein A (PspA), one of the major virulence factors of SPN, is used to design a multi-epitope vaccine.MethodsFor designing the vaccine, the sequence of PspA was retrieved, and then, phylogenetic analysis was performed. Several CTL epitopes, HTL epitopes, and LBL epitopes of PspA were all predicted by using several bioinformatics tools. After checking the antigenicity, allergenicity, and toxicity scores, the best epitopes were selected for the vaccine construction, and then, physicochemical and immunological properties were analyzed. Subsequently, vaccine 3D structure prediction, refinement, and validation were performed. Molecular docking, molecular dynamic simulation, and immune simulation were performed to ensure the binding between HLA and TLR4. Finally, codon adaptation and in silico cloning were performed to transfer into a suitable vector.ResultsThe constructed multi-epitope vaccine showed a strong binding affinity with the receptor molecule TLR4. Analysis of molecular dynamic simulation, C-immune simulation, codon adaptation, and in silico cloning validated that our designed vaccine is a suitable candidate against SPN.ConclusionThe in silico analysis has proven the vaccine as an alternative medication to combat against S. pneumoniae. The designated vaccine can be further tested in the wet lab, and a novel vaccine can be developed.

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Regulation of the pspA virulence factor and essential pcsB murein biosynthetic genes by the phosphorylated VicR (YycF) response regulator in Streptococcus pneumoniae.

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