Dataset Information

Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae.

ABSTRACT: Previous studies have shown that the transcriptional regulator PsaR regulates the expression of the PsaR regulon consisting of genes encoding choline binding protein (PcpA), the extracellular serine protease (PrtA), and the Mn2+-uptake system (PsaBCA), in the presence of manganese (Mn2+), zinc (Zn2+), and cobalt (Co2+). In this study, we explore the Ni2+-dependent regulation of the PsaR regulon. We have demonstrated by qRT-PCR analysis, metal accumulation assays, ?-galactosidase assays, and electrophoretic mobility shift assays that an elevated concentration of Ni2+ leads to strong induction of the PsaR regulon. Our ICP-MS data show that the Ni2+-dependent expression of the PsaR regulon is directly linked to high, cell-associated, concentration of Ni2+, which reduces the cell-associated concentration of Mn2+. In vitro studies with the purified PsaR protein showed that Ni2+ diminishes the Mn2+-dependent interaction of PsaR to the promoter regions of its target genes, confirming an opposite effect of Mn2+ and Ni2+ in the regulation of the PsaR regulon. Additionally, the Ni2+-dependent role of PsaR in the regulation of the PsaR regulon was studied by transcriptome analysis.

SUBMITTER: Manzoor I

PROVIDER: S-EPMC4643063 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae.

Manzoor Irfan I Shafeeq Sulman S Kuipers Oscar P OP

PloS one 20151112 11

Previous studies have shown that the transcriptional regulator PsaR regulates the expression of the PsaR regulon consisting of genes encoding choline binding protein (PcpA), the extracellular serine protease (PrtA), and the Mn2+-uptake system (PsaBCA), in the presence of manganese (Mn2+), zinc (Zn2+), and cobalt (Co2+). In this study, we explore the Ni2+-dependent regulation of the PsaR regulon. We have demonstrated by qRT-PCR analysis, metal accumulation assays, β-galactosidase assays, and elec ...[more]

PMID: 26562538

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Project description:Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Project description:BackgroundStreptococcus pneumoniae is a respiratory pathogen causing severe lung infection that may lead to complications such as bacteremia. Current polysaccharide vaccines have limited serotype coverage and therefore cannot provide maximal and long-term protection. Global efforts are being made to develop a conserved protein vaccine candidate. PrtA, a pneumococcal surface protein, was identified by screening a pneumococcal genomic expression library using convalescent patient serum. The prtA gene is prevalent and conserved among S. pneumoniae strains. Its protective efficacy, however, has not been described. Mucosal immunization could sensitize both local and systemic immunity, which would be an ideal scenario for preventing S. pneumoniae infection.MethodsWe immunized BALB/c mice intranasally with a combination of a PrtA fragment (amino acids 144-1041) and Th17 potentiated adjuvant, curdlan. We then measured the T-cell and antibody responses. The protective efficacy conferred to the immunized mice was further evaluated using a murine model of acute pneumococcal pneumonia and pneumococcal bacteremia.ResultsThere was a profound antigen-specific IL-17A and IFN-? response in PrtA-immunized mice compared with that of adjuvant control group. Even though PrtA-specific IgG and IgA titer in sera was elevated in immunized mice, only a moderate IgA response was observed in the bronchoalveolar lavage fluid. The PrtA-immunized antisera facilitated the activated murine macrophage, RAW264.7, to opsonophagocytose S. pneumoniae D39 strain; however, PrtA-specific immunoglobulins bound to pneumococcal surfaces with a limited potency. Finally, PrtA-induced immune reactions failed to protect mice against S. pneumoniae-induced acute pneumonia and bacterial propagation through the blood.ConclusionsImmunization with recombinant PrtA combined with curdlan produced antigen-specific antibodies and elicited IL-17A response. However, it failed to protect the mice against S. pneumoniae-induced infection.

Dataset Information

Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae.

Publications

Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae.

Similar Datasets

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