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PhoP/Q regulated genes in Salmonella typhi identification of melittin sensitive mutants
S J Baker1, C Daniels, R Morona
1Department of Microbiology and Immunology, University of Adelaide, Australia.
Abstract:
Many of the genes (pags (phoP activated genes) and prgs (phoP repressed genes)) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival of Salmonella typhimurium in murine macrophages and pathogenesis in mice. Although a great deal is known about the S. typhimurium phoP/Q regulon, little has been done with the human specific pathogen S. typhi, prompting us to investigate S. typhi phoP/Q regulated genes. Isogenic phoP12 (null) and phoP24 (constitutive) strains were constructed in S. typhi Ty2 and S. typhimurium C5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested that S. typhi and S. typhimurium may have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of a phoP12 mutant, creating lacZ-gene fusions, and screening for Lac+ or Lac- colonies. A mobilizable plasmid carrying the phoP24 mutant gene was conjugated into these insertion mutants. Those that changed from Lac- to Lac+ were inferred to be pag::MudJ insertions and those that changed from Lac+ to Lac- were inferred to be prg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termed pqa (PhoPQ activated) and pqr (PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. The pqa/pqr::MudJ mutations were transduced into S. typhi phoP+ and phoP24 strains by Vi-l phage transduction. Characterization of the mutants (Southern blot analysis, beta-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.
Insights
This study identifies novel Salmonella typhi genes regulated by PhoP/Q proteins, crucial for bacterial survival and pathogenesis. Researchers discovered two new mutants sensitive to antimicrobial peptides, advancing our understanding of S. typhi virulence.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- The PhoP/Q regulatory system is vital for Salmonella typhimurium survival in macrophages and mouse pathogenesis.
- Limited research exists on the PhoP/Q regulon in the human-specific pathogen Salmonella typhi.
- Understanding S. typhi's PhoP/Q system is crucial for addressing typhoid fever.
Purpose of the Study:
- To investigate and identify genes regulated by the PhoP/Q system in Salmonella typhi.
- To compare the PhoP/Q regulons of S. typhi and S. typhimurium.
- To characterize novel PhoP/Q-regulated genes in S. typhi.
Main Methods:
- Construction of isogenic phoP null and constitutive mutant strains in S. typhi and S. typhimurium.
- Comparative whole-cell protein analysis using SDS-PAGE.
- Development of a genetic procedure using MudJ transposon mutagenesis and lacZ-gene fusions to identify PhoP/Q-regulated genes.
- Confirmation of mutations via Southern blot analysis and beta-galactosidase assays.
- Assessment of antimicrobial peptide sensitivity and mouse virulence.
Main Results:
- Significant differences observed in the number and molecular mass of PhoP/Q-regulated proteins between S. typhi and S. typhimurium.
- Identification of five novel PhoP/Q-regulated genes in S. typhi, designated pqa (PhoPQ activated) and pqr (PhoPQ repressed).
- Two identified mutants exhibited sensitivity to the antimicrobial peptide melittin.
Conclusions:
- S. typhi possesses distinct PhoP/Q-regulated genes and regulatory mechanisms compared to S. typhimurium.
- The identified pqa/pqr genes are important components of the S. typhi PhoP/Q regulon.
- The sensitivity of two mutants to melittin suggests a role for PhoP/Q-regulated genes in antimicrobial peptide resistance in S. typhi.