ABSTRACT
Yersinia pestis, a Gram negative bacterium, causes bubonic and pneumonic plague. Emerging antibiotic resistance in clinical isolates is driving a need to develop novel antibiotics to treat infection by this transmissible and highly virulent pathogen. Proteins required for viability, so called essential genes, are attractive potential therapeutic targets, however, confirmation of essentiality is problematic. For the first time, we report the development of a system that allows the rapid determination of Y. pestis gene essentiality through mutagenesis and inducible expression of a plasmid borne copy of the target gene. Using this approach, we have confirmed the uridine monophosphate kinase PyrH as an essential protein in Y. pestis. This methodology and the tools we have developed will allow the confirmation of other putative essential genes in this dangerous pathogen, and facilitate the identification of novel targets for antimicrobial development.
Subject(s)
Gene Expression Regulation, Bacterial/drug effects , Genes, Essential , Yersinia pestis/genetics , Animals , Disease Models, Animal , Female , Gene Expression , Gene Knockout Techniques , Mice, Inbred BALB C , Microbial Viability , Nucleoside-Phosphate Kinase/genetics , Plague , Plasmids , Virulence , Yersinia pestis/physiologyABSTRACT
Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains. Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.
Subject(s)
Bacterial Toxins/chemistry , Type C Phospholipases/chemistry , Animals , Aspartic Acid/genetics , Aspartic Acid/physiology , Bacterial Toxins/genetics , Bacterial Toxins/pharmacology , Binding Sites , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Line , Cell Membrane/metabolism , Cricetinae , Isoleucine/genetics , Leucine/genetics , Liposomes/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Phenylalanine/genetics , Phenylalanine/physiology , Phospholipids/metabolism , Type C Phospholipases/genetics , Type C Phospholipases/pharmacology , Tyrosine/genetics , Tyrosine/physiologyABSTRACT
Attenuated mutants of Salmonella typhimurium are being evaluated as delivery systems for a variety of heterologous vaccine antigens. Gene promoters which are induced in vivo can direct the stable expression of genes encoding these antigens. We have investigated the utility of the phoP, ompC, pagC and lacZ gene promoters for expression of the Y. pestis F1-antigen in S. typhimurium SL3261 (aroA). After i.g. (intragastric) dosing the highest level of spleen colonisation was found with recombinant Salmonella expressing F1-antigen from the phoP gene promoter, and this recombinant was most effective in inducing serum and mucosal antibody responses. The use of the pagC gene promoter to direct expression of F1-antigen resulted in the induction of serum and mucosal antibody responses even though the recombinant Salmonella were unable to colonise spleen tissues suggesting that colonisation of these tissues is not essential for the induction of antibody responses.
Subject(s)
Antibodies, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Salmonella typhimurium/immunology , Yersinia pestis/immunology , Animals , Bacterial Proteins/genetics , Escherichia coli , Gene Expression Regulation, Bacterial , Immunity, Mucosal , Immunoglobulin A/biosynthesis , Mice , Peyer's Patches/immunology , Plasmids , Promoter Regions, GeneticABSTRACT
The growth of an aroA mutant of Salmonella typhimurium (SL3261) in minimal medium containing 0.5 M NaCl resulted in the intracellular accumulation of 2.2 micromol trehalose/mg total protein. The vacuum drying of these bacteria in the presence of trehalose allowed the recovery of 35% of the viable cells that were present before drying. In contrast, bacteria cultured in control medium accumulated 0.4 micromol trehalose/mg total protein and only 5% of the viable cells were recovered after vacuum drying with trehalose. Similar results were obtained when S. typhimurium SL3261, expressing the vaccine antigen (F1-antigen) of Yersinia pestis, was cultured in minimal medium with or without 0.5 M NaCl and dried in the presence of trehalose. Although these results indicate the potential for trehalose stabilisation of vaccine strains of S. typhimiurium, growth in minimal medium containing 0.5 M NaCl resulted in the loss of invasion competence of the bacteria.
Subject(s)
Salmonella Vaccines/immunology , Salmonella typhimurium/immunology , Trehalose/biosynthesis , Animals , Cell Line , Female , Glucosyltransferases/genetics , Mice , Mice, Inbred BALB C , Phosphoric Monoester Hydrolases/genetics , Salmonella Vaccines/chemistry , Salmonella typhimurium/growth & developmentABSTRACT
The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.
Subject(s)
Clostridium Infections/microbiology , Clostridium perfringens/enzymology , Clostridium/enzymology , Type C Phospholipases/toxicity , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Cloning, Molecular , Clostridium Infections/enzymology , Enzyme Activation , Mice , Molecular Sequence Data , Phospholipids/metabolism , Sequence Alignment , Substrate Specificity , Type C Phospholipases/genetics , Type C Phospholipases/metabolismABSTRACT
A reporter system was constructed to enable the study of gene expression in Clostridium perfingens. The system was based on plasmid shuttle vector pJIR410, which contained the C. perfringens erythromycin resistance gene. The vector was modified by the introduction of a DNA fragment comprising the open reading frame of the C. perfringens chloramphenicol acetyltransferase gene and flanking transcriptional terminators. The presence of a unique restriction site, engineered into the extreme 5' end of the open reading frame enabled a promoter region to be inserted to form an in-fram transcriptional fusion with catP. The system was tested by inserting the promoter region of the alpha-toxin gene of C. perfringens. The production of chloramphenicol acetyltransferase in C. perfringens was monitored during growth and the pattern of expression was shown to reflect levels of plc mRNA and alpha-toxin in the parent strain.
