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
Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/physiology , Francisella/genetics , Francisella/pathogenicity , Transcription Factor RelA/genetics , Transcription Factor RelA/physiology , Animals , Base Sequence , Biofilms/growth & development , Cell Line , DNA Primers/genetics , DNA, Bacterial/genetics , Female , Francisella/growth & development , Francisella/physiology , Genes, Bacterial , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/microbiology , Guanosine Pentaphosphate/biosynthesis , Guanosine Tetraphosphate/biosynthesis , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Mutation , Stress, Physiological , Virulence/genetics , Virulence/physiologyABSTRACT
In 1991, five different total joint replacement surgeons performed 337 primary total knee replacements and 250 primary total hip replacements. They revised 25 total knee replacements and 73 total hip replacements. The average length of stay was 6.6 days for the primary total knee arthroplasty and 7.5 days for knee revisions. For total hip replacement, average stay was 5.9 days for primary surgery and 6 days for revisions. The operative time required for each of the five surgeons to complete a total knee replacement increased 55%, 3%, 38%, 72% and 38%; whereas for the total hip replacement, time increased 87%, 77%, 84% and 58%. Overall, there was a 41% increase in operating time for revision total knee replacements and a 77% increase for revision total hip replacement. Allowable charges by Medicare in 1993 for a primary knee and hip replacement were $1,298 and $1,363, respectively. Revision total knee replacement increased 24.3% ($1,613) and revision total hip replacement increased 30.8% ($1,782) in the state of Indiana. These figures do not encourage those surgeons who are capable of doing total joint replacements to revise other surgeons' problems.
