Project description:To better understand host/phage interactions and the genetic bases of phage resistance in a model system relevant to potential phage therapy, we isolated several spontaneous mutants of the USA300 S. aureus clinical isolate NRS384 that were resistant to phage K. Six of these had a single missense mutation in the host rpoC gene, which encodes the RNA polymerase beta prime subunit. To examine the hypothesis that the mutations in the host RNA polymerase affect the transcription of phage genes, we performed RNA-seq analysis on total RNA samples collected from NRS384 wild-type (WT) and rpoC G17D mutant cultures infected with phage K, at different time points after infection. Infection of the WT host led to a steady increase of phage transcription relative to the host. Our analysis allowed us to define different early, middle, and late phage genes based on their temporal expression patterns and group them into transcriptional units. Predicted promoter sequences defined by conserved -35, -10, and in some cases extended -10 elements were found upstream of early and middle genes. However, sequences upstream of late genes did not contain clear, complete, canonical promoter sequences, suggesting that factors in addition to host RNA polymerase are required for their regulated expression. Infection of the rpoC G17D mutant host led to a transcriptional pattern that was similar to the WT at early time points. However, beginning at 20 minutes after infection, transcription of late genes (such as phage structural genes and host lysis genes) was severely reduced. Our data indicate that the rpoCG17D mutation prevents the expression of phage late genes, resulting in a failed infection cycle for phage K. In addition to illuminating the global transcriptional landscape of phage K throughout the infection cycle, these studies can inform our investigations into the bases of phage K’s control of its transcriptional program as well as mechanisms of phage resistance.
Project description:We analyzed RNA-Seq data of two Staphylococcus aureus strains, Newman and SH1000, infected by Kayvirus phage K. Staphylococcus virus K is used in the phage therapy, its genome is 148 kb long consisting of dsDNA with long terminal repeats, and encodes 233 ORFs and 4 tRNAs. The sampling times 0, 2, 5, 10, 20, and 30 minutes after infection were chosen based on the growth characteristics of the phage K at the two S. aureus strains. From the RNA-Seq data we determined transcriptional profile of the phage K and its hosts, which allowed us to identify differentially expressed genes, ncRNAs, and promotor and terminator sites. Transcription of the phage K genes starts immediately after the infection of bacterial cells and we found a gradual take-over by phage K transcripts in the infected cells. The temporal transcriptional profile of phage K was similar in both strains and the relative expression of phage K genes shows three distinct transcript types – early, middle, and late based on the time they reach maximum expression. The bacterial response to phage K infection is similar to the general stress response. It includes the upregulation of nucleotide, amino acid and energy synthesis and transporter genes and the downregulation of transcription factors. The expression of particular virulence genes involved in adhesion and immune system evasion as well as prophage integrases were marginally affected. This work unveils the versatile nature of phage K infection leading to its broad host range
Project description:The aim of the study was to investigate the resistance mechanism of Staphylococcus aureus towards lytic phages of the genus Kayvirus and the role of the membrane-anchored protein (primary accession Q2FYE0) designated PdpSau encoded by Staphylococcus aureus prophages. PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed 10 min after the infection leading to prophage-activated cell death. The LC-MS/MS analysis, as one of the methods, was used for protein detection and to find out whether this protein is predominantly presented in membranes. These findings are relevant for the advancement of phage therapy.
Project description:Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage Φ6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage Φ6 as a model system in evolutionary biology, Pph resistance to phage Φ6 remains poorly characterized. To investigate differences between phage Φ6 resistant Pseudomonas syringae pathovar phaseolicola strains, we performed expression analysis of super and non piliated strains of Pseudomonas syringae to determine the genetic cause of resistance to viral infection.
Project description:Staphylococcus aureus is a Gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Two phages are known to be important for staphylococcal virulence: the beta-hemolysin (hlb) converting phage and the Panton-Valentine Leukocidin (PVL) converting phage. The hlb-converting phage is found in more than 90% of clinical isolates of S. aureus. This phage produces exotoxins and immune modulatory molecules, which inhibit human innate immune responses. The PVL-converting phage produces the two-component exotoxin PVL, which can kill human leucocytes. This phage is wide-spread among community-associated methicillin resistant S. aureus (CA-MRSA). It also shows strong association with soft tissue infections and necrotizing pneumonia. Several lines of evidence suggest that staphylococcal prophages increase bacterial virulence not only by providing virulence factors but also by altering bacterial gene expression: 1) Transposon insertion into prophage regulatory genes, but not into the genes of virulence factors, reduced S. aureus killing of Caenorhabditis elegans.; 2) Although the toxins and immune modulatory molecules encoded by the hlb- converting phages do not function in the murine system, deletion of ϕNM3, the hlb-converting phage in S. aureus Newman, reduced staphylococcal virulence in the murine abscess formation model. 3) In a preliminary microarray experiment, prophages in S. aureus Newman altered the expression of more than 300 genes. In this research proposal, using microarray and high-throughput quantitative RT-PCR (qRT-PCR) technologies, we will identify the effects of the two important staphylococcal phages on the gene expression of S. aureus in both in vitro and in vivo conditions. This project is intended to be completed within one year. All the data – microarray, qRT-PCR and all the primer sequences- will be made available to public 6 month after completion. Data from this project will help us to understand the role of prophages in the S. aureus pathogenesis and can lead to development of a strategy to interfere with the pathogenesis process. Following strains were grown in TSA broth: Staphylococcus aureus USA300 (reference) Staphylococcus aureus USA300 with deletion of ϕSa2usa (Query) Staphylococcus aureus USA300 with deletion of ϕSa3usa (Query) Staphylococcus aureus USA300 Prophage-free mutant (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa2mw (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa3usa (Query) strain: Staphylococcus aureus USA300 Prophage-free mutant lysogenized with both ϕSa2mw and ϕSa3usa (Query) RNA samples were harvested at early log, midlog and stationary phase.Samples were hybridized on aminosilane coated slides with 70-mer oligos.
Project description:In the face of escalating challenges posed by infections with multiple drug-resistant (MDR) pathogens, the use of bacteriophages in therapeutic applications has emerged as a critically relevant and often sole alternative to traditional antibiotics. The most promising results of phage therapy have been demonstrated in the case of Staphylococcus aureus. It should be noted that the S. aureus population is characterized by a high degree of clonality. Strains belonging to sequence type 239 are among the main epidemic strains of methicillin-resistant S. aureus, identified worldwide. Many ST239 strains are characterized by MDR and resistance to virulent bacteriophages, and can become a serious problem in medicine. The purpose of this study was to investigate the resistance of clinical strains of S. aureus SA191REV of ST239 to bacteriophages of the Herelleviridae family using systems biology methods, starting from microbiological data on the interaction of strains with bacteriophages to transcriptomic and proteomic analysis. As results, the main changes in the cells of the resistant strain caused by phage infection are related to the energy metabolism in the cell. Significant changes were found in the functioning of the tricarboxylic acid cycle, glycolytic process, and glucose metabolic process. The most likely mechanism of resistance in the case of SA191REV strains is the premature death of infected cells due to the type I toxin-antitoxin system, as well as the hyperexpression of the lrgA and lrgB genes.
Project description:Staphylococcus aureus is a Gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Two phages are known to be important for staphylococcal virulence: the beta-hemolysin (hlb) converting phage and the Panton-Valentine Leukocidin (PVL) converting phage. The hlb-converting phage is found in more than 90% of clinical isolates of S. aureus. This phage produces exotoxins and immune modulatory molecules, which inhibit human innate immune responses. The PVL-converting phage produces the two-component exotoxin PVL, which can kill human leucocytes. This phage is wide-spread among community-associated methicillin resistant S. aureus (CA-MRSA). It also shows strong association with soft tissue infections and necrotizing pneumonia. Several lines of evidence suggest that staphylococcal prophages increase bacterial virulence not only by providing virulence factors but also by altering bacterial gene expression: 1) Transposon insertion into prophage regulatory genes, but not into the genes of virulence factors, reduced S. aureus killing of Caenorhabditis elegans.; 2) Although the toxins and immune modulatory molecules encoded by the hlb- converting phages do not function in the murine system, deletion of NM3, the hlb-converting phage in S. aureus Newman, reduced staphylococcal virulence in the murine abscess formation model. 3) In a preliminary microarray experiment, prophages in S. aureus Newman altered the expression of more than 300 genes. In this research proposal, using microarray and high-throughput quantitative RT-PCR (qRT-PCR) technologies, we will identify the effects of the two important staphylococcal phages on the gene expression of S. aureus in both in vitro and in vivo conditions. This project is intended to be completed within one year. All the data – microarray, qRT-PCR and all the primer sequences- will be made available to public 6 month after completion. Data from this project will help us to understand the role of prophages in the S. aureus pathogenesis and can lead to development of a strategy to interfere with the pathogenesis process. Staphylococcus aureus subsp.aureus strain Newman (reference) and Staphylococcus aureus subsp.aureus strain Newman yhcR knockout(query) were grown in TSA broth.Samples were grown under aerobic and anaerobic conditions and RNA samples harvested at mid log, stationary, and log phases.Samples were hybridized on aminosilane coated slides with 70-mer oligos.