Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies

ABSTRACT Staphylococcus aureus is a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence of S. aureus resistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome of S. aureus and used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important for S. aureus fitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important for S. aureus proliferation at sites of infection, which may represent new therapeutic targets

A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival.https://doi.org/10.1186/1750-1172-8-10

Elevated activity of the large form of ADAR1 in vivo: Very efficient RNA editing occurs in the cytoplasm

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae