Project description:The molecular mechanism by which a mycobacterial virus escapes the host bacterial defense and kills the human pathogen Mycobacterium tuberculosis is greatly unclear. Here we report that the gene gp48 of mycobacteriophage A4ZJ24, encoding a metallophosphoesterase-like protein, is required for the killing of M. tuberculosis, but not for M. smegmatis. Gp48 is expressed in the early stage of phage infection and disrupts the mycobacterial chromosomal DNA, and thus silences the expression of multiple anti-phage defensive genes, which only exist in the genome of M. tuberculosis but not in M. smegmatis. The gp48-deleted phage can normally adsorp and invade into M. tuberculosis, however, it does not prevent from activating anti-phage genes, resulting in a loss of its genome DNA replication ability in M. tuberculosis. This study identifies a phage’s metallophosphoesterase as a new determinant of the viral host range and discovered a previously unknown molecular mechanism for mycobacteriophages to kill M. tuberculosis. Our study fills a major gap in current knowledge of the interaction between mycobacterial viruses and M. tuberculosis.
Project description:Virulent bacteriophages (or phages) are viruses that specifically infect and lyse a bacterial host. When multiple phages co-infect a bacterial host, the extent of lysis, dynamics of bacteria-phage and phage-phage interactions are expected to vary. The objective of this study is to identify the factors influencing the interaction of two virulent phages with different Pseudomonas aeruginosa growth states (planktonic, an infected epithelial cell line, and biofilm) by measuring the bacterial time-kill and individual phage replication kinetics. A single administration of phages effectively reduced P. aeruginosa viability in planktonic conditions and infected human lung cell cultures, but phage-resistant variants subsequently emerged. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and meropenem antibiotic. In contrast, adherent biofilms showed tolerance to phage and/or meropenem, suggesting a spatiotemporal variation in the phage-bacterial interaction. The kinetics of adsorption of each phage to P. aeruginosa during single- or co-administration were comparable. However, the phage with the shorter lysis time depleted bacterial resources early and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage. The extent and strength of this phage-phage competition and genetic loci conferring phage resistance, are, however, P. aeruginosa genotype dependent. Nevertheless, adding phages sequentially resulted in their unimpeded replication with no significant increase in bacterial host lysis. These results highlight the interrelatedness of phage-phage competition, phage resistance and specific bacterial growth state (planktonic/biofilm) in shaping the interplay among P. aeruginosa and virulent phages.
Project description:Genomic material isolated from purified phage YerA41 lysate was shown to contain RNA. YerA41 phage lysate was RNase treated to remove phage-external RNA and total RNA was then isolated from the phage preparate using Qiagen Rneasy mini kit. The isolated RNA was sequenced to elucidate its origin. The results suggested that the RNA originated from intact ribosomes of the host bacterium that contaminated the phage lysate.
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.