The Systems Biology Of Mycobacteriophage

Mycobacterium, a genus of Actinobacteria, is notorious for its two pathogenic specices, M.leprae which causes leprae and M.tuberculosis which leads to tuberculosis. Since the ministry of health put forward the goal that basical eradication of leprosy would be accomplished at the end of20th Century, leprosy had got valid control in our country. However, as a result of the deteriorating resistant M.tuberculosis, HIV-TB co-infection, and instable BCG vaccine, tuberculosis spread again.’Stop TB’is a big problem faced by scientists and health workers.The study about mycobacteriophage, the viruse of mycobacterial hosts, began in1950s. With the deepening study, mycobacteriophages have been source to genetic tools for Mycobacteria, scaffold for novel M.tuberculosis diagnostics, and proteins of mycobacteriophages also can be used as lead compound of new antimicrobial drug. Unfortunately, all the patent right of genetic tools for Mycobacteria and diagnostic tools for M.tuberculosis belongs to European and American scientists. For making innovations in genetic and diagnostic tools of own intellectual property, isolation mycobacteriophage from China soil is imperative. In addition, the interaction between mycobacteriophages and their hosts, the function of some host shut-off proteins and the potential impact of prophages on mycobacterial evolution and virulence, all of them offer the theory foundation for the control of tuberculosis using mycobacteriophages. Based on the above points, the paper mainly studies the following parts:At first, we gain a mycobacteriophage SWU1and its genome sequence, which was isolated from a soil sample in China and classified to Cluster A2. We are interested in the particular bull’s eye plaques of SWU1and try to explain the genetic basis of SWU1plaque through a genomic comparative analysis of SWU1and L5that forms very common turbid plaques. We also confirm that the genomes sequence of S. rugosus ATCC BAA-974and M. rhodesiae JS60prophage are similar to SWU1through Blast-P. Cluster A2mycobacteriophages, which are isolated from different locales, provides considerable new light on mycobacteriophage diversity and its evolution.In the process of isolation SWU1, we found that current mycobacteriophage identification procedure is time-consuming and based on genomic sequence. To circumvent this problem, we describe a general duty method, which can be used for choosing specific and conserved gene as molecular clock for specific mycobacteriophage.68sequenced mycobacteriophages are subject to analysis. Firstly, we obtained the core-genome of mycobacteriophages, which was divided into six groups based on homology. Secondly, we found that one kind of gene belonging to group A （TM4gene23-like） was conserved among all mycobacteriophages, and the primers targeting such genes can be designed to interrogate the diversity of mycobacteriophages. Mycobacteriophage subcluster A2-specific primer pairs were test and considered to be feasible.Bacteriophages are viruses that specifically destroy bacteria. During their co-evolution, phages have developed strategy to usurp bacterial host machinery to replicate. Interaction between bacteriophages and bacterial host might represent rich potential targets/leads source to tackle the antibiotic resistance crisis in medicine. In this study, RNA sequencing technologies （RNA-Seq） was firstly used to identify differentially expressed host genes during phage infection and features of the SWU1transcriptional program, using our isolated mycobacteriophage SWU1and host bacterium M.smegmatis MC2155as model. Our objective was to gain insights into host defense strategy mobilized to respond to phage infection. Our results suggest that the host triggered sophisticated response involving signalling, transcription factor, host cell energetics, and the synthesis of the cell wall. The corresponding temporal patterns of SWTJ1transcription were also unveiled by RNA-seq data. The SWU1early transcripts are most possible effectors for host gene expression changes.After the interaction between SWU1and M.smegmatis MC2155is revealed, we want to know that how the one protein functions in the process of phage infection. We are interested in mycobacteriophage D29gp34.1. Bioinformatics analysis showed that D29gp34.1contained DUF2637superfamily. Expression of D29gp34.1leaded to smooth colony and the absence of wrinkle in the recombinant M.smegmatis. And D29gp34.1confers M. smegmatis resistance to TM4. Adsorption and injection of TM4concluded that the phage-TM4-resistant phenotype did not result from the prevention of phage adsorption and injection. Further studies show that D29gp34.1is a toxin protein in E. coli, and is a novel host shut-off protein. And protein truncation test （PTT） show that a peptide fragment （26-100aa） of D29gp34.1could inhibit the bacteria growth, but a fragment （40-100aa） could not. We think that this peptide fragment can be developed as lead compound of novel drug.Though ill-defined, mycobacterium prophages may assume as-yet unknown important roles. To better understand the potential impact of prophages on mycobacterial evolution and virulence, we investigate all prophage-like elements in mycobacterium genomes. In brief, we present here thirty-three mycobacterioprophages mined from sequenced mycobacterial genomes, the WGS databases, and some published literatures. Eleven prophages were newly identified prophages from complete genome database; five prophages were from the WGS databases; seventeen prophages were reported with specific sequence information. The genome sequences, gene contents of eleven newly identified prophages were analyzed. Comparative genomic analysis revealed that one full-length mycobacterioprophage phi172₂belonged to cluster A and one group having recognizable sequence similarity was verified and contained four small prophage-like elements, including the phiMmcs₁, phiMkms₁, phiBN44₁, and phiMCAN₁. To our knowledge, this represents the first systematic analysis of mycobacterioprophages.