Preliminary Research On Potential Value For Tuberculosis Therapy Of Mycobacteriophage

ObjectiveNowadays, the global tuberculosis (TB) control is still a seriouschallenge, of which two major problems are a quite long course ofchemotherapy and lots of drug-resistant mycobacterium tuberculosis (M.tuberculosis) infection. Studies have confirmed that a few ofmycobacteriophages can effectively infect and lyse M. tuberculosis, evendrug-resistant M.tuberculosis. Particularly effective are mycobacteriophagecocktails that simultaneously destroy various strains of M. tuberculosis andreduce the mutation frequency of phage-resistant M. tuberculosis.Cheeringly, these results also have been demonstrated thatmycobacteriophage TM4, its tail tape measure protein contains motif3domain, can specifically identify and infect dormant cells ofMycobacterium smegmatis (M. smegmatis) and this motif3domain canlikely hydrolyze peptidoglycan, which is similar to that of resuscitationpromoting factor (Rpf). Base on above studies, we plan to continume toscreen an ideal mycobacteriophage for developing mycobacterio- phage-based anti-TB therapies and further investigate whethermycobacteriophage TM4, containing a motif3domain, can effectivelyresuscitate dormant cells of M. tuberculosis or not.Methods1Potential value for anti-tuberculosis of mycobacteriophage Bo41.1Determination of mycobacteriophage Bo4lytic ability at variouspH levels in M. smegmatis mc21551.2The lytic ability of mycobacteriophage Bo4was surveyed in vitro,and all data were analyzed using SPSS17.0, and differences achievingvalues of P <0.05were considered statistically significant.2Bioinformatics analysis of mycobacteriophage Bo4genome2.1The genome size, base composition and GC content ofmycobacteriophage Bo4were analyzed using Editseq software in DNAStarpackage. TRF (Tandem Repeat Finder) software was used to search fortandem repeat and tRNAscan software was used to search for tRNA region.2.2Glimmer3.0was used to predict where the protein-coding genes ofmycobacteriophage Bo4are and identify putative open reading frames.2.3Mycobacteriophage Bo4genome was comparatively analyzed withBLAST and the best hit was used to produce a dot plot.2.4All genes of mycobacteriophage Bo4were comparatively analyzedwith BLAST-m8(e-value1e-5), and the best hit was used to produce a synteny diagram using the Perl programming language.2.5Phylogenetic analysis was constructed in the Clustal W programby the neighbor-joining method and drawn using the TreeView software.3The potential of mycobacteriophageTM4in resuscitating dormantcells of M. tuberculosis.3.1Dormant cells of M. tuberculosis were induced by prolonged closedculture of log-phase M. tuberculosis H37Rv at37℃.3.2Drug sensitivity test, M. tuberuclosis culture and electronmicroscope were used to test the model of dormant cells of M. tuberculosis.3.3Mycobacteriophage TM4was amplified by double-layer agar platemethod in different multiplicity of infection (MOI).3.4M. tuberculosis colony count and electron microscope were used todetect whether the dormant cells of M. tuberculosis had been resuscitatedby mycobacteriophage TM4.Results1Potential value for anti-tuberculosis of mycobacteriophage Bo41.1Mycobacteriophage Bo4can lyse M. smegmatis mc2155in bothacidic (pH5.0) and neutral environments (pH7.0)1.2We infected the M. tuberculosis H37Rv strain with Bo4andevaluated its ability to kill M. tuberculosis over time in vitro by comparingthe CFUs between these groups. We observed a significant difference between the Bo4-infected and non-infected control bacteria at severaldifferent time (1day,3days and5days, respectively).2Bioinformatics analysis of mycobacteriophage Bo4genome2.1We subjected the Bo4genome to bioinformatic analysis and foundthat it consisted of39318base pairs (bp) with a G+C content of66.76%.While no tRNA or tmRNA genes were found.2.2We also identified58ORFs by Glimmer3.0. Of the58ORFsfound in the Bo4genome,19were similar to genes with annotatedfunctions in the GenBank database,35showed homology touncharacterized proteins, and the remaining4did not match any sequencedgenes in the database. Finally, annotation of mycobacteriophage Bo4genome indicated that it was a lytic bacteriophage and did not contain anyharmful genes through Blastp.2.3The two closest relatives of Bo4are found to bemycobacteriophage BPs and Angel. There is high identity (identity=93.10%) and clear synteny among genes encoding the virion structure andassembly functions.2.4Complete genome sequencing of mycobacteriophage Bo4showedhigh nucleotide identity (mean identity>90%) with cluster Gmycobacteriophages，suggesting that mycobacteriophage Bo4belongs tocluster G. 2.5Although mycobacteriophage Bo4exhibited a strong similarity tomycobacteriophage Halo and BPs at the nucleotide level, phylogeneticanalysis showed that Bo4was distantly related to thesemycobacteriophages and was actually most closely related tomycobacteriophage Legendre. This data indicates that Bo4and Legendremay share a common ancestor.3The potential of mycobacteriophage TM4in resuscitating dormantcells of M. tuberculosis.3.1Establishment and test of the model for dormant cells of M.tuberculosis.When M. tuberculosis was closed culture for18days at37℃, the bluetubes became colorless, suggesting these tubes had turned anaerobic.Partial M. tuberculosis was still culturable after culturing50days, whichwas investigated by innoculating on Lowenstein-Jensen culture medium for4weeks at37℃. Along with further closed culture, all of M. tuberculosisturned non-culturable until culturing93days. There were lots of dormantcells of M. tuberculosis with thick wall were watched through electronmicroscope after closed culture for180days at37℃.3.2The preparation of mycobacteriophage TM4Mycobacteriophage TM4was amplified by double-layer agar platemethod and then small plate plaque assay was used to measure the titer of mycobacteriophage TM4stock. Finally, the titer of mycobacteriophageTM4was2.1×1010PFU/mL.3.3Resuscitation of dormant cells of M. tuberculosis3.3.1The amount of M. tuberculosis at the bottom of the tube in Rpf Egroup was more than that of the other groups through naked eye afterculturing3days at37℃, while the amount of M. tuberculosis at the bottomin TM4group did not increased obviously until culturing for8days.3.3.2M. tuberculosis was still non-culturable in these three groupsafter culturing1days, which was investigated by innoculating onLowenstein-Jensen culture medium for4weeks at37℃. Along with furtherculture, part of M. tuberculosis turned culturable, the concentration ofresuscitated M. tuberculosis in control, TM4and Rpf E group was0(colony-forming unit, CFU)/mL,0.7×102CFU/mL and2×104CFU/mL,respectively after culturing6days. More and more M. tuberculosis becameculturable with further culture, and the concentration of resuscitated M.tuberculosis in control, TM4and Rpf E group increased to3.4×101CFU/mL,1.68×107CFU/mL and2.1×109CFU/mL, respectively afterculturing14days.3.3.3Various shapes of M. tuberculosis for culturing17days in thesethree groups were watched through electron microscope. There were lots ofthin-wall M. tuberculosis, a few of dormant cells of M. tuberculosis with thick wall and cell pieces in TM4group; whereas the field of view of Rpf Egroup was filled with plentiful thin-wall M. tuberculosis; as we excepted, alot of thick-wall M. tuberculosis and a few thin wall M. tuberculosis werewatched in control group.ConclusionsMycobacteriophage Bo4can lyses M. tuberculosis to effectively stopgrowth, which was confirmed by phage antimicrobial activity. Additionally,annotation of the Bo4genome indicated that it was a lytic bacteriophageand did not contain any harmful genes that increased mycobacterialvirulence or decreased human immunity, making it an ideal candidate fordeveloping mycobacteriophage-based anti-TB therapies. As tomycobacteriophage TM4, we confirm it can resuscitate dormant cells of M.tuberculosis, even the infeced M. tuberculosis cells can be lysed along withthe mature progeny phages releasing.