Towards Second Generation of Fluoromycobacteriophages for Detection and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis

ESTEFANIA URDANIZ; LILIANA RONDON; GRAHAM F HATFULL; MARIANA PIURI

Buenos Aires

Simposio; 3er Encuentro Internacional sobre enfermedades olvidadas XV Simposio sobre control epidemiológico de enfermedades transmitidas por vectores; 2012

Fundación Mundo Sano

Introduction: Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis. In Argentina there are about 12,000 cases and one thousand deaths per year. The emergence of drug resistant strains severely complicates treatment. There is a need for new diagnostic approaches that combine speed (time-to-detection), sensitivity, specificity, biosafety, rapid and accurate determination of resistance to the commonly used anti-tuberculosis drugs at a low cost to be applied in developing countries where the incidence of TB is high. Mycobacteriophages are excellent candidates for the development of diagnostic tools since they efficiently and specifically infect and replicate in Mycobacteria. We recently described the development of Fluoromycobacteriophages ? reporter phages containing a fluorescent reporter gene ? that provide a simple means of revealing the metabolic state of M. tuberculosis cells, and therefore their response to antibiotics. Fluorescence can be detected easily by fluorescent microscopy or by flow cytometry. The assay is responsive to antibiotics, and fluorescence is maintained for at least two weeks following fixation, increasing biosafety and facilitating storage or transportation of samples. Fluoromycobacteriophages have promising attributes in the research laboratory, and our goal is to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples. Materials and methods: Our strategy was to begin with TM4 or one of its replication-proficient derivatives and to construct a lysis defective mutant.  We proposed to do so by deletion of the essential lysin A gene (gene 29), while propagating the mutant in a complementing strain. To generate the desired TM4 mutant we used the BRED recombineering approach. To do so, a dsDNA substrate was generated that had 100 bp of homology flanking the region to be deleted. This substrate was co-electroporated in M. smegmatis expressing the recombineering functions with phAE81 DNA and plated in an infectious center assay. About 25 plaques were screened  by PCR using oligos flanking the deleted region or that only amplify a product in the mutant. To isolate the desired mutant, about 500 plaques were patched on a lawn of M. smegmatis wt and M. smegmatis expressing Corndog lysin A.  We hypothesize that the reporter gene expression in the fluoromycobacteriophages is also limited due to lack of optimization of codon usage and ribosome binding sites. In consequence, we constructed an optimized version of mCherrybomb gene. We decided to replace the RBS present in the Hsp60 promoter with the putative RBS of gp9 of Mycobacteriophage TM4, the major capside protein.We were able to add a Strep II tag (STAG) sequence (8 aa?s and two extras used as a linker) to the C-terminal of the TM4 capsid protein. M. smegmatis cells were infected with STAG- gfp? or gfp? and fixed with paraformaldehyde. An aliquot was incubated with Strep-Tactin coated magnetic beads (that binds the STAG) and two different strategies were compared: elution from beads followed by recovery of phage-cells complexes and visualization by fluorescent microscopy or direct visualization of complexes without elution in the presence of the beads.Results: We made a deletion of the essential gene (gene 29) as it was detected in the primary plaques. After screening of hundreds of plaques we could not isolate the mutant. The impossibility of recovery of the lysA mutant in TM4 could be explained by a polar effect on genes downstream of gene 29. On the other hand, mycobacterial cells transformed with the new mCherrybomb construct developed a purple colour and were brighter than the previous version.  By western blot, ELISA and inmuno-electron microscopy using antibodies that specifically recognize the STAG, we found that a subset of the capsid subunits within each virion contained the tag. These partially tagged virions were used for isolation of phage-cell complexes.Conclusion: The construction of these optimized versions of Fluoromycobacteriophages will facilitate the testing of specific protocols for sputum processing to achieve efficient phage infection of mycobacterial cells directly in these samples. Together, these developments will result in a simple, rapid, and specific diagnostic test for tuberculosis.