Mycobacteriophage Meru: Isolation and Characterization of a Novel Mycobacteriophage

Discussion

Isolation and characterization of the mycobacteriophage Meru reveals that the virus possesses a variety of distinctive features: plaques which demonstrate consistent mixed morphology and a high degree of turbidity, restriction digest patterns that indicate a notable similarity to Cluster A mycobacteriophages, a lack of lysogen immunity to any of the other phages in the VCU Phage Lab course, and a host-range that indicates varied plating efficiency between different bacterial strains of M. smegmatis. Using the results of the procedures utilized to extensively describe Meru, one can suggest rudimentary connections to other sequenced phages that have similar digest, immunity-testing and host range outcomes. In turn, these connections raise the possibility that Meru holds relatable benefits, as these other viruses do, to the contemporary avenues of mycobacteriophage research.

Based on a comparative analysis between the Meru digest and the virtual digests of sequenced mycobacteriophages, it appears that the Meru belongs in the “A” Cluster of phages. Several of the phages in this category show similar band patterns, including the lack of cutting by the ClaI enzyme and noticeable cutting of the phage DNA by the HaeIII enzyme. It is interesting to note that Cluster A phages tend to be the most genetically similar out of all the clusters (Hatful, 2006). In fact, as the most numerous phage genome cluster, Cluster A contains seven mycobacteriophages that are more closely related to each other than to other phages; these include L5, the first sequenced mycobacteriophage genome [28], D29 [30], Bxb1 [29], Bxz2 [6], Che12, Bethlehem, and U2 (Hatful, 2006). Still, only a complete genetic sequence can confirm the inclusion of Meru in this group and suggest potential evolutionary connections.

Cross immunity testing indicated that the Meru lysogen is not immune to any of the other mycobacteriophage from the Virginia Commonwealth University course. In other words, evidence of lysis was seen where all other phage samples were spotted during cross-immunity experimentation. This indicates a dissimilarity in repressor genes between the phages, and suggests that Meru is unique from other isolated mycobacteriophage. Consequently, genomic sequencing will reveal further details regarding the repressor gene and its location, in addition to information regarding the other components of Meru’s genetic makeup. It is possible that Meru shares some genetic similarities, such as repressor location, with other temperate phages on M. smegmatis, including mycobacteriophage Bxb1 and L5 (Mycobacteriophage Database). Observations made by comparing genomic sequences might indicate that Meru can be used for similar purposes as these two phages. For example, L5’s repressor gene, gene 71, can be used as a selectable marker for genetic transformation of mycobacteria (Donnelly, 1993). Consequently, the phage can play a vital role in the construction of recombinant BCG vaccines. Meru’s repressor gene might also exhibit similar applications to the field of mycobacterial genetics.

In addition, host range results indicate that the plating efficiency of Meru on M. smegmatis is not limited to the mc²155 strain. In fact, the presence of more turbid plaques on this strain compared to the ATCC strain for the later spottings indicates that Meru might have varied infection efficiency between multiple strains of M. smegmatis and possibly between bacterial species entirely. A host range test that includes various types of bacteria, such as M. ulcerans, M. tuberculosis, M. bovis, M. avium, M. marinum, M. scrofulaceum, M. fortuitum and M. chelonae, would allow more substantial conclusions to be reached concerning the behavior of the mycobacteriophage Meru on pathogenic strains of Mycobacterium. It is possible that Meru might be added to a cluster of four phages, namely D29, TM4, L5 and Bxz2, who seemed to have the broadest host range, with at least three of these phages forming plaques on all of the slow-growing species, except for M. marinum and one strain of M. scrofulaceum.” (Rybniker, 2005). The genomes of these four phages have detectable sequence similarity and, interestingly enough, three out of four are part of the aforementioned Cluster A group (Mycobacteriophage Database). Hence, a complete genomic sequence for Meru that has notable similarity with the other phages in this grouping might indicate similar plating efficiencies on different bacterial lawns, and can aid in the accurate identification of the correct cluster group for this mycobacteriophage.

As an extension of research related to host-range testing, the infection strength of mycobacteriophage on one particular bacterial species is a key focus that has the potential to revolutionize the medical field. The species in question is that of M. tuberculosis, the bacteria that was responsible for killing around 1.5 million people and sickening almost 9 million total in 2010 (CDC: http://www.cdc.gov/tb/statistics/default.htm). In light of the relationship of mycobacteria such as M. tuberculosis to human survival and the difficulties that have prevented successful genetic manipulation, mycobacteriophages are especially appealing subjects for discovery, genomic characterization, and manipulation (Hatful, 2010). However, phage therapy is limited by the fact that phages are specific to micororganisms, need to be in the same environment as the pathogen to infect it, and may be impermeable to membranes (Broxmeyer, 2002). Still, research studies have shown that some mycobacteriophages are, in fact, adept at infecting the tuberculosis bacteria, such as DS-GA and TM4 (Sula, 1981). Furthermore, Che12 is a temperate Chennai phage infecting Mycobacterium tuberculosis, whose genome is very similar to those of mycobacteriophage L5 and D29 (Gomathi, 2007). It is interesting to note that Meru, a potential Cluster A phage itself, might join both Che12 and L5 in that group, indicating the potential for similarities to exist between genes related to lysogeny as well as phage attachment sites.

As mentioned, a full genetic sequence of the genome of the mycobacteriophage Meru is needed in order to effectively and precisely assess any relationships between the virus and existent Cluster A phages. Only then is it possible to justifiably classify patterns in plating strength and immunity, as well as positions and functions of specific genes, to any specific phage phamily. Nonetheless, the isolation and initial characterization of novel mycobacteriophage such as Meru serves as a crucial foundational point for investigation into the future genetic and therapeutic utility of these viruses.

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References

Broxmeyer, Lawrence, et al., 2002, Killing of Mycobacterium Avium and

Mycobacterium Tuberculosis by a Mycobacteriophage Delivered by a Nonvirulent Mycobacterium: A Model for Phage therapy of Intracellular Bacterial Pathogens, Journal of Infectious Diseases, v. 186.8, p. 1155-1160.

Centers for Disease Control. (2001). Tuberculosis: Data and Statistics. Retrieved from http://www.cdc.gov/tb/statistics/default.htm

Donnelly, Jacobs, and Hatfull, 1993, Superinfection Immunity of Mycobacteriophage L5: Applications for Genetic Transformation of Mycobacteria, Molecular Microbiology, v. 7.3, p. 407-417.

Hatfull, G.F., et al., 2008, Comparative Genomics of the Mycobacteriophages: Insights into Bacteriophage Evolution, Research in Microbiology, v. 159.5, p. 332-339.

Hatfull, Graham F, et al., 2006, Exploring the Mycobacteriophage Metaproteome: Phage Genomics as an Educational Platform, PLOS Genetics, v. 2.6, e92-e92.

Hatfull, Graham F, 2010, Mycobacteriophages: Genes and Genomes, Annual Review of Microbiology, v. 64, p.3 31-356.

Henry, Marine, et al., 2010, In Silico Analysis of Ardmore, a Novel Mycobacteriophage Isolated from Soil, Gene, v. 453.1-2, p. 9-23.

McNerney, R, 1999, TB: the Return of the Phage. A Review of Fifty Years of Mycobacteriophage Research, The International Journal of Tuberculosis and Lung Disease, v. 3.3, p. 179-184.

Mycobacteriophage Database Home. Web. 07 Dec. 2011.Retrieved from: http://phagesdb.org.

Rybniker, Jan, Kramme, and Small, 2006, Host Range of 14 Mycobacteriophages in Mycobacterium Ulcerans and Seven other Mycobacteria including Mycobacterium Tuberculosis--Application for Identification and Susceptibility Testing, Journal of Medical Microbiology, v. 55.1, p. 37-42.

Sampson, Timothy, et al., 2009, Mycobacteriophages BPs, Angel and Halo: Comparative Genomics Reveals a Novel Class of Ultra-small Mobile Genetic Elements, Microbiology, v. 155.9, p. 2962-2977.

Sula, Sulov, and Stolcpartov, 1981. Therapy of Experimental Tuberculosis in Guinea Pigs with Mycobacterial Phages DS-6A, GR-21 T, My-327, Czechoslovak Medicine, v. 4.4, p. 209-214.