Cameron Sargent and Dr. Julianne Grose, Microbiology and Molecular Biology

Introduction

Once the leading cause of death in the United States, tuberculosis still burdens the world as the second deadliest infectious disease worldwide, latently infecting one-third of the world population and causing 1.5 million deaths in 2013 [1]. Tuberculosis is particularly lethal largely because the pathogen Mycobacterium tuberculosis has several characteristics that make detecting, treating, and studying the disease unusually hard. Among these traits, M. tuberculosis has a complex cell wall that limits the effect of many antibiotics and makes genetic manipulation of the bacterium, a necessity for effective research, nearly impossible through conventional transformation techniques [2]. Recently, Howard Hughes Medical Institute (HHMI) initiated a nationwide project called Phage Hunters with an aim of finding an alternative solution to fighting tuberculosis through the discovery and analysis of bacteriophages, viruses that attack specific bacterial hosts. Viruses are relatively simple systems that can be used to kill, detect, and introduce genetic material into bacteria, so researching viruses and their interactions with hosts provides a powerful approach to fighting infectious diseases. Techniques for studying, detecting, and treating tuberculosis with phages have been proposed or tested with promising results, but a greater understanding of phages is still necessary before these techniques can be used in clinical applications [2].

My project targeted a critical component of understanding phage-host interactions using the B4 mycobacteriophages and Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis. After infecting their host, phages can either replicate immediately via the lytic cycle or insert their genome into the host chromosome until propagating at a later time via the lysogenic cycle. As such, phage lysogeny can be utilized not only as a means of killing bacteria but also as a source of molecular machinery for introducing foreign DNA into the bacterial genome. Understanding the key proteins involved in lysogeny is thus crucial to using phages to produce desired benefits in clinical and laboratory settings. Using comparative genomics and protein folding software, I previously identified a protein unique to the B4 subcluster of the mycobacteriophage population that appears to function as a recombination directionality factor (RDF), a protein that helps remove the phage genome from the host to initiate viral replication. Despite strong results from computer-based analysis, however, the data I collected prior to this project were insufficient to confirm my hypothesis about the protein function, necessitating a series of wetlab experiments to more fully elucidate the role of this protein. Through these experiments, I found further evidence for the temperate nature of the B4 phages that supported my hypothesis, allowed me to complete an Honors Thesis, and helped me contribute to the end goal of using phages in combatting tuberculosis.

Methodology

Cultures of M. smegmatis mc²155 and high titer lysates of mycobacteriophages Adawi, Bane1, Bane2, D29, and Serpentine were created from frozen stocks stored on campus; Adawi, Bane1, and Bane2 were selected as representatives of the B4 subcluster based on availability, and D29 and Serpentine were used for controls. Liquid cultures of M. smegmatis were grown in Middlebrook 7H9 Liquid Medium: Complete. High titer lysates were produced by infecting M. smegmatis liquid culture with each phage, incubating for 48 hours, and removing bacteria using 0.45 μm filters. All incubation occurred at 37°C. Wet-lab reagents were made according to the Phage Hunters lab notebook provided by HHMI; protocols are also located on www.phagesdb.org.

For lysogeny spot tests, 0.5 mL of M. smegmatis liquid culture were plated using 4.5 mL of 1X Middlebrook Top Agar on Middlebrook 7H10 Agar plates. After the agar set, 4 μL of each phage stock were dropped onto the agar and allowed to absorb before inverting and incubating the plates for two days. Once distinct plaques were noticeable, a sterile loop or pipet tip was used to streak from each plaque onto fresh plates. Plates were incubated for two days, after which single colonies were incubated in 5 mL 7H9 liquid medium for two days. Each lysogen culture was then plated using 0.5 mL culture with 4.5 mL 1X top agar as before. Once the agar set, 4 μL of each phage stock and of the Serpentine stock were dropped onto their respective plates and allowed to absorb before inverting and incubating the plates for two days; a spot test plate of all phages on normal M. smegmatis stock was simultaneously incubated as a control.

Colony PCR was also used to determine if phage DNA could be found in bacteria as confirmation that prophages were indeed inserted into the host chromosomes. Individual colonies were picked from streak plates and boiled in 50 μL ddH2O for 10 minutes, while 2 μL of undiluted phage stocks were boiled in 50 μL ddH2O for 10 minutes for control reactions. Each PCR reaction contained 2.5 μL 10X reaction buffer, 0.5 μL 10 mM dNTPs, 19 μL ddH2O, 0.5 μL each of forward and reverse primers, 0.5 μL Taq, and 2 μL boiled template. PCR was ran for 35 cycles, with each cycle running at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min. PCR products were analyzed using 1% and 2% agarose gels and 100 bp DNA ladders.

Results

When M. smegmatis was infected with Adawi, Bane1, and Bane2, the phages appeared to have established lysogeny in the host, confirming their temperate nature. When phages establish lysogeny, the prophage expresses genes that prevent superinfection from other similar phages, causing bacteria that contain a prophage to resist lysis when exposed to the original phage or a close relative. Replicate spot tests revealed that, after infection by each B4 phage and recovery incubation, M. smegmatis was not lysed by the original phage of infection in ensuing spot tests. Meanwhile, lysis did occur when Serpentine, a lytic control phage, was spot tested. Spot tests of Serpentine also confirmed that Serpentine is lytic, as it could not establish superinfection resistance against itself. The regrowth of bacteria within the plaque on a spot test and the emergence of small plaques in plated lysogens were also observed; although both of these were observed infrequently, their occurrence did provide further support to the conclusion that the B4 phages are indeed lysogenic.

PCR results also indicated that Adawi, Bane1, and Bane2 were able to establish lysogens when infecting M. smegmatis. PCR was used to amplify the putative RDF sequence in each reaction; BLASTn search also confirmed that the amplification primers would not anneal well with any sites in the M. smegmatis genome, ensuring the reliability of results. Gel electrophoresis of the PCR products revealed bands of the desired length in reactions from all three phages, suggesting that B4 phages are able to establish lysogeny. Negative controls were also included in the PCR, using both primer sets and templates of M. smegmatis stock and M. smegmatis infected with D29 and with Serpentine; no bands were observed in any of these reactions. PCR analysis was also replicated several times, and the presence of the RDF sequences in lysogens was confirmed in roughly half of those reactions.

Discussion

The combined results of superinfection studies and confirmation of lysogens by PCR suggest that the B4 mycobacteriophages are temperate and can establish lysogeny upon infection of M. smegmatis. Due to the continual tweaking of protocol and some inconsistencies in results, however, the lysogeny experiments were not adequately conclusive. Given more time, many of these issues could have been resolved, and a clearer, more resolute conclusion can be made. A robust protocol for the lysogeny spot tests had finally been developed, a definitive lytic control phage was requested from another university, and protocols using UV radiation and mitomycin C had been identified as additional tests for confirming lysogeny. Given these new tools, we are confident that the temperate nature of the B4 phages should be able to be confirmed irrefutably. Originally, plans were also made to directly analyze the role of the putative RDF in phage replication. One of the proposed experiments was to clone the putative RDF into an expression vector and transform it into a specially designed strain of Escherichia coli that, based on the rate of removal of two selective markers, would indicate whether the protein was indeed involved in recombination; the specific E. coli strain and protocol were already prepared by my mentor. Site-specific removal of the unique B4 protein using the BRED procedure to confirm the role of the protein in lysogeny was also planned [3]. However, time constraints did not allow for a completion of these experiments either.

Conclusion

Despite time constraints and other complications, this study was able to gather evidence in favor of our hypothesis that the B4 phages are temperate. This conclusion provides valuable insight regarding lysogenic phage replication. Since the gene sequence of the putative RDF was not found to be similar to known RDF sequences through BLASTn searches, this protein appears to be a novel RDF protein. Because no sequences similar to any known integrases were found in the B4 phages either, the presence of an RDF and the observation of lysogeny in B4 phages reveal that these phages employ a distinct type of integrase that is yet to be discovered. The finding of a novel class of integrase would be a major contribution to virology, but more experimentation is first required to more conclusively characterize the B4 phages as temperate and to determine the role of the putative B4 RDF.

Sources

1. “Tuberculosis” Media Centre: Fact Sheets. World Health Organization, Oct. 2014. Web. 28 Oct. 2014. < http:// www.who.int/mediacentre/factsheets/fs104/en/>.
2. E. J. Stella, A. I. de la Iglesia, H. R. Morbidoni, Mycobacteriophages as versatile tools for genetic manipulation of mycobacteria and development of simple methods for diagnosis of mycobacterial diseases. Revista Argentina de microbiologia 41, 45 (Jan-Mar, 2009).
3. L. J. Marinelli et al., BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PloS one 3, e3957 (2008).