Temperature Effects on a M. tuberculosis-infectious Subset of Mycobacteriophages

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Dr. Joseph Stukey, Biology

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Mycobacteriophages are viruses that infect mycobacterial hosts. Over 1300 mycobacteriophages have been organized into at least 34 distinct groupings or clusters based on genomic sequence similarity. Some mycobacteriophages from Clusters A and K can also infect Mycobacterium tuberculosis, a distinction of potential medical importance. Recently, Hope College SEA-PHAGES students have been isolating predicted Cluster K phages at a higher frequency (≥ 2x) after changing the isolation temperature from 37°C to 32°C. Additionally, these phages were unable to propagate at 42°C. PCR analysis supported a Cluster K classification for many predicted Cluster K phages isolated at 32°C, but for only one of the phages (Ruthiejr) isolated at 37°C. Interestingly, Ruthiejr does propagate at 42°C. We hypothesized that Cluster K phages may have a relative growth advantage at lower temperatures. We investigated temperature-dependent growth properties of several known and PCR-supported Cluster K mycobacteriophages. We examined phage thermostability, adsorption rate, reproductive cycle time (latent period), and burst size. Stability at 42°C appeared phage-dependent and was not always consistent with growth temperature profiles and/or host adsorption kinetics. For example, phages Bella96 and Krueger, both growth-defective at 42°C, also displayed reduced thermostability and host adsorption kinetics at 42°C compared to lower temperatures. In contrast, phages Polymorphads and Hyperbowlee, also growth defective at 42°C, were nonetheless stable at 42°C. Notably, Hyperbowlee also showed almost no host adsorption at 42°C. One-step growth analysis of D29 (control), Bella96, and Krueger showed impaired growth of the Cluster K phages at ≥ 37°C compared to D29. These results now suggest that Cluster K phages may have a growth disadvantage at temperatures ≥ 32°C. Our findings provide insight into the growth behavior and temperature sensitivity of Cluster K phages and may lead to discoveries about M. smegmatis and M. tuberculosis infection by mycobacteriophages.

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