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

Abstract

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