A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

Authors: KUGADAS, ABIRAMI - University Of Minnesota; LAMONT, ELISE - University Of Minnesota; Bannantine, John; SHOYAMA, FERNANDA - University Of Minnesota; BRENNER, EVAN - University Of Minnesota; JANAGAMA, HARISH - Texas A&M University; SREEVATSAN, SRINAND - University Of Minnesota

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2016
Publication Date: 8/22/2016
Citation: Kugadas, A., Lamont, E., Bannantine, J.P., Shoyama, F., Brenner, E., Janagama, H., Sreevatsan, S. 2016. A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival. Frontiers in Cellular and Infection Microbiology. doi: 10.3389/fcimb.2016.00085.

Interpretive Summary: Mycobacteria are able to survive inside macrophage cells of animals and humans that are specifically designed to kill bacteria. One of the ways these specialized cells kill bacteria is by creating an acidic envrionment in the compartment harboring the bacteria. That compartment is termed a lysossome. Mycobacterium avium subspecies paratuberculosis causes Johne's disease in cattle and sheep and it is one of the bacterial species that can survive in this compartment. In this paper, we show that the bacteria survives not by withstanding the acidic environment, but rather by stopping the cell from creating that environment. Specifically, we show this capability is attributed to a single gene, MAP0403, which is a serine protease. When we clone this gene into a bacterium that is easily killed in the lysosome, it now has the ability to survive. Finally, we found additional genes that are highly expressed in acidic enviroments that may also contribute to MAP's suvivability. This work is of interest to researchers in the field and stakeholders in the dairy industry.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen that persists inside host macrophages despite severe oxidative stress and nutrient deprivation. Intrabacterial pH homeostasis is vital to pathogenic mycobacteria to preserve cellular biological processes and stability of nucleic acids and proteins. The basic mechanism by which MAP regulates intrabacterial pH is not completely understood. We hypothesize that a serine protease of Mycobacterium avium subspecies paratuberculosis (MAP), encoded by MAP0403, aids in the resistance to phagosomal acidification and is critical for survival in macrophages. We observed significantly increased expression of MAP0403 within macrophages by qRT-PCR, and found that Bafilomycin treatment to block phagosomal acidification ablated this upregulation. The highest levels of MAP0403 expression correlated with peak phagosome acidification in macrophages. The non-pathogenic species Mycobacterium smegmatis is acid-susceptible and cannot persist in the acidified phagosome, and we cloned the open reading frame for MAP0403 into M. smegmatis mc2 155 to better understand the serine protease’s function. Compared with controls, M. smegmatis mc2 155 transformants carrying the MAP serine protease show increased survival during in vitro acid stress and in monocyte derived macrophages. Further, we show that serine protease-carrying M. smegmatis transformants are able to maintain intrabacterial pH when exposed to an acidic medium (pH~5), while controls failed to do so. In silico predictions found a set of protein-coding genes co-expressed with MAP0403 or potentially interacting with the protein, and these genes were analyzed by microarray during phagosomal acidification and confirmed separately by qPCR. These data show that this set of associated genes is upregulated in the acidified phagosome. Thus, our studies suggest that MAP serine protease MAP0403 is part of a MAP resistance response towards the conditions encountered during phagosomal acidification.