Abstract
The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.
Highlights
A universal theme among bacteria is the ability to persist, replicate, and expand their territory despite which environment they occupy
Mycobacterium avium subsp. paratuberculosis (MAP) K-10 was allowed to invade MDMS for 10–120 min post-infection (p.i.) and phagosomes were subsequently assessed for acidification using LysoTracker Blue, a fluorescent, acidotropic probe, that emits upon protonation of its basic amine
Phagosome acidification continued for 1 h (Figure 1A); the acidification process completed by 2 h p.i
Summary
A universal theme among bacteria is the ability to persist, replicate, and expand their territory despite which environment they occupy. Bacteria and other microbe resistance to and survival in acid has allowed for the development of diagnostics and drugs as well as the understanding of host responses that regulate acid stress. Investigations into acid stress have elucidated host components of the mycobacterial disease process, such as the acidification of M. tuberculosis containing phagosomes (pH = 4.5–4.8) during infection (Sprick, 1956). It is important to note that the first-line drug in TB, pyrazinamide, becomes active due to acidification of the mycobacteria containing phagosome (Zhang et al, 2014). It is all the more surprising why few studies have explored mechanisms of acid resistance in mycobacteria. It is likely that similar acid resistance mechanisms exist in other mycobacterial species
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