Measurable Genomic Changes in Mycobacterium avium subsp. hominissuis after Long-Term Adaptation in Acanthamoeba lenticulata and Reduced Persistence in Macrophages

Free-living amoebae are ubiquitous in aquatic environments and act as environmental reservoirs for nontuberculous mycobacteria. Mycobacterium avium subsp. hominissuis recovered from Acanthamoeba has been demonstrated to be more virulent in both human and murine models. Here, we investigate the persistence of M. avium subsp. hominissuis after short-term (2 weeks) and long-term (42 weeks) coculture in Acanthamoeba lenticulata. We hypothesize that A. lenticulata-adapted M. avium subsp. hominissuis demonstrates phenotypic and genomic changes facilitating intracellular persistence in naive Acanthamoeba and human macrophages. M. avium subsp. hominissuis CFU in coculture with A. lenticulata were recorded every 2 weeks for up to 60 weeks. While A. lenticulata-associated M. avium subsp. hominissuis CFU did not significantly change across 60 weeks of coculture, longer adaptation time in amoebae reduced the colony size. Isolates recovered after 2 or 42 weeks of amoeba coculture were referred to as “early-adapted” and “late-adapted” M. avium subsp. hominissuis, respectively. Whole-genome sequencing was performed on amoeba-adapted isolates with pan-genome comparisons to the original M. avium subsp. hominissuis isolate. Next, amoeba-adapted isolates were assessed for their persistence in A. lenticulata, Acanthamoeba castellanii, and human THP-1 macrophages. Multiplex cytokine/chemokine analyses were conducted on THP-1 culture supernatants. Compared to the original isolate, counts of late-adapted M. avium subsp. hominissuis were reduced in Acanthamoeba and, contrary to expectations, lower counts were also observed in THP-1 macrophages, with concomitant decreases in tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and macrophage inflammatory protein 1 beta (MIP-1β), suggesting that host adaptation may influence the inflammatory properties of M. avium. IMPORTANCE Short-term interaction between Acanthamoeba and M. avium has been demonstrated to increase infectivity in human and murine models of infection, establishing the paradigm that amoebae “train” M. avium in the environment by selecting for phenotypes capable of enduring in human cells. We investigated this phenomenon further by determining the consequence of long-term amoeba adaptation on M. avium subsp. hominissuis persistence in host cells. We monitored genomic changes across long-term Acanthamoeba coculture and report significant changes to the M. avium subsp. hominissuis genome in response to amoeba adaptation and reduced colony size. Furthermore, we examined isolates cocultured with A. lenticulata for 2 or 42 weeks and provide biological evidence that long-term coculture in amoebae reduces M. avium persistence in human macrophages.