Measurable genomic changes in subsp. after long-term adaptation in and reduced persistence in macrophages.

Free-living amoebae are ubiquitous in aquatic environments and act as environmental reservoirs for nontuberculous mycobacteria. subsp. recovered from has been demonstrated to be more virulent in both human and murine models. Here, we investigate the persistence of subsp. after short-term (2 weeks) and long-term (42 weeks) co-culture in We hypothesize that adapted subsp. demonstrate phenotypic and genomic changes facilitating intracellular persistence in naïve and human macrophages. subsp. CFU in co-culture with were recorded every 2 weeks up to 60 weeks. While -associated subsp. CFU did not significantly change across 60 weeks of co-culture, longer adaptation time in amoebae reduced colony size. Isolates recovered after 2 or 42 weeks of amoebae co-culture were referred as "early-adapted" and "late-adapted" subsp. respectively. Whole genome sequencing was performed on amoebae-adapted isolates with pan-genome comparisons to the original subsp. isolate. Next, amoebae-adapted isolates were assessed for their persistence in 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 subsp. were reduced in and contrary to expectations, lower counts were also observed in THP-1 macrophages with concomitant decrease in TNFa, IL-6, and MIP-1b suggesting that host adaptation may influence the inflammatory properties of Short-term interaction between and has been demonstrated to increase infectivity in human and murine models of infection, establishing the paradigm that amoebae "train" in the environment by selecting for phenotypes capable of enduring in human cells. We investigate this phenomenon further by determining the consequence of long-term amoebae adaptation on subsp. persistence in host cells. We monitored genomic changes across long-term co-culture and report significant changes to the subsp. genome in response to amoebae-adaptation and reduced colony size. Furthermore, we examined isolates co-cultured with for 2 or 42 weeks and provide biological evidence that long-term co-culture in amoebae reduces persistence in human macrophages.