Chronic lung infection – Investigation of the mechanisms of adaptation during chronic colonisation: A study on Mycobacterium abscessus and Burkholderia cenocepacia

Chronic infection by opportunistic pathogens is a major contributor to mortality in people with cystic fibrosis (CF). These infections are caused by antimicrobial resistant (AMR) pathogens such as Burkholderia cepacia complex (Bcc) and the emerging pathogen, Mycobacterium abscessus which causes recalcitrant infections with high antibiotic resistance. M. abscessus adapts over time of colonisation to the conditions in the CF lung, hampering effective treatment. The mechanisms underlying this pathoadaptation are poorly understood and could be key for developing future therapies. One of the most striking pressures present in the CF lung is hypoxia. Previous work in the McClean lab identified that chronic infection isolates of Bcc displayed upregulation of proteins encoded by the low oxygen activated locus (lxa), which suggesting that adaptation during chronic infection in the CF lung could be linked to the hypoxic response. Interestingly, the DosR regulon which is activated in hypoxic conditions in M. abscessus shares several genes and gene functions with the lxa locus including USPs and proteins such as α-crystallin and phosphofructokinase. This led to the aim of this thesis which was to explore the role of hypoxia in driving bacterial adaptation to the CF lung. Four sequential isolate pairs of M. abscessus from CF patients were examined for adaptive changes over time of infection. Genomic analysis confirmed that these isolate pairs were sequential. Significant changes in phenotype, such as increased host cell attachment to CF bronchial epithelial cells and increased intracellular survival in macrophages, were observed in late infection isolates, indicative of adaptation to the CF lung environment. Late isolates exhibited adapted proteomes, in particular displaying increased abundance of proteins with roles in intracellular survival and antibiotic resistance. Early infection isolates were then exposed to hypoxia in a controlled oxygen chamber to determine whether hypoxia exposure was driving some of the adaptations observed. Early patient isolates from two CF patients, Patient 4 and Patient 7 showed phenotypic adaptations to 6% oxygen over 14-21 days in pilot studies, while the proteome of Patient 7 isolates was significantly impacted over the 14- days of exposure, with 18 proteins significantly increased in abundance and 67 significantly decreased in abundance. These data indicated that long term hypoxia exposure had a considerable impact on M. abscessus and may be involved in its adaptation in the CF lung environment. Two common adaptations were observed in late infection isolates and hypoxia-adapted isolates: increased abundance of proteins with roles in oxidative stress and intracellular survival; and increased abundance of proteins with roles in antibiotic and drug resistance. The recapitulation of changes seen in chronic infection isolates following hypoxia exposure emphasises the contribution of hypoxia in shaping bacterial adaptation amongst the other stresses and pressures experienced in chronic pulmonary infection. This validated our hypothesis that hypoxia is a key player driving the adaptation of bacterial species in the CF lung. The mechanism of action of a potential regulator of the switch from acute to chronic infection in Bcc, Bnr1, was also evaluated by assessing its binding partners. Two histone-like proteins, HctB, an outer membrane localised histone H1-like protein, and a second putative histone-like protein (BCAM1012 were identified, suggesting that Bnr1 may function as a DNA mimic protein. A further interaction with the protein translocase SecA may transport the Bnr1-HctB complex to the membrane where it causes numerous effects such as changes in cell morphology. Overall, the data presented in this thesis has increased our understanding of the mechanisms of adaptation used by M. abscessus and Bcc to adapt to the CF lung environment and the role of hypoxia in driving this adaptation process.