|Abstract or Summary
- Respiratory infections caused by nontuberculous mycobacteria (NTM), especially Mycobacterium avium, can lead to progressive, recurrent disease that is refractory to therapy. Bacterial biofilms are intrinsically resistant to a variety of stressors and pressures, including host killing mechanisms and antibiotic therapy. Though it is becoming increasingly evident that NTM biofilms are important for infection, it is not currently known how these biofilms evade the immune response in an immunocompetent individual. Furthermore, what distinguishes biofilm-associated bacteria from planktonic bacteria is also mostly unknown. The main goals of this dissertation were to investigate the interaction between the host and the NTM biofilm, as well as to further identify and characterize the biofilm itself.
In Chapter 2, I developed an in vitro model to investigate the interaction between an established MAH biofilm and surveilling macrophages, mimicking a scenario of how these cells would encounter NTM in the airway. Using this model, I was able to assess multiple aspects of this interaction including bacterial killing, activation of macrophages, and apoptosis. My results show the biofilm eliciting a unique hyper-stimulation from the macrophages that results in attenuation of bacterial killing and early, atypical TNFα-driven apoptosis. Interestingly, UV-killed biofilms elicited similar responses from macrophages, suggesting that acellular biofilm matrix components could be contributing to this unique response.
In Chapter 3, I discovered that M. avium and other NTM can contain substantial amounts of extracellular DNA (eDNA) in their biofilm matrix and supernatant. Utilizing scanning electron microscopy and immuno-gold labelling, I was able to visualize the eDNA in these biofilms, yielding insight into the structural scaffolding-like properties the eDNA possesses. I determined that the eDNA was genomic in origin, and did not contain a specific exported sequence. By using DNA targeted enzymatic digestion with DNase I, I showed that eDNA is integral for biofilm establishment, persistence, and tolerance to clinically used antibiotics.
The work in Chapter 3 led me to hypothesize that eDNA is potentially secreted by active mechanisms, which contrasts the widely accepted idea that eDNA in bacterial biofilms results from cell autolysis. In Chapter 4, I conducted an in-depth analysis into the regulation and production of eDNA. I designed a model for fluorescently quantifying eDNA export in real time in undisturbed biofilms, which allowed investigation into triggers responsible for eDNA. When these results were combined with biofilm surface proteomics and a comprehensive transposon library screen for eDNA deficient mutants, my data suggests bicarbonate as a novel trigger for eDNA and identified many genes involved with the mechanism of eDNA export. Interesting components include an undescribed FtsK/SpoIIIE-like DNA exporting pore, multiple bicarbonate-interacting carbonic anhydrases, and a unique genomic region in MAH and other eDNA-producing NTM that could be implicated in biofilm formation and eDNA production.
Cumulatively, the work presented in this dissertation significantly advances the information known about NTM biofilms, and how they interact with and persist in the host respiratory tract. Current therapies can be ineffective at treating NTM respiratory infections, even after very long, multidrug treatment regimens. Since there is evidence to be implicated between NTM biofilms and respiratory infection, the more we can learn about the physiology of these biofilms, the more targets we can have to direct new anti-virulence therapies toward. Both bicarbonate sensing and eDNA production could become valuable anti-virulence targets for NTM infection.