(558) Identifying the mechanisms of regulation of biofilm formation in Mycobacterium abscessus, a CF clinical pathogen.

Background: Chronic pulmonary infection by bacteria is the primary cause of respiratory failure and death amongst Cystic Fibrosis (CF) patients. Pulmonary infections caused by nontuberculous mycobacteria (NTM), especially those of the archetypal, rapidly growing mycobacterial (RGM) pathogen Mycobacterium abscessus (MABS), are notoriously difficult to treat, demonstrating persistent infections recalcitrant to antibiotic therapy. Here we are interested in investigating physiological mechanisms by which MABS evade immune clearance and increase antibiotic tolerance. Biofilm formation allows chronic pulmonary bacterial pathogens to persist in the face of environmental and host stressors. A critical early step in NTM biofilm formation is cell-cell adhesion, characterized by the formation of cellular aggregates in liquid culture. MABS CF isolates classified as rough colony variants contain mutations to disrupt the production of glycopeptidolipids (GPLs), key mycomembrane components. Cell surface remodeling is a known way that MABS adapt to the host lung; however, the role of GPL regulation on MABS biofilm formation has not been fully explored. In addition, CF sputum is characterized by pockets of hypoxia, which is known to drive NTM into dormancy. By use of the model species for RGM, Mycobaterium smegmatis, we are exploring the connection between NTM biofilm formation and the hypoxic dormancy response. The two-component DosSR dormancy response system senses low oxygen environments using the DosS sensor and, through the DosR response regulator, promotes transcriptional changes to increase survival under hypoxia. These include activation of a ribosome stabilizing protein, RafH, which is essential for ribosome hibernation during hypoxic dormancy. We hypothesize that hypoxic conditions activate Dos, which in turn downregulates GPL production and modification through an unknown Dos regulon component. In this model, GPL downregulation exposes underlying membrane adhesins and increases membrane hydrophobicity resulting in increased cell-cell adhesion and an increase in biofilm formation.

Methods: To elucidate how these systems regulate biofilm formation and dispersal, we have created a library of mutants with disruption of key GPL biosynthesis and Dos regulon components. We use an in vitro aggregation assay that approximately quantifies the aggregate and planktonic cell population separately to track aggregation throughout culture maturity, allowing for a better understanding of the factors which influence the early stages of biofilm formation.

Results: By varying culture volumes, we have observed that decreasing culture oxygenation increases culture aggregation. In addition, we have identified that mutation of the Dos operon component, RafH, results in an aggregation defect like the DosR knockout mutation, as well as a novel re-aggregation phenotype. Finally, we have determined that a GPL deficient mutant of Mycobacterium smegmatis constitutively aggregates.

Conclusions: By identifying key regulatory components of M.smegmatis biofilm formation and how they shape early biofilm formation, we hope to identify targets for novel therapeutics to prevent or disperse biofilms. Our future experiments will focus on exploring how the mycomembrane lipid profile changes throughout aggregation dynamics, as well as more controlled and quantifiable oxygenation experiments by use of a bioreactor.

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