Exopolysaccharide regulation in Aggregatibacter actinomycetemcomitans

ABSTRACT A hallmark of the oral bacterium Aggregatibacter actinomycetemcomitans’ biology and infectivity are its ability to form tenacious biofilms. Biofilm formation in the organism is attributed to attachment via surface proteins and exopolysaccharides (EPS). The EPS of A. actinomycetemcomitans (Aa), also referred to as PNAG, is often

overlooked as a virulence factor of bacterial pathogenesis and only considered for its role in biofilm formation. PNAG plays a critical role in the evasion from macrophages, oral colonization, and bone loss. However, very little information is available for the regulation of PNAG production in Aa. In other PNAG-producing bacteria,

such as E. coli, the synthesis of PNAG is regulated by the second messenger, cyclic-di-GMP or the post- translational carbon storage regulator CsrA. Aa does not produce cyclic-di-GMP due to the absence of the genes responsible for its synthesis in the Aa genome and the role of CsrA is unknown in Aa. In the absence of such

regulation, in anaerobic environment, Aa could produce excessive amount of PNAG which is a highly energy consuming process and can potentially reduce fitness. A quorum sensing two-component system, QseBC, functions as a global regulator of virulence in Aa but does not regulate PNAG genes AapgaABCD. Thus, there

is a knowledge gap regarding regulation of PNAG in Aa. In search of novel mechanisms of regulation, we have identified two proteins, AaFlp-1 (a unique Aa pili protein) and AaDcuB (an ABC succinate transporter involved in fumarate respiration), both of which impacted PNAG production. Succinate in an anaerobic environment

contributes to dysbiosis of the oral microbiota and chemotaxis. Further, exogenous addition of succinate has been shown to accelerate periodontal disease and Aa’s ability to expel succinate through fumarate respiration and AaDcuB might be critical in disease initiation. These two proteins, apparently unrelated, appear to control PNAG production either independently or in a

coordinated manner. Our overarching hypothesis is that AaFlp-1 and AaDcuB proteins regulate the PNAG production in Aa either directly or indirectly. Such impact by AaFlp-1 and AaDcuB has not been reported in other PNAG-producing bacteria such as E. coli or S. epidermidis and these two proteins offer a unique opportunity to

study the novel regulatory mechanism(s) operational in Aa. Our immediate goal is to test the overarching hypotheses that AaFlp-1 and AaDcuB proteins regulate the PNAG production in Aa and that the production of PNAG is unique in Aa. The following aims are proposed: Specific Aim 1. Decipher the unique crosstalk mechanism between AaFlp-1 and PNAG production in Aa.

Hypothesis: AaFlp-1 is a signal for the induction of PNAG production in Aa. Specific Aim 2: Determine the mechanism by which AaDcuB regulates the production of PNAG in Aa. Hypothesis: AaDcuB protein impacts the production of PNAG through the fumarate respiration.