Molecular & environmental regulation of biofilm determinant expression in Aggregatibacter actinomycetemcomitans

The periodontopathogen Aggregatibacter actinomycetemcomitans form biofilms that adhere tenaciously to abiotic surfaces. Fimbriae, lipopolysaccharide (LPS) and extracellular polymeric substance (EPS) contribute to the formation of biofilm by this organism.;In specific aim one of this dissertation, we sought to understand the biofilm behaviors of A. actinomycetemcomitans under different environmental conditions that it would encounter in the oral cavity and to provide evidence for differential gene expression in response to those changes. The transcription and translation of biofilm determinants: fimbriae, LPS and EPS genes in rough and smooth strains of A. actinomycetemcomitans were determined in aerobic or anaerobic conditions, and in nutrient and nutrient-stress medium, and in iron-supplemented and iron chelated growth conditions. Each of the biofilm determinants was significantly up-regulated in the rough strains compared to their expression in smooth strain. Selected biofilm determinants expression was increased under anaerobic conditions and in nutrient-stress medium at transcriptional level. Iron-chelated conditions increased the transcription and translation of EPS and fimbriae as compared to their expression in presence of iron.;The gene loci of fimbriae, LPS and EPS do not contain any iron regulatory motifs in their promoters. Therefore, the role of biofilm determinants regulated by iron via sRNA molecules was sought in specific aim two. Four putative sRNA molecules, JA01-JA04, in A. actinomycetemcomitans and their potential mRNA targets, including known biofilm determinants were identified using a bioinformatics approach. All four sRNA molecules were highly expressed under iron-chelated conditions. Since the ferric uptake regulator (Fur) protein is a global regulator of iron metabolism in many bacterial species, we tested its role in regulation of these sRNAs. The Electrophoretic Mobility Shift Assay (EMSA) was used to demonstrate the specificity of Fur binding to sRNA promoters. We further characterized the regulation of these sRNA molecules by the Fur protein using a fur mutant strain. We showed that the expression of these sRNA molecules was highly expressed in the fur::kan mutant strain when compared to its counterpart wild-type strain regardless of environmental iron concentration. Although expression of biofilm determinant genes and iron-uptake genes were up-regulated at transcriptional level in fur::kan mutant strain, we did not observe any discernible changes in biofilm mass under normal iron concentrations by fur::kan mutant strain compared to wild-type strain.;Our in silico analysis showed that JA01 sRNA significantly base pairs with mRNA from biofilm determinant genes. Upon mutation of JA01 sRNA, transcript levels from several biofilm determinant genes (tadV, tadC, pgaC, rmlB, lbgB) and iron-regulated (hemU, afuA) genes were significantly down-regulated under normal iron concentration. Biofilm formation by JA01::kan mutant strain was minimal in normal TSBY broth; however, under iron-restricted conditions biofilm formation by JA01::kan strain was significantly enhanced. JA03 sRNA extensively base pairs with the biofilm determinant gene (tadG) and iron-regulated genes (hitC, ltxD and murD). In iron-replete conditions, JA03 sRNA overexpression resulted in significant repression of the iron acquisition gene hitC, the EPS-encoding gene pgaC, the peptidoglycan-encoding gene murD and the leukotoxin gene ltxD. The JA03 sRNA over-expression strain formed large, minimally adherent microcolonies in broth cultures and failed to form biofilms on microtiter plates. Thus, JA03 sRNA may be involved in regulating biofilm formation, either directly or indirectly.;Our results indicate that selected sRNA molecules are regulated by the global regulator, Fur, to sense the environment and fine--tune the expression of certain set of genes involved in iron homeostasis and colonization.