Novel iron-regulated sRNAs involved in biofilm formation of Aggregatibacter actinomycetemcomitans

Biofilm determinants, including fimbriae, extracellular polysaccharide (EPS), and lipopolysaccharide (LPS), of the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) are influenced by environmental factors such as nutrient stress, pH, oxygen and iron concentration. Mechanisms explaining how iron influences biofilm formation of Aa have yet to be elucidated. In other bacterial species iron influences biofilm formation via short non-coding RNA molecules called small regulatory RNA (sRNA). Objective: To determine the mechanism behind iron regulation of biofilm formation by Aa through identifying novel sRNA molecules that target biofilm determinants and iron-regulated genes. Method: Potential iron-regulated sRNA molecules were identified using bioinformatics by focusing on three characteristics previously used to identify sRNA in other bacteria: (1). sRNA are present in intergenic regions of a genome (2). A FUR box sequence (NAT(A/T)ATNAT(A/T)ATNAT(A/T)ATN) is present in the promoter region of sRNA and (3). sRNAs end with a r-independent terminator. RNA was isolated from Aa grown in medium with and without iron supplementation or chelation. The effect of iron on predicted sRNA molecules was determined by RT-PCR, real-time qPCR and the FURTA assay. Potential targets of the sRNA were determined using targetRNA, a computational tool for identifying mRNA targets for sRNA. Results: Several novel sRNA molecules were identified through in silico analysis. RT-PCR and real-time PCR results indicated that four of these molecules were significantly expressed under iron-depleted medium as compared to iron-replete medium. FUR regulation of sRNAs was supported by positive results of the FURTA. Identified sRNAs regulatory mRNA targets included genes involving in biofilm formation (fimbriae, EPS, LPS), iron storage, oxidative stress response and anaerobic respiration. Conclusion: The discovery of several iron-regulated sRNAs suggests that Aa has evolved an array of iron-dependent sRNA regulatory mechanisms to control the biofilm formation, iron uptake/ utilization and the oxidative stress response. Grant number: DE09838 (to F.A.S)