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"Aggregatibacter actinomycetemcomitans VIRULENCE FACTORS -A STUDY OF HOST MICROBE INTERACTIONS IN A NON-HUMAN PRIMATE MODEL"

by
Vandana Sampathkumar
Oral Biology Program
B.D.S. 2007, SRM University, India


Thesis Advisor: Dr. Daniel H. Fine
Professor
Department of Oral Biology

Wednesday, April 13, 2016
10:00 A.M., Room: B723, Dental School


Abstract

Aggregatibacter actinomycetemcomitans (Aa), a pathobiont, is considered to be the etiological agent of localized aggressive periodontitis (LAP). LAP is characterized by massive gum tissue destruction and tooth loss caused by Aa virulence factors. Rodent models have been used to study the role of various virulence factors of Aa. However, so far there is no real world model to study initial Aa colonization. In this study we have developed a rhesus (Rh) monkey colonization model to study initial colonization and periodontal disease progression. Our goals are (i) to establish a colonization model in Rh monkeys; (ii) to study the role of LuxS, a quorum sensing molecule and leukotoxin, LtxA, a toxin involved in subgingival immune evasion (iii) and to ascertain the role of LtxA on colonization in vivo. The non-human primates are the real world exceptional model to study LAP because of the similar anatomical features and oral ecological factors. In our first Rh colonization study, human (IDH781) and Rh derived wild type Aa (Rh 4.35) were inoculated into the oral cavity of Rh monkeys to study their colonization efficacies. The results showed that Rh 4.35 colonized the oral cavity whereas IDH781 did not. In our second study, comparison of colonization efficiencies of ltxA (RhAa-VS2) and luxS (RhAa-VS4) knock-outs revealed that RhAa-VS2 did not colonize at all whereas RhAa-VS4 had higher colonization efficiency when compared to RhAa-VS2. Based on the results from second colonization study, an artificially created LtxA hyper producing strain (RhAa- ltxP 530) was used to test the role of LtxA on colonization. As expected, RhAa- ltxP 530 colonized better than RhAa-VS2 suggesting that LtxA affected colonization. However, in vitro studies showed that the effect was indirect and a result of decreased expression of adherence genes. Further, to understand the differential expression of ltxA in wild type minimal LtxA producer and a LtxA hyper-producer, we carried out a series of deletions in ltxA promoter region. Our work indicated that the region between 298-397 in the 530 bp region contained a negative regulatory element that affects leukotoxin expression. We have identified that this region possesses a potential weak terminator that minimizes LtxA production in the wild type minimal producer strains.


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