Search

The human microbiome has important roles in maintaining health, but dysbiosis of the microbiota can lead to disease. Polymicrobial interactions can result in synergy, producing disease that is worse than the sum of the respective single species infections. Despite this significant impact, synergy is understudied due to the complexity of polymicrobial interactions. Periodontitis is a microbiome-associated disease, and is one of the most common infectious diseases worldwide. Therefore, we have used periodontal disease as a model to study polymicrobial synergy. I have used two complementary approaches to study polymicrobial infections. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans exhibits synergy with streptococci in model murine infections. Because polymicrobial interactions are dependent on organisms’ abilities to sense their environments, I have examined the genetic regulatory mechanisms used by A. actinomycetemcomitans to interact with its environment. Through Northern blot analyses and biochemical approaches, I show that A. actinomycetemcomitans uses non-coding RNAs to regulate amino acid transport. Taking a comparative genomics approach, I demonstrate that A. actinomycetemcomitans DNA uptake systems are evolutionarily linked to genome defense. To describe host-influenced changes in gene expression, I develop a new technique to transcriptionally profile A. actinomycetemcomitans in a murine abscess infection, thereby revealing the importance of specific fermentative and anaerobic respiratory genes for in vivo survival. The long-term goal is to use these studies as a basis to characterize genetic regulatory mechanisms mediating synergy in polymicrobial A. actinomycetemcomitans infections with streptococci and other oral microbes. As a second approach to study polymicrobial infections, I analyze gene expression of healthy and diseased human plaque communities from aggressive periodontitis patients. Profiling ribosome content of healthy and diseased communities, I show that disease communities adopt similar less diverse population structures distinct from healthy populations. In addition to changes in population composition, using community transcriptional profiling I show that a keystone species within diseased communities up-regulates expression of genes involved in making the oral inflammatory molecule butyrate. These studies demonstrate for the first time that microbiome based diseases are marked by gene expression changes in addition to compositional changes.

The dental pathogen A. actinomycetemcomitans, a Gram- negative organism, has been associated with aggressive forms of periodontitis. A. actinomycetemcomitans requires iron to grow. In the host, iron-binding proteins such as transferrin, lactoferrin, hemoglobin, and ferritin, maintain a low free-iron concentration. Microorganisms, however, have evolved complex systems to efficiently harvest iron. The iron acquisition systems encoded in the genome of A. actinomycetemcomitans are likely tailored towards the specific survival strategies needed to survive in the oral biofilm. The objectives of this project were to determine the growth of A. actinomycetemcomitans in different iron sources and to examine the expression of the various iron uptake systems encoded by this organism under these conditions. A. actinomycetemcomtians was grown in chemically defined media (CDM) with or without an iron chelator (dipyridyl (DIP), and supplemented with various iron forms to determine growth. The growth of A. actinomycetemcomitans was decreased in a dose-dependent manner when cultured in CDM without exogenous ferrous sulfate supplemented with DIP (CDIDIP). Growth of A. actinomycetemcomitans was restored most efficiently when CDMIDIP was supplemented with hemin, less so with ferric citrate, and ferric chloride, and not with ferrous sulfate. Using these growth conditions, we examined the differential regulation of the numerous iron uptake systems encoded III the A. actinomycetemcomitans genome.