The colonization of the human colon by Bacteroides spp. is a fundamental requirement for the establishment and maintenance of a normal, healthy intestinal flora and a disruption in this commensal relationship has a great impact on health and disease. In order for Bacteroides fragilis to colonize the human colon, they need to compete efficiently for the available nutrients with other microorganisms of the microflora and host nutritional defenses. Among these essential nutrients required by Bacteroides spp. are iron and heme. We postulate that Bacteroides play an important role in scavenging non-absorbed intestinal iron not only for their own physiological requirements for colonization of the large intestine but also to store iron, making it less accessible to other microorganisms in the colon. It is our goal in this research proposal to determine if non-absorbed iron in the large intestine is taken up by B. fragilis ferrous iron transport system (FeoAB) and stored into the iron storage protein ferritin (FtnA) in a non-toxic-form as a model system to test our hypothesis that B. fragilis makes iron unavailable to intestinal pathogenic bacteria. The long-term objective of this study is therefore to investigate in more detail the way B. fragilis, a commensal component of the intestinal microflora acquire and store iron during intestinal colonization. To accomplish these objectives, the following aims will be pursued: [PARAGRAPH] Aim 1: We will determine the role of B. fragilis Feo(AB) in iron uptake and intestinal colonization. 1A. Determination of ferrous iron uptake. 1B. Role and regulation of feo(A/B) in the colonization of the intestinal tract.. 1B1: Colonization of gnotobiotic rat intestinal tract with B. fragilis feo(A/B) mutant. 1B2: We will assess the expression of feo(A/B) mRNA in vivo by Real-Time RT-PCR. 1C. Role of iron acquisition in competitive gut colonization. Dual-association of gnotobiotic rats with B. fragilis mutants and E. coli. [PARAGRAPH] Aim 2: We will analyze the role of B. fragilis FtnA in iron-storage and colonization of intestinal tract. 2A. Delineate the role of FtnA in intestinal colonization 2B. We will analyze the role of FtnA in total iron content of B. fragilis. 2C. Determine the regulation of B. fragilis ftnA mRNA in vivo.
The novel aspect of this research project resides in the elucidation of the iron acquisition and storage mechanisms in the anaerobe B. fragilis and their role in bacterial intestinal colonization and survival. Our hypothesis is that intestinal commensal bacteria, such as B. fragilis group protect the intestinal mucosa against colonization of pathogenic bacteria by scavenging non-absorbed free iron in the colon. This study is aimed to provide new information that may lead to new prebiotics and probiotics therapeutic strategies to control and manipulate the intestinal microflora population.
