Date of Award

7-1-1997

Degree Type

Thesis

University or Center

Clark Atlanta University(CAU)

Degree Name

M.S.

Department

Biology

First Advisor

Caroline A. Genco, Ph.D

Abstract

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. Here, the isolation and characterization of a transpositional insertion mutant of P. gingivalis A7436 (designated MSM-3) which is defective in the utilization and transport of hemin, is reported. P. gingivalis MSM-3 was initially selected on the basis of its non-pigmented colony phenotype on anaerobic blood agar plates following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplies with hemin as a sole source of iron; however growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown under hemin-replete or hemin-deplete conditions bound and transported less [14C] hemin and [59Fe] hemin than did the parental strain A7436. At 4 hr, P. gingivalis MSM-3 grown under hemin-replete conditions transported only 10,000 pmol of hemin per mg total cellular protein, or 14% of the amount transported by P. gingivalis A7436 grown under similar conditions. Unlike P. gingivalis A7436, hemin binding and transport by P. gingivalis MSM-3 were not tightly regulated by hemin or iron. Examination of P. gingivalis MSM-3 cultures by electron microscopy revealed an overproduction of membrane vesicles, and determination of the dry weight of purified vesicles indicated that P. gingivalis MSM-3 produced twice the amount of membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 were found to express higher hemagglutination titers, as well as enhanced hemolytic and arginine-X-specific protease activities. In vivo studies revealed that P. gingivalis MSM-3 was more infectious and invasive than the parent strain, as indicated by secondary lesion formation and death in experimental mice.

Genetic analysis has revealed that while Tn4351 has inserted into a non-coding region (60 bp downstream from a known P. gingivalis insertion sequence element, IS1126), its insertion into the host chromosome resulted in the mobility of IS 1126. We have determined that P. gingivalis MSM-3 contains two additional copies of IS1126 as compared to the parent strain. The insertion site of one of the additional IS1126 elements has been sequenced. Genetic analysis reveals that it contains numerous open reading frames and does not provide evidence of an open reading frame which may encode for a gene involved in iron acquisition. However, approximately 1 kb downstream of the insertion site, a 1.014 kb open reading frame (designated hemB) has been located. Analysis has revealed that this open reading frame shares homology with several genes that encode for hemin/iron receptors or iron-regulated outer membrane proteins.

Taken together, the results presented in this study provide evidence for the coordinate regulation of P. gingivalis virulence factors by hemin. Additionally, the capability of mobilization of an insertion sequence element, a phenomenon not previously reported in P. gingivalis, has been demonstrated. Further characterization of hemB and the second additional IS1126 element's insertion site may provide evidence for the role of specific proteins involved in hemin/iron acquisition by P. gingivalis.

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