Characterization of a Tn4351-generated hemin uptake mutant of Porphyromonas gingivalis: evidence for the coordinate regulation of virulence factors by hemin.

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. We report here on 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. P. gingivalis MSM-3 was selected on the basis of its nonpigmented phenotype on anaerobic blood agar following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplied with hemin as a sole source of iron; however, growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown in either hemin-replete or hemin-depleted conditions bound and transported less [14C]hemin or [59Fe]hemin than did the parent strain. At 4 h, P. gingivalis MSM-3 grown in hemin-replete conditions transported only 10,000 pmol of hemin per mg of protein, or 14% of the amount transported by P. gingivalis A7436. 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 as much membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 also were found to express increased hemolytic and trypsin-like protease activities compared with the parent strain. When inoculated into subcutaneous chambers implanted in mice, P. gingivalis MSM-3 was highly infectious and more invasive than the parent strain, as indicated by secondary lesion formation and death. Taken together, these results indicate that the decreased transport of hemin by P. gingivalis MSM-3 results in the increased expression of several virulence factors which may be coordinately regulated by hemin.