Role of Porphyromonas gingivalis FeoB2 in metal uptake and oxidative stress protection

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is a recognized periodontopathogen. It exhibits a high degree of aerotolerance and is able to survive in host cells, indicating that efficient oxidative stress protection mechanisms must be present in this organism. Manganese homeostasis plays a major role in oxidative stress protection in a variety of organisms; however, the transport and role of this metal in P. gingivalis is not well understood. Analysis of the genome of P. gingivalis W83 revealed the presence of two genes encoding homologs of a ferrous iron transport protein, FeoB1 and FeoB2. FeoB2 has been implicated in manganese accumulation in P. gingivalis. We sought to determine the role of the FeoB2 protein in metal transport as well as its contribution to resistance to oxygen radicals. Quantitative reverse transcriptase PCR analyses demonstrated that expression of feoB2 is induced in the presence of oxygen. The role of FeoB2 was investigated using an isogenic mutant strain deficient in the putative transporter. We characterized the FeoB2-mediated metal transport using ⁵⁵Fe²⁺ and ⁵⁴Mn²⁺. The FeoB2-deficient mutant had dramatically reduced rates of manganese uptake (0.028 pmol/min/10⁷ bacteria) compared with the parental strain (0.33 pmol/min/10⁷ bacteria) (after 20 min of uptake using 50 nM of ⁵⁴Mn²⁺). The iron uptake rates, however, were higher in the mutant strain (0.75 pmol/min/10⁷ bacteria) than in the wild type (0.39 pmol/min/10⁷ bacteria). Interestingly, reduced survival rates were also noted for the mutant strain after exposure to H₂O₂ and to atmospheric oxygen compared to the parental strain cultured under the same conditions. In addition, in vitro infection of host cells with the wild type, the FeoB2-deficient mutant, and the same-site revertant revealed that the mutant had a significantly decreased capability for intracellular survival in the host cells compared to the wild-type strain. Our results demonstrate that feoB2 encodes a major manganese transporter required for protection of the bacterium from oxidative stress generated by atmospheric oxygen and H₂O₂. Furthermore, we show that FeoB2 and acquisition of manganese are required for intracellular survival of P. gingivalis in host cells.