Nguyen et al. [11] reported that the last five C-terminal residues (KVIVK) of RgpB play a significant
role in the post-translational modification/proteolytic processing and exportation of proteins to the outer membrane. To determine whether the last five C-terminal residues (K340VLVP344) of HBP35 play a role similar to that of RgpB, we constructed an hbp35 deletion of K340-P344 mutant and found that the mutant showed no diffuse bands but only 33-and 31-kDa proteins, which may have been generated by degradation of HBP35 protein accumulating in the cell (Figure 8). The result suggests that the last five C-terminal residues have an important role in the transport of HBP35 protein to the cell surface. Figure 8 Immunoblot analysis of cell extracts of various P. gingivalis strains with anti-HBP35 antibody. Lane 1, 33277; lane 2, KDP164 (hbp35 insertion mutant); lane see more 3, KDP167 (hbp35 deletion of K340-P344 mutant). Discussion As P. gingivalis requires heme as the source of iron and protoporphyrin IX, a heme binding and transport system is essential for the microorganism
to survive. Recently, several TonB-linked outer membrane receptors for heme utilization, including HmuR, Tlr, IhtA and HemR, SHP099 price have been reported [4]. The ability to store heme in bacterial cells appears to provide a nutritional advantage for survival of the bacterium in the iron-limited environment of a healthy
gingival crevice [17]. In fact, heme can bind the P. gingivalis cell surface and may then be transported into the cell by an energy-dependent process [18]. Shibata Autophagy activator et al. [7] found that purified rHBP35 protein (Q22-P344) could bind hemin but not hemoglobin or lactoferrin. HBP35 was suggested to possess a putative heme binding sequence (Y50CPGGK55), however, we found in this study that hemin could bind the mutant rHBP35 (Q22-P344 with C48S and C51S) and the truncated rHBP35 (M135-P344) (Figure 4B), selleck chemicals llc indicating that the hemin binding site is located between M135 and P344. The hbp35 mutants grew more slowly than the wild type in hemin-depleted conditions and even in the condition with a sufficient hemin concentration (5 μg/ml), indicating that HBP35 protein plays a role in hemin utilization in various hemin levels. The truncated HBP35 proteins of 27-and 29-kDa, which were derived from a 3′-portion of the hbp35 gene, were mainly located in the cytoplasm/periplasm fraction. This finding together with the fact that there is no signal peptide region in the two proteins suggests that these proteins are located in the cytoplasm and contribute to the intracellular storage of heme as does bacterioferritin (Figure 6). Similar protein expression has been found in Neisseria meningitidis: two forms of PilB protein are produced from the pilB gene.
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