In the current study we showed that known
In the current study, we showed that known inhibitors of the F-ATPase and ionophores affect the growth of P. gingivalis and its plasma membrane ATPase, suggesting that the membrane ATPase can be a target for anti-periodontitis agents. We also identified stilbenoids that strongly inhibit the growth of P. gingivalis.
Materials and methods
Results and discussion
Conflicts of interest
Role of intracellular pH regulation Eukaryotic M871 are highly compartmentalized to provide optimized environments for distinct cellular functions. A critical factor determining the functions of organelles is the local pH, i.e., the amount of available protons. Protonation and deprotonation alter the net charges of biological surfaces and are important for protein structure and function. Whereas the pH is near neutral in the cytosol and ER (endoplasmic reticulum, pH 7.2), it is alkaline within the mitochondrial matrix (pH ca. 8.0) (Casey et al., 2010). In contrast, compartments of the secretory and endocytic pathways progressively acidify along the cis-trans axis and from early endosomes to lysosomes. In both pathways, luminal pH may reach values as low as 4.5, providing optimal conditions for enzymes that mediate either post-translational modifications and processing of proteins in the secretory pathway or degradation within endolysosomes. Acidification is central for protein sorting in both pathways, e.g., by mediating receptor-ligand dissociation and, hence, receptor recycling. Acidification also provides the basis for membrane fusion and fission events (Aniento et al., 1996, Gu and Gruenberg, 2000, Ungermann et al., 1999, Wu et al., 2001). During phagocytosis, microorganisms are captured by an invagination of the plasma membrane resulting in the formation of a phagosome. This compartment has initially a near-neutral pH and it lacks the machinery to clear the enclosed microorganisms. During the further maturation process, the lumen of phagosomes is rapidly and strongly acidified and phagosomes fuse with endosomes and eventually lysosomes generating a hydrolytically competent phagolysosome. The low pH activates enzymes which mediate some of the killing and degradation of microbes (Haas, 2007, Levin et al., 2016). Acidic conditions may also directly affect the growth of bacteria and promote the generation of hydrogen peroxide (detailed below). The main driving force for strong acidification is a large multiprotein complex, the vacuolar ATPase (V-ATPase) (Cotter et al., 2015) which is enriched on lysosome and phago(lyso)some membranes.
V-ATPase as a driving force of intra-organelle acidification V-ATPase belongs to a family of ATP-dependent proton pumps. It consists of 14 different subunits (Cotter et al., 2015) (Fig. 1). Similar as with F-type and A-type ATPases, a rotary mechanism couples ATP hydrolysis within a peripheral V1 sector to the transport of protons, against the electrochemical gradient, through a membrane-integral VO sector. At the molecular level, two protons are pumped from the cytosol for each hydrolyzed ATP (Johnson et al., 1982) allowing, in theory, to decrease luminal pH values to below 3.0 (Grabe et al., 2000, Kettner et al., 2003). Such low pH values are never reached in living cells because several cellular factors regulate the actual extent and velocity of acidification: Proton pumping generates an electrochemical potential difference across the membrane (positive charge on the luminal side) which, if continued permanently, would first decelerate and eventually inhibit further acidification. To prevent this inhibition the generated voltage is neutralized by import of anions and/or export of cations. As for regulation by anions an influx of cytosolic chloride is mediated by members of the chloride channel (CLC) family, CLCN3 through CLCN7, which are 2 Cl−-for-1 H+ antiporters found on endosomes and lysosomes (Mindell, 2012, Stauber and Jentsch, 2013). Furthermore, CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride transporter that may also participate in phagosome acidification (Di et al., 2006, Haggie and Verkman, 2007). Regulation of luminal pH by cations, on the other hand, can be achieved by an efflux of monovalent cations which counteracts the V-ATPase-mediated electrochemical potential (Steinberg et al., 2010). Two members of the TRP (transient receptor potential) channel family, TRPML1 (Bach et al., 1999, Pryor et al., 2006, Soyombo et al., 2006) and TRPM2 (Di et al., 2017), are potential cation exporters but show diverging influence on luminal pH depending on the cell type.