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  • neurotensin receptor Phagosomes acquire some hydrolases earl

    2022-11-09

    Phagosomes acquire some hydrolases early during their journey from the cell surface to lysosomes. An example for such hydrolase is cathepsin H which is most concentrated in the early phagosome at a pH of approximately 6.3 (Claus et al., 1998), the pH optimum of enzyme activity (Schwartz and Barrett, 1980). Most hydrolases are localized to late endosomes and lysosomes which maintain a pH between 4.5 and 5.5. This acidic environment ensures optimal hydrolase activities and can only be generated through V-ATPase-mediated proton translocation (Hackam et al., 1997). But which is the main killer of microbes within phagosomes? Is it neurotensin receptor itself or rather acid hydrolases or both or rather acidification plus other factors? To determine the effect of acidification on ingested pathogens experimentally, most investigators use either bafilomycin A1 or concanamycin A as potent V-ATPase blockers or the weak bases methylamine or chloroquine (Fig. 2b) to raise intra-phagosome pH or (phago)lysosome pH. An increased pH often resulted in less killing, e.g., of E. coli DH5α (Frankenberg et al., 2008), Mycobacterium smegmatis (Anes et al., 2006) or of Staphylococcus aureus (Bidani et al., 2000). Such reduced killing is usually ascribed to the sensitivity of microorganisms to low pH. This is a comprehensible but surprising conclusion considering that many microorganisms that thrive in and on humans are quite acid-tolerant, for example E. coli (Richard and Foster, 2007) or Mycobacterium tuberculosis (Vandal et al., 2009). Actually, the pH in even strongly acidified phagolysosomes is not dramatically low: A pH of 4.5 is about that of sour milk (van Slyke and Baker, 1918) which again is a microbial product. Most microbes would be expected not to be dramatically sensitive to this level of acidity. Stomach acid, on the other hand, at a pH of 1, contains 5000 times the proton concentration of lysosomes and acts as a powerful disinfectant. Not surprisingly, a genetically or pharmacologically caused deficiency in proper stomach acidification comes with higher infection risks (Martinsen et al., 2005). Given that many microbes can survive pH 2–3 for some time (Martinsen et al., 2005) it must be suspected that the hasty conclusion ‘killing is by direct effects of acid on the pathogen’ neglects that killing may normally occur by combined action of many potential killing factors that are regulated by the lysosome’s proton content such as the effects of protons… Furthermore, drugs used in analysis may have effects on the ingested microorganisms themselves in addition to the targeted host cell. For example parasitic protists of the genera Toxoplasma, Leishmania, Plasmodium and others possess V-ATPases which control cytosol pH homeostasis and the acidification of digestive vacuoles. These V-ATPases and acidification can be inhibited by agents such as bafilomycin A1 and the ionophore monensin (Elandalloussi et al., 2005, Moreno et al., 1998, Saliba and Kirk, 1999). Testing of the antimicrobial effects of such pH modulators on Plasmodium falciparum has been consequently considered a valid approach to the development of anti-malarial drugs (van Schalkwyk et al., 2010). Either or all of these and other acidification-related effects may cooperate in killing. In fact, it is not even clearly established that encounter of a given microorganism with lysosome contents is necessarily microbicidal, although it can be, as has been shown by in vitro incubations of specific microorganisms with purified lysosome contents (Jena et al., 2011, Styrt and Klempner, 1988). Yet it is clear that sensitivities to lysosome contents can vary strongly between microbial species (Rest et al., 1977, Styrt and Klempner, 1988). The major argument put forward to support the apparent hazardousness of acidic lysosomes is that mutations in central microbial virulence factor genes often force the mutant-containing phagosome to fuse with lysosomes and this comes with killing. But this is a chicken-and-egg argument: Is it primarily killing of the microorganism which secondarily leads to fusion of a formerly privileged compartment with lysosomes or is it that the mutant’s phagosome fuses with lysosomes because a central virulence determinant is missing and which has resulted in killing? An argument sometimes used to support the view ‘first fusion, then killing’ stems from the observation that macrophage activation by interferon-γ leads to increase of V-ATPase concentration (Jutras et al., 2008) and to more microorganisms being targeted to (phago)lysosomes hence more readily killed (Bosedasgupta and Pieters, 2014, Schaible et al., 1998). However, macrophage activation comes with the up- and downregulation of around 1000 host genes (Ehrt et al., 2001) so that it is hard to single out the one compound or event during immune activation which has the highest killing impact.