Abstract
Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.
Highlights
Phagocytosis is the process by which particles are recognized, bound to the surface of cells and internalized into a plasma membrane-derived intracellular vacuole, or phagosome
A similar scenario was reported when the virulence of Aeromonas spp was analyzed using D. discoideum model [50], i.e. Dictyostelium was incapable of growing on wild-type bacteria or on any of the nonvirulent mutants of Aeromonas tested when the assays were performed on standard medium (SM) medium
Since the richness of the growth medium affects the threshold at which a bacteria is permissive for Dictyostelium [42], we tested a range of dilutions of HL5 to determine the medium where only Kp52145 remains nonpermissive (Figure 1B)
Summary
Phagocytosis is the process by which particles are recognized, bound to the surface of cells and internalized into a plasma membrane-derived intracellular vacuole, or phagosome. Phagocytosis is a special feature of the so-called professional phagocytes, i.e. polymorphonuclear leukocytes ( known as neutrophils), dendritic cells, monocytes and macrophages. There are D. discoideum axenic strains that can feed by phagocytosis and by macropynocytosis of liquid nutrients [2]. Of special interest is the fact that Dictyostelium cytoskeleton architecture is similar to that found in mammalian cells. The process of particle uptake in Dictyostelium is similar to macrophage phagocytosis [1]. The fact that the strategies evolved to counteract mammalian professional phagocytes are considered essential to establish an infection has led to the notion that Dictyostelium amoebae could be used as host model to measure virulence [3,4]. The genome of D. discoideum strain AX4 has been sequenced [5]
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