Survival and persistence of Enterobacter species in human macrophages

Background Enterobacter species are opportunistic Gram-negative bacteria associated with morbidity and mortality worldwide due to multiple antibiotic resistance that also extends to last-resort antibiotics; they belong to the ESKAPE pathogens, a group of bacterial species highlighted by the WHO as the most dangerous pathogens worldwide. Surprisingly, very little is known about the infection biology of Enterobacter species. Preliminary work To bridge this knowledge gap, we have investigated the intracellular trafficking of a subset of Enterobacter clinical isolates, including colistin-resistant strains, within human macrophages, and determined the macrophage response to the intracellular infection. Phagocytosis of 11 clinical isolates representing E. cloacae, E. bugandensis, E. kobei, E. xiangfangensis, E. roggenkampii, E. hoffmannii, and E. ludwigii was investigated in primary human macrophages. Intracellular bacterial trafficking was followed by confocal ?uorescence microscopy; intracellular bacterial replication was assessed by bacterial enumeration and a ?uorescence dilution approach to follow bacterial cell division over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection and by determining cleavage of the proinflammatory markers caspase-1, gasdermin D and pro-interleukin-1ß. Our preliminary results indicate that the Enterobacter isolates did not replicate in human macrophages, but exhibited long-term survival within a modi?ed late phagolysosome compartment. Survival did not correlate with colistin resistance, lipopolysaccharide modi?cations, or bacterial pathogenicity in the Galleria mellonella infection model. Remarkably, intracellular bacteria induced low levels of macrophage cytotoxicity that correlated with absence of cleavage of proinflammatory markers in infected macrophages. Hypothesis Since clinical isolates from Enterobacter species isolates can persist without replication inside human macrophages with minimal effects on cell viability and inflammation we hypothesise that these bacteria can hideout within macrophages to escape innate immune responses and antibiotics. Aims To address this hypothesis, this proposal will investigate human macrophage-Enterobacter interactions by pursuing 3 aims: (i)detailed characterisation of the Enterobacter-containing vacuole(EcV) in macrophages by state-of-the-art confocal microscopy. (ii)identification of genes required for intramacrophage survival by high-throughput screens(TnSeq) and a candidate gene-based approach by targeted gene deletions and functional analyses. (iii)elucidating how macrophages respond to intracellular Enterobacter infection and how is their cell death downregulated using metabolic analyses, FACS and global transcriptomics. All experiments will involve relevant clinical isolates of different Enterobacter species obtained from the British Society for Antimicrobial Chemotherapy(BSAC) collection and primary human macrophages from blood donors. Timeliness and Impact Our research has implications in the clinical outcome of patients that cannot readily clear Enterobacter infections, which can potentially lead to prolonged intracellular survival and infection relapse. We anticipate our research will lead to the discovery of bacterial gene targets and macrophage pathways that can be exploited to design host-directed therapeutic interventions to complement conventional antimicrobial treatments.