RESUMO
!!NCR1!! presents a great challenge to antimycobacterial therapy due to its innate resistance against most antibiotics. M. abscessus is able to grow intracellularly in human macrophages, suggesting that intracellular models can facilitate drug discovery. Thus, we have developed two host cell models: human macrophages for use in a new high-content screening method for M. abscessus growth and a Dictyostelium discoideum infection model with the potential to simplify downstream genetic analysis of host cell factors. A screen of 568 antibiotics for activity against intracellular M. abscessus led to the identification of two hit compounds with distinct growth inhibition. A collection of 317 human kinase inhibitors was analyzed, with the results yielding three compounds with an inhibitory effect on mycobacterial growth, strengthening the notion that host-directed therapy can be applied for M. abscessus.
Assuntos
Antibacterianos/farmacologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/patogenicidade , Macrófagos/microbiologia , Mycobacterium abscessus/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Células THP-1RESUMO
By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.