Community-Acquired Methicillin-Resistant Staphylococcus Aureus (CA-MRSA) USA300 Perturbs Acquisition Of Lysosomal Hydrolases And Requires Phagosomal Acidification For Survival In A Human Macrophage Cell Line

Community-acquired Methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA300 is a major cause of invasive drug-resistant skin and soft tissue infections in humans. Although S. aureus is a well-recognized extracellular pathogen, recent reports that USA300 survives inside host macrophages suggest that the intramacrophage environment may be a niche for persistent infection. Intramacrophage survival requires bacteria to avoid destruction in the phagosome; however, mechanisms by which USA300 evades phagosomal defenses are unclear. We examined the fate of the USA300-containing phagosome in human THP-1 macrophages by evaluating phagosomal acidification and maturation, and by testing the impact of phagosomal conditions on bacterial viability. Utilizing confocal microscopy, we discovered that the USA300-containing phagosome acidified rapidly, and colocalized with the late endosome and lysosome protein LAMP-1. Interestingly, significantly fewer phagosomes containing live USA300 associated with lysosomal hydrolyses cathepsin D and β-glucuronidase than those containing dead bacteria, suggesting that USA300 harbors the ability to perturb lysosomal fusion during macrophage infection. We then examined the impact of phagosomal acidification on USA300 intracellular viability and found that inhibition of acidification significantly impairs USA300 survival, as well as negatively impacts virulence gene regulator agr expression. Together, these results suggest that USA300 survives inside macrophages by altering phagolysosome formation, as well as relying on vacuolar acidification as a trigger for virulence

Robotic fluidic coupling and interrogation of multiple vascularized organ chips

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an ???interrogator??? that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood???brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling