Disruption of staphylococcal aggregation protects against lethal lung injury
Jaime L. Hook, … , Sunita Bhattacharya, Jahar Bhattacharya
Jaime L. Hook, … , Sunita Bhattacharya, Jahar Bhattacharya
Published March 1, 2018; First published February 12, 2018
Citation Information: J Clin Invest. 2018;128(3):1074-1086. https://doi.org/10.1172/JCI95823.
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Research Article Cell biology Pulmonology

Disruption of staphylococcal aggregation protects against lethal lung injury

Abstract

Infection by Staphylococcus aureus strain USA300 causes tissue injury, multiorgan failure, and high mortality. However, the mechanisms by which the bacteria adhere to, then stabilize on, mucosal surfaces before causing injury remain unclear. We addressed these issues through the first real-time determinations of USA300-alveolar interactions in live lungs. We found that within minutes, inhaled USA300 established stable, self-associated microaggregates in niches at curved, but not at flat, regions of the alveolar wall. The microaggregates released α-hemolysin toxin, causing localized alveolar injury, as indicated by epithelial dye loss, mitochondrial depolarization, and cytosolic Ca2+ increase. Spread of cytosolic Ca2+ through intercellular gap junctions to adjoining, uninfected alveoli caused pulmonary edema. Systemic pretreatment with vancomycin, a USA300-cidal antibiotic, failed to protect mice infected with inhaled WT USA300. However, vancomycin pretreatment markedly abrogated mortality in mice infected with mutant USA300 that lacked the aggregation-promoting factor PhnD. We interpret USA300-induced mortality as having resulted from rapid bacterial aggregation in alveolar niches. These findings indicate, for the first time to our knowledge, that alveolar microanatomy is critical in promoting the aggregation and, hence, in causing USA300-induced alveolar injury. We propose that in addition to antibiotics, strategies for bacterial disaggregation may constitute novel therapy against USA300-induced lung injury.

Authors

Jaime L. Hook, Mohammad N. Islam, Dane Parker, Alice S. Prince, Sunita Bhattacharya, Jahar Bhattacharya

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Figure 2

USA300 MAs are stable and solute impermeable.

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USA300 MAs are stable and solute impermeable. (A–E) Images (A–D) were ob...
(A–E) Images (A–D) were obtained at the indicated times after bacterial instillation. A shows a vehicle-pretreated alveolus (alv), delineated by calcein red (CR) fluorescence in the epithelium (red), that contains an MA (arrow) of GFP-labeled WT USA300 (green) in an alveolar niche. Small clusters of 1–3 bacteria (arrowheads) are shown on the flat alveolar surface. B and C show the dashed region at high magnification. D is a high-magnification view from a different alveolus that was pretreated with CFTR inhibitor. Epithelial fluorescence was digitally removed in B–D. Note, small clusters were removed in C but not D. Scale bars: 20 (A) and 5 (B–D) μm. Bars (E) show group data for bacterial removal 4 hours after bacterial microinstillation. SC, small clusters; Veh, vehicle; CFinh, CFTR inhibitor. Bars: mean ± SEM; n = 3 lungs (10 clusters quantified per lung); *P < 0.05 as indicated using 2-tailed t test. (F) Bars show fluorescence of the indicated microinstilled bacteria. Washout was given 30 minutes after each microinstillation. WT, WT USA300; phnD–, PhnD-deficient USA300. For fourth and fifth bars, WT was preincubated with the indicated antibodies. Bars: mean ± SEM; n = 3 lungs (5 MAs quantified per lung); *P < 0.05 vs. left bar using ANOVA with Bonferroni correction. (G) Confocal images show epithelium (red) of 2 alveolar niches containing MAs (arrows) of microinstilled USA300 (green). USA300 are GFP-labeled WT (top) and calcein green–loaded (CG-loaded) phnD– (bottom). One hour after USA300 instillation, Alexa Fluor (AF; blue) was microinstilled in alveoli. In the right images, MA fluorescence was digitally removed and replaced by outlines. Open arrow highlights Alexa Fluor penetrance of the MA formed by phnD–. Scale bars: 8 μm. Replicated in 3 lungs.