Actin-Fence Enhancement Protects against Extra-Pulmonary Infection of Pseudomonas Aeruginosa.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology(2022)
Abstract
Lung-endothelial membrane damage underlies acute lung injury (ALI), a major cause of mortality. ALI occurs after extra-pulmonary infection by Gram-negative bacteria. However, damage-inducing bacteria-endothelial interactions are unclear. Endothelial uptake of blood-borne bacteria might induce cell-membrane pores (CMPs), leading to membrane damage. Since the cytoskeletal involvement in this membrane damage remains unclear, we explored the role played by the membrane stabilizing, cell cortical f-actin layer (PMID: 33749665). We induced extra-pulmonary infection in mice, by intraperitoneal (i.p.) inoculation of fluorescent (GFP-labelled) Pseudomonas aeruginosa (GFP-PA) (3.5x10 cfu). In one group (n=5), after 40 hours we carried out real-time confocal microscopy (RCM) of the lung to determine GFP-PA interactions with calcein-red (CR)-loaded lung endothelium, per our reported approaches (PMID: 33749665). In another group, we carried out single-blind assessments of survival in mice given no therapy (n=10), or the actin fence enhancer, TAT-V12Rac1 (PMID: 33749665) by intravenous injection 7 hours after PA inoculation (n=10). RCM of live lung microvessels revealed GFP-PA as green particles attached to the lung-endothelial surface. Therefore, bacteria released from the peritoneum, reached the lung by the blood route. Confocal sections taken through the cell interior revealed internalized bacteria as yellow particles, reflecting the merged fluorescence of green (bacteria) and red (cytosol). Imaging indicated slow loss of CR fluorescence over the time course of an hour. As we reported, this slow loss of cytosolic dye indicates CMP formation (PMID: 22561462). In the survival assay, 80% of the mice died within 3 days of PA inoculation (p<0.01 versus PBS control). By contrast, in the group given TAT-V12Rac1, mortality was markedly less at 30% (p<0.01 versus bacteria alone). We show for the first time, that within 2 days of i.p. inoculation, GFP-PA internalized in lung endothelium, causing CMP formation. A day later there was high mortality that however, could be majorly abrogated by enhancing the endothelial actin fence by therapy with intravenous TAT-V12Rac1. Thus, the CMPs may have promoted endothelial damage, an effect that was mitigated by fence enhancement. We suggest, the actin fence is a major protective factor against bacteria-induced endothelial damage. Actin fence enhancement might be viable therapy against ALI due to septicemia.
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Key words
pseudomonas aeruginosa,infection,protects
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