The opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments. ExoU is a phospholipase which causes destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosain vitro than other antimicrobial classes. Their overuse over the past decade, however, has resulted in the emergence of antibiotic resistance. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered that tobramycin disrupted T3SS expression and reduced both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-t cells at concentrations below the minimal inhibitory concentration (MIC). The fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and did not inhibit and may have increased the cytotoxic effects of ExoS and ExoU.

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Infection from the opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments and lysosomal degradation. ExoU is a potent phospholipase which causes rapid destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitro than other antimicrobial classes. However, their overuse over the past decade has caused alarming rates of antibiotic resistance to emerge. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered tobramycin disrupted T3SS expression and inhibited both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-T cells even at concentrations below the minimal inhibitory concentrations (MIC). Fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and in particular did not subvert the cytotoxic effects of ExoS and ExoU.

In vascular smooth muscle cells (VSMCs), the diffusive second messenger, 3'-5'-cyclic adenosine monophosphate (cAMP), relays crucial blood vessel-widening 'relaxation' signals from surface receptors to downstream effectors. These in turn modulate multiple proteins to relax VSMCs and increase arterial blood flow. Different cAMP-coupled receptors are believed to modulate distinct populations of proteins, leading to variation in the speed, degree and longevity of relaxation in response to different vasodilators, although this has never been fully investigated. In humans, genetic variations in proteins that control cAMP production and degradation are associated with variability in vascular reactivity and susceptibility to hypertension, but mechanistically we know surprisingly little about cAMP signalling pathways in VSMCs. Here we use quantitative phosphoproteomics to map spatial and temporal protein phosphorylation patterns in human coronary smooth muscle cells (HCASMC) in response to different vasodilators. Primary HCASMC were stimulated with either adenosine (10μM), isoprenaline (1μM), calcitonin gene-related peptide (CGRP; 10nM) or epoprostenol (10nM) for 2 mins, 10 mins or 1 hour. Quantitative phosphoproteomics exploiting Tandem Mass Tag (TMT)-based isobaric labelling was used to correlate, in a high-throughput unbiased manner, changes in phosphorylation of all cellular proteins. Four biological replicates were analyzed. Functional enrichment analysis was performed with the DAVID bioinformatics database. 721 phosphopeptides with at least one site of phosphorylation were identified (all time points). 329 of these peptides had a significant p-value (p<0.05) when compared to untreated cells. 90 p-value significant phosphopeptides were identified for adenosine, 72 for isoprenaline, 68 for CGRP and 70 for epoprostenol, with typically 20-30 phosphopeptides shared between different vasodilators. Consistent with vasodilatory function, all agonists phosphorylated proteins associated with actin binding activity at early time points, with a general shift to proteins involved in RNA binding following prolonged exposure. Distinct spatio-temporal phosphorylation patterns were also evident for different vasodilators: For epoprostenol, phosphorylation activity peaked in the nucleus at 2 mins before declining, while nuclear activity induced by all other vasodilators increased over time. Isoprenaline-induced phosphorylation appeared confined to the plasma membrane, cytoskeleton and cytoplasmic regions at all time points, whereas adenosine, CGRP and epoprostanol induced additional activity in the Golgi apparatus, endoplasmic reticulum and mitochondria at 2-10 mins.Our results demonstrate vasodilator-specific signalling compartmentation in HCASMCs, likely controlled by the ‘funnelling’ of cAMP to discrete, localised effectors. Future work should define the role of specific phosphodiesterases in shaping this signalling and determine whether these patterns are Supported by BBSRC Award BB/V002767/1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.