Pseudomonas-Derived Pyocyanin Links Oxidative Stress and Keratin 6 Expression to Wound Healing.

Clinical Implications•Infection-driven inflammation can be life threatening if the body fails to clear microbial pathogens.•Bacterial colonization is enhanced in several skin diseases, including epidermolysis bullosa, and aggravates disease severity.•Cytoskeletal keratin 6A is upregulated upon pyocyanin stimulation.•Keratin 6A−derived antimicrobial peptides can control bacterial infections. •Infection-driven inflammation can be life threatening if the body fails to clear microbial pathogens.•Bacterial colonization is enhanced in several skin diseases, including epidermolysis bullosa, and aggravates disease severity.•Cytoskeletal keratin 6A is upregulated upon pyocyanin stimulation.•Keratin 6A−derived antimicrobial peptides can control bacterial infections. The opportunistic pathogen Pseudomonas aeruginosa (PA) causes acute and chronic infections of most epithelial tissues, including the epidermis, in addition to the heart and the CNS, in particular upon tissue damage. In fact, a study found that 56% of patients suffering from epidermolysis bullosa were infected with PA (Levin et al., 2021Levin L.E. Shayegan L.H. Lucky A.W. Hook K.P. Bruckner A.L. Feinstein J.A. et al.Characterization of wound microbes in epidermolysis bullosa: results from the epidermolysis bullosa clinical characterization and outcomes database.Pediatr Dermatol. 2021; 38: 119-124Crossref PubMed Scopus (14) Google Scholar). The ability of PA to cause life-threatening diseases results from its high potential to develop multidrug resistance, to release a range of virulence factors, and to form biofilms (Ruffin and Brochiero, 2019Ruffin M. Brochiero E. Repair process impairment by Pseudomonas aeruginosa in epithelial tissues: major features and potential therapeutic avenues.Front Cell Infect Microbiol. 2019; 9: 182Crossref PubMed Scopus (56) Google Scholar). The blue, reduction−oxidative−active phenazine pyocyanin (PCN) is an important virulence factor in the pathogenesis of PA-triggered lung diseases. Its cytotoxic effects are in part mediated by its ability to form ROS such as superoxide and hydrogen peroxide (Ruffin and Brochiero, 2019Ruffin M. Brochiero E. Repair process impairment by Pseudomonas aeruginosa in epithelial tissues: major features and potential therapeutic avenues.Front Cell Infect Microbiol. 2019; 9: 182Crossref PubMed Scopus (56) Google Scholar). The skin protects the body against various chemical and physical stresses, including microbial pathogens, and has the ability to repair wounds that result from trauma, burns, or genetic disease. Skin damage initiates a complex program that aims at rapid wound closure and includes keratinocytes (KCs), fibroblasts, immune cells, and components of the extracellular matrix (Eming et al., 2014Eming S.A. Martin P. Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation.Sci Transl Med. 2014; 6: 265sr6Crossref PubMed Scopus (1728) Google Scholar). Wound-proximal KCs transiently change their differentiation program, enhance proliferation and migration, and adapt their intercellular and cell−matrix junctions and their cytoskeleton to close the wound. The upregulation of cytoskeletal keratin (K) 6, K16, and K17 represents a major event in the wound healing response (Wang et al., 2018Wang F. Chen S. Liu H.B. Parent C.A. Coulombe P.A. Keratin 6 regulates collective keratinocyte migration by altering cell-cell and cell-matrix adhesion.J Cell Biol. 2018; 217: 4314-4330Crossref PubMed Scopus (54) Google Scholar). Although many lines of evidence show that PA infections negatively affect epithelial integrity and repair processes, the molecular mechanisms by which PA virulence factors affect epidermal wounding and repair remain incompletely understood. In their recent study, Ghatak et al., 2023Ghatak S. Hemann C. Boslett J. Singh K. Sharma A. El Masry M.S. et al.Bacterial pyocianin inducible keratin 6A accelerates closure of epithelial defect under conditions of mitochondrial dysfunction.J Invest Dermatol. 2023; 143: 2052-2064. e5Abstract Full Text Full Text PDF Google Scholar investigate the response of human HaCaT KCs to pyocyanin exposure. Unlike previous studies (Ruffin and Brochiero, 2019Ruffin M. Brochiero E. Repair process impairment by Pseudomonas aeruginosa in epithelial tissues: major features and potential therapeutic avenues.Front Cell Infect Microbiol. 2019; 9: 182Crossref PubMed Scopus (56) Google Scholar), they find increased KC proliferation and migration, in line with accelerated wound closure in monolayer-based wound closure assays in vitro and in diabetic mice in vivo. In line with its importance in wounding and repair, the authors show pyocyanin-mediated mitochondrial stress and upregulation of K6A and provide evidence that K6A enhances KC migration. To unravel the effect of PCN on the cellular proteome, stable isotope labeling by amino acids in cell culture−based mass spectrometry of immortalized human HaCaT KCs was performed. To that end, two cell lines were grown for six passages in media containing heavy (13C, 15N) and light (12C, 14N) isotopes, respectively, followed by PCN or vehicle treatment for 5 days. A subsequent mass spectrometry−based analysis identified 74 upregulated and 59 downregulated proteins. Ingenuity-based pathway analysis revealed upregulated proteins involved in cell metabolism and mitochondrial respiration. Notably, cytoskeletal keratins, including K6A, which represent major proteins in epidermal KCs (Feng et al., 2013Feng X. Zhang H. Margolick J.B. Coulombe P.A. Keratin intracellular concentration revisited: implications for keratin function in surface epithelia.J Invest Dermatol. 2013; 133: 850-853Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), were not reported. Proteins involved in processes such as DNA replication, inflammatory responsive elements, amino acid synthesis, and NADPH through pentose−phosphate shunt pathway were downregulated. Given its well-known role in oxidative stress generation and guided by proteome analysis, the authors first turned to the analysis of mitochondria. Upon entry into cells, PCN stimulates ROS production, in particular superoxide and hydrogen peroxide, inflicting damage on major cell constituents, including mitochondria. As one might predict, elevated ROS lowered the glutathione (GSH)/GSH disulfide (GSSG) ratio in response to PCN exposure. Functional studies revealed a decrease in maximal mitochondrial respiration, accompanied by a 3.5-fold decrease in the intracellular adenosine triphosphate/adenosine diphosphate ratio, which is consistent with mitochondrial protein leak. The increase in the mitochondrial fission regulator DRP1 and the decrease in the fusion mediator OPA1 are in line with more fragmented mitochondria, as revealed by image analysis. Elevated GSSG is subject to the activity of GSSG reductase for the regeneration of GSH at the expense of NADPH. In agreement, PCN treatment caused depletion of reducing equivalents NAD and NADPH. Lower NADPH levels are also consistent with the downregulation of NADPH-regenerating pathways noted in the proteomic analysis. At the same time, nonmitochondrial respiration, including glycolytic activity, was highly increased. The extensive disturbance of cellular reduction−oxidation balance upon the addition of PCN to HaCaT KCs raises the question of whether antioxidant transcription factor NRF2 signaling is involved (Hiebert and Werner, 2023Hiebert P. Werner S. Targeting NRF2 to promote epithelial repair.Biochem Soc Trans. 2023; 51: 101-111Crossref PubMed Scopus (1) Google Scholar). If confirmed, this could also explain the accelerated wound closure noted in diabetic mice and provide a clue toward mechanisms governing the upregulation of K6A, which is a bona fide NRF2 target (Yang et al., 2017Yang L. Fan X. Cui T. Dang E. Wang G. Nrf2 promotes keratinocyte proliferation in psoriasis through up-regulation of keratin 6, keratin 16, and keratin 17.J Invest Dermatol. 2017; 137: 2168-2176Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Several clinical studies reported delayed re-epithelialization of PA-infected wounds (Ruffin and Brochiero, 2019Ruffin M. Brochiero E. Repair process impairment by Pseudomonas aeruginosa in epithelial tissues: major features and potential therapeutic avenues.Front Cell Infect Microbiol. 2019; 9: 182Crossref PubMed Scopus (56) Google Scholar). To selectively investigate the contribution of PCN to wound closure, an in vitro wound closure assay using HaCaT cells grown in monolayer culture was performed. In agreement with increased glycolysis, which is known to promote migration, PCN treatment accelerated in vitro wound closure significantly. The accelerated wound gap closure was largely due to increased cell proliferation, as revealed by 5-ethynyl-2'-deoxyuridine incorporation, which increased from 5% in control to ∼60% in PCN-treated cells. Although faster wound closure at first may come as a surprise, it may be explained by different experimental conditions, such as a two-dimensional culture of immortalized KCs versus three-dimensional skin wounds and the addition of PCN alone compared with a mixture of virulence factors. Unexpectedly, the increase in proliferation is not reflected in the pathway analysis, which rather suggested a decreased proliferation. Future work, using primary KCs grown as skin equivalents in air−liquid culture, might help to resolve the different findings. Successful wound closure also depends on the polarized arrangement of epithelial intercellular junctions. Immunofluorescence-based analysis of tight junction−constituent proteins (ZO-1, ZO-2, claudins 1 and 4, occludin, JAM-A, tricellulin) and of adherens junction proteins (E-cadherin and β-catenin) indicated downregulation of these proteins upon PCN. Assuming that the apparent downregulation of the proteins mentioned earlier might be due to oxidative stress, the authors added catalase or SOD1 to PCN-treated HaCaT cells. This rescued the level of the proteins mentioned earlier, at least by immunofluorescence analysis. Beyond their fundamental role in maintaining tissue architecture, several keratins are crucial for the regulation of inflammation and wound repair (Hobbs et al., 2012Hobbs R.P. Lessard J.C. Coulombe P.A. Keratin intermediate filament proteins - novel regulators of inflammation and immunity in skin.J Cell Sci. 2012; 125: 5257-5258Crossref PubMed Scopus (30) Google Scholar; Roth et al., 2012Roth W. Kumar V. Beer H.D. Richter M. Wohlenberg C. Reuter U. et al.Keratin 1 maintains skin integrity and participates in an inflammatory network in skin through interleukin-18.J Cell Sci. 2012; 125: 5269-5279Crossref PubMed Scopus (126) Google Scholar; Yang et al., 2017Yang L. Fan X. Cui T. Dang E. Wang G. Nrf2 promotes keratinocyte proliferation in psoriasis through up-regulation of keratin 6, keratin 16, and keratin 17.J Invest Dermatol. 2017; 137: 2168-2176Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). K6, K16, and K17 represent hyperproliferation and wound repair−associated keratins (Yang et al., 2017Yang L. Fan X. Cui T. Dang E. Wang G. Nrf2 promotes keratinocyte proliferation in psoriasis through up-regulation of keratin 6, keratin 16, and keratin 17.J Invest Dermatol. 2017; 137: 2168-2176Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), prompting the authors to investigate their state upon PCN treatment. Although not reported in the proteome study, the wound- and disease-associated keratin K6A was upregulated in PCN-treated cells, on the basis of immunofluorescence staining. Surprisingly, migration of K6A-overexpressing KCs was increased, in stark contrast to that of K6A-deficient mouse KCs, which showed enhanced KC migration (Wang et al., 2018Wang F. Chen S. Liu H.B. Parent C.A. Coulombe P.A. Keratin 6 regulates collective keratinocyte migration by altering cell-cell and cell-matrix adhesion.J Cell Biol. 2018; 217: 4314-4330Crossref PubMed Scopus (54) Google Scholar). In addition, overexpression of K6A was also associated with the depletion of apical junctional proteins except for JAM-A. In light of numerous reports characterizing epidermal keratins as inhibitors of migration, the findings reported in this study require more experiments, which should address desmosomes, the major keratin attachment sites at epithelial intercellular junctions. Unfortunately, the distribution and composition of the keratin−desmosome complex, which is crucial for KC cohesion, differentiation, and wound healing (Hatzfeld et al., 2017Hatzfeld M. Keil R. Magin T.M. Desmosomes and intermediate filaments: their consequences for tissue mechanics.Cold Spring Harb Perspect Biol. 2017; 9: a029157Crossref PubMed Scopus (81) Google Scholar), was not examined. Finally, to study the impact of PCN on in vivo wound healing, it was topically applied to wounds in an established diabetic mouse model. Over a course of 12 days, this revealed a twofold faster wound closure upon PCN treatment, accompanied by an elevated K6A immunofluorescence signal. In addition to forming an intracellular cytoskeleton that governs cytoarchitecture and cell migration, regulated proteolysis of K6A by the ubiquitin−proteasome system gives rise to C-terminal glycine-rich fragments that have bactericidal and cytoprotective activities (Chan et al., 2018Chan J.K.L. Yuen D. Too P.H. Sun Y. Willard B. Man D. et al.Keratin 6A reorganization for ubiquitin-proteasomal processing is a direct antimicrobial response.J Cell Biol. 2018; 217: 731-744Crossref PubMed Scopus (20) Google Scholar). Generation and release of these K6A-derived antimicrobial peptides (KAMPs) from corneal KCs is triggered by bacterial ligands and has been shown to alleviate bacterial keratitis (Sun et al., 2023Sun Y. Chan J. Bose K. Tam C. Simultaneous control of infection and inflammation with keratin-derived antibacterial peptides targeting TLRs and co-receptors.Sci Transl Med. 2023; 15eade2909Crossref Scopus (2) Google Scholar). In light of the upregulation of K6A by PCN, it will be highly interesting to investigate whether it remains confined intracellularly or whether KAMPs are produced and released upon PCN. If so, this might explain the improved wound healing upon PCN treatment in vitro and in the diabetic wound mouse model. Niclas Groh: http://orcid.org/0009-0006-6725-5333 Thomas M. Magin: http://orcid.org/0000-0001-7016-2409 The authors state no conflict of interest. Bacterial Pyocyanin Inducible Keratin 6A Accelerates Closure of Epithelial Defect under Conditions of Mitochondrial DysfunctionJournal of Investigative DermatologyVol. 143Issue 10PreviewRepair of epithelial defect is complicated by infection and related metabolites. Pyocyanin (PYO) is one such metabolite that is secreted during Pseudomonas aeruginosa infection. Keratinocyte (KC) migration is required for the closure of skin epithelial defects. This work sought to understand PYO–KC interaction and its significance in tissue repair. Stable Isotope Labeling by Amino acids in Cell culture proteomics identified mitochondrial dysfunction as the top pathway responsive to PYO exposure in human KCs. Full-Text PDF