Skeletal muscle mitochondrial dysfunction mediated by Pseudomonas aeruginosa quorum-sensing transcription factor MvfR: reversing effects with anti-MvfR and mitochondrial-targeted compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum-sensing transcription factor MvfR (multiple virulence factor regulator) by interrogating, 5 days post-infection, its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs adenosine triphosphate generation, oxidative phosphorylation, and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR-mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients. IMPORTANCE: Skeletal muscle, pivotal for many functions in the human body, including breathing and protecting internal organs, contains abundant mitochondria essential for maintaining cellular homeostasis during infection. The effect of Pseudomonas aeruginosa (PA) infections on skeletal muscle remains poorly understood. Our study delves into the role of a central quorum-sensing transcription factor, multiple virulence factor regulator (MvfR), that controls the expression of multiple acute and chronic virulence functions that contribute to the pathogenicity of PA. The significance of our study lies in the role of MvfR in the metabolic perturbances linked to mitochondrial functions in skeletal muscle and the effectiveness of the novel MvfR inhibitor and the mitochondrial-targeted peptide SS-31 in alleviating the mitochondrial disturbances caused by PA in skeletal muscle. Inhibiting MvfR or interfering with its effects can be a potential therapeutic strategy to curb PA virulence.

Skeletal Muscle Mitochondrial Dysfunction Mediated by Pseudomonas aeruginosa Quorum Sensing Transcription Factor MvfR: Reversing Effects with Anti-MvfR and Mitochondrial-Targeted Compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum sensing (QS) transcription factor MvfR by interrogating five days post-infection its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs ATP generation, oxidative phosphorylation (OXPHOS), and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR- mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients.

Quorum-Sensing Signaling Molecule 2-Aminoacetophenone Mediates the Persistence of Pseudomonas aeruginosa in Macrophages by Interference with Autophagy through Epigenetic Regulation of Lipid Biosynthesis.

Macrophages are crucial components of the host's defense against pathogens. Recent studies indicate that macrophage functions are influenced by lipid metabolism. However, knowledge of how bacterial pathogens exploit macrophage lipid metabolism for their benefit remains rudimentary. We have shown that the Pseudomonas aeruginosa MvfR-regulated quorum-sensing (QS) signaling molecule 2-aminoacetophenone (2-AA) mediates epigenetic and metabolic changes associated with this pathogen's persistence in vivo. We provide evidence that 2-AA counteracts the ability of macrophages to clear the intracellular P. aeruginosa, leading to persistence. The intracellular action of 2-AA in macrophages is linked to reduced autophagic functions and the impaired expression of a central lipogenic gene, stearoyl-CoA desaturase 1 (Scd1), which catalyzes the biosynthesis of monounsaturated fatty acids. 2-AA also reduces the expression of the autophagic genes Unc-51-like autophagy activating kinase 1 (ULK1) and Beclin1 and the levels of the autophagosomal membrane protein microtubule-associated protein 1, light chain 3 isoform B (LC3B) and p62. Reduced autophagy is accompanied by the reduced expression of the lipogenic gene Scd1, preventing bacterial clearance. Adding the SCD1 substrates palmitoyl-CoA and stearoyl-CoA increases P. aeruginosa clearance by macrophages. The impact of 2-AA on lipogenic gene expression and autophagic machinery is histone deacetylase 1 (HDAC1) mediated, implicating the HDAC1 epigenetic marks at the promoter sites of Scd1 and Beclin1 genes. This work provides novel insights into the complex metabolic alterations and epigenetic regulation promoted by QS and uncovers additional 2-AA actions supporting P. aeruginosa sustainment in macrophages. These findings may aid in designing host-directed therapeutics and protective interventions against P. aeruginosa persistence. IMPORTANCE This work sheds new light on how P. aeruginosa limits bacterial clearance in macrophages through 2-aminoacetophenone (2-AA), a secreted signaling molecule by this pathogen that is regulated by the quorum-sensing transcription factor MvfR. The action of 2-AA on the lipid biosynthesis gene Scd1 and the autophagic genes ULK1 and Beclin1 appears to secure the reduced intracellular clearance of P. aeruginosa by macrophages. In support of the 2-AA effect on lipid biosynthesis, the ability of macrophages to reduce the intracellular P. aeruginosa burden is reinstated following the supplementation of palmitoyl-CoA and stearoyl-CoA. The 2-AA-mediated reduction of Scd1 and Beclin1 expression is linked to chromatin modifications, implicating the enzyme histone deacetylase 1 (HDAC1), thus opening new avenues for future strategies against this pathogen's persistence. Overall, the knowledge obtained from this work provides for developing new therapeutics against P. aeruginosa.

Interconnections of Pseudomonas aeruginosa Quorum-Sensing Systems in Intestinal Permeability and Inflammation.

Quorum sensing (QS) is a highly conserved microbial communication mechanism based on the production and sensing of secreted signaling molecules. The recalcitrant pathogen Pseudomonas aeruginosa is a problematic nosocomial pathogen with complex interconnected QS systems controlling multiple virulence functions. The relevance of QS in P. aeruginosa pathogenesis is well established; however, the regulatory interrelationships of the three major QS systems, LasR/LasI, MvfR (PqsR)/PqsABCD, and RhlR/RhlI, have been studied primarily in vitro. It is, therefore, unclear how these relationships translate to the host environment during infection. Here, we use a collection of P. aeruginosa QS mutants of the three major QS systems to assess the interconnections and contributions in intestinal inflammation and barrier function in vivo. This work reveals that MvfR, not LasR or RhlR, promotes intestinal inflammation during infection. In contrast, we find that P. aeruginosa-driven murine intestinal permeability is controlled by an interconnected QS network involving all three regulators, with MvfR situated upstream of LasR and RhlR. This study demonstrates the importance of understanding the interrelationships of the QS systems during infection and provides critical insights for developing successful antivirulence strategies. Moreover, this work provides a framework to interrogate QS systems in physiologically relevant settings. IMPORTANCE Pseudomonas aeruginosa is a common multidrug-resistant bacterial pathogen that seriously threatens critically ill and immunocompromised patients. Intestinal colonization by this pathogen is associated with elevated mortality rates. Disrupting bacterial communication is a desirable anti-infective approach since these systems coordinate multiple acute and chronic virulence functions in P. aeruginosa. Here, we investigate the role of each of the three major communication systems in the host intestinal functions. This work reveals that P. aeruginosa influences intestinal inflammation and permeability through distinct mechanisms.

A PREVENTIVE TOOL FOR PREDICTING BLOODSTREAM INFECTIONS IN CHILDREN WITH BURNS.

ABSTRACT: Introduction: Despite significant advances in pediatric burn care, bloodstream infections (BSIs) remain a compelling challenge during recovery. A personalized medicine approach for accurate prediction of BSIs before they occur would contribute to prevention efforts and improve patient outcomes. Methods: We analyzed the blood transcriptome of severely burned (total burn surface area [TBSA] ≥20%) patients in the multicenter Inflammation and Host Response to Injury ("Glue Grant") cohort. Our study included 82 pediatric (aged <16 years) patients, with blood samples at least 3 days before the observed BSI episode. We applied the least absolute shrinkage and selection operator (LASSO) machine-learning algorithm to select a panel of biomarkers predictive of BSI outcome. Results: We developed a panel of 10 probe sets corresponding to six annotated genes ( ARG2 [ arginase 2 ], CPT1A [ carnitine palmitoyltransferase 1A ], FYB [ FYN binding protein ], ITCH [ itchy E3 ubiquitin protein ligase ], MACF1 [ microtubule actin crosslinking factor 1 ], and SSH2 [ slingshot protein phosphatase 2 ]), two uncharacterized ( LOC101928635 , LOC101929599 ), and two unannotated regions. Our multibiomarker panel model yielded highly accurate prediction (area under the receiver operating characteristic curve, 0.938; 95% confidence interval [CI], 0.881-0.981) compared with models with TBSA (0.708; 95% CI, 0.588-0.824) or TBSA and inhalation injury status (0.792; 95% CI, 0.676-0.892). A model combining the multibiomarker panel with TBSA and inhalation injury status further improved prediction (0.978; 95% CI, 0.941-1.000). Conclusions: The multibiomarker panel model yielded a highly accurate prediction of BSIs before their onset. Knowing patients' risk profile early will guide clinicians to take rapid preventive measures for limiting infections, promote antibiotic stewardship that may aid in alleviating the current antibiotic resistance crisis, shorten hospital length of stay and burden on health care resources, reduce health care costs, and significantly improve patients' outcomes. In addition, the biomarkers' identity and molecular functions may contribute to developing novel preventive interventions.

Tackling recalcitrant Pseudomonas aeruginosa infections in critical illness via anti-virulence monotherapy.

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

The Role of Common Solvents against Pseudomonas aeruginosa-Induced Pathogenicity in a Murine Burn Site Infection Model.

Dimethyl sulfoxide (DMSO) and polyethylene glycols (PEGs) are frequently used as potent excipients in pharmaceutical formulations. However, these agents also have an interesting antimicrobial and anti-inflammatory profile that could interfere with the efficacy testing of anti-infective compounds when the latter are solubilized in DMSO or PEGs. Here, we demonstrate the antimicrobial and anti-inflammatory effects of DMSO-PEG400 in a murine Pseudomonas aeruginosa infection model, aiming to draw attention to the appropriate selection of solvents for difficult-to-solubilize anti-infectives. IMPORTANCE Our study demonstrates the antimicrobial and anti-inflammatory effects of the combination of DMSO and PEG400 against Pseudomonas aeruginosa in vitro and in vivo in a murine infection model of heightened intestinal permeability. The aim of this study is to draw attention to the appropriate selection of solvents for difficult-to-solubilize anti-infective compounds, to avoid interference with the assay or system tested. This is an extremely important consideration, since potential antimicrobial and anti-inflammatory effects of the solvent vehicle are detrimental to research studies on the efficacy of new anti-infective agents, given that the vehicle effect can mask the effect of the tested compounds. Our results can therefore be of great value to the scientific community, as they can guide researchers in the future to avoid this significant pitfall that can cost substantial amounts of money and valuable time during investigations of the effects of novel, difficult-to-solubilize antimicrobial compounds.

Antimicrobial Peptide Dendrimers and Quorum-Sensing Inhibitors in Formulating Next-Generation Anti-Infection Cell Therapy Dressings for Burns.

Multidrug resistance infections are the main cause of failure in the pro-regenerative cell-mediated therapy of burn wounds. The collagen-based matrices for delivery of cells could be potential substrates to support bacterial growth and subsequent lysis of the collagen leading to a cell therapy loss. In this article, we report the development of a new generation of cell therapy formulations with the capacity to resist infections through the bactericidal effect of antimicrobial peptide dendrimers and the anti-virulence effect of anti-quorum sensing MvfR (PqsR) system compounds, which are incorporated into their formulation. Anti-quorum sensing compounds limit the pathogenicity and antibiotic tolerance of pathogenic bacteria involved in the burn wound infections, by inhibiting their virulence pathways. For the first time, we report a biological cell therapy dressing incorporating live progenitor cells, antimicrobial peptide dendrimers, and anti-MvfR compounds, which exhibit bactericidal and anti-virulence properties without compromising the viability of the progenitor cells.

Characterization of antibiotic resistance profiles in Pseudomonas aeruginosa isolates from burn patients.

OBJECTIVE: To investigate the prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) producing extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria. METHODS: Between April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR). RESULTS: Among the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for six groups of bla genes encoding ESBL-type enzymes, namely blaCTX-M2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Out of the 28 ESBL-producing strains, 23 (82.14%) were blaCTX-M2 positive; 18 (38.29%) were blaPER positive, and 16 (34.04%) were blaTEM positive, while 5 (17.9%) were co-harboring blaCTX-M2, blaTEM, and blaPER genes. The blaSHV, blaVEB, and blaGES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the blaNDM-1, blaIMP, blaVIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the oprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 11 distinct PA clones among the blaCTX-M2 positive strains. CONCLUSION: This is the first study to report the presence of CTX-M2-producing PA in the North Africa region and the first to detect blaCTX-M2-positive and blaPER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains to this part of the world. Identification of bacterial genotypic alterations that confer antibiotic resistance is critical in determining the most effective antimicrobial strategies to be employed. Therefore, our findings could potentially facilitate clinical decision making regarding the antibiotics of choice for the treatment of burn patients that suffer from PA infections in Algeria.

Targeting the gut to prevent sepsis from a cutaneous burn.

Severe burn injury induces gut barrier dysfunction and subsequently a profound systemic inflammatory response. In the present study, we examined the role of the small intestinal brush border enzyme, intestinal alkaline phosphatase (IAP), in preserving gut barrier function and preventing systemic inflammation after burn wound infection in mice. Mice were subjected to a 30% total body surface area dorsal burn with or without intradermal injection of Pseudomonas aeruginosa. Mice were gavaged with 2000 units of IAP or vehicle at 3 and 12 hours after the insult. We found that both endogenously produced and exogenously supplemented IAP significantly reduced gut barrier damage, decreased bacterial translocation to the systemic organs, attenuated systemic inflammation, and improved survival in this burn wound infection model. IAP attenuated liver inflammation and reduced the proinflammatory characteristics of portal serum. Furthermore, we found that intestinal luminal contents of burn wound-infected mice negatively impacted the intestinal epithelial integrity compared with luminal contents of control mice and that IAP supplementation preserved monolayer integrity. These results indicate that oral IAP therapy may represent an approach to preserving gut barrier function, blocking proinflammatory triggers from entering the portal system, preventing gut-induced systemic inflammation, and improving survival after severe burn injuries.

Multi-Biomarker Prediction Models for Multiple Infection Episodes Following Blunt Trauma.

Severe trauma predisposes patients to multiple independent infection episodes (MIIEs), leading to augmented morbidity and mortality. We developed a method to identify increased MIIE risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections' detection after their establishment. Applying machine learning algorithms to genome-wide transcriptome data from 128 adult blunt trauma patients' (42 MIIE cases and 85 non-cases) leukocytes collected ≤48 hr of injury and ≥3 days before any infection, we constructed a 15-transcript and a 26-transcript multi-biomarker panel model with the least absolute shrinkage and selection operator (LASSO) and Elastic Net, respectively, which accurately predicted MIIE (Area Under Receiver Operating Characteristics Curve [AUROC] [95% confidence intervals, CI]: 0.90 [0.84-0.96] and 0.92 [0.86-0.96]) and significantly outperformed clinical models. Gene Ontology and network analyses found various pathways to be relevant. External validation found our model to be generalizable. Our unique precision medicine approach can be applied to a wide range of patient populations and outcomes.

Denver and Marshall scores successfully predict susceptibility to multiple independent infections in trauma patients.

Trauma patients are at risk of repeated hospital-acquired infections, however predictive scores aiming to identify susceptibility to such infections are lacking. The objective of this study was to investigate whether commonly employed disease-severity scores can successfully predict susceptibility to multiple independent infectious episodes (MIIEs) among trauma patients. A secondary analysis of data derived from the prospective, longitudinal study "Inflammation and the Host Response to Injury" ("Glue Grant") was performed. 1,665 trauma patients, older than 16, were included. Patients who died within seven days from the time of injury were excluded. Five commonly used disease-severity scores [Denver, Marshall, Acute Physiology and Chronic Health Evaluation II (APACHE II), Injury Severity Score (ISS), and New Injury Severity Score (NISS)] were examined as independent predictors of susceptibility to MIIEs. The latter was defined as two or more independent infectious episodes during the index hospital stay. Multivariable logistic regression was used for the statistical analysis. 22.58% of the population was found to be susceptible to MIIEs. Denver and Marshall scores were highly predictive of the MIIE status. For every 1-unit increase in the Denver or the Marshall score, there was a respective 15% (Odds Ratio:1.15; 95% CI: 1.07-1.24; p < 0.001) or 16% (Odds Ratio:1.16; 95% CI: 1.09-1.24; p < 0.001) increase in the odds of MIIE occurrence. APACHE II, ISS, and NISS were not independent predictors of susceptibility to MIIEs. In conclusion, the Denver and Marshall scores can reliably predict which trauma patients are prone to MIIEs, prior to any clinical sign of infection. Early identification of these individuals would potentially allow the implementation of rapid, personalized, preventative measures, thus improving patient outcomes and reducing healthcare costs.

Intestinal alkaline phosphatase targets the gut barrier to prevent aging.

Gut barrier dysfunction and gut-derived chronic inflammation play crucial roles in human aging. The gut brush border enzyme intestinal alkaline phosphatase (IAP) functions to inhibit inflammatory mediators and also appears to be an important positive regulator of gut barrier function and microbial homeostasis. We hypothesized that this enzyme could play a critical role in regulating the aging process. We tested the role of several IAP functions for prevention of age-dependent alterations in intestinal homeostasis by employing different loss-of-function and supplementation approaches. In mice, there is an age-related increase in gut permeability that is accompanied by increases in gut-derived portal venous and systemic inflammation. All these phenotypes were significantly more pronounced in IAP-deficient animals. Oral IAP supplementation significantly decreased age-related gut permeability and gut-derived systemic inflammation, resulted in less frailty, and extended lifespan. Furthermore, IAP supplementation was associated with preserving the homeostasis of gut microbiota during aging. These effects of IAP were also evident in a second model system, Drosophilae melanogaster. IAP appears to preserve intestinal homeostasis in aging by targeting crucial intestinal alterations, including gut barrier dysfunction, dysbiosis, and endotoxemia. Oral IAP supplementation may represent a novel therapy to counteract the chronic inflammatory state leading to frailty and age-related diseases in humans.

Associations between clinical characteristics and the development of multiple organ failure after severe burns in adult patients.

To determine the association between potential risk factors and multiple organ failure (MOF) in severe burn adult patients, we performed a secondary analysis of data from the "Inflammation and the Host Response to Injury" database, which included patients from six burn centers in the United States between 2003 and 2009. Three hundred twenty-two adult patients (aged ≥16 years) with severe burns (≥20.0% total body surface area [TBSA]) were included. MOF was defined according to the Denver score. Potential risk factors were analyzed for their association with MOF. Models were built using multivariable logistic regression analysis. Eighty-eight patients (27.3%) developed MOF during the study period. We found that TBSA, age, and inhalation injury were significant risk factors for MOF. This predictive model showed good performance, with the total area under the receiver operating characteristic curve being 0.823. Moreover, among patients who developed MOF, inhalation injury was significantly associated with the development of MOF in the acute phase (within three days of injury) (adjusted odds ratio 3.1; 95% confidence interval 1.1-8.3). TBSA, age, lactate, and Denver score within 24h were associated with the late phase development of MOF. Thus, we have identified key risk factors for the onset of MOF after severe burn injury. Our findings contribute to the understanding of individualized treatment and will potentially allow for efficient allocation of resources and a lower threshold for admission to an intensive care unit, which can prevent the development of MOF and eventually reduce mortality.

Pseudomonas aeruginosa Quorum Sensing Molecule Alters Skeletal Muscle Protein Homeostasis by Perturbing the Antioxidant Defense System.

Skeletal muscle function is compromised in many illnesses, including chronic infections. The Pseudomonas aeruginosa quorum sensing (QS) signal, 2-amino acetophenone (2-AA), is produced during acute and chronic infections and excreted in human tissues, including the lungs of cystic fibrosis patients. We have shown that 2-AA facilitates pathogen persistence, likely via its ability to promote the formation of bacterial persister cells, and that it acts as an interkingdom immunomodulatory signal that epigenetically reprograms innate immune functions. Moreover, 2-AA compromises muscle contractility and impacts the expression of genes involved in reactive oxygen species (ROS) homeostasis in skeletal muscle and in mitochondrial functions. Here, we elucidate the molecular mechanisms of 2-AA's impairment of skeletal muscle function and ROS homeostasis. Murine in vivo and differentiated C2C12 myotube cell studies showed that 2-AA promotes ROS generation in skeletal muscle via the modulation of xanthine oxidase (XO) activity, NAD(P)H oxidase2 (NOX2) protein level, and the activity of antioxidant enzymes. ROS accumulation triggers the activity of AMP-activated protein kinase (AMPK), likely upstream of the observed locations of induction of ubiquitin ligases Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx), and induces autophagy-related proteins. The protein-level perturbation in skeletal muscle of silent mating type information regulation 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), and uncoupling protein 3 (UCP3) is rescued by the antioxidant N-acetyl-l-cysteine (NAC). Together, these results unveil a novel form of action of a QS bacterial molecule and provide molecular insights into the 2-AA-mediated skeletal muscle dysfunction caused by P. aeruginosaIMPORTANCEPseudomonas aeruginosa, a bacterium that is resistant to treatment, causes serious acute, persistent, and relapsing infections in humans. There is increasing evidence that bacterial excreted small molecules play a critical role during infection. We have shown that a quorum sensing (QS)-regulated excreted small molecule, 2-AA, which is abundantly produced by P. aeruginosa, promotes persistent infections, dampens host inflammation, and triggers mitochondrial dysfunction in skeletal muscle. QS is a cell-to-cell communication system utilized by bacteria to promote collective behaviors. The significance of our study in identifying a mechanism that leads to skeletal muscle dysfunction, via the action of a QS molecule, is that it may open new avenues in the control of muscle loss as a result of infection and sepsis. Given that QS is a common characteristic of prokaryotes, it is possible that 2-AA-like molecules promoting similar effects may exist in other pathogens.

Targeting bacterial quorum sensing shows promise in improving intestinal barrier function following burn­site infection.

Burn­site infections, commonly due to Pseudomonas aeruginosa, have been associated with deranged intestinal integrity, allowing bacteria and their products to translocate from the gut to the circulatory system. The P. aeruginosa quorum sensing (QS) transcription factor MvfR (PqsR) controls the expression of numerous virulence factors, and the synthesis of several toxic products. However, the role of QS in intestinal integrity alterations, to the best of our knowledge, has not been previously investigated. Using a proven anti­MvfR, anti­virulence agent, the in vivo results of the present study revealed that inhibition of MvfR function significantly decreased Fluorescein Isothiocyanate­Dextran (FITC­Dextran) flow from the intestine to the systemic circulation, diminished bacterial translocation from the intestine to mesenteric lymph nodes (MLNs), and improved tight junction integrity in thermally injured and infected mice. In addition, the MvfR antagonist administration alleviates the intestinal inflammation, as demonstrated by reduced ileal TNF­α and fecal lipocalin­2 concentrations. In addition, it is associated with lower levels of circulating endotoxin and decreased P. aeruginosa dissemination from the burn wound to the ileum. Collectively, these results hold great promise that the inhibition of this QS system mitigates gut hyperpermeability by attenuating the derangement of morphological and immune aspects of the intestinal barrier, suggesting that MvfR function is crucial in the deterioration of intestinal integrity following P. aeruginosa burn­site infection. Therefore, an anti­virulence approach targeting MvfR, could potentially offer a novel therapeutic approach against multi­drug resistant P. aeruginosa infections following thermal injuries. Since this approach is targeting virulence pathways that are non­essential for growth or viability, our strategy is hypothesized to minimize the development of bacterial resistance, and preserve the beneficial enteric microbes, while improving intestinal integrity that is deranged as a result of burn and infection.

Magnetization transfer contrast MRI in GFP­tagged live bacteria.

Green fluorescent protein (GFP) is a widely utilized molecular reporter of gene expression. However, its use in in vivo imaging has been restricted to transparent tissue mainly due to the tissue penetrance limitation of optical imaging. Magnetization transfer contrast (MTC) is a magnetic resonance imaging (MRI) methodology currently utilized to detect macromolecule changes such as decrease in myelin and increase in collagen content. MTC MRI imaging was performed to detect GFP in both in vitro cells and in an in vivo mouse model to determine if MTC imaging could be used to detect infection from Pseudomonas aeruginosa in murine tissues. It was demonstrated that the approach produces values that are protein specific and concentration dependent. This method provides a valuable, non­invasive imaging tool to study the impact of novel antibacterial therapeutics on bacterial proliferation and perhaps viability within the host system, and could potentially suggest the modulation of bacterial gene expression within the host when exposed to such compounds.

Effects of a small, volatile bacterial molecule on Pseudomonas aeruginosa bacteria using whole cell high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and genomics.

In the present study, high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was applied to live Pseudomonas aeruginosa (PA) bacterial cells to determine the metabolome of this opportunistic Gram-negative human pathogen, and in particular, its response to the volatile aromatic low molecular weight signaling molecule, 2-aminoacetophenone (2-AA). Multi-dimensional HRMAS NMR is a promising method which may be used to determine the in vivo metabolome of live intact bacterial cells; 2-AA is produced by PA and triggers the emergence of phenotypes that promote chronic infection phenotypes in in vitro and in vivo (animal) models. In the present study, we applied one-dimensional and two-dimensional proton (1H) HRMAS NMR to PA cells which were grown with or without 2-AA in order to examine the associations between metabolites and cellular processes in response to 2-AA. We also compared whole-genome transcriptome profiles of PA cells grown with or without 2-AA and found that 2-AA promoted profound metabolic changes in the PA cells. By comparing the whole-genome transcriptome profiles and metabolomic analysis, we demonstrated that 2-AA profoundly reprogramed the gene expression and metabolic profiles of the cells. Our in vivo 1H HRMAS NMR spectroscopy may prove to be a helpful tool in the validation of gene functions, the study of pathogenic mechanisms, the classification of microbial strains into functional/clinical groups and the testing of anti-bacterial agents.