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The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P . aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P . aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P . aeruginosa ; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P . aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

Modification of the host lipidome via secreted enzymes is an integral, but often overlooked aspect of bacterial pathogenesis. In the current era of prevalent antibiotic resistance, knowledge regarding critical host pathogen lipid interactions has the potential for use in developing novel antibacterial agents. While most studies to date on this matter have focused on specific lipids, or select lipid classes, this provides an incomplete picture. Modern methods of untargeted lipidomics have the capacity to overcome these gaps in knowledge and provide a comprehensive understanding of the role of lipid metabolism in the pathogenesis of infections. In an attempt to determine the role of lipid modifying enzymes produced by staphylococci, we exposed bovine heart lipids, a standardized model for the mammalian lipidome, to spent medium from staphylococcal cultures, and analyzed lipid molecular changes by MS/MSALL shotgun lipidomics. We elucidate distinct effects of different staphylococcal isolates, including 4 clinical isolates of the pathogenic species Staphylococcus aureus, a clinical isolate of the normally commensal species S. epidermidis, and the non-pathogenic species S. carnosus. Two highly virulent strains of S. aureus had a more profound effect on mammalian lipids and modified more lipid classes than the other staphylococcal strains. Our studies demonstrate the utility of the applied untargeted lipidomics methodology to profile lipid changes induced by different bacterial secretomes. Finally, we demonstrate the promise of this lipidomics approach in assessing the specificity of bacterial enzymes for mammalian lipid classes. Our data suggests that there may be a correlation between the bacterial expression of lipid-modifying enzymes and virulence, and could facilitate the guided discovery of lipid pathways required for bacterial infections caused by S. aureus and thereby provide insights into the generation of novel antibacterial agents.

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S . aureus protein, and we propose the annotation S . aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S 66 -A, D 167 -A, S 168 -A, and H 301 -A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S . aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

Arachidonic acid metabolites resulting from the cyclooxygenase (COX), lipoxygenase, and cytochrome P450 oxidase enzymatic pathways play pro- and anti-inflammatory roles in allergic airway inflammation (AAI) and asthma. Expression of COX-2 and soluble epoxide hydrolase (sEH) are elevated in allergic airways and their enzymatic products (e.g., prostaglandins and diols of epoxyeicosatrienoic acids, respectively) have been shown to participate in the pathogenesis of AAI. Here, we evaluated the outcome of inhibiting the COX-2 and sEH enzymatic pathways with a novel dual inhibitor, PTUPB, in -induced AAI. Allergen-challenged mice were administered with 10 or 30 mg/kg of PTUPB, celecoxib (selective COX-2 inhibitor), -TUCB (selective sEH inhibitor) or vehicle daily by gavage and evaluated for various features of AAI. PTUPB and -TUCB at 30 mg/kg, but not celecoxib, inhibited eosinophilic infiltration and significantly increased levels of anti-inflammatory EETs in the lung tissue of allergen-challenged mice. -TUCB significantly inhibited allergen-induced IL-4 and IL-13, while a less pronounced reduction was noted with PTUPB and celecoxib. Additionally, -TUCB markedly inhibited eotaxin-2, an eosinophil-specific chemokine, which was only marginally reduced by PTUPB and remained elevated in celecoxib-treated mice. PTUPB or -TUCB administration reversed allergen-induced reduction in levels of various lipid mediators in the lungs, with only a minimal effect noted with celecoxib. Despite the anti-inflammatory effects, PTUPB or -TUCB did not reduce allergen-induced airway hyperresponsiveness (AHR). However, development of structural changes in the allergic airways, such as mucus hypersecretion and smooth muscle hypertrophy, was significantly inhibited by both inhibitors. Celecoxib, on the other hand, inhibited only airway smooth muscle hypertrophy, but not mucus hypersecretion. In conclusion, dual inhibition of COX-2 and sEH offers no additional advantage relative to sEH inhibition alone in attenuating various features associated with -induced AAI, while COX-2 inhibition exerts only moderate or no effect on several of these features. Dual sEH/COX-2 inhibition may be useful in treating conditions where eosinophilic inflammation co-exists with pain-associated inflammation.

Diet is major modifiable risk factor for cardiovascular disease that can influence the immune status of the individual and contribute to persistent low-grade inflammation. In recent years, there has been an increased appreciation of the role of polyunsaturated fatty acids (PUFA) in improving immune function and reduction of systemic inflammation via the modulation of pattern recognition receptors (PRR) on immune cells. Extensive research on the use of bioactive lipids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their metabolites have illustrated the importance of these pro-resolving lipid mediators in modulating signaling through PRRs. While their mechanism of action, bioavailability in the blood, and their efficacy for clinical use forms an active area of research, they are found widely administered as marine animal-based supplements like fish oil and krill oil to promote health. The focus of this review will be to discuss the effect of these bioactive fatty acids and their metabolites on immune cells and the resulting inflammatory response, with a brief discussion about modern methods for their analysis using mass spectrometry-based methods.

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S. aureus protein, and we propose the annotation S. aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S.sup.66 -A, D.sup.167 -A, S.sup.168 -A, and H.sup.301 -A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S. aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

The clinical outcome of allogeneic hematopoietic stem cell transplantation (SCT) may be influenced by the metabolic status of the recipient following conditioning, which in turn may enable risk stratification with respect to the development of transplant-associated complications such as graft vs. host disease (GVHD). To better understand the impact of the metabolic profile of transplant recipients on post-transplant alloreactivity, we investigated the metabolic signature of 14 patients undergoing myeloablative conditioning followed by either human leukocyte antigen (HLA)-matched related or unrelated donor SCT, or autologous SCT. Blood samples were taken following conditioning and prior to transplant on day 0 and the plasma was comprehensively characterized with respect to its lipidome and metabolome via liquid chromatography/mass spectrometry (LCMS) and gas chromatography/mass spectrometry (GCMS). A pro-inflammatory metabolic profile was observed in patients who eventually developed GVHD. Five potential pre-transplant biomarkers, 2-aminobutyric acid, 1-monopalmitin, diacylglycerols (DG 38:5, DG 38:6), and fatty acid FA 20:1 demonstrated high sensitivity and specificity towards predicting post-transplant GVHD. The resulting predictive model demonstrated an estimated predictive accuracy of risk stratification of 100%, with area under the curve of the ROC of 0.995. The likelihood ratio of 1-monopalmitin (infinity), DG 38:5 (6.0), and DG 38:6 (6.0) also demonstrated that a patient with a positive test result for these biomarkers following conditioning and prior to transplant will be at risk of developing GVHD. Collectively, the data suggest the possibility that pre-transplant metabolic signature may be used for risk stratification of SCT recipients with respect to development of alloreactivity.

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistance in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within the multilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a Δ mutant unable to assemble the T3SS needle traversed as efficiently as wild-type , while a Δ mutant 'constitutively on' for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes (Δ mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible , but not if the effector genes were also deleted (Δ Δ ). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced -complemented Δ mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of pathogenesis and has relevance to development of therapeutics targeting the T3SS system.

is a Gram-negative opportunistic pathogen able to cause life- and sight-threating infections. Once considered an extracellular pathogen, numerous studies have shown it can survive intracellularly. Previously, we showed that inside cells can diversify into distinct subpopulations in vacuoles and the cytoplasm. Here, we report that the transition from vacuoles to cytoplasm requires collaboration with the extracellular subpopulation, through Ca influx enabled by their type III secretion system (T3SS) translocon pore proteins. Moreover, we show that collaboration among subpopulations can contribute to disseminating intracellular bacteria in a mouse infection model. This study provides the basis for future studies to investigate how cooperation of extracellular and intracellular bacteria within the host may contribute to disease progression and persistence.

There is a lack of biomarkers for pre-kidney transplant immune risk stratification to avoid over- or under-immunosuppression. Since the circulating lipidome is integrally involved in inflammation, we hypothesized that the lipidome may provide biomarkers that are helpful in the prediction of antibody-mediated rejection. We used mass spectrometry to detect the plasma lipidome in samples collected over 1 year post-kidney transplant from a prospective, observational cohort of adult kidney transplant recipients (KTR), classified in two groups, one with antibody mediated rejection (AMR) and the other with stable graft function (SC). We used linear discriminant analysis to generate predictive models of rejection. A lipid-only model generated from samples taken on day of transplant (T1) revealed a seven lipid classifier (lysophosphatidylethanolamine and phosphatidylcholine species) with misclassification rate of 8.9% [AUC = 0.95 (95% CI = 0.84-0.98), R2 = 0.63]. A clinical model [(using donor specific antibody (DSA) and panel reactive antibody (PRA)] was inferior with a misclassification rate of 15.6% [AUC = 0.82 (95% CI = 0.69-0.93), R2 = 0.41]. A combined model using four lipid classifiers and DSA improved the AUC further to 0.98 (95% CI = 0.89-1.0, R2 = 0.83) with a misclassification of only 2.2%. The polyunsaturated phospholipid subspecies that discriminated the two groups were much lower in the AMR group when compared to the SC group. While the lipidomic profile changed significantly among SC patients on serial sampling post-transplant, such changes were not seen in AMR patients. After taking serial lipidomic changes overtime in SC patients in to account, the AMR group still showed sustained decreased levels of specific lipids at the time of AMR. These findings suggest that a lack of anti-inflammatory polyunsaturated phospholipids could identify patients at a higher risk of AMR at the time of transplant.