Shigella flexneri remodeling and consumption of host lipids during infection.

IMPORTANCE: Bacterial pathogens have vastly distinct sites that they inhabit during infection. This requires adaptation due to changes in nutrient availability and antimicrobial stress. The bacterial surface is a primary barrier, and here, we show that the bacterial pathogen Shigella flexneri increases its surface decorations when it transitions to an intracellular lifestyle. We also observed changes in bacterial and host cell fatty acid homeostasis. Specifically, intracellular S. flexneri increased the expression of their fatty acid degradation pathway, while the host cell lipid pool was significantly depleted. Importantly, bacterial proliferation could be inhibited by fatty acid supplementation of host cells, thereby providing novel insights into the possible link between human malnutrition and susceptibility to S. flexneri.

Shigella flexneri Adapts to Niche-Specific Stresses through Modifications in Cell Envelope Composition and Decoration.

Shigella flexneri is the primary causative agent of worldwide shigellosis. As the pathogen transverses the distinct niches of the gastrointestinal tract it necessitates dynamic adaptation strategies to mitigate host antimicrobials such as dietary fatty acids (FAs) and the bile salt, deoxycholate (DOC). This study investigates the dynamics of the S. flexneri cell envelope, by interrogating adaptations following FA or DOC exposure. We deciphered the effects of FAs and DOC on bacterial membrane fatty acid and lipopolysaccharide (LPS) compositions. We identified novel LPS-based strategies by the pathogen to support resistance to these host compounds. In particular, expression of S. flexneri very-long O antigen (VL-Oag) LPS was found to play a central role in stress mitigation, as VL-Oag protects against antimicrobial FAs, but its presence rendered S. flexneri susceptible to DOC stress. Collectively, this work underpins the importance for S. flexneri to maintain appropriate regulation of cell envelope constituents, in particular VL-Oag LPS, to adequately adapt to diverse stresses during infection.

Penicillin Binding Protein 7/8 Is a Potential Drug Target in Carbapenem-Resistant Acinetobacter baumannii.

Limited therapeutic options dictate the need for new classes of antimicrobials active against carbapenem-resistant Acinetobacter baumannii. Presented data confirm and extend penicillin binding protein 7/8 (PBP 7/8) as a high-value target in the CR A. baumannii strain HUMC1. PBP 7/8 was essential for optimal growth/survival of HUMC1 in ex vivo human ascites and in a rat subcutaneous abscess model; in a mouse pneumonia model, the absence of PBP 7/8 decreased lethality 11-fold. The loss of PBP 7/8 resulted in increased permeability, sensitivity to complement, and lysozyme-mediated bactericidal activity. These changes did not appear to be due to alterations in the cellular fatty acid composition or capsule production. However, a decrease in lipid A and an increase in coccoidal cells and cell aggregation were noted. The compromise of the stringent permeability barrier in the PBP 7/8 mutant was reflected by an increased susceptibility to several antimicrobials. Importantly, expression of ampC was not significantly affected by the loss of PBP 7/8 and serial passage of the mutant strain in human ascites over 7 days did not yield revertants possessing a wild-type phenotype. In summary, these data and other features support PBP 7/8 as a high-value drug target for extensively drug-resistant and CR A. baumannii. Our results guide next-stage studies; the determination that the inactivation of PBP 7/8 results in an increased sensitivity to lysozyme enables the design of a high-throughput screening assay to identify small molecule compounds that can specifically inhibit PBP 7/8 activity.

Characterizing the role of phosphatidylglycerol-phosphate phosphatases in Acinetobacter baumannii cell envelope biogenesis and antibiotic resistance.

The dissemination of multi-drug resistant Acinetobacter baumannii threatens global healthcare systems and necessitates the development of novel therapeutic options. The Gram-negative bacterial cell envelope provides a first defensive barrier against antimicrobial assault. Essential components of this multi-layered complex are the phospholipid-rich membranes. Phosphatidylglycerol phosphate (PGP) phosphatases are responsible for a key step in the biosynthesis of a major phospholipid species, phosphatidylglycerol (PG), but these enzymes have also been implicated in the biogenesis of other cell envelope components. Our bioinformatics analyses identified two putative PGP candidates in the A. baumannii genome, PgpA and PgpB. Phospholipid analyses of isogenic pgpA mutants in two distinct A. baumannii strains revealed a shift in the desaturation levels of phosphatidylethanolamine (PE) phospholipid species, possibly due to the activation of the phospholipid desaturase DesA. We also investigated the impact of the inner membrane phosphatases on other cell envelope components, which revealed a role of PgpB in the maintenance of the A. baumannii peptidoglycan layer, and consequently carbapenem resistance. Collectively, this work provides novel insights into the roles of PGP phosphatases on the global lipidomic landscape of A. baumannii and their interconnectivity with the biogenesis of other cell envelope components. The non-essentiality of these candidates exemplifies metabolic versatility of A. baumannii, which is believed to be key to its success as global pathogen.

Identification of the Shigella flexneri Wzy Domain Modulating WzzpHS-2 Interaction and Detection of the Wzy/Wzz/Oag Complex.

Shigella flexneri implements the Wzy-dependent pathway to biosynthesize the O antigen (Oag) component of its surface lipopolysaccharide. The inner membrane polymerase WzySF catalyzes the repeat addition of undecaprenol-diphosphate-linked Oag (Und-PP-RUs) to produce a polysaccharide, the length of which is tightly regulated by two competing copolymerase proteins, WzzSF (short-type Oag; 10 to 17 RUs) and WzzpHS-2 (very-long-type Oag; >90 RUs). The nature of the interaction between WzySF and WzzSF/WzzpHS-2 in Oag polymerization remains poorly characterized, with the majority of the literature characterizing the individual protein constituents of the Wzy-dependent pathway. Here, we report instead a major investigation into the specific binding interactions of WzySF with its copolymerase counterparts. For the first time, a region of WzySF that forms a unique binding site for WzzpHS-2 has been identified. Specifically, this work has elucidated key WzySF moieties at the N- and C-terminal domains (NTD and CTD) that form an intramolecular pocket modulating the WzzpHS-2 interaction. Novel copurification data highlight that disruption of residues within this NTD-CTD pocket impairs the interaction with WzzpHS-2 without affecting WzzSF binding, thereby specifically disrupting polymerization of longer polysaccharide chains. This study provides a novel understanding of the molecular interaction of WzySF with WzzSF/WzzpHS-2 in the Wzy-dependent pathway and, furthermore, detects the Wzy/Wzz/Und-PP-Oag complex for the first time. Beyond S. flexneri, this work may be extended to provide insight into the interactions between protein homologues expressed by related species, especially members of Enterobacteriaceae, that produce dual Oag chain length determinants. IMPORTANCE Shigella flexneri is a pathogen causing significant morbidity and mortality, predominantly devastating the pediatric age group in developing countries. A major virulence factor contributing to S. flexneri pathogenesis is its surface lipopolysaccharide, which is comprised of three domains: lipid A, core oligosaccharide, and O antigen (Oag). The Wzy-dependent pathway is the most common biosynthetic mechanism implemented for Oag biosynthesis by Gram-negative bacteria, including S. flexneri. The nature of the interaction between the polymerase, WzySF, and the polysaccharide copolymerases, WzzSF and WzzpHS-2, in Oag polymerization is poorly characterized. This study investigates the molecular interplay between WzySF and its copolymerases, deciphering key interactions in the Wzy-dependent pathway that may be extended beyond S. flexneri, providing insight into Oag biosynthesis in Gram-negative bacteria.

Influence of left ventricular unloading on pediatric post-cardiotomy veno-arterial extracorporeal life support outcomes.

Background: The effectiveness of veno-arterial extracorporeal life support (V-A ECLS) in treating neonatal and pediatric patients with complex congenital heart disease (CHD) and requiring cardio-circulatory assistance is well-known. Nevertheless, the influence of left ventricle (LV) distension and its countermeasure, namely LV unloading, on survival and clinical outcomes in neonates and children treated with V-A ECLS needs still to be addressed. Therefore, the aim of this study was to determine the effects of LV unloading on in-hospital survival and complications in neonates and children treated with V-A ECLS. Methods: The clinical outcomes of 90 pediatric patients with CHD under 16 years of age supported with V-A ECLS for post-cardiotomy cardiogenic shock (CS) were retrospectively reviewed in relationship with the presence or absence of an active LV unloading strategy. Results: The patient cohort included 90 patients (age 19.6 ± 31.54 months, 64.4% males), 42 of whom were vented with different techniques (38 with atrial septostomy (AS) or left atria cannula, two with cannula from LV apex, 1 with intra-aortic balloon pump (IABP), and one with pigtail across the aortic valve). The LV unloading strategy significantly increased the in-hospital survival (odds ratio [OR] = 2.74, 95% CI 1.06-7.08; p = 0.037). On the contrary, extracorporeal cardiopulmonary resuscitation decreased the related survival (OR = 0.32, 95% CI 1.09-0.96; p = 0.041). The most common complications were infections (28.8%), neurological injury (26%), and bleeding (25.6%). However, these did not differently occur in venting and no-venting groups. Conclusion: In pediatric patients with CHD supported with V-A ECLS for post-cardiotomy CS, the LV unloading strategy was associated with increased survival.

Microcirculatory Alterations in Critically Ill Patients with COVID-19-Associated Acute Respiratory Distress Syndrome.

BACKGROUND: Presently, a number of specific observations have been performed on microcirculatory function in a coronavirus disease-19 (COVID-19) setting. We hypothesized that, in the critically ill, endothelial dysfunction secondary to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the subsequent inflammation and coagulopathy may lead to microcirculatory alterations, further exacerbated by the hypoxemic state. A dysfunctional microcirculation may represent the hidden motor underlying the development of COVID-19's clinical manifestations. METHODS: A single center, prospective, observational study. We analyzed bedside sublingual microcirculation in twenty-four consecutive COVID-19-associated acute respiratory distress syndrome (ARDS) patients mechanically ventilated in an Intensive Care Unit (ICU), together with macro-hemodynamics, clinical parameters, echocardiography, and laboratory data at a single time-point after ICU admission. All participants were recruited between March and May 2020. RESULTS: The microcirculatory pattern was characterized by increased values of total vessel density and perfused vessel density, a reduced value of proportion of perfused vessels and microvascular flow index, and high values of heterogeneity index. The duration of mechanical ventilation before microcirculation assessment was inversely associated with the proportion of perfused vessels (p = 0.023). Within the macro-hemodynamic parameters, the right ventricle end-diastolic diameter was inversely associated with proportion of perfused vessels and microvascular flow index (p = 0.039 and 0.014, respectively) and directly associated with the heterogeneity index (p = 0.033). CONCLUSIONS: In COVID-19-associated ARDS patients, the microcirculation showed impaired quality of flow parameters coupled with a high vessel density.

Detection of a disulphide bond and conformational changes in Shigella flexneri Wzy, and the role of cysteine residues in polymerase activity.

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein WzySF polymerises Oag repeat units, whilst two co-polymerase proteins, WzzSF and WzzpHS-2, together interact with WzySF to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of WzySF transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of WzySF, or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ WzySF arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe WzySF conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including WzzSF and WzzpHS-2, and extrinsic factors, such as temperature. Site-directed mutagenesis of WzySF cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent analyses highlighted both the structural and functional importance of WzySF cysteines. Substitution of WzySF cysteine residues significantly decreased biosynthesis of the VL-Oag modal length, without disruption to S-Oag production. This phenotype was corroborated in the absence of co-polymerase competition for WzySF interaction. These data suggest WzySF cysteine substitutions directly impair the interaction between Wzy/WzzpHS-2, without altering the Wzy/WzzSF interplay, and in combination with structural data, we propose that the N- and C-termini of WzySF are arranged in close proximity, and together may form the unique WzzpHS-2 interaction site.

Stimulating versus non-stimulating catheter for lumbar plexus continuous infusion after total hip replacement.

BACKGROUND: This study was aimed to investigate whether stimulating catheters for continuous lumbar plexus block reduce local anesthetic consumption after hip arthroplasty if compared with traditional non-stimulating catheters. METHODS: Seventy-two ASA I-III, 18-82-year-old, undergoing primary hip replacement (THA) for osteoarthritis with spinal anesthesia were randomized into two groups: Stim group (stimulating catheter, N.=36) and Nonstim group (non-stimulating catheter, N.=36). After surgery, 15 mL of mepivacaine 1% were administered in both groups through the catheter. An electronic pump was connected to deliver ropivacaine 0.2% (3 mL/h, bolus 3 mL, lock out 15 min) for the first 72 h. Patients were given ketorolac 30 mg IV every 8 h, acetaminophen 1g IV every 8 h and oxycodone 10 mg per os for rescue analgesia. Primary outcome was postoperative local anesthetic consumption. Numerical Rating Scale (NRS), complications, both quadriceps and obturator strength measurements, and opioid requirement were also registered. Mixed effect models (random intercept) were built for repeated measures over time. A difference between groups was considered statistically significant if P<0.05. RESULTS: Local anesthetic consumption and NRS were comparable between groups. Patients in the Nonstim group required significant more rescue opioid analgesia compared with the Stim group during the first 36 h (P=0.002). Quadriceps and adductor muscle strength was equally preserved in the two groups. CONCLUSIONS: The study showed comparable local anesthetic consumption, pain scores and muscle strength preservation between the two groups. The stimulating catheter allowed a significant, although underpowered, reduction in opioid consumption.

The combined effects of the microcirculatory status and cardiopulmonary bypass on platelet count and function during cardiac surgery.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass is associated with important changes in the microcirculation, usually attributed to endothelial dysfunction. Another common finding of cardiac surgery is postoperative thrombocytopenia and platelet loss of function. OBJECTIVE: To investigate the association between microvascular flow pattern and postoperative changes in platelet count and function in cardiac surgery patients. METHODS: Twelve adult cardiac surgery patients received microvascular circulation (sidestream darkfield sublingual mucosa analysis) and platelet count and function (multiple electrode aggregometry ADPtest and TRAPtest) assessment before and after cardiopulmonary bypass. RESULTS: After cardiopulmonary bypass, sublingual microcirculation showed a significantly (P = 0.001) decreased microvascular flow index and increased heterogeneity index (P = 0.006). Platelet function significantly decrease after cardiopulmonary bypass both at ADPtest (P = 0.011) and TRAPtest (P = 0.002). Preoperative patterns of poor microvascular perfusion (low perfused vessels density and total vessels density) were significantly associated with lower values of post-cardiopulmonary bypass platelet function (ADPtest, P = 0.009, TRAPtest, P = 0.031) and count (P = 0.048). CONCLUSIONS: A preoperative disturbance of the microcirculation is associated with a greater postoperative platelet dysfunction. Endothelial damage, chemical and mechanical stimuli are the possible link between the two patterns.