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Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis , a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI. Catheter-associated urinary tract infections can often lead to secondary bloodstream infections, and catheter-induced bladder inflammation. In this work, authors utilise murine models to probe defective fibrinolysis drives extravascular fibrin formation, potentially predisposing hosts to severe CAUTI.

Background The goal of this study is to determine the presence of platelet dysfunction in patients with traumatic brain injury (TBI). The mechanisms underlying the coagulopathy associated with TBI remain elusive. The question of platelet dysfunction in TBI is unclear. Methods This was a prospective observational study conducted at Memorial Hospital of South Bend, IN, and Denver Health Medical Center, CO. A total of 50 patients sustaining TBI, and not under treatment with anticoagulants or platelet inhibitors, were analyzed utilizing modified thromboelastography (TEG) with platelet mapping (TEG/PM), along with standard coagulation tests. Results Compared to normal controls, patients with severe TBI had a significantly increased percentage of platelet ADP and arachidonic acid (AA) receptor inhibition. Furthermore, the percentage of ADP inhibition distinguished between survivors and non-survivors in patients with TBI (Mann–Whitney test, P  = 0.035). ADP inhibition correlates strongly with severity of TBI (Mann–Whitney test, P  = 0.014), while AA inhibition did not. Conclusion These data indicate that early platelet dysfunction is prevalent after severe TBI, can be measured in a point-of-care setting using TEG/PM, and correlates with mortality. The mechanism responsible for this platelet dysfunction and associated implications for TBI management remains to be defined.

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/-) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

Group A Streptococcus (GAS, Streptococcus pyogenes ) is a Gram-positive human pathogen that employs several secreted and surface-bound virulence factors to manipulate its environment, allowing it to cause a variety of disease outcomes. One such virulence factor is Streptolysin S (SLS), a ribosomally-produced peptide toxin that undergoes extensive post-translational modifications. The activity of SLS has been studied for over 100 years owing to its rapid and potent ability to lyse red blood cells, and the toxin has been shown to play a major role in GAS virulence in vivo . We have previously demonstrated that SLS induces hemolysis by targeting the chloride-bicarbonate exchanger Band 3 in erythrocytes, indicating that SLS is capable of targeting host proteins to promote cell lysis. However, the possibility that SLS has additional protein targets in other cell types, such as keratinocytes, has not been explored. Here, we use bioinformatics analysis and chemical inhibition studies to demonstrate that SLS targets the electroneutral sodium-bicarbonate cotransporter NBCn1 in keratinocytes during GAS infection. SLS induces NF-κB activation and host cytotoxicity in human keratinocytes, and these processes can be mitigated by treating keratinocytes with the sodium-bicarbonate cotransport inhibitor S0859. Furthermore, treating keratinocytes with SLS disrupts the ability of host cells to regulate their intracellular pH, and this can be monitored in real time using the pH-sensitive dye pHrodo Red AM in live imaging studies. These results demonstrate that SLS is a multifunctional bacterial toxin that GAS uses in numerous context-dependent ways to promote host cell cytotoxicity and increase disease severity. Studies to elucidate additional host targets of SLS have the potential to impact the development of therapeutics for severe GAS infections.

Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It has been speculated that PAI-1 may regulate cardiac fibrosis by inactivating urokinase-type plasminogen activator (uPA) and ultimately plasmin (Pm) generation. However, the in vivo role of PAI-1 in inactivating uPA and limiting the generation of Pm during cardiac fibrosis remains to be established. The objective of this study was to determine if the cardioprotective effect of PAI-1 is mediated through its ability to directly regulate urokinase -mediated activation of plasminogen (Pg). An Angiotensin II (AngII)-aldosterone (Ald) infusion mouse model of hypertension was utilised in this study. Four weeks after AngII-Ald infusion, PAI-1-deficient (PAI-1 −/− ) mice developed severe cardiac fibrosis. However, a marked reduction in cardiac fibrosis was observed in PAI-1 −/− /uPA −/− double knockout mice that was associated with reduced inflammation, lower expression levels of TGF-β and proteases associated with tissue remodeling, and diminished Smad2 signaling. Moreover, total ablation of cardiac fibrosis was observed in PAI-1 −/− mice that express inactive plasmin (Pm) but normal levels of zymogen Pg (PAI-1 −/− /Pg S743A/S743A ). Our findings indicate that PAI-1 protects mice from hypertension-induced cardiac fibrosis by inhibiting the generation of active Pm.

The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC-/-/PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and in vivo, topical treatment with activated PC led to mucosal healing and amelioration of colitis. Taken together, these findings demonstrate that the PC pathway is a unique system involved in controlling intestinal homeostasis and inflammation by regulating epithelial barrier function.

A murine genetic model of LDL-cholesterol- (LDL-C-) driven atherosclerosis, based on complete deficiencies of both the LDL-receptor (Ldlr-/-) and key catalytic component of an apolipoprotein B-edisome complex (Apobec1-/-), which converts apoB-100 to apoB-48, has been extensively characterized. These gene deficiencies allow high levels of apoB-100 to be present and inefficiently cleared, thus leading to very high levels of LDL-C in mice on a normal diet. Many key features of atherosclerotic plaques observed in human familial hypercholesterolemia are found in these mice as they are allowed to age through 72 weeks. The general characteristics include the presence of high levels of LDL-C in plasma and macrophage-related fatty streak formation in the aortic tree, which progressively worsens with age. More specifically, plaque found in the aortic sinuses contains a lipid core with relatively high numbers of macrophages and a smooth muscle cell α-actin- and collagen-containing cap, which thins with age. These critical features of plaque progression suggest that the Ldlr-/-/Apobec1-/- mouse line presents a superior model of LDL-C-driven atherosclerosis.

Acute coagulopathy is a serious complication of traumatic brain injury (TBI) and is of uncertain etiology because of the complex nature of TBI. However, recent work has shown a correlation between mortality and abnormal hemostasis resulting from early platelet dysfunction. The aim of the current study was to develop and characterize a rodent model of TBI that mimics the human coagulopathic condition so that mechanisms of the early acute coagulopathy in TBI can be more readily assessed. Studies utilizing a highly reproducible constrained blunt-force brain injury in rats demonstrate a strong correlation with important postinjury pathological changes that are observed in human TBI patients, namely, diminished platelet responses to agonists, especially adenosine diphosphate (ADP), and subarachnoid bleeding. Additionally, administration of a direct thrombin inhibitor, preinjury, recovers platelet functionality to ADP stimulation, indicating a direct role for excess thrombin production in TBI-induced early platelet dysfunction.

Bacteriocins hold unprecedented promise as a largely untapped source of antibiotic alternatives in the age of multidrug resistance. Here, we describe the first approach to systematically design variants of a novel AS-48 bacteriocin homologue, which we have termed safencin AS-48, from Bacillus safensis, to gain insights into engineering improved activity of bacteriocins. A library of synthetic peptides in which systematic amino acid substitutions to vary the periodicity and abundance of polar, acidic, aliphatic, and hydrophobic residues were generated for a total of 96 novel peptide variants of a single bacteriocin candidate. Using this method, we identified nine synthetic safencin (syn-safencin) variants with broad and potent antimicrobial activities with minimal inhibitory concentrations (MIC) as low as 250 nM against E. coli, P. aeruginosa, X. axonopodis, and S. pyogenes with minimal cytotoxicity to mammalian cells. It is anticipated that the strategies we have developed will serve as general guides for tuning the specificity of a given natural bacteriocin compound for therapeutic specificity.