The inhibitory effect of LPS on the expression of GPR81 lactate receptor in blood-brain barrier model in vitro.

BACKGROUND: Lipopolysaccharide (LPS) is one of the main constituents of the cell wall of gram-negative bacteria. As an endotoxin, LPS induces neuroinflammation, which is associated with the blood-brain barrier impairment. Lactate is a metabolite with some significant physiological functions within the neurovascular unit/blood-brain barrier (BBB). Accumulation of extracellular and cerebrospinal fluid lactate is a specific feature of bacterial meningitis. However, the role of lactate production, transport, and sensing by lactate receptors GPR81 in the pathogenesis of bacterial neuroinflammation is still unknown. METHODS: In this study, we analyzed effects of LPS on the expression of GPR81 and MCT-1 and proliferation of cerebral endothelial cells in the BBB model in vitro. We used molecular profiling methods to measure the expression of GPR81, MCT-1, IL-1ß, and Ki67 in the cerebral endothelium after treatment with different concentrations of LPS followed by measuring the level of extracellular lactate, transendothelial electric resistance, and permeability of the endothelial cell layer. RESULTS: Our findings showed that exposure to LPS results in neuroinflammatory changes associated with decreased expression of GPR81 and MCT-1 in endothelial cells, as well as overproduction of IL-1ß and elevation of lactate concentrations in the extracellular space in a dose-dependent manner. LPS treatment reduced JAM tight junction protein expression in cerebral endothelial cells and altered BBB structural integrity in vitro. CONCLUSION: The impairment of lactate reception and transport might contribute to the alterations of BBB structural and functional integrity caused by LPS-mediated neuroinflammation.

CagA-mediated pathogenesis of Helicobacter pylori.

Helicobacter pylori has been described as the main etiologic agent of gastric cancer, causing a considerable rate of mortality and morbidity in human population across the world. Although the infection mainly begins asymptomatically, but simply develops to peptic ulcer, chronic gastritis, lymphoma of the gastric mucosa and eventually adenocarcinoma. The major pathological feature of H. pylori infection is due to the activity of the cytotoxin-associated gene A (CagA), a 125-140 kDa protein encoded by the cag pathogenicity island (cagPAI). CagA is also known as the first bacterial onco-protein, ranking the H. pylori-mediated adenocarcinoma as the second most deadly cancer type worldwide. Upon cytoplasmic translocation CagA undergoes interacting with numerous proteins in phosphorylation dependent and independent manners within the gastric epithelial cells. The profound effect of CagA on multiple intracellular pathways causes major consequences such as perturbation of intracellular actin trafficking, stimulation of inflammatory responses and disruption of cellular tight junctions. Such activities of CagA further participate in development of the hummingbird phenotype and gastric cancer. This review is sought to provide a structural and functional analysis of the CagA protein with focus on demonstrating the molecular basis of the mechanism of CagA intracellular translocation and its interaction with intracellular targets.

Determination of the efflux pump-mediated resistance prevalence in Pseudomonas aeruginosa, using an efflux pump inhibitor.

In gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl beta-naphtylamide (PAbetaN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256-0.25 microg/ml) with a fixed concentration of PAbetaN (50 microg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAbetaN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAbetaN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.

The antimicrobial potential of a new derivative of cathelicidin from Bungarus fasciatus against methicillin-resistant Staphylococcus aureus.

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32-128 µg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 µg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.

The Middle Fragment of Helicobacter pylori CagA Induces Actin Rearrangement and Triggers Its Own Uptake into Gastric Epithelial Cells.

Cytotoxin-associated gene product A (CagA) is a major virulence factor secreted by Helicobacter pylori. CagA activity in the gastric epithelium is associated with higher risk of gastric cancer development. Bacterial type IV secretion system (T4SS)-mediated translocation of CagA into the cytosol of human epithelial cells occurs via a poorly understood mechanism that requires CagA interaction with the host membrane lipid phosphatidylserine (PS) and host cell receptor integrin α5ß1. Here we have characterized the isolated recombinant middle fragment of CagA (CagA-M) that contains the positively-charged PS-binding region (aa 613-636) and a putative ß1 integrin binding site, but lacks the EPIYA region, secretion signal peptide and the CagA multimerization motif. We show that CagA-M, when immobilized on latex beads, is capable of binding to, and triggering its own uptake into, gastric epithelial cells in the absence of infection with cagA-positive H. pylori. Using site-directed mutagenesis, fluorescent and electron microscopy, and highly-specific inhibitors, we demonstrate that the cell-binding and endocytosis-like internalization of CagA-M are dependent on (1) binding to PS; (2) ß1 integrin activity; and (3) actin dynamics. Interaction of CagA-M with the host cells is accompanied by the development of long filopodia-like protrusions (macrospikes). This novel morphology is different from the hummingbird phenotype induced by the translocation of full-length CagA. The determinants within CagA-M and within the host that are important for endocytosis-like internalization into host cells are very similar to those observed for T4SS-mediated internalization of full-length CagA, suggesting that the latter may involve an endocytic pathway.

Neuroinflammation and Infection: Molecular Mechanisms Associated with Dysfunction of Neurovascular Unit.

Neuroinflammation is a complex inflammatory process in the central nervous system, which is sought to play an important defensive role against various pathogens, toxins or factors that induce neurodegeneration. The onset of neurodegenerative diseases and various microbial infections are counted as stimuli that can challenge the host immune system and trigger the development of neuroinflammation. The homeostatic nature of neuroinflammation is essential to maintain the neuroplasticity. Neuroinflammation is regulated by the activity of neuronal, glial, and endothelial cells within the neurovascular unit, which serves as a "platform" for the coordinated action of pro- and anti-inflammatory mechanisms. Production of inflammatory mediators (cytokines, chemokines, reactive oxygen species) by brain resident cells or cells migrating from the peripheral blood, results in the impairment of blood-brain barrier integrity, thereby further affecting the course of local inflammation. In this review, we analyzed the most recent data on the central nervous system inflammation and focused on major mechanisms of neurovascular unit dysfunction caused by neuroinflammation and infections.

Conformational analysis of isolated domains of Helicobacter pylori CagA.

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea.

The aim of the current study was to detect mutations in the gyrA gene of quinolone-resistant Salmonella spp. isolates recovered in Tehran, Iran. Between April 2008 and September 2009, 174 Salmonella spp. were collected and assayed for quinolone resistance and detection of gyrA mutations. Isolates identified as Salmonella enterica were tested for susceptibility by the disk diffusion method. Polymerase chain reaction (PCR) amplification and sequencing of the gyrA gene segment encoding the quinolone resistance-determining region (QRDR) were performed for the nalidixic acid-resistant isolates. Amongst the 174 recovered Salmonella spp. isolates, 89 were resistant to nalidixic acid, of which 9 were resistant to enrofloxacin; 10 isolates had reduced susceptibility to nalidixic acid. None of the isolates were resistant to ciprofloxacin, but a single isolate showed reduced susceptibility. Twelve types of amino acid replacement were found in the QRDR region of GyrA, namely the previously described substitutions in positions 83 and 87 as well as five new substitutions Leu41-Pro, Arg47-Ser, Ser111-Thr, Ala118-Thr and Asp147-Gly. Double substitutions in both positions 83 and 87 were not identified. A Gly133-Glu substitution was identified in a single S. enterica serotype Typhi isolate.