26 research outputs found
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Amino acids other than glutamate affect the expression of the GAD system in Listeria monocytogenes enhancing acid resistance
The Glutamate Decarboxylase (GAD) system is important for survival of L. monocytogenes and other microorganisms under acidic conditions. Environmental conditions influence the function of the GAD system. Until now, the only conditions known to lead to increased transcription of the GAD system are the stationary phase in rich media and anoxic conditions. Previously, we showed that transcription of the GAD system requires unidentified compounds other than glutamate present in rich media. Following a test looking at various compounds we identified for first time that peptone, tryptone and casamino acids activate the GAD system under oxic conditions suggesting that amino acid(s) other than glutamate and/or peptides are important for the above process. The defined medium, where the GAD system is inactive, once it is supplemented with the above compounds results in an active intracellular and extracellular GAD system and increased acid resistance. Through functional genomics we show that these compounds are required for GadD2 activity and although we previously showed that GadD3 is active part of the intracellular GAD system, the supplementation did not activate this gene. The above is explained by the fact that only gadD2 transcription was upregulated by these compounds while the transcription of gadD1 and gadD3 remained unaffected. Together our results show that the L. monocytogenes GadD2 decarboxylase is activated in the presence of amino acids or peptides other than glutamate, a finding that has important implications for acid tolerance and food safety
The Vibrio parahaemolyticus Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis
<p>Abstract</p> <p>Background</p> <p><it>Vibrio parahaemolyticus </it>is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to <it>V. parahaemolyticus</it>, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.</p> <p>Results</p> <p><it>V. parahaemolyticus </it>deployed its TTSS1 to induce activation of the JNK, p38 and ERK MAPK in human epithelial cells. VP1680 was identified as the TTSS1 effector protein responsible for MAPK activation in Caco-2 cells and the activation of JNK and ERK by this protein was important in induction of host cell death. <it>V. parahaemolyticus </it>actively induced IL-8 secretion in a response mediated by TTSS1. A role for VP1680 and for the ERK signalling pathway in the stimulation of IL-8 production in epithelial cells by <it>V. parahaemolyticus </it>was established. Interestingly, TTSS2 inhibited IL-8 mRNA transcription at early stages of interaction between the bacterium and the cell.</p> <p>Conclusions</p> <p>This study demonstrated that <it>V. parahaemolyticus </it>activates the three major MAPK signalling pathways in intestinal epithelial cells in a TTSS1-dependent manner that involves the TTSS1 effector VP1680. Furthermore VP1680 and JNK and ERK activation were needed for maximal cytotoxicity of the bacterium. It was shown that <it>V. parahaemolyticus </it>is a strong inducer of IL-8 secretion and that induction reflects a balance between the effects of TTSS1 and TTSS2. Increases in IL-8 secretion were mediated by TTSS1 and VP1680, and augmented by ERK activation. These results shed light on the mechanisms of bacterial pathogenesis mediated by TTSS and suggest significant roles for MAPK signalling during infection with <it>V. parahaemolyticus</it>.</p
High power, low frequency ultrasound: meniscal tissue interaction and ablation characteristics
This study evaluates high power low frequency ultrasound transmitted via a flat vibrating probe tip as an alternative technology for meniscal debridement in the bovine knee. An experimental force controlled testing rig was constructed using a 20kHz ultrasonic probe suspended vertically from a load cell. Effect of variation in amplitude of distal tip displacement (242-494”m peak-peak) settings and force (2.5-4.5N) on tissue removal rate (TRR) and penetration rate (PR) for fifty-two bovine meniscus samples was analyzed. Temperature elevation in residual meniscus was measured by embedded thermocouples and histological analysis. As amplitude or force increases, there is a linear increase in TRR (Mean: 0.9 to 11.2mg/s) and PR (Mean: 0.08 to 0.73mm/s). Maximum mean temperatures of 84.6°C and 52.3°C were recorded in residual tissue at 2mm and 4mm from the ultrasound probe-tissue interface. There is an inverse relationship between both amplitude and force, and temperature elevation, with higher settings resulting in less thermal damage
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Loss of SigB in Listeria monocytogenes strains EGD-e and 10403S leads to hypersensitivity to hydrogen peroxide in stationary phase under aerobic conditions
SigB is the main stress gene regulator in L. monocytogenes affecting the expression of more than 150 genes and thus contributing in multiple stress resistance. Despite its clear role in most stresses, its role in oxidative stress is uncertain as results accompanying the loss of sigB range from hyperresistance to hypersensitivity. Previously, these differences have been attributed to strain variation. In this study, we show conclusively that in contrast to all other stresses, loss of sigB results in hyperresistance against H2O2 (more than 8 log CFU ml-1 compared to the wild type) in aerobically-grown stationary phase cultures of 10403S and EGD-e.. Furthermore, growth at 30°C resulted in higher resistance to oxidative stress than at 37°C. Oxidative stress resistance seemed to be higher with higher levels of oxygen. Under anaerobic conditions, loss of SigB in 10403S did not affect survival against H2O2 while in EGD-e it resulted in a sensitive phenotype. During exponential phase, minor differences occurred as expected due to the absence of sigB transcription. Catalase tests were performed under all conditions and stronger catalase results corresponded well with higher survival underpinning the important role of catalase in this phenotype. Furthermore, we assessed the catalase activity in protein lysates which corresponded with the catalase tests and survival. In addition, RT-PCR showed no differences in transcription between the wild type and the ÎsigB in various oxidative stress genes. Further investigation of the molecular mechanism behind this phenotype and its possible consequences for the overall phenotype of L. monocytogenes are underway
Mild stress conditions during laboratory culture promote the proliferation of mutations that negatively affect Sigma B activity in Listeria monocytogenes
In Listeria monocytogenes, the full details of how stress signals are integrated into the ÏB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (ÏB). These mutants were tested for acid tolerance, a trait that is known to be under ÏB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking ÏB (ÎsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for ÏB activation, independently of the transposon location. Reduced ÏB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of ÏB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting ÏB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype.This project has received funding from the European Unionâs Horizon 2020 research-and-innovation program under Marie SkĆodowska-Curie grant agreement no. 721456. Jialun Wu was funded by the Department of Agriculture, Food and the Marine (17/F/244)
Different carbon sources result in differential activation of sigma B and stress resistance in Listeria monocytogenes
Listeria monocytogenes is an important food-borne pathogen that is ubiquitous in the environment. It is able to utilize a variety of carbon sources, to produce biofilms on food-processing surfaces and to survive food preservationâassociated stresses. In this study, we investigated the effect of three common carbon sources, namely glucose, glycerol and lactose, on growth and activation of the general stress response Sigma factor, SigB, and corresponding phenotypes including stress resistance. A fluorescent reporter coupled to the promoter of lmo2230, a highly SigB-dependent gene, was used to determine SigB activation via quantitative fluorescence spectroscopy. This approach, combined with Western blotting and fluorescence microscopy, showed the highest SigB activation in lactose grown cells and lowest in glucose grown cells. In line with this observation, lactose grown cells showed the highest resistance to lethal heat and acid stress, the highest biofilm formation, and had the highest adhesion/invasion capacity in Caco-2-derived C2Bbe1 cell lines. Our data suggest that lactose utilisation triggers a strong SigB dependent stress response and this may have implications for the resistance of L. monocytogenes along the food chain
Gene expression analysis in human osteoblasts exposed to dexamethasone identifies altered developmental pathways as putative drivers of osteoporosis
BACKGROUND: Osteoporosis, a disease of decreased bone mineral density represents a significant and growing burden in the western world. Aging population structure and therapeutic use of glucocorticoids have contributed in no small way to the increase in the incidence of this disease. Despite substantial investigative efforts over the last number of years the exact molecular mechanism underpinning the initiation and progression of osteoporosis remain to be elucidated. This has meant that no significant advances in therapeutic strategies have emerged, with joint replacement surgery being the mainstay of treatment. METHODS: In this study we have used an integrated genomics profiling and computational biology based strategy to identify the key osteoblast genes and gene clusters whose expression is altered in response to dexamethasone exposure. Primary human osteoblasts were exposed to dexamethasone in vitro and microarray based transcriptome profiling completed. RESULTS: These studies identified approximately 500 osteoblast genes whose expression was altered. Functional characterization of the transcriptome identified developmental networks as being reactivated with 106 development associated genes found to be differentially regulated. Pathway reconstruction revealed coordinate alteration of members of the WNT signaling pathway, including frizzled-2, frizzled-7, DKK1 and WNT5B, whose differential expression in this setting was confirmed by real time PCR. CONCLUSION: The WNT pathway is a key regulator of skeletogenesis as well as differentiation of bone cells. Reactivation of this pathway may lead to altered osteoblast activity resulting in decreased bone mineral density, the pathological hallmark of osteoporosis. The data herein lend weight to the hypothesis that alterations in developmental pathways drive the initiation and progression of osteoporosis