24 research outputs found
Role of Type III secretory effectors EspF and SopB in enteric pathogenesis of Escherichia coli and Salmonella enterica serovar Typhimurium
The EspF protein is translocated into host cells by the type III secretion system of
enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF
sequences differ between EPEC and EHEC serotypes in terms of the number of
SH3-binding polyproline rich repeats and specific residues in these regions as well
as residues in the amino domain involved in cellular localization. In this study we
have compared the capacity of different espF alleles to inhibit: (i) bacterial
phagocytosis by macrophages; (ii) translocation through an M-cell co-culture
system; (iii) uptake by and translocation through cultured bovine epithelial cells.
The espFO157 allele was significantly less effective at inhibiting phagocytosis and
also had reduced capacity to inhibit E. coli translocation through a human-derived in
vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast,
espFO157 was the most effective allele at restricting bacterial uptake into and
translocation through primary epithelial cells cultured from the bovine terminal
rectum, the predominant colonisation site of EHEC O157 in cattle and a site
containing M-like cells. As functional differences could not be simply assigned to
variation in established interactions of EspF with Sorting Nexin 9 and N-WASP,
yeast-2-hybrid screening was used to identify additional host proteins that may
interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was
identified from this screen. Mad2L2 was then demonstrated to interact with EspF
variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays.
While Mad2L2 has been shown to be targeted by the non homologous Shigella
effector protein IpaB to limit epithelial cell turnover, we presume that EspF
interactions with this protein may indicate a similar function to promote EPEC and
EHEC colonization.
The final section of work addressed whether bacterial interactions can actually
induce M-cell differentiation on follicle-associated epithelium. The work focused on
bovine rectal primary cell cultures interacting with Salmonella enterica serovar
Typhimurium. The type III secreted protein, SopB, was required for Salmonella to:
III
(i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii)
transform a subset of epithelial cells to a cell type that phenotypically and
functionally resembles specialized antigen sampling M cells; (iii) induce RANKL
and downstream RelB dependent NFkB signaling. The work suggests that
Salmonella may induce this cellular transformation to promote its invasion and
colonization of intestinal mucosa
Inflammasome Activation in Bovine Peripheral Blood-Derived Macrophages Is Associated with Actin Rearrangement
Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) Interacts with Escherichia coli Effector Protein EspF
Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli are considered emerging zoonotic pathogens of worldwide distribution. The pathogenicity of the bacteria is conferred by multiple virulence determinants, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and effector proteins, including the multifunctional secreted effector protein (EspF). EspF sequences differ between EPEC and EHEC serotypes in terms of the number and residues of SH3-binding polyproline-rich repeats and N-terminal localization sequence. The aim of this study was to discover additional cellular interactions of EspF that may play important roles in E. coli colonization using the Yeast two-hybrid screening system (Y2H). Y2H screening identified the anaphase-promoting complex inhibitor Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) as a host protein that interacts with EspF. Using LUMIER assays, MAD2L2 was shown to interact with EspF variants from EHEC O157:H7 and O26:H11 as well as EPEC O127:H6. MAD2L2 is targeted by the non-homologous Shigella effector protein invasion plasmid antigen B (IpaB) to halt the cell cycle and limit epithelial cell turnover. Therefore, we postulate that interactions between EspF and MAD2L2 serve a similar function in promoting EPEC and EHEC colonization, since cellular turnover is a key method for bacteria removal from the epithelium. Future work should investigate the biological importance of this interaction that could promote the colonization of EPEC and EHEC E. coli in the host
Epidemiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt
Background Salmonella is one of major causes of foodborne outbreaks globally.
This study was conducted to estimate the prevalence, typing and antibiotic
susceptibilities of Salmonella enterica serovars isolated from 41 broiler
chicken farms located in Kafr El-Sheikh Province in Northern Egypt during
2014–2015. The clinical signs and mortalities were observed. Results In total
615 clinical samples were collected from broiler flocks from different organs
(liver, intestinal content and gall bladder). Salmonella infection was
identified in 17 (41%) broiler chicken flocks and 67 Salmonella isolates were
collected. Recovered isolates were serotyped as 58 (86.6%) S. enterica serovar
Typhimurium, 6 (9%) S. enterica serovar Enteritidis and 3 (4.5%) were non-
typable. The significant high mortality rate was observed only in 1-week-old
chicks. sopE gene was detected in 92.5% of the isolates which indicating their
ability to infect humans. All S. enterica serovar Enteritidis isolates were
susceptible to all tested antimicrobials. The phenotypically resistant S.
enterica serovar Typhimurium isolates against ampicillin, tetracycline,
sulphamethoxazole and chloramphenicol were harbouring BlaTEM, (tetA and tetC),
(sul1 and sul3) and (cat1 and floR), respectively. The sensitivity rate of S.
enterica serovar Typhimurium to gentamycin, trimethoprim/sulphamethoxazole and
streptomycin were 100, 94.8, 89.7%, respectively. The silent streptomycin
antimicrobial cassettes were detected in all Salmonella serovars. A class one
integron (dfrA12, orfF and aadA2) was identified in three of S. enterica
serovar Typhimurium strains. Conclusions To the best of our knowledge, this
study considered first report discussing the prevalence, genotyping,
antibiotic susceptibility and public health significance of S. enterica
serovars in broilers farms of different ages in Delta Egypt. Further studies
are mandatory to verify the location of some resistance genes that are within
or associated with the class one integron
Ameliorative effect of heat-killed lactobacillus plantarum L.137 and/or Aloe vera against colitis in mice
Inflammatory bowel disease (IBD) is one of the predominant intestinal diseases associated with chronic inflammation and ulceration of the colon. This study explored the ameliorative effect of Aloe vera extract (Aloe) and/or heat-killed Lactobacillus plantarum L.137 (HK L.137) on dextran sodium sulfate (DSS)-induced colitis in mice. Aloe and/or HK L.137 were supplied for 9 days and the mice were challenged with DSS for 7 days. The DSS group demonstrated bloody diarrhea, colitis of high histologic grade, increased nuclear factor-kappa B (NF-κB) p65, inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α, and decreased IL-10 expression. These alterations were dwindled in DSS-induced mice treated with Aloe and HK L.137 separately. Aloe and HK L.137 together have augmented the therapeutic e_ect of each other. In conclusion, our findings demonstrated that Aloe and/or HK L.137 ameliorated DSS-induced colitis by promoting the secretion of anti-inflammatory cytokines and suppressing pro-inflammatory mediators. This study indicated that A. vera may function synergistically with HK L.137 to confer an e_ective strategy to prevent colitis
Correction: Ameliorative Effect of Heat-Killed Lactobacillus plantarum L.137 and/or Aloe vera against Colitis in Mice (Processes, (2020), 8, 2, (225), 10.3390/pr8020225)
In the original publication [1], there was a mistake in Figure 5 where subfigure 5E was accidentally replaced by an incorrect image. The corrected Figure 5 appears below. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated
The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components
Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively
Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides
A subset of Gram negative bacterial pathogens use a type 3 secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides, telithromycin and subsequently solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-minimum inhibitory (sub-MIC) concentrations, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Pre-incubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin and there was significant preferential killing of E. coli O157 when added to epithelial cells pre-exposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide may traffic back into the bacteria via the T3SS from accumulated levels in epithelial cells. Considering that neither ketolide induces the SOS response, non-toxic members of this class of antibiotic, such as solithromycin, should be considered for future testing and trials in relation to EHEC infections. These antibiotics may also have broader significance for treating other pathogenic bacteria, including intracellular bacteria, that express a T3SS.Fil: Fernández Brando, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. University of Edinburgh; Reino UnidoFil: Yamaguchi, Nao. University of Edinburgh; Reino UnidoFil: Tahoun, Amin. University of Edinburgh; Reino Unido. Kafrelsheikh University; EgiptoFil: McAteer, Sean P.. University of Edinburgh; Reino UnidoFil: Gillespie, Trudi. University of Edinburgh; Reino UnidoFil: Wang, Dai. University of Edinburgh; Reino Unido. Xiamen University; ChinaFil: Argyle, Sally A.. University of Edinburgh; Reino UnidoFil: Palermo, Marina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gally, David L.. University of Edinburgh; Reino Unid
Development Automobile Engine Hood Latch
V diplomové práci se zabývá konstrukčním návrhem automobilového zámku, který má hlavní komponenty v „plastovém“ provedení (rohatka, západka, tělesa). Tyto plastové díly jsem vhodně navrhl a optimalizoval na požadované zatížení. Zároveň srovnávám plastový zámek a ocelový zámek přední kapoty, srovnávám jak cenu zámků, tak hmotnost a zatížení zámků. V krátkosti porovnávám automobilové zámky přední kapoty v současném automobilovém průmyslu. Také se zabývám etapou výroby prototypového vzorku v současnosti.The diploma thesis deals with the design of the car lock, which main components such as claw, pawl and housing are made of plastic. My main goal was to optimized these plastic parts for a certain amount of power. The following part shows a comparison of plastic lock and the steel lock of front bonnet. The following part shows a comparison of the plastic lock and the steel lock of the front bonnet. The comparison is made in terms of the price, weight and load of the locks. The diploma thesis also briefly deals with the comparison of car locks of car locks of front bonnets in the current automotive industry and the stage of prototype sample production nowadays.340 - Katedra výrobních strojů a konstruovánívelmi dobř
Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella
International audienceFlagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response