INTRODUCTION: Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Salmonella Typhimurium and Yersinia enterocolitica remains unknown. METHODS: To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during Salmonella infection. RESULTS: Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon Salmonella infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during Salmonella infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. DISCUSSION: The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during Salmonella infection.
- MeSH
- autofagie MeSH
- deubikvitinasy metabolismus MeSH
- endopeptidasy genetika MeSH
- endozomální třídící komplexy pro transport genetika MeSH
- lidé MeSH
- Salmonella typhimurium metabolismus MeSH
- salmonelóza * MeSH
- thiolesterasa ubikvitinu genetika metabolismus MeSH
- ubikvitinace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.
- MeSH
- cytokiny imunologie metabolismus MeSH
- krční mandle imunologie mikrobiologie MeSH
- lidé MeSH
- maso MeSH
- nemoci prasat imunologie mikrobiologie MeSH
- prasata MeSH
- Salmonella enteritidis metabolismus fyziologie MeSH
- Salmonella typhimurium metabolismus fyziologie MeSH
- salmonelová infekce u zvířat imunologie mikrobiologie MeSH
- Sus scrofa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
BACKGROUND: Within the last decade, macrophages have been shown to be capable of differentiating toward a classically activated phenotype (M1) with a high antimicrobial potential or an alternatively activated phenotype (M2). Some pathogens are capable of interfering with differentiation in order to down-regulate the anti-microbial activity and enhance their survival in the host. RESULTS: To test this ability in Salmonella enterica serovar Typhimurium, we infected porcine alveolar macrophages with wild-type Salmonella Typhimurium and its isogenic mutants devoid of two major pathogenicity islands, SPI-1 and SPI-2. The induction of genes linked with M1 or M2 polarization was determined by quantification of gene expression by RT-qPCR. The ΔSPI-1 mutant induced a high, dose-dependent M1 response but a low M2 response in infected macrophages. On the other hand, wild-type Salmonella Typhimurium induced a low M1 response but a high, dose-dependent M2 response in infected macrophages. The response to ΔSPI-2 mutant infection was virtually the same as the wild-type strain. CONCLUSIONS: We therefore propose that Salmonella Typhimurium DT104 studied here can polarize macrophages towards the less bactericidal M2 phenotype and that this polarization is dependent on the type III secretion system encoded by SPI-1.
- MeSH
- alveolární makrofágy cytologie fyziologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- cytokiny metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- prasata MeSH
- regulace genové exprese u bakterií fyziologie MeSH
- Salmonella typhimurium genetika metabolismus MeSH
- signální transdukce MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The role of MicA (repressing small regulatory non-coding RNAs of two Salmonella porins) was determined in virulence of Salmonella enterica serovar Typhimurium. Transcriptional analysis revealed that the expression of the micA gene is driven by a single σ(E)-dependent promoter, micAp. Its activity increased towards stationary phase; in exponential phase, the activity was induced by several stresses by a DegS-dependent mechanism. Although phenotypic analysis revealed no significant differences between wild-type and the micA mutant strains, in vivo studies showed that this mutant is more virulent in the mouse model.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- bakteriální RNA genetika metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nekódující RNA genetika metabolismus MeSH
- regulace genové exprese u bakterií MeSH
- Salmonella typhimurium genetika metabolismus patogenita MeSH
- salmonelóza mikrobiologie MeSH
- sekvence nukleotidů MeSH
- sigma faktor genetika metabolismus MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression.
- MeSH
- alveolární makrofágy imunologie metabolismus MeSH
- cytokiny genetika metabolismus MeSH
- genomové ostrovy * MeSH
- nemoci prasat imunologie mikrobiologie MeSH
- prasata MeSH
- Salmonella enteritidis genetika MeSH
- Salmonella typhimurium genetika metabolismus MeSH
- salmonelová infekce u zvířat imunologie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529 genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence, but not a total absence of such clones in poultry flocks.
- MeSH
- alantoin metabolismus MeSH
- drůbež MeSH
- kyselina močová metabolismus MeSH
- mnohočetná bakteriální léková rezistence MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nemoci drůbeže mikrobiologie patologie MeSH
- Salmonella enteritidis genetika metabolismus patogenita MeSH
- Salmonella typhimurium genetika metabolismus patogenita MeSH
- salmonelová infekce u zvířat mikrobiologie patologie MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of H(2)O(2) on the induction of ciprofloxacin (CFL) resistant mutants of Salmonella enterica subsp. enterica serovar Typhimurium was evaluated and determinants of CFL resistance in the mutants were analyzed. Factors associated with CFL resistance in H(2)O(2)-induced mutants included (i) mutations in gyrA gene, predominantly (63 %) Asp(87)-->Asn and less (37 %) Ser(83)-->Phe substitutions, (ii) mutations in the regulatory genes of MarRAB or SoxRS or in the individual structural genes of these operons. Such mutations are induced by H(2)O(2) in a much lower extent. Reduced OmpF expression simultaneously with enhanced efflux was detected only in one mutant strain and 20 % of mutant strains had increased CFL efflux from the cells.
- MeSH
- antibakteriální látky farmakologie metabolismus MeSH
- bakteriální léková rezistence MeSH
- bakteriální proteiny genetika metabolismus MeSH
- ciprofloxacin farmakologie metabolismus MeSH
- DNA gyráza genetika metabolismus MeSH
- missense mutace MeSH
- mutageneze MeSH
- peroxid vodíku farmakologie MeSH
- Salmonella typhimurium genetika metabolismus účinky léků MeSH
Using transcriptional promoter fusions, we investigated the expression of selected SPI-1 and SPI-2 genes of Salmonella enterica serovar Typhimurium (S. Typhimurium). Promoters of genes related to the invasion of the epithelial cell (hilA, hilC, hilD, invF, sicA, sopA, sopB and sopE2) were active in... ( view more ) Luria-Bertani (LB) medium and LB with butyrate but were suppressed by bile salts and in glucose minimal (M9) medium. Genes related to S. Typhimurium intracellular survival (phoP, ssrA, ssaB, ssaG, sifA, sifB and pipB) were characterized by their expression in stationary phase in LB and M9 medium. Activity of phoP and ssrA promoters indicated that these might be expressed inside the gut. SPI-1 genes were expressed on the transition to stationary phase while SPI-2 genes were expressed in stationary phase. Among SPI-1 genes, those with regulatory functions preceded in expression the effector genes and sop genes were expressed in the order of sopA, sopB and sopE2, showing hierarchy in the expression of S. Typhimurium virulence genes.
- MeSH
- exprese genu fyziologie genetika imunologie MeSH
- finanční podpora výzkumu jako téma MeSH
- financování vládou MeSH
- genetické techniky trendy využití MeSH
- luciferasy genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí využití MeSH
- promotorové oblasti (genetika) imunologie účinky léků MeSH
- reportérové geny genetika MeSH
- Salmonella typhimurium genetika imunologie metabolismus MeSH
- transkripční faktory Sp genetika MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Od r. 1997 do r. 2000 sa v SR izolovalo S kmeňov Salmonella enterica sérovar Typhimurium DT104 zo sporadických prípadov humánnej salmonelózy. 5 kmeňov bolo multirezistentných a 3 kmene holi citlivé na antibiotiká. Študovali sme vplyv kultivačného média (kompletně Miieller-Hinton, syntetické - Staples) na rast, povrchová hydrofobicitu a motility uvedených izolátov. Hydrofobicita hola hodnotená metódami adherencie na hydrokarbón xylén (BATH), v sofno-agregačnom taste síranu amónneho (SAT) a adherenciou na plast. Nárast testovaných kmeňov za 24 h bol vyšší po kultivácii v kompletnom médiu, ale Salmonely orali vysoký nárast aj v syntetickom médiu, kde jediným zdrojom C a N bol asparagín. Zo študovaných charakteristik, kultivačné prostredie najvýraznejšie ovplyvnilo adherenciu izolátov na xylén, kedy po kultivácii v syntetickom médiu holi všetky izoláty, okrem 2, hydrofóbne. Tiež motility kmeňov hola vyššia po raste v tomto médiu. Naopak kultivácia v kompletnom médiu tieto vlastnosti izolátov potlačila. Kultivačné prostredie neovplyvnilo adherenciu izolátov na plast, a len slabý vplyv sa pozoroval na sofno-agregačné schopnosti študovaných kmeňov.
