RESUMO
Staphylococcus aureus, a versatile Gram-positive bacterium, is the main cause of bone and joint infections (BJI), which are prone to recurrence. The inflammasome is an immune signaling platform that assembles after pathogen recognition. It activates proteases, most notably caspase-1 that proteolytically matures and promotes the secretion of mature IL-1ß and IL-18. The role of inflammasomes and caspase-1 in the secretion of mature IL-1ß and in the defence of S. aureus-infected osteoblasts has not yet been fully investigated. We show here that S. aureus-infected osteoblast-like MG-63 but not caspase-1 knock-out CASP1 -/- MG-63 cells, which were generated using CRISPR-Cas9 technology, activate the inflammasome as monitored by the release of mature IL-1ß. The effect was strain-dependent. The use of S. aureus deletion and complemented phenole soluble modulins (PSMs) mutants demonstrated a key role of PSMs in inflammasomes-related IL-1ß production. Furthermore, we found that the lack of caspase-1 in CASP1 -/- MG-63 cells impairs their defense functions, as bacterial clearance was drastically decreased in CASP1 -/- MG-63 compared to wild-type cells. Our results demonstrate that osteoblast-like MG-63 cells play an important role in the immune response against S. aureus infection through inflammasomes activation and establish a crucial role of caspase-1 in bacterial clearance.
Assuntos
Caspase 1/genética , Caspase 1/imunologia , Inflamassomos/imunologia , Osteoblastos/microbiologia , Staphylococcus aureus/patogenicidade , Sistemas CRISPR-Cas , Linhagem Celular , Deleção de Genes , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Células THP-1RESUMO
Staphylococcus aureus (S. aureus) is a highly versatile Gram-positive bacterium that is carried asymptomatically by up to 30% of healthy people, while being a major cause of healthcare-associated infections, making it a worldwide problem in clinical medicine. The adaptive evolution of S. aureus strains is demonstrated by its remarkable capacity to promptly develop high resistance to multiple antibiotics, thus limiting treatment choice. Nowadays, there is a continuous demand for an alternative to the use of antibiotics for S. aureus infections and a strategy to control the spread or to kill phylogenetically related strains. In this scenario, bacteriocins fit as with a promising and interesting alternative. These molecules are produced by a range of bacteria, defined as ribosomally synthesized peptides with bacteriostatic or bactericidal activity against a wide range of pathogens. This work reviews ascertained the main antibiotic-resistance mechanisms of S. aureus strains and the current, informative content concerning the applicability of the use of bacteriocins overlapping the use of conventional antibiotics in the context of S. aureus infections. Besides, we highlight the possible application of these biomolecules on an industrial scale in future work.
Assuntos
Bacteriocinas , Infecções Estafilocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias Gram-Positivas , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureusRESUMO
The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis.
Assuntos
Toxinas Bacterianas/biossíntese , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Interleucinas/genética , Staphylococcus aureus/patogenicidade , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Bovinos , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica , Teste de Complementação Genética , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Interleucinas/imunologia , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/patologia , Transdução de Sinais , Especificidade da Espécie , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , VirulênciaRESUMO
Staphylococcus aureus is a gram-positive bacterium responsible for a wide range of infections. Host cell cycle alteration is a sophisticated mechanism used by pathogens to hijack the defense functions of host cells. We previously demonstrated that S. aureus MW2 (USA400) bacteria induced a G2/M phase transition delay in HeLa cells. We demonstrate here that this activity is triggered by culture supernatant compounds. Using size exclusion chromatography of the MW2 supernatant, followed by mass spectroscopy analysis of corresponding peaks, we identified phenol-soluble modulin α (PSMα) peptides as the likely candidates for this effect. Indeed, synthetic PSMα1 and PSMα3 caused a G2/M phase transition delay. The implication of PSMα in cell cycle alteration was confirmed by comparison of S. aureus Los Angeles County clone (LAC) wild-type with the isogenic mutant LAC∆psmα, which lacks the psmα operon encoding PSMα1-4. PSMα-induced G2/M transition delay correlated with a decrease in the defensin genes expression suggesting a diminution of antibacterial functions of epithelial cells. By testing the supernatant of S. aureus human clinical isolates, we found that the degree of G2/M phase transition delay correlated with PSMα1 production. We show that PSMs secreted by S. aureus alter the host cell cycle, revealing a newly identified mechanism for fostering an infection.
Assuntos
Toxinas Bacterianas/farmacologia , Meios de Cultivo Condicionados/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fenol/química , Staphylococcus aureus/fisiologia , Western Blotting , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Células HeLa , Humanos , Infecções Estafilocócicas/microbiologia , Espectrometria de Massas em TandemRESUMO
S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Mastite Bovina/microbiologia , Staphylococcus aureus/fisiologia , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Doença Crônica , Feminino , Proteoma , Staphylococcus aureus/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent invasion of bMEC by two S. aureus bovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively. L. casei strains affected adhesion and/or internalization of S. aureus in a strain-dependent manner. Interestingly, L. casei CIRM-BIA 667 reduced S. aureus Newbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two other L. casei strains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate of S. aureus was not affected by L. casei. It should be noted that L. casei was internalized at a low rate but survived in bMEC cells with a better efficiency than that of S. aureus RF122. Inhibition of S. aureus adhesion was maintained with heat-killed L. casei, whereas contact between live L. casei and S. aureus or bMEC was required to prevent S. aureus internalization. This first study of the antagonism of LAB toward S. aureus in a mammary context opens avenues for the development of novel control strategies against this major pathogen.
Assuntos
Antibiose , Células Epiteliais/microbiologia , Lacticaseibacillus casei/fisiologia , Staphylococcus aureus/patogenicidade , Animais , Bovinos , Endocitose , Células Epiteliais/fisiologia , Mastite Bovina/microbiologia , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Introduction: The mechanisms underlying innate immune memory (trained immunity) comprise epigenetic reprogramming of transcriptional pathways associated with alterations of intracellular metabolism. While the mechanisms of innate immune memory carried out by immune cells are well characterized, such processes in non-immune cells, are poorly understood. The opportunistic pathogen, Staphylococcus aureus, is responsible for a multitude of human diseases, including pneumonia, endocarditis and osteomyelitis, as well as animal infections, including chronic cattle mastitis that are extremely difficult to treat. An induction of innate immune memory may be considered as a therapeutic alternative to fight S. aureus infection. Methods: In the current work, we demonstrated the development of innate immune memory in non-immune cells during S. aureus infection employing a combination of techniques including Enzyme-linked immunosorbent assay (ELISA), microscopic analysis, and cytometry. Results: We observed that training of human osteoblast-like MG-63 cells and lung epithelial A549 cells with ß-glucan increased IL-6 and IL-8 production upon a stimulation with S. aureus, concomitant with histones modifications. IL-6 and IL-8 production was positively correlated with an acetylation of histone 3 at lysine 27 (H3K27), thus suggesting epigenetic reprogramming in these cells. An addition of the ROS scavenger N-Acetylcysteine, NAC, prior to ß-glucan pretreatment followed by an exposure to S. aureus, resulted in decreased IL-6 and IL-8 production, thereby supporting the involvement of ROS in the induction of innate immune memory. Exposure of cells to Lactococcus lactis resulted in increased IL-6 and IL-8 production by MG-63 and A549 cells upon a stimulation with S. aureus that was correlated with H3K27 acetylation, suggesting the ability of this beneficial bacterium to induce innate immune memory. Discussion: This work improves our understanding of innate immune memory in non-immune cells in the context of S. aureus infection. In addition to known inducers, probiotics may represent good candidates for the induction of innate immune memory. Our findings may help the development of alternative therapeutic approaches for the prevention of S. aureus infection.
Assuntos
Imunidade Inata , Infecções Estafilocócicas , Feminino , Humanos , Animais , Bovinos , Espécies Reativas de Oxigênio , Staphylococcus aureus , Imunidade Treinada , Interleucina-8 , Interleucina-6RESUMO
Staphylococcus aureus is a major etiological agent of mastitis in ruminants. We report here the genome sequence of bovine strain Newbould 305, isolated in the 1950s in a case of bovine mastitis and now used as a model strain able to reproducibly induce chronic mastitis in cows.
Assuntos
Genoma Bacteriano , Mastite Bovina/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Animais , Bovinos , Feminino , Dados de Sequência Molecular , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Staphylococcus aureus is an opportunistic pathogen that causes a range of devastating diseases including chronic osteomyelitis, which partially relies on the internalization and persistence of S. aureus in osteoblasts. The identification of the mechanisms of the osteoblast response to intracellular S. aureus is thus crucial to improve the knowledge of this infectious pathology. Since the signal from specifically infected bacteria-bearing cells is diluted and the results are confounded by bystander effects of uninfected cells, we developed a novel model of long-term infection. Using a flow cytometric approach we isolated only S. aureus-bearing cells from mixed populations that allows to identify signals specific to intracellular infection. Here we present an in-depth analysis of the effect of long-term S. aureus infection on the transcriptional program of human osteoblast-like cells. After RNA-seq and KEGG and Reactome pathway enrichment analysis, the remodeled transcriptomic profile of infected cells revealed exacerbated immune and inflammatory responses, as well as metabolic dysregulations that likely influence the intracellular life of bacteria. Numerous genes encoding epigenetic regulators were downregulated. The later included genes coding for components of chromatin-repressive complexes (e.g., NuRD, BAHD1 and PRC1) and epifactors involved in DNA methylation. Sets of genes encoding proteins of cell adhesion or neurotransmission were also deregulated. Our results suggest that intracellular S. aureus infection has a long-term impact on the genome and epigenome of host cells, which may exert patho-physiological dysfunctions additionally to the defense response during the infection process. Overall, these results not only improve our conceptual understanding of biological processes involved in the long-term S. aureus infections of osteoblast-like cells, but also provide an atlas of deregulated host genes and biological pathways and identify novel markers and potential candidates for prophylactic and therapeutic approaches.
Assuntos
Osteomielite , Infecções Estafilocócicas , Epigênese Genética , Humanos , Osteomielite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , TranscriptomaRESUMO
Staphylococcus aureus is a major etiological agent of mastitis in ruminants. We report here the genome sequences of two ovine strains that were isolated from gangrenous (strain O11) and subclinical (strain O46) ewe mastitis. Both strains belong to the same clonal complex. Despite this close genotypic relationship, the two isolates were shown to reproducibly induce highly divergent types of infections, either severe (O11) or mild (O46) mastitis, in an experimental ewe model.
Assuntos
Genoma Bacteriano , Mastite/veterinária , Doenças dos Ovinos/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Animais , Feminino , Mastite/microbiologia , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , OvinosRESUMO
Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland.
Assuntos
Proteínas de Bactérias/imunologia , Mastite/veterinária , Proteoma/imunologia , Doenças dos Ovinos/imunologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Animais , Proteínas de Bactérias/genética , Cromatografia Líquida/veterinária , Eletroforese em Gel Bidimensional/veterinária , Feminino , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Mastite/imunologia , Mastite/microbiologia , Camundongos , Proteoma/genética , Proteoma/metabolismo , Testes Sorológicos/veterinária , Ovinos , Doenças dos Ovinos/microbiologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Espectrometria de Massas em Tandem/veterináriaRESUMO
Galactofuranose (Galf) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus. The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the AfUGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.
Assuntos
Aspergillus fumigatus/fisiologia , Adesão Celular , Galactose/análogos & derivados , Galactose/metabolismo , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/ultraestrutura , Linhagem Celular , Células Epiteliais/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactose/biossíntese , Deleção de Genes , Humanos , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Microscopia Eletrônica de Varredura , Micélio/ultraestrutura , Esporos Fúngicos/crescimento & desenvolvimento , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/biossínteseRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
BACKGROUND: Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response. RESULTS: The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1beta antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to A. fumigatus points to the biological significance of described phenomena. CONCLUSION: Our findings provide evidence that respiratory epithelium might play an important role in the immune response during Aspergillus infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Células Epiteliais/imunologia , beta-Defensinas/imunologia , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Linhagem Celular , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Hifas/imunologia , Hifas/patogenicidade , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esporos Fúngicos/imunologia , Esporos Fúngicos/patogenicidade , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
Exfoliative toxins (ETs) are secreted virulence factors produced by staphylococci. These serine proteases specifically cleave desmoglein 1 (Dsg1) in mammals and are key elements in staphylococcal skin infections. We recently identified a new et gene in S. aureus O46, a strain isolated from ovine mastitis. In the present study, we characterized the new et gene at a genetic level and the enzymatic activity of the deduced protein. The S. aureus O46 genome was re-assembled, annotated and compared with other publicly available S. aureus genomes. The deduced amino acid sequence of the new et gene shared 40%, 53% and 59% sequence identity to those of ETA, ETB and ETD, respectively. The new et gene shared the same genetic vicinity and was similar in other S. aureus strains bearing this gene. The recombinant enzyme of the new et gene caused skin exfoliation in vivo in neonatal mice. The new et-gene was thus named ete, encoding a new type (type E) of exfoliative toxin. We showed that ETE degraded the extracellular segments of Dsg1 in murine, ovine and caprine epidermis, as well as in ovine teat canal epithelia, but not that in bovine epidermis. We further showed that it directly hydrolyzed human and swine Dsg1 as well as murine Dsg1α and Dsg1ß, but not canine Dsg1 or murine Dsg1γ. Molecular modeling revealed a correlation between the preferred orientation of ETE docking on its Dsg1 cleavage site and species-specific cleavage activity, suggesting that the docking step preceding cleavage accounts for the ETE species-specificity. This new virulence factor may contribute to the bacterial colonization on the stratified epithelia in certain ruminants with mastitis.
Assuntos
Especificidade de Hospedeiro , Staphylococcus aureus/metabolismo , Toxinas Biológicas/metabolismo , Sequência de Aminoácidos , Animais , Espaço Extracelular/metabolismo , Genoma Bacteriano/genética , Hidrólise , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Ruminantes/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/fisiologia , Toxinas Biológicas/químicaRESUMO
Staphylococcus aureus causes serious medical problems in human and animals. Here we show that S. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AX phosphorylation, a marker of DNA double strand breaks (DSBs), in human cervix cancer HeLa and osteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1-4), while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens the H2AX phosphorylation thus counteracting the DNA damage. This DNA damage is mediated by reactive oxygen species (ROS), which promotes oxidation of guanine forming 7,8-dihydro-8-oxoguanine (8-oxoG). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signaling pathway. An examination of S. aureus strains, isolated from the same patient during acute initial and recurrent bone and joint infections (BJI), showed that recurrent strains produce lower amounts of Lpls, induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest an involvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findings redefine our understanding of mechanisms of S. aureus-host interaction and suggest that the balance between the levels of PSMα and Lpls expression impacts the persistence of the infection.
Assuntos
Dano ao DNA , Staphylococcus aureus/patogenicidade , Acetilcisteína/farmacologia , Artrite Infecciosa/microbiologia , Toxinas Bacterianas/farmacologia , Linhagem Celular Tumoral , Reparo do DNA , Etoposídeo/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Ilhas Genômicas , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa/microbiologia , Histonas/análise , Interações Hospedeiro-Patógeno , Humanos , Lipoproteínas/farmacologia , Osteíte/microbiologia , Osteoblastos/microbiologia , Estresse Oxidativo , Fosforilação , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio , Infecções Estafilocócicas/microbiologiaRESUMO
Staphylococcus aureus is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. S. aureus naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that S. aureus Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs in vitro with a designated protein content. Purified S. aureus N305-secreted EVs were not cytotoxic when tested in vitro on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live S. aureus N305. The in vivo immune response to purified S. aureus N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live S. aureus N305, heat-killed S. aureus N305 and to S. aureus lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live S. aureus induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, S. aureus N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary S. aureus N305-secreted EVs was comparable to that of heat-killed S. aureus N305 and, partly, by LTA. These results demonstrated that S. aureus N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined in vitro and in vivo data suggest that EVs are worth to be investigated to better understand the S. aureus pathogenesis and are relevant tools to develop strategies against bovine S. aureus mastitis.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Vesículas Extracelulares/imunologia , Glândulas Mamárias Humanas/patologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/imunologia , Animais , Bovinos , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mastite Bovina/patologia , Camundongos , Neutrófilos/imunologia , Infecções Estafilocócicas/patologiaRESUMO
[This corrects the article on p. 208 in vol. 7, PMID: 28589102.].
RESUMO
Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.
Assuntos
Bactérias/patogenicidade , Fenômenos Fisiológicos Bacterianos , Toxinas Bacterianas/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Eucarióticas/microbiologia , Toxina Adenilato Ciclase/toxicidade , Animais , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/toxicidade , Toxina da Cólera/toxicidade , Células Eucarióticas/efeitos dos fármacos , Exotoxinas/toxicidade , Interações Hospedeiro-Parasita , Humanos , Leucocidinas/toxicidade , Macrolídeos/toxicidade , Toxina Shiga/toxicidade , Transdução de Sinais , Fatores de Virulência/toxicidadeRESUMO
The cell cycle is an ordered set of events, leading to cell growth and division into two daughter cells. The eukaryotic cell cycle consists of interphase (G1, S, and G2 phases), followed by the mitotic phase and G0 phase. Many bacterial pathogens secrete cyclomodulins that interfere with the host cell cycle. In Staphylococcus aureus four cyclomodulins have been described so far that all represent toxins and are secreted into the culture supernatant. Here we show that the membrane-anchored lipoprotein-like proteins (Lpl), encoded on a genomic island called νSaα, interact with the cell cycle of HeLa cells. By comparing wild type and lpl deletion mutant it turned out that the lpl cluster is causative for the G2/M phase transition delay and also contributes to increased invasion frequency. The lipoprotein Lpl1, a representative of the lpl cluster, also caused G2/M phase transition delay. Interestingly, the lipid modification, which is essential for TLR2 signaling and activation of the immune system, is not necessary for cyclomodulin activity. Unlike the other staphylococcal cyclomodulins Lpl1 shows no cytotoxicity even at high concentrations. As all Lpl proteins are highly conserved there might be a common function that is accentuated by their multiplicity in a tandem gene cluster. The cell surface localized Lpls' suggests a correlation between G2/M phase transition delay and host cell invasion.