RESUMO
Staphylococcus aureus is a major pathogen associated with important diseases in humans and animals. Macrophages are a key component of the innate immune response to S. aureus infection and play a major role in disease outcomes. To investigate the adaptive evolution of S. aureus in response to macrophages, we developed an experimental infection assay. S. aureus strains representing major human epidemic clones were passaged many times in a macrophage cell line, accumulating mutations in an array of genomic loci. Phenotypic analysis revealed the emergence of a lineage exhibiting increased survival in macrophages and human blood, and resistance to vancomycin. The evolved lineage exhibited a previously undescribed small colony variant (SCV) phenotype characterized by hyper-pigmentation, which resulted from a missense mutation in rsbW. Notably, the novel SCV was a conditional adaptive trait that was unstable in nutrient-replete conditions in vitro, rapidly converting from hyper-pigmented SCV to a non-pigmented large colony variant via spontaneous sigB deletion events. Importantly, we identified similar deletions in the genome sequences of a limited number of clinical S. aureus isolates from public databases, indicating that related events may occur during clinical infection. Experimental infection of zebrafish did not reveal a difference in virulence between parent and novel SCV but demonstrated an in vivo fitness cost for the compensatory sigB deletion events. Taken together, we report an experimental evolutionary approach for investigating bacterial innate immune cell interactions, revealing a conditional adaptation that promotes S. aureus survival in macrophages and resistance to vancomycin. IMPORTANCE: Staphylococcus aureus is an important human bacterial pathogen. The host response to S. aureus involves the production of innate immune cells such as macrophages which are important for fighting infection. Here we report a new model of experimental evolution for studying how S. aureus can evade killing by macrophages. We identified a novel adaptive phenotype that promotes survival in macrophages and blood and resistance to antibiotics. The phenotype is lost rapidly upon growth in nutrient-rich conditions via disruption of the alternative sigma factor sigB, revealing a conditional niche-specific fitness advantage. Genomic analysis of clinical isolates suggests similar adaptations may occur during human infections. Our model may be used broadly to identify adaptations of S. aureus to the innate immune response.
Assuntos
Macrófagos , Infecções Estafilocócicas , Staphylococcus aureus , Peixe-Zebra , Staphylococcus aureus/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Macrófagos/microbiologia , Macrófagos/imunologia , Humanos , Animais , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/imunologia , Peixe-Zebra/microbiologia , Fenótipo , Viabilidade Microbiana , Antibacterianos/farmacologia , Adaptação Fisiológica/genética , Linhagem Celular , Camundongos , Genoma Bacteriano , Evolução MolecularRESUMO
Monocyte chemoattractant protein-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase that has been described as a key negative modulator of inflammation. MCPIP1 also controls numerous tumor-related processes, such as proliferation, apoptosis and differentiation. In this study, we utilized a zebrafish model to investigate the role of Mcpip1 during embryogenic development. Our results demonstrated that during embryogenesis, the expression of the zc3h12a gene encoding Mcpip1 undergoes dynamic changes. Its transcript levels gradually increase from the 2-cell stage to the spherical stage and then decrease rapidly. We further found that ectopic overexpression of wild-type Mcpip1 but not the catalytically inactive mutant form resulted in an embryonic lethal phenotype in zebrafish embryos (24 hpf). At the molecular level, transcriptomic profiling revealed extensive changes in the expression of genes encoding proteins important in the endoplasmic reticulum stress response and in protein folding as well as involved in the formation of primary germ layer, mesendoderm and endoderm development, heart morphogenesis and cell migration. Altogether, our results demonstrate that the expression of zc3h12a must be tightly controlled during the first cell divisions of zebrafish embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper embryo development.
Assuntos
Fatores de Transcrição , Peixe-Zebra , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas Quimioatraentes de Monócitos , Diferenciação Celular , Ribonucleases/genética , Ribonucleases/metabolismo , Desenvolvimento Embrionário/genéticaRESUMO
People with activated PI3 kinase delta syndrome 1 (APDS1) suffer from immune deficiency and severe bronchiectasis. APDS1 is caused by dominant activating mutations of the PIK3CD gene that encodes the PI3 kinase delta (PI3Kδ) catalytic subunit. Despite the importance of innate immunity defects in bronchiectasis, there has been limited investigation of neutrophils or macrophages in APDS1 patients or mouse models. Zebrafish embryos provide an ideal system to study neutrophils and macrophages. We used CRISPR-Cas9 and CRISPR-Cpf1, with oligonucleotide-directed homologous repair, to engineer zebrafish equivalents of the two most prevalent human APDS1 disease mutations. These zebrafish pik3cd alleles dominantly caused excessive neutrophilic inflammation in a tail-fin injury model. They also resulted in total body neutrophilia in the absence of any inflammatory stimulus but normal numbers of macrophages. Exposure of zebrafish to the PI3Kδ inhibitor CAL-101 reversed the total body neutrophilia. There was no apparent defect in neutrophil maturation or migration, and tail-fin regeneration was unimpaired. Overall, the finding is of enhanced granulopoeisis, in the absence of notable phenotypic change in neutrophils and macrophages.
Assuntos
Bronquiectasia , Peixe-Zebra , Animais , Camundongos , Humanos , Peixe-Zebra/genética , Fosfatidilinositol 3-Quinases , Mutação , NeutrófilosRESUMO
In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.
Assuntos
Doenças dos Peixes , Microglia/imunologia , Doenças Neuroinflamatórias , Infecções por Vírus de RNA , Animais , Comportamento Animal , Encéfalo/imunologia , Encéfalo/patologia , Encéfalo/virologia , Ingestão de Alimentos , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/patologia , Doenças dos Peixes/virologia , Expressão Gênica , Comportamento de Doença , Locomoção , Macrófagos/imunologia , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/veterinária , Doenças Neuroinflamatórias/virologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/patologia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , Carga Viral , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.
Assuntos
Galanina/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/patogenicidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Galanina/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno , Mediadores da Inflamação/metabolismo , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium marinum/imunologia , Transdução de Sinais , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/imunologia , Transcriptoma , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Staphylococcus aureus infects â¼30% of the human population and causes a spectrum of pathologies ranging from mild skin infections to life-threatening invasive diseases. The strict host specificity of its virulence factors has severely limited the accuracy of in vivo models for the development of vaccines and therapeutics. To resolve this, we generated a humanised zebrafish model and determined that neutrophil-specific expression of the human C5a receptor conferred susceptibility to the S. aureus toxins PVL and HlgCB, leading to reduced neutrophil numbers at the site of infection and increased infection-associated mortality. These results show that humanised zebrafish provide a valuable platform to study the contribution of human-specific S. aureus virulence factors to infection in vivo that could facilitate the development of novel therapeutic approaches and essential vaccines.
Assuntos
Staphylococcus aureus , Fatores de Virulência , Animais , Humanos , Receptor da Anafilatoxina C5a/genética , Staphylococcus aureus/genética , Virulência , Fatores de Virulência/genética , Peixe-ZebraRESUMO
Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci.Abbreviations: ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification.
Assuntos
Autofagia , Espaço Intracelular/microbiologia , Neutrófilos/microbiologia , Staphylococcus aureus/fisiologia , Animais , Animais Geneticamente Modificados , Cinética , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Fagocitose , Fagossomos/metabolismo , Agregados Proteicos , Proteína Sequestossoma-1/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Macroautophagy/autophagy functions to degrade cellular components and intracellular pathogens. Autophagy receptors, including SQSTM1/p62, target intracellular pathogens. Staphylococcus aureus is a significant pathogen of humans, especially in immunocompromise. S. aureus may use neutrophils as a proliferative niche, but their intracellular fate following phagocytosis has not been analyzed in vivo. In vitro, SQSTM1 can colocalize with intracellular Staphylococcus aureus, but whether SQSTM1 is beneficial or detrimental in host defense against S. aureus in vivo is unknown. Here we determine the fate and location of S. aureus within neutrophils throughout zebrafish infection. We show Lc3 and Sqstm1 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil and that Lc3 marking of bacterial phagosomes within neutrophils may precede bacterial degradation. Finally, we show Sqstm1 is important for controlling cytosolic bacteria, demonstrating for the first time a key role of Sqstm1 in autophagic control of S. aureus in neutrophils.Abbreviations: AR: autophagy receptor; CFU: colony-forming unit; CHT: caudal hematopoietic tissue; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; LWT: london wild-type: lyz: lysozyme; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification; UBD: ubiquitin binding domain.
Assuntos
Autofagia/fisiologia , Neutrófilos/metabolismo , Proteína Sequestossoma-1/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Macrófagos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Staphylococcus aureus , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Macrófagos , Monócitos , Tuberculose/tratamento farmacológico , Peixe-ZebraRESUMO
Intracellular pathogens such as Salmonella depend on their molecular virulence factors to evade host defense responses like autophagy. Using a zebrafish systemic infection model, we have previously shown that phagocytes, predominantly macrophages, target Salmonella Typhimurium by an autophagy-related pathway known as Lc3-associated phagocytosis (LAP), which is dependent on the host protein Rubicon. Here, we explore the influence of Salmonella virulence factors on pathogenicity in the zebrafish model and induction of LAP as a defense response. We investigated five mutant strains that all could trigger GFP-Lc3 recruitment as puncta or rings around single bacteria or bacterial clusters, in a Rubicon-dependent manner. We found that S. Typhimurium strains carrying mutations in PhoP or PurA, responsible for adaptation to the intracellular environment and efficient metabolism of purines, respectively, are attenuated in the zebrafish model. However, both strains show increased virulence when LAP is inhibited by knockdown of Rubicon. Mutations in type III secretion systems 1 and 2, SipB and SsrB, which are important for invading and replicating in non-phagocytic cells, did not affect the ability to establish successful infection in the zebrafish model. This observation is in line with our previous characterization of this infection model revealing that macrophages actively phagocytose the majority of S. Typhimurium. In contrast to SipB mutants, SsrB mutants were unable to become more virulent in Rubicon-deficient hosts, suggesting that type III system 2 effectors are important for intracellular replication of Salmonella in the absence of LAP. Finally, we found that mutation of FlhD, required for production of flagella, renders S. Typhimurium hypervirulent both in wild type zebrafish embryos and in Rubicon-deficient hosts. FlhD mutation also led to lower levels of GFP-Lc3 recruitment compared with the wild type strain, indicating that recognition of flagellin by the host innate immune system promotes the LAP response. Together, our results provide new evidence that the Rubicon-dependent LAP process is an important defense mechanism against S. Typhimurium.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Interações Hospedeiro-Patógeno , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/metabolismo , Fagossomos/microbiologia , Salmonella typhimurium/imunologia , Fatores de Virulência/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Modelos Animais de Doenças , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Salmonella typhimurium/crescimento & desenvolvimento , Peixe-ZebraRESUMO
Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.
Assuntos
Antígenos de Superfície/imunologia , Enterococcus faecalis/patogenicidade , Infecções por Bactérias Gram-Positivas/imunologia , Infecções por Bactérias Gram-Positivas/microbiologia , Muramidase/imunologia , Polissacarídeos/imunologia , Virulência , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/imunologia , Infecções por Bactérias Gram-Positivas/metabolismo , Muramidase/metabolismo , Mutagênese , Mutação , Polissacarídeos/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologiaRESUMO
The neutrophil enzyme myeloperoxidase (MPO) is a major enzyme made by neutrophils to generate antimicrobial and immunomodulatory compounds, notably hypochlorous acid (HOCl), amplifying their capacity for destroying pathogens and regulating inflammation. Despite its roles in innate immunity, the importance of MPO in preventing infection is unclear, as individuals with MPO deficiency are asymptomatic with the exception of an increased risk of candidiasis. Dysregulation of MPO activity is also linked with inflammatory conditions such as atherosclerosis, emphasising a need to understand the roles of the enzyme in greater detail. Consequently, new tools for investigating granular dynamics in vivo can provide useful insights into how MPO localises within neutrophils, aiding understanding of its role in preventing and exacerbating disease. The zebrafish is a powerful model for investigating the immune system in vivo, as it is genetically tractable, and optically transparent. To visualise MPO activity within zebrafish neutrophils, we created a genetic construct that expresses human MPO as a fusion protein with a C-terminal fluorescent tag, driven by the neutrophil-specific promoter lyz. After introducing the construct into the zebrafish genome by Tol2 transgenesis, we established the Tg(lyz:Hsa.MPO-mEmerald,cmlc2:EGFP)sh496 line, and confirmed transgene expression in zebrafish neutrophils. We observed localisation of MPO-mEmerald within a subcellular location resembling neutrophil granules, mirroring MPO in human neutrophils. In Spotless (mpxNL144) larvae-which express a non-functional zebrafish myeloperoxidase-the MPO-mEmerald transgene does not disrupt neutrophil migration to sites of infection or inflammation, suggesting that it is a suitable line for the study of neutrophil granule function. We present a new transgenic line that can be used to investigate neutrophil granule dynamics in vivo without disrupting neutrophil behaviour, with potential applications in studying processing and maturation of MPO during development.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Neutrófilos/enzimologia , Peroxidase/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde/genética , Humanos , Larva/genética , Larva/metabolismo , Proteínas Luminescentes/genética , Microscopia Confocal , Microscopia de Fluorescência , Peroxidase/genética , Transgenes/genética , Peixe-Zebra/genética , Proteína Vermelha FluorescenteRESUMO
Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection. Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type.
Assuntos
Modelos Animais de Doenças , Macrófagos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagocitose/genética , Salmonelose Animal , Salmonella typhimurium/imunologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Autofagia/fisiologia , Bacteriemia/genética , Bacteriemia/imunologia , Bacteriemia/microbiologia , Bacteriemia/patologia , Embrião não Mamífero , Proteínas Associadas aos Microtúbulos/genética , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/genética , Salmonelose Animal/imunologia , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
All bacterial infections occur within a polymicrobial environment, from which a pathogen population emerges to establish disease within a host. Emphasis has been placed on prevention of pathogen dominance by competing microflora acting as probiotics1. Here we show that the virulence of the human pathogen Staphylococcus aureus is augmented by native, polymicrobial, commensal skin flora and individual species acting as 'proinfectious agents'. The outcome is pathogen proliferation, but not commensal. Pathogenesis augmentation can be mediated by particulate cell wall peptidoglycan, reducing the S. aureus infectious dose by over 1,000-fold. This phenomenon occurs using a range of S. aureus strains and infection models and is not mediated by established receptor-mediated pathways including Nod1, Nod2, Myd88 and the NLPR3 inflammasome. During mouse sepsis, augmentation depends on liver-resident macrophages (Kupffer cells) that capture and internalize both the pathogen and the proinfectious agent, leading to reduced production of reactive oxygen species, pathogen survival and subsequent multiple liver abscess formation. The augmented infection model more closely resembles the natural situation and establishes the role of resident environmental microflora in the initiation of disease by an invading pathogen. As the human microflora is ubiquitous2, its role in increasing susceptibility to infection by S. aureus highlights potential strategies for disease prevention.
Assuntos
Bactérias/metabolismo , Peptidoglicano/farmacologia , Sepse/microbiologia , Pele/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Modelos Animais de Doenças , Humanos , Inflamassomos/metabolismo , Células de Kupffer/citologia , Células de Kupffer/imunologia , Macrófagos/metabolismo , Camundongos , Microbiota , Peptidoglicano/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/crescimento & desenvolvimento , Simbiose , VirulênciaRESUMO
The study of the dynamics that occur during the course of a bacterial infection has been attempted using several methods. Here we discuss the construction of a set of antibiotic-resistant, otherwise-isogenic Staphylococcus aureus strains that can be used to observe the progress of systemic disease in a mouse model at various time-points postinfection. The strains can likewise be used to study the progression of infection in other animal infection models, such as the zebrafish embryo. Furthermore, the use of antibiotic resistance tags provides a convenient system with which to investigate the effect of antimicrobial chemotherapy during disease.
Assuntos
Interações Hospedeiro-Patógeno , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Carga Bacteriana , Modelos Animais de Doenças , Genes Transgênicos Suicidas , Vetores Genéticos/genética , Camundongos , Mutação , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/virologia , Transdução GenéticaRESUMO
Investigating bacterial dynamics within the infected host has proved very useful for understanding mechanisms of pathogenesis. Here we present the protocols we use to study bacterial dynamics within infected embryonic zebrafish. This chapter encompasses basic techniques used to study bacterial infection within larval zebrafish, including embryonic zebrafish maintenance, injections of morpholino oligonucleotides, intravenous injections of bacterial suspensions, and fluorescence imaging of infected zebrafish. Specific methods for studying bacterial within-host population dynamics are also described.
Assuntos
Infecções Bacterianas/microbiologia , Interações Hospedeiro-Patógeno , Peixe-Zebra/microbiologia , Animais , Carga Bacteriana , Modelos Animais de Doenças , Embrião não Mamífero , Larva , Microscopia de FluorescênciaRESUMO
BACKGROUND: The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq). RESULTS: We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745. CONCLUSIONS: Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.
Assuntos
Enterococcus faecium/genética , Aptidão Genética , Enterococos Resistentes à Vancomicina/genética , Animais , Sangue , Enterococcus faecium/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Genoma Bacteriano , Infecções por Bactérias Gram-Positivas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de RNA , Enterococos Resistentes à Vancomicina/crescimento & desenvolvimento , Peixe-ZebraRESUMO
Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections.
Assuntos
Parede Celular/metabolismo , Enterococcus faecalis/citologia , Enterococcus faecalis/patogenicidade , Infecções por Bactérias Gram-Positivas/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Parede Celular/genética , Enterococcus faecalis/enzimologia , Enterococcus faecalis/genética , Humanos , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Virulência , Peixe-Zebra/microbiologiaRESUMO
Porphyromonas gingivalis (Pg) is a keystone pathogen in the aetiology of chronic periodontitis. However, recent evidence suggests that the bacterium is also able to enter the bloodstream, interact with host cells and tissues, and ultimately contribute to the pathogenesis of cardiovascular disease (CVD). Here we established a novel zebrafish larvae systemic infection model showing that Pg rapidly adheres to and penetrates the zebrafish vascular endothelium causing a dose- and time-dependent mortality with associated development of pericardial oedemas and cardiac damage. The in vivo model was then used to probe the role of Pg expressed gingipain proteases using systemically delivered gingipain-deficient Pg mutants, which displayed significantly reduced zebrafish morbidity and mortality compared to wild-type bacteria. In addition, we used the zebrafish model to show efficacy of a gingipain inhibitor (KYT) on Pg-mediated systemic disease, suggesting its potential use therapeutically. Our data reveal the first real-time in vivo evidence of intracellular Pg within the endothelium of an infection model and establishes that gingipains are crucially linked to systemic disease and potentially contribute to CVD.
Assuntos
Adesinas Bacterianas/metabolismo , Doenças Cardiovasculares/microbiologia , Cisteína Endopeptidases/metabolismo , Endotélio Vascular/microbiologia , Periodontite/microbiologia , Porphyromonas gingivalis/patogenicidade , Adesinas Bacterianas/genética , Animais , Doenças Cardiovasculares/patologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases Gingipaínas , Larva/microbiologia , Porphyromonas gingivalis/genética , Peixe-Zebra/embriologiaRESUMO
Autophagy, a catabolic pathway of lysosomal degradation, acts not only as an efficient recycle and survival mechanism during cellular stress, but also as an anti-infective machinery. The human pathogen Staphylococcus aureus (S. aureus) was originally considered solely as an extracellular bacterium, but is now recognized additionally to invade host cells, which might be crucial for persistence. However, the intracellular fate of S. aureus is incompletely understood. Here, we show for the first time induction of selective autophagy by S. aureus infection, its escape from autophagosomes and proliferation in the cytoplasm using live cell imaging. After invasion, S. aureus becomes ubiquitinated and recognized by receptor proteins such as SQSTM1/p62 leading to phagophore recruitment. Yet, S. aureus evades phagophores and prevents further degradation by a MAPK14/p38α MAP kinase-mediated blockade of autophagy. Our study demonstrates a novel bacterial strategy to block autophagy and secure survival inside the host cell.