RESUMEN
Shigella represents a paraphyletic group of enteroinvasive Escherichia coli. More than 40 Shigella serotypes have been reported. However, most cases within the men who have sex with men (MSM) community are attributed to 3 serotypes: Shigella sonnei unique serotype and Shigella flexneri 2a and 3a serotypes. Using the zebrafish model, we demonstrate that Shigella can establish persistent infection in vivo. Bacteria are not cleared by the immune system and become antibiotic tolerant. Establishment of persistent infection depends on the O-antigen, a key constituent of the bacterial surface and a serotype determinant. Representative isolates associated with MSM transmission persist in zebrafish, while representative isolates of a serotype not associated with MSM transmission do not. Isolates of a Shigella serotype establishing persistent infections elicited significantly less macrophage death in vivo than isolates of a serotype unable to persist. We conclude that zebrafish are a valuable platform to illuminate factors underlying establishment of Shigella persistent infection in humans.
Asunto(s)
Disentería Bacilar , Minorías Sexuales y de Género , Shigella , Humanos , Masculino , Animales , Pez Cebra , Serogrupo , Homosexualidad Masculina , Infección Persistente , Disentería Bacilar/microbiología , Shigella flexneriRESUMEN
Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.
Asunto(s)
Neutrófilos/inmunología , Antígenos O/inmunología , Shigella sonnei/inmunología , Shigella sonnei/patogenicidad , Virulencia/inmunología , Animales , Disentería Bacilar , Humanos , Pez CebraRESUMEN
Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation.
Asunto(s)
Disentería Bacilar/inmunología , Inmunidad Innata/inmunología , Septinas/metabolismo , Animales , Modelos Animales de Enfermedad , Disentería Bacilar/microbiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inflamación/inmunología , Inflamación/microbiología , Mucosa Intestinal/microbiología , Larva/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiología , Shigella flexneri , Pez CebraRESUMEN
The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Larva/genética , Microinyecciones/métodos , Robótica/métodos , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Benchmarking , Modelos Animales de Enfermedad , Embrión no Mamífero/inmunología , Embrión no Mamífero/microbiología , Embrión no Mamífero/ultraestructura , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Larva/inmunología , Larva/microbiología , Larva/ultraestructura , Microscopía Fluorescente , Morfolinos/administración & dosificación , Mycobacterium tuberculosis/inmunología , Trasplante de Neoplasias , Oligonucleótidos Antisentido/administración & dosificación , Staphylococcus epidermidis/inmunología , Células Tumorales Cultivadas/trasplante , Pez Cebra/inmunología , Pez Cebra/microbiologíaRESUMEN
Macroautophagy/autophagy is an essential pro-survival mechanism activated in response to nutrient deficiency. The proper fusion between autophagosomes and lysosomes is a critical step for autophagic degradation. We recently reported that RUNDC1 (RUN domain containing 1) inhibits autolysosome formation via clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding. We showed that RUNDC1 colocalizes with LC3 and associates with mature autophagosomes in cell lines and the zebrafish model. We utilized liposome fusion and in vitro autophagosome-lysosome fusion assays to demonstrate that RUNDC1 inhibits autolysosome formation. Moreover, we found that RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation, which in turn prevents VAMP8 from binding to STX17-SNAP29. Our results demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes and is crucial for zebrafish survival in nutrient-deficient conditions. Here, we summarize our findings and discuss their implications for our understanding of autophagy regulation.
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Autofagosomas , Autofagia , Animales , Autofagosomas/metabolismo , Autofagia/fisiología , Pez Cebra/metabolismo , Factores de Transcripción/metabolismo , Lisosomas/metabolismo , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismoRESUMEN
Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6â h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24â h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection.
Asunto(s)
Disentería Bacilar , Animales , Humanos , Disentería Bacilar/genética , Shigella flexneri/genética , Shigella flexneri/metabolismo , Pez Cebra/genética , Pez Cebra/microbiología , Inflamación/microbiología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The incidence of Hepatocellular carcinoma (HCC) and HCC-related deaths have remarkably increased over the recent decades. It has been reported that ß-catenin activation can be frequently observed in HCC cases. This study identified the integrin-linked kinase-associated phosphatase (ILKAP) as a novel ß-catenin-interacting protein. ILKAP is localized both in the nucleus and cytoplasm and regulates the WNT pathway in different ways. First, it is demonstrated that ILKAP activates the WNT pathway in HCC cells by increasing the protein level of ß-catenin and other proteins associated with the WNT signaling, such as c-Myc and CyclinD1. Next, it is shown that ILKAP promotes the metastasis of HCC both in vitro and in vivo in a zebrafish xenograft model. It is also found that ILKAP dephosphorylates the GSK3ß and CK1, contributing to the reduced ubiquitination of ß-catenin. Furthermore, it is identified that ILKAP functions by mediating binding between TCF4 and ß-catenin to enhance expression of WNT target genes. Taken together, the study demonstrates a critical function of ILKAP in metastasis of HCC, since ILKAP is crucial for the activation of the WNT pathway via stabilization of ß-catenin and increased binding between TCF4 and ß-catenin.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Fosfoproteínas Fosfatasas , Vía de Señalización Wnt , beta Catenina , Animales , Humanos , beta Catenina/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Metástasis de la Neoplasia , Factor de Transcripción 4/metabolismo , Factor de Transcripción 4/genética , Vía de Señalización Wnt/fisiología , Pez Cebra , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismoRESUMEN
Septins are evolutionarily conserved GTP-binding proteins known for their roles in cell division and host defence against Shigella infection. Although septin group members are viewed to function as hetero-oligomeric complexes, the role of individual septins within these complexes or in isolation is poorly understood. Decades of work using mouse models has shown that some septins (including SEPT7) are essential for animal development, while others (including SEPT6) are dispensable, suggesting that some septins may compensate for the absence of others. The zebrafish genome encodes 19 septin genes, representing the full complement of septin groups described in mice and humans. In this report, we characterise null mutants for zebrafish Sept6 (a member of the SEPT6 group) and Sept15 (a member of the SEPT7 group) and test their role in zebrafish development and host defence against Shigella infection. We show that null mutants for Sept6 and Sept15 are both viable, and that expression of other zebrafish septins are not significantly affected by their mutation. Consistent with previous reports using knockdown of Sept2, Sept7b, and Sept15, we show that Sept6 and Sept15 are required for host defence against Shigella infection. These results highlight Shigella infection of zebrafish as a powerful system to study the role of individual septins in vivo.
Asunto(s)
Disentería Bacilar , Septinas , Animales , Disentería Bacilar/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Septinas/genética , Septinas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Apoptosis is a form of regulated cell death essential for tissue homeostasis and embryonic development. Apoptosis also plays a key role during bacterial infection, yet some intracellular bacterial pathogens (such as Shigella flexneri, whose lipopolysaccharide can block apoptosis) can manipulate cell death programs as an important survival strategy. Septins are a component of the cytoskeleton essential for mitochondrial dynamics and host defense, however, the role of septins in regulated cell death is mostly unknown. Here, we discover that septins promote mitochondrial (i.e., intrinsic) apoptosis in response to treatment with staurosporine (a pan-kinase inhibitor) or etoposide (a DNA topoisomerase inhibitor). Consistent with a role for septins in mitochondrial dynamics, septins promote the release of mitochondrial protein cytochrome c in apoptotic cells and are required for the proteolytic activation of caspase-3, caspase-7, and caspase-9 (core components of the apoptotic machinery). Apoptosis of HeLa cells induced in response to infection by S. flexneri ΔgalU (a lipopolysaccharide mutant unable to block apoptosis) is also septin-dependent. In vivo, zebrafish larvae are significantly more susceptible to infection with S. flexneri ΔgalU (as compared to infection with wildtype S. flexneri), yet septin deficient larvae are equally susceptible to infection with S. flexneri ΔgalU and wildtype S. flexneri. These data provide a new molecular framework to understand the complexity of mitochondrial apoptosis and its ability to combat bacterial infection.
RESUMEN
The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.
Asunto(s)
Antiinfecciosos , Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Edición Génica , Bacteriófago P1/genética , Pez Cebra/genética , Shigella flexneri/genética , AnimalesRESUMEN
Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired. IMPORTANCE Enteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America.
Asunto(s)
Disentería Bacilar , Infecciones por Escherichia coli , Shigella , Animales , Humanos , Escherichia coli , Virulencia/genética , Pez Cebra , Sistemas de Secreción Tipo III/genética , Teorema de Bayes , Temperatura , Plásmidos/genética , Shigella/genéticaRESUMEN
Autophagy serves as a pro-survival mechanism for a cell or a whole organism to cope with nutrient stress. Our understanding of the molecular regulation of this fusion event remains incomplete. Here, we identified RUNDC1 as a novel ATG14-interacting protein, which is highly conserved across vertebrates, including zebrafish and humans. By gain and loss of function studies, we demonstrate that RUNDC1 negatively modulates autophagy by blocking fusion between autophagosomes and lysosomes via inhibiting the assembly of the STX17-SNAP29-VAMP8 complex both in human cells and the zebrafish model. Moreover, RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation. This also prevents VAMP8 from binding to STX17-SNAP29. We further identified that phosphorylation of RUNDC1 Ser379 is crucial to inhibit the assembly of the STX17-SNAP29-VAMP8 complex via promoting ATG14 homo-oligomerization. In line with our findings, RunDC1 is crucial for zebrafish in their response to nutrient-deficient conditions. Taken together, our findings demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes by clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding.
RESUMEN
Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Lisosomas/inmunología , Macrófagos/inmunología , Infecciones por Mycobacterium/genética , Receptores CXCR3/genética , Transducción de Señal/inmunología , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/inmunología , Rastreo Celular , Quimiotaxis/genética , Quimiotaxis/inmunología , Embrión no Mamífero , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Larva/inmunología , Larva/microbiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Lisosomas/metabolismo , Lisosomas/microbiología , Lisosomas/ultraestructura , Activación de Macrófagos , Macrófagos/microbiología , Macrófagos/ultraestructura , Mutación , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/microbiología , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Receptores CXCR3/inmunología , Análisis de Secuencia de ARN , Transducción de Señal/genética , Pez Cebra/inmunología , Pez Cebra/microbiología , Proteínas de Pez Cebra/inmunología , Proteína Fluorescente RojaRESUMEN
Phagocytes are highly motile immune cells that ingest and clear microbial invaders, harmful substances, and dying cells. Their function is critically dependent on the expression of chemokine receptors, a class of G-protein-coupled receptors (GPCRs). Chemokine receptors coordinate the recruitment of phagocytes and other immune cells to sites of infection and damage, modulate inflammatory and wound healing responses, and direct cell differentiation, proliferation, and polarization. Besides, a structurally diverse group of atypical chemokine receptors (ACKRs) are unable to signal in G-protein-dependent fashion themselves but can shape chemokine gradients by fine-tuning the activity of conventional chemokine receptors. The optically transparent zebrafish embryos and larvae provide a powerful in vivo system to visualize phagocytes during development and study them as key elements of the immune response in real-time. In this review, we discuss how the zebrafish model has furthered our understanding of the role of two main classes of chemokine receptors, the CC and CXC subtypes, in phagocyte biology. We address the roles of the receptors in the migratory properties of phagocytes in zebrafish models for cancer, infectious disease, and inflammation. We illustrate how studies in zebrafish enable visualizing the contribution of chemokine receptors and ACKRs in shaping self-generated chemokine gradients of migrating cells. Taking the functional antagonism between two paralogs of the CXCR3 family as an example, we discuss how the duplication of chemokine receptor genes in zebrafish poses challenges, but also provides opportunities to study sub-functionalization or loss-of-function events. We emphasize how the zebrafish model has been instrumental to prove that the major determinant for the functional outcome of a chemokine receptor-ligand interaction is the cell-type expressing the receptor. Finally, we highlight relevant homologies and analogies between mammalian and zebrafish phagocyte function and discuss the potential of zebrafish models to further advance our understanding of chemokine receptors in innate immunity and disease.
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Fagocitos/metabolismo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Pez Cebra/inmunología , Animales , Humanos , Inmunidad Innata , Inflamación/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Heridas y Lesiones/inmunologíaRESUMEN
The CXCR3-CXCL11 chemokine-signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging function, while being unable to signal in the classic G-protein-dependent manner. Two copies of the CXCR3 gene in zebrafish, cxcr3.2 and cxcr3.3, are expressed on macrophages and share a highly conserved ligand-binding site. However, Cxcr3.3 has structural characteristics of ACKRs indicative of a ligand-scavenging role. In contrast, we previously showed that Cxcr3.2 is an active CXCR3 receptor because it is required for macrophage motility and recruitment to sites of mycobacterial infection. In this study, we generated a cxcr3.3 CRISPR-mutant to functionally dissect the antagonistic interplay among the cxcr3 paralogs in the immune response. We observed that cxcr3.3 mutants are more susceptible to mycobacterial infection, whereas cxcr3.2 mutants are more resistant. Furthermore, macrophages in the cxcr3.3 mutant are more motile, show higher activation status, and are recruited more efficiently to sites of infection or injury. Our results suggest that Cxcr3.3 is an ACKR that regulates the activity of Cxcr3.2 by scavenging common ligands and that silencing the scavenging function of Cxcr3.3 results in an exacerbated Cxcr3.2 signaling. In human, splice variants of CXCR3 have antagonistic functions and CXCR3 ligands also interact with ACKRs. Therefore, in zebrafish, an analogous regulatory mechanism appears to have evolved after the cxcr3 gene duplication event, through diversification of conventional and atypical receptor variants.
Asunto(s)
Movimiento Celular , Macrófagos/fisiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/fisiología , Receptores CXCR3/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Sistemas CRISPR-Cas , Macrófagos/citología , Macrófagos/microbiología , Mutación , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/patología , Conformación Proteica , Receptores CXCR3/antagonistas & inhibidores , Receptores CXCR3/clasificación , Receptores CXCR3/genética , Pez Cebra/microbiología , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death.
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Autofagia/genética , Macrófagos/metabolismo , Proteínas de la Membrana/deficiencia , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Piroptosis/genética , Tuberculosis/genética , Animales , Muerte Celular , Humanos , Pez CebraRESUMEN
We describe new open source software called QuantiFish for rapid quantitation of fluorescent foci in zebrafish larvae, to support infection research in this animal model. QuantiFish extends the conventional measurements of bacterial load and number of bacterial foci to include measures for dissemination of infection. These are represented by the proportions of bacteria between foci and their spatial distribution. We showcase these measures by comparison of intravenous and hindbrain routes of Mycobacterium marinum infection, which are indistinguishable by measurement of bacterial load and not consistently differentiated by the number of bacterial foci. The intravenous route showed dose dependent dissemination of infection, reflected by increased spatial dispersion of bacteria and lower proportions of bacteria distributed across many foci. In contrast, hindbrain infection resulted in localised disease, limited to a smaller area and higher proportions of bacteria distributed across fewer foci. The application of QuantiFish may extend beyond models of infection, to study other pathologies such as metastatic cancer.
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Larva/microbiología , Microscopía Fluorescente/métodos , Rombencéfalo/microbiología , Pez Cebra/embriología , Animales , Carga Bacteriana , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Procesamiento de Imagen Asistido por Computador , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium marinum , Reconocimiento de Normas Patrones Automatizadas , Programas InformáticosRESUMEN
Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.
Asunto(s)
Embrión no Mamífero/ultraestructura , Imagen Molecular/métodos , Espectrometría Raman/métodos , Imagen de Lapso de Tiempo/métodos , Pez Cebra/anatomía & histología , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/metabolismo , Imagen Molecular/instrumentación , Análisis Multivariante , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum/crecimiento & desarrollo , Mycobacterium marinum/patogenicidad , Espectrometría Raman/instrumentación , Imagen de Lapso de Tiempo/instrumentación , Cicatrización de Heridas/fisiología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Macrófagos/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Fagocitosis/genética , Salmonelosis Animal , Salmonella typhimurium/inmunología , Proteínas de Pez Cebra/fisiología , Pez Cebra , Animales , Animales Modificados Genéticamente , Autofagia/fisiología , Bacteriemia/genética , Bacteriemia/inmunología , Bacteriemia/microbiología , Bacteriemia/patología , Embrión no Mamífero , Proteínas Asociadas a Microtúbulos/genética , Fagocitosis/inmunología , Especies Reactivas de Oxígeno/metabolismo , Salmonelosis Animal/genética , Salmonelosis Animal/inmunología , Salmonelosis Animal/metabolismo , Salmonelosis Animal/microbiología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/inmunología , Pez Cebra/microbiología , Proteínas de Pez Cebra/genéticaRESUMEN
Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model.