RESUMEN
Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.
Asunto(s)
Interacciones Huésped-Patógeno , Infecciones por Klebsiella , Klebsiella pneumoniae , Larva , Microinyecciones , Microscopía Fluorescente , Pez Cebra , Animales , Pez Cebra/microbiología , Klebsiella pneumoniae/inmunología , Microinyecciones/métodos , Larva/microbiología , Larva/inmunología , Microscopía Fluorescente/métodos , Interacciones Huésped-Patógeno/inmunología , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/inmunología , Modelos Animales de EnfermedadRESUMEN
The microbiome can influence cancer development and progression. However, less is known about the role of the skin microbiota in melanoma. Here, we took advantage of a zebrafish melanoma model to probe the effects of Staphylococcus aureus on melanoma invasion. We found that S. aureus produces factors that enhance melanoma invasion and dissemination in zebrafish larvae. We used a published in vitro 3D cluster formation assay that correlates increased clustering with tumor invasion. S. aureus supernatant increased clustering of melanoma cells and was abrogated by a Rho-Kinase inhibitor, implicating a role for Rho-GTPases. The melanoma clustering response was specific to S. aureus but not to other staphylococcal species, including S. epidermidis. Our findings suggest that S. aureus promotes melanoma clustering and invasion via lipids generated by the lipase Sal2 (officially known as GehB). Taken together, these findings suggest that specific bacterial products mediate melanoma invasive migration in zebrafish.
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Melanoma , Invasividad Neoplásica , Staphylococcus aureus , Pez Cebra , Animales , Pez Cebra/microbiología , Melanoma/patología , Melanoma/microbiología , Línea Celular Tumoral , Lípidos/química , Movimiento Celular/efectos de los fármacos , Larva/microbiología , Humanos , Proteínas de Pez Cebra/metabolismo , Agregación Celular/efectos de los fármacosRESUMEN
Acinetobacter baumannii is currently one of the most important opportunistic pathogens causing severe nosocomial infections worldwide. Quorum Sensing (QS) system is a widespread mechanism in bacteria to coordinate group behavior by sensing the density of bacterial populations and affect eukaryotic host cell. In Acinetobacter baumannii, AbaI protein is used as QS molecule synthetase to synthesize N- acyl homoserine lactones (AHLs). Currently, QS has made great progress in the study of drug resistance, but there is still a lack of complete understanding of its damage to host cells after adhesion and invasion. Thus, in this study, we examined the effects of abaI mutant (ΔabaI) on the functions of adhesion and invasion, cell viability, inflammation, apoptosis in A. baumannii infected A549 cells, to evaluate the effects of ΔabaI in a zebrafish model. We found the group infected with ΔabaI increased cell viability, reduced adhesion and invasion, cell injury, inflammatory cytokine production and apoptosis. By RNA-Seq, we explored the possibility that abaI stimulated A549 cells inflammation by A. baumannii infection via TLR4/MAPK signaling pathway. In addition, the ΔabaI significantly reduced pathogenicity and recruitment to neutrophils in zebrafish. These observations suggest that abaI plays a major role in A. baumannii infection.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Inflamación , Percepción de Quorum , Pez Cebra , Animales , Pez Cebra/microbiología , Acinetobacter baumannii/patogenicidad , Humanos , Infecciones por Acinetobacter/microbiología , Células A549 , Modelos Animales de Enfermedad , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Adhesión Bacteriana/efectos de los fármacos , Interacciones Huésped-Patógeno , Ligasas/metabolismo , Ligasas/genéticaRESUMEN
The zebrafish Danio rerio has become a popular model host to explore disease pathology caused by infectious agents. A main advantage is its transparency at an early age, which enables live imaging of infection dynamics. While multispecies infections are common in patients, the zebrafish model is rarely used to study them, although the model would be ideal for investigating pathogen-pathogen and pathogen-host interactions. This may be due to the absence of an established multispecies infection protocol for a defined organ and the lack of suitable image analysis pipelines for automated image processing. To address these issues, we developed a protocol for establishing and tracking single and multispecies bacterial infections in the inner ear structure (otic vesicle) of the zebrafish by imaging. Subsequently, we generated an image analysis pipeline that involved deep learning for the automated segmentation of the otic vesicle, and scripts for quantifying pathogen frequencies through fluorescence intensity measures. We used Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, three of the difficult-to-treat ESKAPE pathogens, to show that our infection protocol and image analysis pipeline work both for single pathogens and pairwise pathogen combinations. Thus, our protocols provide a comprehensive toolbox for studying single and multispecies infections in real-time in zebrafish.
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Procesamiento de Imagen Asistido por Computador , Pseudomonas aeruginosa , Pez Cebra , Pez Cebra/microbiología , Animales , Procesamiento de Imagen Asistido por Computador/métodos , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/diagnóstico por imagen , Acinetobacter baumannii/patogenicidad , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Klebsiella pneumoniae/patogenicidad , Oído Interno/microbiología , Oído Interno/diagnóstico por imagen , Aprendizaje ProfundoRESUMEN
A meta-analytic approach deciphered the taxonomic profile of the zebrafish gut microbiota at different developmental stages. Data (16S rDNA) were systematically searched in databases, selecting those with intestine samples of fish not exposed to a particular treatment or challenge (e.g., pathogens, dietetic tests, xenobiotics, etc.) and obtaining 340 samples to be processed. Results revealed marked differences between the developmental phases. Proteobacteria was the dominant phylum in the larval phase, with a relative abundance of 90%, while the rest of the phyla did not exceed 2%. Vibrio, Aeromonas, Plesiomonas, Pseudomonas, Shewanella, and Acinetobacter were the dominant genera in this phase. Transitional changes were observed after the larvae stage. Proteobacteria still registered high abundance (48%) in the juvenile phase, but Fusobacteria (40%) and Bacteriodota (5.9%) registered considerable increases. Genera, including Cetobacterium, Plesiomonas, Aeromonas, Vibrio, and Flavobacterium, dominated this stage. The phyla Proteobacteria (48%) and Fusobacteria (35%) were strongly established in the adult phase. Cetobacterium was registered as the most abundant genus, followed by Aeromonas, Acinetobacter, Plesiomonas, Vibrio, and ZOR0006 (Firmicutes; 6%). In conclusion, the composition of the intestinal microbiota of zebrafish is consistently determined by two primary phyla, Proteobacteria and Fusobacteria; however, this composition varies depending on the developmental stage. Cetobacterium and Aeromonas are the most relevant genera in juveniles and adults. Finally, these results reveal a consistent pattern of certain bacterial groups in the zebrafish microbiota that could help shape gnotobiotic models (colonized with a specific known bacterial community) or synthetic microbiota (in vitro assembly of microbes), among other approaches.
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Microbioma Gastrointestinal , Pez Cebra , Animales , Pez Cebra/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Proteobacteria/aislamiento & purificación , Proteobacteria/genética , ARN Ribosómico 16S/genética , Larva/microbiología , Modelos BiológicosRESUMEN
There is an increasing need for new treatment regimens to combat antibiotic-resistant strains of bacteria. Staphylococcus aureus is a clinically important, opportunist pathogen that has developed resistance to a range of antibiotics. The zebrafish larval model of systemic disease has been increasingly utilized to elucidate S. aureus virulence mechanisms and host-pathogen interactions. Here, we outline how this model can be used to investigate the effects of different antibiotics alone and in combination against S. aureus.
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Antibacterianos , Modelos Animales de Enfermedad , Larva , Infecciones Estafilocócicas , Staphylococcus aureus , Pez Cebra , Animales , Pez Cebra/microbiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Staphylococcus aureus/efectos de los fármacos , Larva/microbiología , Larva/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Quimioterapia Combinada , Interacciones Huésped-Patógeno/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
Human exposure to Vibrio vulnificus, a gram-negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease-causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1-BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.
Asunto(s)
Vibriosis , Vibrio vulnificus , Pez Cebra , Vibrio vulnificus/patogenicidad , Vibrio vulnificus/genética , Vibrio vulnificus/aislamiento & purificación , Animales , Pez Cebra/microbiología , Virulencia/genética , Vibriosis/microbiología , Factores de Virulencia/genética , Modelos Animales de Enfermedad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Microbiología AmbientalRESUMEN
Interbacterial competition is known to shape the microbial communities found in the host, however the interplay between this competition and host defense are less clear. Here, we use the zebrafish hindbrain ventricle (HBV) as an in vivo platform to investigate host responses to defined bacterial communities with distinct forms of interbacterial competition. We found that antibacterial activity of the type VI secretion system (T6SS) from both Vibrio cholerae and Acinetobacter baylyi can induce host inflammation and sensitize the host to infection independent of any individual effector. Chemical suppression of inflammation could resolve T6SS-dependent differences in host survival, but the mechanism by which this occurred differed between the two bacterial species. By contrast, colicin-mediated antagonism elicited by an avirulent strain of Shigella sonnei induced a negligible host response despite being a more potent bacterial killer, resulting in no impact on A. baylyi or V. cholerae virulence. Altogether, these results provide insight into how different modes of interbacterial competition in vivo affect the host in distinct ways.
Asunto(s)
Sistemas de Secreción Tipo VI , Vibrio cholerae , Pez Cebra , Animales , Pez Cebra/microbiología , Sistemas de Secreción Tipo VI/metabolismo , Vibrio cholerae/patogenicidad , Acinetobacter , Virulencia , Interacciones Huésped-Patógeno , Antibiosis/fisiología , Rombencéfalo/microbiología , Rombencéfalo/metabolismoRESUMEN
Candida auris is an emerging fungal pathogen with several concerning qualities. First recognized in 2009, it has arisen in multiple geographically distinct genomic clades nearly simultaneously. C. auris strains are typically multidrug resistant and colonize the skin much better than most other pathogenic fungi; it also persists on abiotic surfaces, enabling outbreaks due to transmission in health care facilities. All these suggest a biology substantially different from the 'model' fungal pathogen, Candida albicans and support intensive investigation of C. auris biology directly. To uncover novel virulence mechanisms in this species requires the development of appropriate animal infection models. Various studies using mice, the definitive model, are inconsistent due to differences in mouse and fungal strains, immunosuppressive regimes, doses, and outcome metrics. At the same time, developing models of skin colonization present a route to new insights into an aspect of fungal pathogenesis that has not been well studied in other species. We also discuss the growing use of nonmammalian model systems, including both vertebrates and invertebrates, such as zebrafish, C. elegans, Drosophila, and Galleria mellonella, that have been productively employed in virulence studies with other fungal species. This review will discuss progress in developing appropriate animal models, outline current challenges, and highlight opportunities in demystifying this curious species.
Asunto(s)
Candida auris , Modelos Animales de Enfermedad , Animales , Candida auris/patogenicidad , Candida auris/genética , Virulencia , Candidiasis/microbiología , Ratones , Humanos , Invertebrados/microbiología , Vertebrados/microbiología , Pez Cebra/microbiología , Caenorhabditis elegans/microbiologíaRESUMEN
Early-life exposure to environmental toxicants like Benzo[a]pyrene (BaP) is associated with several health consequences in vertebrates (i.e., impaired or altered neurophysiological and behavioral development). Although toxicant impacts were initially studied relative to host physiology, recent studies suggest that the gut microbiome is a possible target and/or mediator of behavioral responses to chemical exposure in organisms, via the gut-brain axis. However, the connection between BaP exposure, gut microbiota, and developmental neurotoxicity remains understudied. Using a zebrafish model, we determined whether the gut microbiome influences BaP impacts on behavior development. Embryonic zebrafish were treated with increasing concentrations of BaP and allowed to grow to the larval life stage, during which they underwent behavioral testing and intestinal dissection for gut microbiome profiling via high-throughput sequencing. We found that exposure affected larval zebrafish microbiome diversity and composition in a manner tied to behavioral development: increasing concentrations of BaP were associated with increased taxonomic diversity, exposure was associated with unweighted UniFrac distance, and microbiome diversity and exposure predicted larval behavior. Further, a gnotobiotic zebrafish experiment clarified whether microbiome presence was associated with BaP exposure response and behavioral changes. We found that gut microbiome state altered the relationship between BaP exposure concentration and behavioral response. These results support the idea that the zebrafish gut microbiome is a determinant of the developmental neurotoxicity that results from chemical exposure.
Asunto(s)
Conducta Animal , Benzo(a)pireno , Microbioma Gastrointestinal , Larva , Pez Cebra , Animales , Pez Cebra/microbiología , Benzo(a)pireno/toxicidad , Microbioma Gastrointestinal/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Larva/efectos de los fármacos , Larva/microbiologíaRESUMEN
Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1ß and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.
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Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas , Pez Cebra , Animales , Humanos , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/genética , Mycobacterium marinum , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pez Cebra/microbiología , Masculino , FemeninoRESUMEN
Staphylococcus aureus is a pathogenic bacterium responsible for a broad spectrum of infections, including cutaneous, respiratory, osteoarticular, and systemic infections. It poses a significant clinical challenge due to its ability to develop antibiotic resistance. This resistance limits therapeutic options, increases the risk of severe complications, and underscores the urgent need for new strategies to address this threat, including the investigation of treatments complementary to antibiotics. The evaluation of novel antimicrobial agents often employs animal models, with the zebrafish embryo model being particularly interesting for studying host-pathogen interactions, establishing itself as a crucial tool in this field. For the first time, this study presents a zebrafish embryo model for the in vivo assessment of bacteriophage efficacy against S. aureus infection. A localized infection was induced by microinjecting either methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA). Subsequent treatments involved administering either bacteriophage, vancomycin (the reference antibiotic for MRSA), or a combination of both via the same route to explore potential synergistic effects. Our findings indicate that the bacteriophage was as effective as vancomycin in enhancing survival rates, whether used alone or in combination. Moreover, bacteriophage treatment appears to be even more effective in reducing the bacterial load in S. aureus-infected embryos post-treatment than the antibiotic. Our study validates the use of the zebrafish embryo model and highlights its potential as a valuable tool in assessing bacteriophage efficacy treatments in vivo.
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Antibacterianos , Staphylococcus aureus Resistente a Meticilina , Terapia de Fagos , Infecciones Estafilocócicas , Vancomicina , Pez Cebra , Animales , Pez Cebra/microbiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/virología , Terapia de Fagos/métodos , Vancomicina/farmacología , Vancomicina/uso terapéutico , Infecciones Estafilocócicas/terapia , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Modelos Animales de Enfermedad , Embrión no Mamífero/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
Mycobacterium tuberculosis (Mtb) as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular Mtb and Mycobacterium avium in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone in vivo. In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections. IMPORTANCE: Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of Mtb and Mycobacterium avium in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an in vivo tuberculosis model based on Mycobacterium marinum infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.
Asunto(s)
Amiodarona , Autofagia , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Pez Cebra , Amiodarona/farmacología , Autofagia/efectos de los fármacos , Animales , Pez Cebra/microbiología , Humanos , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Modelos Animales de Enfermedad , Mycobacterium avium/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/microbiologíaRESUMEN
Edwardsiella piscicida is an acute marine pathogen that causes severe damage to the aquaculture industry worldwide. The pathogenesis of E. piscicida is dependent mainly on the type III secretion system (T3SS) and type VI secretion system (T6SS), both of which are critically regulated by EsrB and EsrC. In this study, we revealed that fatty acids influence T3SS expression. Unsaturated fatty acids (UFAs), but not saturated fatty acids (SFAs), directly interact with EsrC, which abolishes the function of EsrC and results in the turn-off of T3/T6SS. Moreover, during the in vivo colonization of E. piscicida, host fatty acids were observed to be transported into E. piscicida through FadL and to modulate the expression of T3/T6SS. Furthermore, the esrCR38G mutant blocked the interaction between EsrC and UFAs, leading to dramatic growth defects in DMEM and impaired colonization in HeLa cells and zebrafish. In conclusion, this study revealed that the interaction between UFAs and EsrC to turn off T3/T6SS expression is essential for E. piscicida infection.
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Proteínas Bacterianas , Edwardsiella , Infecciones por Enterobacteriaceae , Ácidos Grasos Insaturados , Enfermedades de los Peces , Sistemas de Secreción Tipo III , Sistemas de Secreción Tipo VI , Pez Cebra , Animales , Edwardsiella/genética , Edwardsiella/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/genética , Infecciones por Enterobacteriaceae/microbiología , Humanos , Células HeLa , Pez Cebra/microbiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Sistemas de Secreción Tipo VI/metabolismo , Sistemas de Secreción Tipo VI/genética , Ácidos Grasos Insaturados/metabolismo , Enfermedades de los Peces/microbiología , Regulación Bacteriana de la Expresión GénicaRESUMEN
Zebrafish are popular research organisms selected for laboratory use due in part to widespread availability from the pet trade. Many contemporary colonies of laboratory zebrafish are maintained in aquaculture facilities that monitor and aim to curb infections that can negatively affect colony health and confound experiments. The impact of laboratory control on the microbial constituents associated with zebrafish in research environments compared to the pet trade are unclear. Diseases of unknown causes are common in both environments. We conducted a metatranscriptomic survey to broadly compare the zebrafish-associated microbes in pet trade and laboratory environments. We detected many microbes in animals from the pet trade that were not found in laboratory animals. Cohousing experiments revealed several transmissible microbes including a newly described non-enveloped, double-stranded RNA virus in the Birnaviridae family we name Rocky Mountain birnavirus (RMBV). Infections were detected in asymptomatic animals from the pet trade, but when transmitted to laboratory animals RMBV was associated with pronounced antiviral responses and hemorrhagic disease. These experiments highlight the pet trade as a distinct source of diverse microbes that associate with zebrafish and establish a paradigm for the discovery of newly described pathogenic viruses and other infectious microbes that can be developed for study in the laboratory.
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Pez Cebra , Animales , Pez Cebra/virología , Pez Cebra/microbiología , Enfermedades de los Peces/virología , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/transmisión , Mascotas/virología , Mascotas/microbiología , Animales de Laboratorio/virología , Animales de Laboratorio/microbiología , AcuiculturaRESUMEN
The α-Gal syndrome (AGS) is an IgE-mediated tick borne-allergy that results in delayed anaphylaxis to the consumption of mammalian meat and products containing α-Gal. Considering that α-Gal-containing microbiota modulates natural antibody production to this glycan, this study aimed to evaluate the influence on tick salivary compounds on the gut microbiota composition in the zebrafish (Danio rerio) animal model. Sequencing of 16â¯S rDNA was performed in a total of 75 zebrafish intestine samples, representing different treatment groups: PBS control, Ixodes ricinus tick saliva, tick saliva non-protein fraction (NPF), tick saliva protein fraction (PF), and tick saliva protein fractions 1-5 with NPF (F1-5). The results revealed that treatment with tick saliva and different tick salivary fractions, combined with α-Gal-positive dog food feeding, resulted in specific variations in zebrafish gut microbiota composition at various taxonomic levels and affected commensal microbial alpha and beta diversities. Metagenomics results were corroborated by qPCR, supporting the overrepresentation of phylum Firmicutes in the tick saliva group, phylum Fusobacteriota in group F1, and phylum Cyanobacteria in F2 and F5 compared to the PBS-control. qPCRs results at genus level sustained significant enrichment of Plesiomonas spp. in groups F3 and F5, Rhizobium spp. in NPF and F4, and Cloacibacterium spp. dominance in the PBS control group. This study provides new results on the role of gut microbiota in allergic reactions to tick saliva components using a zebrafish model of AGS. Overall, gut microbiota composition in response to tick saliva biomolecules may be associated with allergic reactions to mammalian meat consumption in AGS.
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Hipersensibilidad a los Alimentos , Microbioma Gastrointestinal , Saliva , Pez Cebra , Animales , Saliva/microbiología , Saliva/inmunología , Pez Cebra/microbiología , Hipersensibilidad a los Alimentos/microbiología , Hipersensibilidad a los Alimentos/inmunología , ARN Ribosómico 16S/genética , Carne , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Metagenómica , Proteínas y Péptidos Salivales/inmunología , Proteínas y Péptidos Salivales/metabolismo , Ixodes/microbiología , Modelos Animales de EnfermedadRESUMEN
Vitamin B12 (B12) serves as a critical cofactor within mycobacterial metabolism. While some pathogenic strains can synthesize B12 de novo, others rely on host-acquired B12. In this investigation, we studied the transport of vitamin B12 in Mycobacterium marinum using B12-auxotrophic and B12-sensitive strains by deleting metH or metE, respectively. These two enzymes rely on B12 in different ways to function as methionine synthases. We used these strains to select mutants affecting B12 scavenging and confirmed their phenotypes during growth experiments in vitro. Our analysis of B12 uptake mechanisms revealed that membrane lipids and cell wall integrity play an essential role in cell envelope transport. Furthermore, we identified a potential transcription regulator that responds to B12. Our study demonstrates that M. marinum can take up exogenous B12 and that altering mycobacterial membrane integrity affects B12 uptake. Finally, during zebrafish infection using B12-auxotrophic and B12-sensitive strains, we found that B12 is available for virulent mycobacteria in vivo.IMPORTANCEOur study investigates how mycobacteria acquire essential vitamin B12. These microbes, including those causing tuberculosis, face challenges in nutrient uptake due to their strong outer layer. We focused on Mycobacterium marinum, similar to TB bacteria, to uncover its vitamin B12 absorption. We used modified strains unable to produce their own B12 and discovered that M. marinum can indeed absorb it from the environment, even during infections. Changes in the outer layer composition affect this process, and genes related to membrane integrity play key roles. These findings illuminate the interaction between mycobacteria and their environment, offering insights into combatting diseases like tuberculosis through innovative strategies. Our concise research underscores the pivotal role of vitamin B12 in microbial survival and its potential applications in disease control.
Asunto(s)
Membrana Externa Bacteriana , Mycobacterium marinum , Vitamina B 12 , Pez Cebra , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Vitamina B 12/metabolismo , Animales , Pez Cebra/microbiología , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Permeabilidad de la Membrana Celular , Transporte Biológico , Membrana Celular/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiologíaRESUMEN
Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.
Asunto(s)
Vida Libre de Gérmenes , Larva , Modelos Animales , Pez Cebra , Animales , Pez Cebra/microbiología , Larva/microbiología , Larva/crecimiento & desarrollo , Femenino , MasculinoRESUMEN
The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.
Asunto(s)
Proteínas Bacterianas , Edwardsiella , Infecciones por Enterobacteriaceae , Enfermedades de los Peces , Regulación Bacteriana de la Expresión Génica , Sulfuro de Hidrógeno , Estrés Fisiológico , Edwardsiella/genética , Edwardsiella/patogenicidad , Sulfuro de Hidrógeno/metabolismo , Animales , Virulencia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Enterobacteriaceae/microbiología , Enfermedades de los Peces/microbiología , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Especies Reactivas de Oxígeno/metabolismo , Operón , Péptidos Catiónicos Antimicrobianos/farmacología , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/genética , Pez Cebra/microbiologíaRESUMEN
Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.