RESUMO
The gut microbiota is known to play a role in regulating host metabolism, yet the mechanisms underlying this regulation are not well elucidated. Our study aimed to characterize the differences in gut microbiota compositions and their roles in iron absorption between wild-type (WT) and CD163/pAPN double-gene-knockout (DKO) weaned piglets. A total of 58 samples along the entire digestive tract were analyzed for microbial community using 16S rRNA gene sequencing. The colonic microbiota and their metabolites were determined by metagenomic sequencing and untargeted liquid chromatography-mass spectrometry (LC-MS), respectively. Our results showed that no alterations in microbial community structure and composition were observed between DKO and WT weaned piglets, with the exception of colonic microbiota. Interestingly, the DKO piglets had selectively increased the relative abundance of the Leeia genus belonging to the Neisseriaceae family and decreased the Ruminococcaceae_UCG_014 genus abundance. Functional capacity analysis showed that organic acid metabolism was enriched in the colon in DKO piglets. In addition, the DKO piglets showed increased iron levels in important tissues compared with WT piglets without any pathological changes. Pearson's correlation coefficient indicated that the specific bacteria such as Leeia and Ruminococcaceae_UCG_014 genus played a key role in host iron absorption. Moreover, the iron levels had significantly (P < 0.05) positive correlation with microbial metabolites, particularly carboxylic acids and their derivatives, which might increase iron absorption by preventing iron precipitation. Overall, this study reveals an interaction between colonic microbiota and host metabolism and has potential significance for alleviating piglet iron deficiency. IMPORTANCE Iron deficiency is a major risk factor for iron deficiency anemia, which is among the most common nutritional disorders in piglets. However, it remains unclear how the gut microbiota interacts with host iron absorption. The current report provides the first insight into iron absorption-microbiome connection in CD163/pAPN double knockout piglets. The present results showed that carboxylic acids and their derivatives contributed to the absorption of nonheme iron by preventing ferric iron precipitation.
Assuntos
Microbioma Gastrointestinal , Animais , Suínos , Microbioma Gastrointestinal/fisiologia , RNA Ribossômico 16S/genética , Antígenos CD , Colo/microbiologiaRESUMO
Like obligate intracellular parasites, viruses co-opt host cell resources to establish productive infections. Polyamines are key aliphatic molecules that perform important roles in cellular growth and proliferation. They are also needed for the successful multiplication of various viruses. Little is known about the effects of polyamines on Arteriviridae infections. Here, porcine reproductive and respiratory syndrome virus (PRRSV), an economically prominent porcine virus, was used to investigate virus-polyamine interactions. We found that PRRSV infection significantly downregulated the levels of cellular polyamines. Using an inhibitor or specific short interfering RNAs (siRNAs) of ornithine decarboxylase 1, a key anabolic enzyme involved in the classical de novo biosynthesis of polyamines, we found that polyamine depletion abrogated PRRSV proliferation, and this effect was recoverable by adding exogenous spermidine and spermine, but not putrescine to the cells, suggesting that the host inhibits polyamine biosynthesis to restrict PRRSV proliferation. Further analysis revealed that the expression level of spermidine-spermine acetyltransferase 1 (SAT1), a catabolic enzyme that reduces spermidine and spermine levels, was upregulated during PRRSV infection, but conversely, SAT1 had an inhibitory effect on PRRSV reproduction. Our data show that polyamines are important molecules during PRRSV-host interactions, and polyamines and their biosynthetic pathways are potential therapeutic targets against PRRSV infection.
Assuntos
Acetiltransferases/genética , Interações entre Hospedeiro e Microrganismos , Poliaminas/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Espermidina/metabolismo , Espermina/metabolismo , Acetiltransferases/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica , Rim/citologia , Suínos , Regulação para Cima , Replicação ViralRESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.
Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.
Assuntos
Antígenos CD13/deficiência , Infecções por Coronavirus/prevenção & controle , Coronavirus/patogenicidade , Gastroenterite Suína Transmissível/prevenção & controle , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Receptores de Superfície Celular/deficiência , Vírus da Gastroenterite Transmissível/patogenicidade , Animais , Animais Geneticamente Modificados , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Composição Corporal , Antígenos CD13/genética , Antígenos CD13/imunologia , Coronavirus/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Suscetibilidade a Doenças , Gastroenterite Suína Transmissível/genética , Gastroenterite Suína Transmissível/imunologia , Gastroenterite Suína Transmissível/virologia , Técnicas de Silenciamento de Genes , Interações entre Hospedeiro e Microrganismos , Indústria de Embalagem de Carne , Fenótipo , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Sus scrofa/genética , Suínos , Vírus da Gastroenterite Transmissível/imunologia , Aumento de PesoRESUMO
Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.