RESUMO
The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein with receptor recognition ability that plays an important role in the infection of cells by NDV. An alignment of NDV HN protein sequences of different genotypes showed that vaccine strains of NDV, such as the LaSota strain, generally have an HN protein of 577 amino acids. In comparison, the HN protein of the V4 strain has 616 amino acids, with 39 more amino acids at the C-terminus. In this study, we generated a recombinant NDV (rNDV) with a 39-amino-acid truncation at the HN C-terminus based on the full-length cDNA clone of the V4 strain. This rNDV, named rV4-HN-tr, displayed thermostability similar to that of the parental V4 strain. However, growth kinetics and pathogenicity analysis suggested that rV4-HN-tr is more virulent than the V4 strain. Notably, the C-terminus of HN affected the ability of the virus to adsorb onto cells. Structural predictions further suggested that the C-terminus of HN may obstruct the sialic acid binding site. Immunization of chickens with rV4-HN-tr induced a 3.5-fold higher level of NDV-specific antibodies than that obtained with the V4 strain and provided 100% immune protection against NDV challenge. Our study suggests that rV4-HN-tr is a thermostable, safe, and highly efficient vaccine candidate against Newcastle disease.
Assuntos
Doença de Newcastle , Vacinas Virais , Animais , Vírus da Doença de Newcastle , Galinhas , Virulência , Neuraminidase/genética , Hemaglutininas/genética , Proteína HN/genética , Proteína HN/metabolismo , Vacinas Virais/genética , Anticorpos Antivirais , AminoácidosRESUMO
Mycoplasma bovis (M. bovis) is one of the important pathogens which may cause bovine respiratory disease syndrome (BRDS), and results in huge economic losses for yaks (Bos gaurus) breeding industry. However, there is limited information about M. bovis in yaks. In our study, 145 nasal mucus samples from yaks with pneumonia were collected to clarify. Bacteriological determination was carried out through biochemical identification and Polymerase Chain Reaction (PCR) detection. And ten strains of Mycoplasma bovis (M. bovis) were found from collected samples. Then, the growth curve of isolated strains was determined by the change of optical density (OD630), pH value and Color Change Cnit (CCU). K-B disk method was also used for antimicrobial susceptibility testing. Results of colony morphology and biochemical testing were consistent with the biological characters of M. bovis. The nucleotide sequences of uvrC specific gene and 16S rRNA gene among the 10 strains were highly homologous. The growth curve assay showed that the isolates cultured in PPLO medium were in lag phase for 24 h, entered stable period in 42 h, and entered decline phase after 78 h. The isolates were found resistant to macrolides, aminoglycosides and lincomycin at various degrees, but they were sensitive or moderately sensitive to doxycycline and kanamycin under antimicrobial susceptibility analysis. In conclusion, the results provided certain reference for the follow-up research and guiding for the treatment of M. bovis in yaks.
Assuntos
Doenças dos Bovinos , Infecções por Mycoplasma , Mycoplasma bovis , Animais , Antibacterianos/farmacologia , Bovinos , Macrolídeos/farmacologia , Infecções por Mycoplasma/veterinária , Mycoplasma bovis/genética , RNA Ribossômico 16S/genéticaRESUMO
Riemerellosis, a Riemerella anatipestifer infection, can cause meningitis, pericarditis, parahepatitis, and airsacculitis in ducks, leading to serious economic losses in the duck meat industry. However, the molecular mechanism of the pathogenesis and virulence factors of this infection are poorly understood. In the present study, we created a mutant strain RA-YMΔCas9 using trans-conjugation. Bacterial virulence tests indicated that the median lethal dose (LD50) of RA-YMΔCas9 was 5.01â¯×â¯107â¯CFU, significantly lower than that of the RA-YM strain, which was 1.58â¯×â¯105â¯CFU. The distribution and blood bacterial load from the infection groups showed no significant difference in the brain between the RA-YMΔCas9 mutant and the wild-type RA-YM strains, however, the number of mutant strains were significantly reduced in the liver, heart, and blood. Animal immunization experiments demonstrated that the intranasal administration of RA-YMΔCas9 in ducklings provided 80% protection after challenge with the wild-type strain, showing potential use as a live mucosal vaccine. RNAseq analysis indicated that Cas9 protein played a regulatory role in gene expression. This study is the first to report on the involvement of Cas9 in the regulation and pathogenesis of R. anatipestifer, and provides a theoretical basis for the development of relevant genetic engineering vaccines.
Assuntos
Bacteriemia/veterinária , Doenças das Aves/microbiologia , Proteína 9 Associada à CRISPR/metabolismo , Infecções por Flavobacteriaceae/veterinária , Regulação Bacteriana da Expressão Gênica , Riemerella/patogenicidade , Fatores de Virulência/metabolismo , Animais , Animais Recém-Nascidos , Bacteriemia/microbiologia , Bacteriemia/patologia , Doenças das Aves/patologia , Proteína 9 Associada à CRISPR/deficiência , Patos , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/patologia , Dose Letal Mediana , Riemerella/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Riemerella anatipestifer (RA), the causative agent of infectious serositis that targets ducklings and other poultry, secretes protein via the type IX secretion system (T9SS). The proteins transported by T9SS are located on the bacterial cell surface or secreted into the extracellular milieu. In this study, a sprA deletion mutant was constructed encoding a core protein of T9SS to investigate its influence on outer membrane protein expression and its role in virulence. Compared with the wild-type RA-YM strain, the deletion mutant ΔsprA failed to digest gelatin, showed the same growth rate in the logarithmic phase and exhibited greater sensitivity to the bactericidal activity of duck sera, whereas the complemented strain restored these phenotypes. The outer membrane proteome of RA-YM and the ΔsprA mutant were analyzed by Tandem Mass Tags, which revealed 198 proteins with predicted localization to the cell envelope. Sixty-three of these proteins were differentially expressed in the outer membrane, with 43 up-regulated and 20 down-regulated. Among the twelve outer membrane proteins which were secreted by T9SS, four proteins were up-regulated and one protein was down-regulated. Animal experiments demonstrated that the median lethal dose of the mutant strain ΔsprA was about 500 times higher than that of the wild-type RA-YM strain, and bacterial loads in blood, brain, heart, liver and spleen of the ΔsprA-infected ducks were significantly reduced. Our results indicate that the SprA is a virulence-associated factor of RA, and its absence results in altered abundance of outer membrane proteins, and secretion disorders associated with some of the T9SS effector proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Patos/microbiologia , Infecções por Flavobacteriaceae/veterinária , Regulação Bacteriana da Expressão Gênica , Doenças das Aves Domésticas/microbiologia , Riemerella/genética , Animais , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/patologia , Deleção de Genes , Doenças das Aves Domésticas/patologia , Riemerella/patogenicidade , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs.
Assuntos
Leucócitos/virologia , Microdomínios da Membrana/metabolismo , Receptores Virais/biossíntese , Vaccinia virus/fisiologia , Tropismo Viral , Ligação Viral , Células Cultivadas , HumanosRESUMO
African swine fever (ASF) and postweaning multisystemic wasting syndrome (PMWS) are acute infectious diseases caused by the African swine fever virus (ASFV) and porcine circovirus type 2 (PCV2). At present, there are no effective vaccines for the prevention of ASFV. PMWS, which is harmful to the domestic and even the world pig industry, is difficult to cure and has a high mortality. So, developing simple, inexpensive, and accurate analytical methods to detect and effectively diagnose ASFV and PCV2 can be conducive to avoid ASFV and PCV2 infection. CRISPR has become a potentially rapid diagnostic tool due to recent discoveries of the trans-cleavage properties of CRISPR type V effectors. Herein, we report the visual detection based on CRISPR-Cas12a (cpf1), which is more convenient than fluorescence detection. Through in vitro cleavage target DNA activation, Cas12a can trans-cleavage ssDNA G-quadruplex. TMB/H2O2 and Hemin cannot be catalyzed by cleavaged G-DNA to produce green color products. This protocol is useful for the detection of ASFV and PCV2 with high sensitivity. This method can enable the development of visual and label-free ASFV and PCV2 detection and can be carried out in the field without relying on instruments or power. This method can complete nucleic acid detection at 37 °C without using other instruments or energy. Our research has expanded the application of Cas12a and laid the foundation for the field's rapid detection of viral nucleic acid in future.
RESUMO
Duck infectious serositis, also known as Riemerella anatipestifer disease, infects domestic ducks, geese, and turkeys and wild birds. However, the regulatory mechanism of its pathogenicity remains unclear. The PhoPR two-component system (TCS) was first reported in Gram-negative bacteria in our previous research and was demonstrated to be involved in virulence and gene expression. Here, DNA affinity purification sequencing (DAP-seq) was applied to further explore the regulation of PhoPR in relation to pathogenicity in R. anatipestifer. A conserved motif was identified upstream of 583 candidate target genes which were directly regulated by PhoP. To further confirm the genes which are regulated by PhoR and PhoP, single-gene-deletion strains were constructed. The results of transcriptome analysis using next-generation RNA sequencing showed 136 differentially expressed genes (DEGs) between the ΔphoP strain and the wild type (WT) and 183 DEGs between the ΔphoR strain and the WT. The candidate target genes of PhoP were further identified by combining transcriptome analysis and DAP-seq, which revealed that the main direct regulons of PhoP are located on the membrane and PhoP is involved in regulating aerotolerance. Using the in vivo duck model, the pathogenicity of ΔphoP and ΔphoR mutants was found to be significantly lower than that of the WT. Together, our findings provide insight into the direct regulation of PhoP and suggest that phoPR is essential for the pathogenicity of R. anatipestifer. The gene deletion strains are expected to be candidate live vaccine strains of R. anatipestifer which can be used as ideal genetic engineering vector strains for the expression of foreign antigens. IMPORTANCE Riemerella anatipestifer is a significant pathogen with high mortality in the poultry industry that causes acute septicemia and infectious polyserositis in ducks, chickens, geese, and other avian species. Previously, we characterized the two-component system encoded by phoPR and found that R. anatipestifer almost completely lost its pathogenicity for ducklings when phoPR was deleted. However, the mechanism of PhoPR regulation of virulence in R. anatipestifer had not been deeply explored. In this study, we utilized DAP-seq to explore the DNA-binding sites of PhoP as a response regulator in the global genome. Furthermore, phoP and phoR were deleted separately, and transcriptomics analysis of the corresponding gene deletion strains was performed. We identified a series of directly regulated genes of the PhoPR two-component system. The duckling model showed that both PhoP and PhoR are essential virulence-related factors in R. anatipestifer.
Assuntos
Infecções por Flavobacteriaceae , Doenças das Aves Domésticas , Animais , Proteínas de Bactérias/metabolismo , Galinhas , Patos/metabolismo , Patos/microbiologia , Infecções por Flavobacteriaceae/veterinária , Infecções por Flavobacteriaceae/microbiologia , Doenças das Aves Domésticas/microbiologia , Vacinas Atenuadas , Virulência/genética , Fatores de Virulência/genética , Genoma BacterianoRESUMO
A simple gold-immunochromatographic assay (GICA) based on indirect reaction format was developed for rapid detection of antibodies against avian infectious bronchitis virus (IBV). The detection time of IBV IgG by the GICA was less than 10 min, whereas the HI test and the enzyme-linked immunosorbent assay (ELISA) require 1-2 hr. Reference sera against newcastle disease virus, infectious bursal disease, avian influenza virus H5 and H9 subtypes were all negatives for anti-IBV antibodies using the GICA. Compared with the HI, the sensitivity of the GICA was 92.11% and specificity was 81.71%. The agreement rate between the 2 tests was 85%. Compared with the ELISA, the sensitivity of the GICA was 92.31% and specificity was 97.06%. The agreement rate between the 2 tests was 95%. The GICA test strip is a reliable and useful tool for the on-site surveillance of anti-IBV antibodies.
Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/isolamento & purificação , Fitas Reagentes , Animais , Doenças das Aves/sangue , Doenças das Aves/imunologia , Doenças das Aves/virologia , Galinhas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Vírus da Bronquite Infecciosa/imunologia , Doença de Newcastle/sangue , Doença de Newcastle/imunologia , Vacinas de Produtos Inativados , Vacinas ViraisRESUMO
Riemerella anatipestifer is the causative agent of duck septicaemia. Determination of R. anatipestifer virulence mechanisms will help us to effectively control this contagious agent. The differentially expressed gene profile of R. anatipestifer in infected duck livers was therefore identified and compared with in vitro cultures by selective capture of transcribed sequences analysis. A total of 48 genes were identified, of which 43 were genes that encode enzymes for amino acid biosynthesis and metabolism, intermediary metabolism, and energy metabolism, or proteins for regulatory adaptive responses, general microbial stress response, transport proteins and secreted proteinases. Five were unknown, novel genes. Eight genes representing the categories were randomly chosen and verified by real-time reverse transcriptase-polymerase chain reaction analysis. All were upregulated by R. anatipestifer in infected duck livers, with changes ranging from 1.44-fold to 4.62-fold compared with in vitro cultures. The results from the present study revealed a gene expression profile of R. anatipestifer in infected duck livers. The unknown but novel genes may be potential novel virulence factors for R. anatipestifer. In conclusion, the data from this study will provide a molecular basis for further study of R. anatipestifer pathogenesis.
Assuntos
Genes Bacterianos , Fígado/metabolismo , Infecções por Pasteurella/veterinária , Pasteurella/genética , Doenças das Aves Domésticas/genética , Animais , Patos , Perfilação da Expressão Gênica , Fígado/microbiologia , Pasteurella/isolamento & purificação , Pasteurella/patogenicidade , Infecções por Pasteurella/genética , Doenças das Aves Domésticas/microbiologia , Fatores de Virulência/genéticaRESUMO
Riemerella anatipestifer is a Gram-negative, pathogenic bacterium, which is harmful to poultry. However, the genomic islands (GIs) in R. anatipestifer are not well-studied. In this study, a 10K genomic island was predicted by the bioinformatics analysis of R. anatipestifer ATCC 11845, which is widely found in other R. anatipestifer genomes. We had first reported the genomic island integration and excision function in R. anatipestifer. We successfully constructed the integration plasmid by using the integrase and 53 bp attP elements. The 10K GI was found integrated at the 53 bp attB located in the Arg-tRNA of the R. anatipestifer RA-YM chromosome. We identified an integrase that helped in the precise integration and excision in R. anatipestifer and elucidated the molecular mechanism of the 10K genomic island integration and excision. Furthermore, we provided a new method for the gene expression and construction of complementary strain.
RESUMO
The S-layer protein CTC surface-display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying the protein of Mycoplasma gallisepticum (MG) agglutinin (pMGA) on the Bt cell surface. By fusing part of pmga1.2 (pmga1.2p) with the surface-anchoring motif of ctc, two recombinant plasmids, pCTC-PMGA1.2P and pCSPMGA1.2P, were constructed. They harboured the fusion genes ctc-pmga1.2p and csa-ctc-pmga1.2p (csa represents csaAB operon, important in anchoring the S-layer protein on the cell surface), respectively. Two recombinant Bt strains were constructed by electro-transferring recombinant plasmids to a Bt plasmid-free derivative strain BMB171. Strains obtained were BCCG (bearing pCTC-PMGA1.2P and the csaAB operon-carrying plasmid pMIL-CSA) and CG (pCSPMGA1.2P). The vegetative cells of both strains were used as antigens for haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA and HI assays showed that recombinant PMGA1.2P proteins were not only displayed on the cell surface of BCCG and CG, but also specific to MG-positive serum. After oral immunization of chickens with spores, both BCCG and CG elicited a humoral response to PMGA1.2P and exhibited immunogenicity, as indicated by serum plate agglutination (SPA) assays. This study suggests the possibility of generating heat-stable and oral vaccines against infectious diseases of fowl with Bt surface-display system.
Assuntos
Aglutininas/metabolismo , Bacillus thuringiensis/metabolismo , Glicoproteínas de Membrana/metabolismo , Mycoplasma gallisepticum/metabolismo , Aglutininas/genética , Animais , Antígenos de Bactérias , Bacillus thuringiensis/genética , Vacinas Bacterianas , Galinhas/microbiologia , DNA Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Engenharia Genética , Hemaglutinação , Glicoproteínas de Membrana/genética , Mycoplasma gallisepticum/imunologia , Plasmídeos , Proteínas RecombinantesRESUMO
The pathogenic process of highly pathogenic avian influenza virus (HPAIV) infection is poorly understood. To explore the differential expression of kidney genes as a result of HPAIV infection, two cDNA libraries were constructed from uninfected and infected kidneys by suppression subtractive hybridization (SSH). Fifteen genes including IFN-stimulated genes (ISG12), lymphocyte antigen 6 complex locus E gene (LY6E), matrix Gla protein gene (MGP), lysozyme gene, haemopoiesis related membrane protein 1 gene, KIAA1259, MGC68696, G6pc-prov protein gene (G6PC), MGC4504, alcohol dehydrogenase gene (ADH), glutathione S-transferase gene (GST), sodium-dependent high-affinity dicarboxylate transporter gene (SDCT), Synaptotagmin XV (SytXV) and two novel genes were found significantly up-regulated or dramatically suppressed. Differential expression of these genes was further identified by Northern blot. Functional analysis indicated that the regulation of their expression might contribute to the pathogenic process of HPAIV infection. In contrast, the increased expression of three IFN-stimulated genes named ISG12, LY6E, and haemopoiesis related membrane protein 1 gene might reflect host defense responses. Further study showed that ISG12 protein failed to directly interact with NS1 protein of HPAIV which expressed simultaneously in the organs where HPAIV replication occurred, by use of BacterioMatch two-hybrid system. Therefore, our findings may provide new insights into understanding the molecular mechanism underlying the pathophysiological process of HPAIV infection in chicken.
Assuntos
Regulação Viral da Expressão Gênica , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/genética , Vírus da Influenza A/patogenicidade , Influenza Aviária/genética , Rim/fisiopatologia , Doenças das Aves Domésticas/virologia , Animais , Galinhas , China , Primers do DNA , Etiquetas de Sequências Expressas , Vírus da Influenza A/genética , Rim/virologia , Doenças das Aves Domésticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas não Estruturais Virais/genéticaRESUMO
Chlamydia trachomatis urogenital serovars primarily replicate in epithelial cells lining the reproductive tract. Epithelial cells recognize Chlamydia through cell surface and cytosolic receptors, and/or endosomal innate receptors such as Toll-like receptors (TLRs). Activation of these receptors triggers both innate and adaptive immune mechanisms that are required for chlamydial clearance, but are also responsible for the immunopathology in the reproductive tract. We previously demonstrated that Chlamydia muridarum (Cm) induces IFN-ß in oviduct epithelial cells (OE) in a TLR3-dependent manner, and that the synthesis of several cytokines and chemokines are diminished in Cm-challenged OE derived from TLR3-/- 129S1 mice. Furthermore, our in vitro studies showed that Cm replication in TLR3-/- OE is more efficient than in wild-type OE. Because TLR3 modulates the release inflammatory mediators involved in host defense during Cm infection, we hypothesized that TLR3 plays a protective role against Cm-induced genital tract pathology in congenic C57BL/6N mice. Using the Cm mouse model for human Chlamydia genital tract infections, we demonstrated that TLR3-/- mice had increased Cm shedding during early and mid-stage genital infection. In early stage infection, TLR3-/- mice showed a diminished synthesis of IFN-ß, IL-1ß, and IL-6, but enhanced production of IL-10, TNF-α, and IFN-γ. In mid-stage infection, TLR3-/- mice exhibited significantly enhanced lymphocytic endometritis and salpingitis than wild-type mice. These lymphocytes were predominantly scattered along the endometrial stroma and the associated smooth muscle, and the lamina propria supporting the oviducts. Surprisingly, our data show that CD4+ T-cells are significantly enhanced in the genital tract TLR3-/- mice during mid-stage Chlamydial infection. In late-stage infections, both mouse strains developed hydrosalpinx; however, the extent of hydrosalpinx was more severe in TLR3-/- mice. Together, these data suggest that TLR3 promotes the clearance of Cm during early and mid-stages of genital tract infection, and that loss of TLR3 is detrimental in the development hydrosalpinx.
Assuntos
Infecções por Chlamydia/genética , Infecções por Chlamydia/microbiologia , Chlamydia muridarum , Suscetibilidade a Doenças , Infecções do Sistema Genital/genética , Infecções do Sistema Genital/microbiologia , Receptor 3 Toll-Like/genética , Animais , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/patologia , Citocinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções do Sistema Genital/metabolismo , Infecções do Sistema Genital/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor 3 Toll-Like/metabolismoRESUMO
A gold-immunochromatographic test-strip kit is used for the detection of IgG antibodies against the nucleocapsid protein of Avian Influenza Virus (AIV). Compared with the "gold standard", i.e. hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) assays, the gold-immunochromatographic test strip has many advantages, such as high specificity, high sensitivity, convenience, is rapid and has low cost. The gold-immunochromatographic test strip provides a unique tool for the on-site surveillance and diagnosis of Avian Influenza.
Assuntos
Anticorpos Antivirais/sangue , Galinhas , Imuno-Histoquímica/veterinária , Vírus da Influenza A/imunologia , Influenza Aviária/imunologia , Animais , Coloide de Ouro/química , Testes de Inibição da Hemaglutinação/métodos , Testes de Inibição da Hemaglutinação/veterinária , Imunodifusão/métodos , Imunodifusão/veterinária , Imunoglobulina G/sangue , Imuno-Histoquímica/métodos , Imuno-Histoquímica/normas , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Influenza Aviária/virologia , Proteínas do Nucleocapsídeo/imunologia , Fitas Reagentes , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Organismos Livres de Patógenos EspecíficosRESUMO
The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YMΔRS09735/RS09740 strain was constructed to investigate the role of the RAYM_RS09735/RAYM_RS09740 TCS in R. anatipestifer virulence and gene regulation. The median lethal dose (LD50) of YMΔRS09735/RS09740 was found to be >1011 CFU, equivalent to that of avirulent bacterial strains. The bacterial abundances of the YMΔRS09735/RS09740 strain in the heart, brain, liver, blood, and spleen were significantly lower than that of the wild-type R. anatipestifer YM strain. Pathological analysis using hematoxylin and eosin staining showed that, compared to the wild-type, the mutant YMΔRS09735/RS09740 strain caused significantly less virulence in infected ducklings. RNAseq and real-time PCR analysis indicated that the RAYM_RS09735/RAYM_RS09740 TCS is a PhoP/PhoR system. This is a novel type of TCS for Gram-negative bacteria. The TCS was also found to be a global regulator of expression in R. anatipestifer, with 112 genes up-regulated and 693 genes down-regulated in the YMΔRS09735/RS09740 strain (~33% genes demonstrated differential expression). In summary, we have reported the first PhoP/PhoR TCS identified in a Gram-negative bacterium and demonstrated that it is involved in virulence and gene regulation in R. anatipestifer.
RESUMO
It has been well characterized that piglets can absorb colostrum IgG across the intestine to neonatal bloodstream and a certain level of IgG has been found in the mucosal secretions of the porcine intestinal tract. However, little is known about how the maternal IgG transport across the intestinal barrier and how IgG enter the lumen of intestinal tract. In this study, we demonstrated that the porcine neonatal Fc receptor (pFcRn) was expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2) as well as in kidney cells (PK-15), and pFcRn was mainly distributed in the apical side of the polarized IPEC-J2 cells. Analyzing the phylogenetic relatedness of this gene we found that swine and human neonatal Fc receptor (FcRn) amino acid sequence are closer than rodents. We also showed that pFcRn mediated bidirectional IgG transport across polarized IPEC-J2 cells and bound to IgG in a pH-dependent manner. Furthermore, pFcRn-transcytosed viral-specific IgG reduced the transmissible gastroenteritis virus (TGEV) yield from the luminal direction by a 50% tissue culture infective dose (TCID50) assay. Our results indicate that pFcRn-dependent bidirectional IgG transport across the intestinal epithelium plays critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces.
Assuntos
Gastroenterite Suína Transmissível/imunologia , Imunidade nas Mucosas , Imunoglobulina G/imunologia , Mucosa Intestinal/imunologia , Receptores Fc/imunologia , Vírus da Gastroenterite Transmissível/fisiologia , Animais , Linhagem Celular , Células Epiteliais/imunologia , Concentração de Íons de Hidrogênio , Mucosa Intestinal/virologia , Jejuno/imunologia , Modelos Animais , SuínosRESUMO
We previously reported that the IFN-ß secreted by Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) was mostly dependent on the TLR3 signaling pathway. To further characterize the mechanisms of IFN-ß synthesis during Chlamydia infection of OE cells in vitro, we utilized specific inhibitory drugs to clarify the roles of IRF3 and NF-κB on both early- and late-phase C. muridarum infections. Our results showed that the pathways involved in the early-phase of IFN-ß production were distinct from that in the late-phase of IFN-ß production. Disruption of IRF3 activation using an inhibitor of TBK-1 at early-phase Chlamydia infection had a significant impact on the overall synthesis of IFN-ß; however, disruption of IRF3 activation at late times during infection had no effect. Interestingly, inhibition of NF-κB early during Chlamydia infection also had a negative effect on IFN-ß production; however, its impact was not significant. Our data show that the transcription factor IRF7 was induced late during Chlamydia infection, which is indicative of a positive feedback mechanism of IFN-ß synthesis late during infection. In contrast, IRF7 appears to play little or no role in the early synthesis of IFN-ß during Chlamydia infection. Finally, we demonstrate that antibiotics that target chlamydial DNA replication are much more effective at reducing IFN-ß synthesis during infection versus antibiotics that target chlamydial transcription. These results provide evidence that early- and late-phase IFN-ß production have distinct signaling pathways in Chlamydia-infected OE cells, and suggest that Chlamydia DNA replication might provide a link to the currently unknown chlamydial PAMP for TLR3.
Assuntos
Chlamydia muridarum/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Interferon beta/biossíntese , Oviductos/citologia , Transdução de Sinais , Animais , Linhagem Celular , Chlamydia muridarum/efeitos dos fármacos , Chlamydia muridarum/genética , Replicação do DNA/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , NF-kappa B/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Fatores de Tempo , Receptor 3 Toll-Like/metabolismoRESUMO
We previously reported that Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) secrete interferon ß (IFN-ß) in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-ß into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-ß synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type (WT) and TLR3-deficient OE cells to ascertain its putative role at early- and late-times during Chlamydia infection. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in WT OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-ß detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared with WT OE cells, which suggest that STAT1 is required for the optimal synthesis of IFN-ß during infection. Real-time quantitative polymerase chain reaction analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 genes in the WT and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR1 was blocked with neutralizing antibody revealed that IFNAR1-mediated signaling was critical to the Chlamydia-induced upregulation in IFN-α gene transcription, but had no role in the Chlamydia-induced upregulation in IFN-ß gene transcription.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia muridarum/imunologia , Células Epiteliais/imunologia , Tubas Uterinas/imunologia , Interferon beta/biossíntese , Fator de Transcrição STAT1/genética , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Infecções por Chlamydia/microbiologia , Células Epiteliais/citologia , Tubas Uterinas/citologia , Feminino , Regulação da Expressão Gênica/imunologia , Fator Regulador 7 de Interferon/biossíntese , Fator Regulador 7 de Interferon/genética , Interferon-alfa/biossíntese , Interferon beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/biossíntese , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/genética , Ativação Transcricional/imunologia , Regulação para CimaRESUMO
UNLABELLED: The pathogenesis of accelerated liver damage in subjects coinfected with hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) remains largely unknown. Recent studies suggest that ongoing chronic liver inflammation is responsible for the liver injury in HCV-infected patients. We aimed to determine whether HIV-1 coinfection altered intrahepatic inflammatory profiles in HCV infection, thereby hastening liver damage. We used a real-time RT-PCR-based array to comparatively analyze intrahepatic inflammation gene profiles in liver biopsy specimens from HCV-infected (nâ=â16), HCV/HIV-1-coinfected (nâ=â8) and uninfected (nâ=â8) individuals. We then used human hepatocytes to study the molecular mechanisms underlying alternations of the inflammatory profiles. Compared with uninfected individuals, HCV infection and HCV/HIV-1 coinfection markedly altered expression of 59.5% and 50.0% of 84 inflammation-related genes tested, respectively. Among these genes affected, HCV infection up-regulated the expression of 24 genes and down-regulated the expression of 26 genes, whereas HCV/HIV-1 coinfection up-regulated the expression of 21 genes and down-regulated the expression of 21 genes. Compared with HCV infection, HCV/HIV-1 coinfection did not dramatically affect intrahepatic gene expression profiles of cytokines and their receptors, but profoundly altered expression of several chemokine genes including up-regulation of the CXCR3-associated chemokines. Human hepatocytes produced these chemokines in response to virus-related microbial translocation, viral protein stimulation, and antiviral immune responses. CONCLUSIONS: HIV-1 coinfection profoundly alters intrahepatic chemokine but not cytokine profiles in HCV-infected subjects. The altered chemokines may orchestrate the tissue-specific and cell-selective trafficking of immune cells and autoimmunity to accelerate liver disease in HCV/HIV-1 coinfection.
Assuntos
Quimiocinas/metabolismo , Coinfecção/virologia , Infecções por HIV/complicações , Infecções por HIV/virologia , Hepatite C/complicações , Mediadores da Inflamação/metabolismo , Fígado/patologia , Adolescente , Adulto , Idoso , Quimiocinas/genética , Estudos de Coortes , Coinfecção/genética , Demografia , Feminino , Regulação da Expressão Gênica , Infecções por HIV/genética , Hepatite C/genética , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Fígado/virologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR3/metabolismo , Adulto JovemRESUMO
Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4⺠T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA), but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA) binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA)-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA)-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why infection of N. gonorrhoeae fails to trigger an effective specific immune response or develop immune memory, and may affect the potent synergy between gonorrhea and HIV-1 infection.