RESUMO
Enteric viruses, including poliovirus, are spread by the fecal-oral route. In order to persist and transmit to a new host, enteric virus particles must remain stable once they are in the environment. Environmental stressors such as heat and disinfectants can inactivate virus particles and prevent viral transmission. It has been previously demonstrated that bacteria or bacterial surface glycans can enhance poliovirus virion stability and limit inactivation from heat or bleach. While investigating the mechanisms underlying bacterially enhanced virion thermal stability, we identified and characterized a poliovirus (PV) mutant with increased resistance to heat inactivation. The M132V mutant harbors a single amino acid change in the VP1 capsid coding that is sufficient to confer heat resistance but not bleach resistance. Although the M132V virus was stable in the absence of bacteria or feces at most temperatures, M132V virus was stabilized by feces at very high temperatures. M132V PV had reduced specific infectivity and RNA uncoating compared with those of wild-type (WT) PV, but viral yields in HeLa cells were similar. In orally inoculated mice, M132V had a slight fitness cost since fecal titers were lower and 12.5% of fecal viruses reverted to the WT. Overall, this work sheds light on factors that influence virion stability and fitness.IMPORTANCE Viruses spread by the fecal-oral route need to maintain viability in the environment to ensure transmission. Previous work indicated that bacteria and bacterial surface polysaccharides can stabilize viral particles and enhance transmission. To explore factors that influence viral particle stability, we isolated a mutant poliovirus that is heat resistant. This mutant virus does not require feces for stability at most temperatures but can be stabilized by feces at very high temperatures. Even though the mutant virus is heat resistant, it is susceptible to inactivation by treatment with bleach. This work provides insight into how viral particles maintain infectivity in the environment.
Assuntos
Proteínas do Capsídeo/genética , Capsídeo/fisiologia , Mutação/genética , Poliovirus/genética , Aminoácidos/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HeLa , Temperatura Alta , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Poliomielite/virologia , RNA Viral/genética , Vírion/genéticaRESUMO
Electron-beam (eBeam) irradiation technology has a variety of applications in modern society. The underlying hypothesis was that eBeam-inactivated Salmonella enterica serovar Enteritidis (SE) cells can serve as a vaccine to control SE colonization and shedding in poultry birds. An eBeam dose of 2.5 kGy (kilograys) was used to inactivate a high-titer (10(8) colony-forming units [CFU]) preparation of SE cells. Microscopic studies revealed that the irradiation did not damage the bacterial cell membranes. The vaccine efficacy was evaluated by administering the eBeam-killed SE cells intramuscularly (1 x 10(6) CFU/bird) into 50-wk-old single comb white leghorn hens. On day 14 postvaccination, the hens were challenged orally with live SE cells (1 x 10(9) CFU) and SE colonization of liver, spleen, ceca, and ovaries determined on day 23. Blood samples were collected on days 0, 14, and 23 postvaccination and the sera were analyzed to quantify SE-specific IgG titers. The vaccinated chickens exhibited significantly (P < 0.0001) higher SE-specific IgG antibody responses and reduced SE ceca colonization (1.46 ± 0.39 logi10 CFU/g) compared to nonvaccinated birds (5.32 ± 0.32 log10 CFU/g). They also exhibited significantly lower SE colonization of the ovaries (1/30), spleen (3/30), liver (4/30), and ceca (7/30) compared to nonvaccinated birds. These results provide empirical evidence that eBeam-based SE vaccines are immunogenic and are capable of protecting chickens against SE colonization. The advantages of eBeam-based vaccine technology are that it is nonthermal, avoids the use of formalin, and can be used to generate inactivated vaccines rapidly to address strain-specific infections in farms or flocks.
Assuntos
Vacinas Bacterianas/imunologia , Galinhas , Muda , Salmonelose Animal/prevenção & controle , Salmonella enteritidis/efeitos da radiação , Animais , Anticorpos Antibacterianos/sangue , Feminino , Imunoglobulina G/sangue , Vacinas de Produtos InativadosRESUMO
Here, we report the draft genomes of 10 Campylobacter strains isolated from the cecal contents of market-age broiler chickens naturally colonized with Campylobacter. Through a comprehensive analysis of these draft genomes, we have unveiled their core genetic elements and several antimicrobial resistance genes.
RESUMO
Poultry act as a major reservoir host for Salmonella and Campylobacter spp., the 2 leading causes of foodborne illnesses globally and in the United States. Preharvest stage interventions to reduce foodborne pathogen carriage in poultry are increasingly informed by consumer preference for antibiotic-free poultry production. The in-feed inclusion of plant-derived antimicrobial compounds is a promising antibiotic alternative strategy to reduce foodborne pathogen load in the broiler chicken gut. Yet, the fate of these phytochemicals through the broiler chicken gastrointestinal tract is unknown. Likewise, while in-feed phytochemicals have been widely demonstrated in challenge models to reduce foodborne pathogen carriage, little is known regarding efficacy to curb natural routes of infection. As such, the aim of the present study was 2-fold. We sought to determine the concentrations of 2 phytochemicals, trans-cinnamaldehyde and caprylic acid, in each region of the chicken gastrointestinal tract following their in-feed inclusion over a 6-wk production period. In addition, we investigated how the in-feed provision of these phytochemicals may protect against environmental acquisition of Campylobacter jejuni and Salmonella spp. Trans-cinnamaldehyde and caprylic acid were detected in crop, gizzard, duodenal, jejunal, and ileal contents. Crop and gizzard concentrations were not significantly (P > 0.05) different. A significant (P < 0.05) decrease in phytochemical concentration was observed in intestinal regions compared to crop and gizzard. Trans-cinnamaldehyde was consistently identified in cecal and colon contents, while caprylic acid was not detectable in these regions. Trans-cinnamaldehyde and caprylic acid were found to reduce (P < 0.05) Salmonella load. Together, our data establish that the in-feed addition of trans-cinnamaldehyde and caprylic acid, 2 phytochemicals that have previously been shown to exert antimicrobial activity against poultry-associated foodborne pathogens, results in detectable concentrations in the broiler chicken gastrointestinal tract. By providing researchers with a gastrointestinal region-by-region map of phytochemical concentrations, the present study is expected to inform the choice of in-feed phytochemicals targeting foodborne pathogen carriage in the broiler chicken gastrointestinal tract.
Assuntos
Acroleína/análogos & derivados , Infecções por Campylobacter , Campylobacter jejuni , Caprilatos , Doenças das Aves Domésticas , Animais , Galinhas , Antibacterianos , Compostos Fitoquímicos , Infecções por Campylobacter/veterinária , Doenças das Aves Domésticas/prevenção & controleRESUMO
Animal agriculture is under pressure to increase efficiency, sustainability, and innovation to meet the demands of a rising global population while decreasing adverse environmental effects. Feed cost and availability are 2 of the biggest hurdles to sustainable production. Current diets depend on sources of grain and animal byproduct protein for essential amino acids which have limited sustainability. Insects have arisen as an attractive, sustainable alternative protein source for animal diets due to their favorable nutrient composition, low space and water requirements, and natural role in animal diets. Additionally, insects are capable of bioremediating waste streams including agricultural and food waste, manure, and plastics helping to increase their sustainability. The insect rearing industry has grown rapidly in recent years and shows great economic potential. However, state-of-the-art research is urgently needed to overcome barriers to adoption in commercial animal diets such as regulatory restrictions, production scale issues, and food safety concerns. To address this need, the USDA Agricultural Research Service "MINIstoc: Model for INsect Inclusion" project was created to bring together diverse scientists from across the world to synergistically advance insect meal production and inclusion in animal diets. Here, we provide a short review of insects as feed while describing the MINIstock project which serves as the inspiration for the Journal of Economic Entomology Special Collection "Insects as feed: sustainable solutions for food waste and animal production practices."
Assuntos
Agricultura , Ração Animal , Insetos , United States Department of Agriculture , Animais , Ração Animal/análise , Estados Unidos , Agricultura/métodos , Dieta , United States National Aeronautics and Space Administration , Criação de Animais DomésticosRESUMO
Campylobacter jejuni is a foodborne pathogen that causes campylobacteriosis globally, affecting ~95 million people worldwide. Most C. jejuni infections involve consuming and/or handling improperly cooked poultry meat. To better understand chicken host factors modulated by Campylobacter colonization, we explored a novel LCMS-based multiomic technology using three experimental groups: (1) negative control, (2) positive control, and (3) eugenol nanoemulsion (EGNE) treatment (supplemented with 0.125% EGNE in the water) of broiler chickens (n = 10 birds/group). Birds in groups two and three were challenged with C. jejuni on day 7, and serum samples were collected from all groups on day 14. Using this multiomic analysis, we identified 1216 analytes (275 compounds, seven inorganics, 407 lipids, and 527 proteins). The colonization of C. jejuni significantly upregulated CREG1, creatinine, and 3-[2-(3-Hydroxyphenyl) ethyl]-5-methoxyphenol and downregulated sphingosine, SP d18:1, high mobility group protein B3, phosphatidylcholines (PC) P-20:0_16:0, PC 11:0_26:1, and PC 13:0_26:2. We found that 5-hydroxyindole-3-acetic acid significantly increased with the EGNE treatment when compared to the positive and negative controls. Additionally, the treatment increased several metabolites when compared to the negative controls. In conclusion, this study revealed several potential targets to control Campylobacter in broiler chickens.
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Obacunone belongs to a class of unique triterpenoids called limonoids, present in Citrus species. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling in Vibrio harveyi and Escherichia coli O157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogen Salmonella enterica serovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone represses Salmonella pathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated through hilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to repress hilA in an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect on S. Typhimurium and may serve as a lead compound for development of antivirulence strategies for S. Typhimurium.
Assuntos
Proteínas de Bactérias/metabolismo , Benzoxepinas/metabolismo , Ilhas Genômicas , Limoninas/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Fatores de Virulência/biossíntese , Células CACO-2 , Citrus/química , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Humanos , Maltose/metabolismo , Análise em Microsséries , Fatores de Virulência/antagonistas & inibidoresRESUMO
BACKGROUND: E. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC. RESULTS: Isolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC25=19.7 and 28.3 µM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid. CONCLUSIONS: Altogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion.
Assuntos
Sistemas de Secreção Bacterianos/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Escherichia coli O157/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Limoninas/farmacologia , Transativadores/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Células CACO-2 , Citrus/química , Células Epiteliais/microbiologia , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/genética , Deleção de Genes , Expressão Gênica/efeitos dos fármacos , Humanos , Limoninas/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/genéticaRESUMO
Approximately 1.35 million human salmonellosis cases are reported in the United States every year, resulting in over 26,000 hospitalizations and 400 deaths. Consumption of contaminated poultry products is one of the leading causes of human salmonellosis. Poultry meat becomes contaminated when feces from an infected bird comes into contact with the carcass during processing. Additional carcasses can then become cross-contaminated along the processing line. While chemicals such as peracetic acid are currently used to kill microbes such as Salmonella, consumers are increasingly calling for more natural alternatives. Our objective for this study was to determine the ability of the phytochemicals garlic and ginger oil to reduce Salmonella prevalence in the processing environment. In a simulated scalding tank environment, dipping contaminated chicken skin samples in a solution containing both garlic and ginger oil reduced Salmonella by up to 2 log CFU. Furthermore, the oils prevented Salmonella growth in the tank solution. The mechanism of action of garlic and ginger was evaluated using the sub-inhibitory concentration of each oil individually. While both were found to decrease autoinducer-2 (AI-2) levels, no effect was seen on expression of 10 genes involved in Salmonella virulence and survival. In total, this work demonstrates the potential of garlic and ginger to reduce Salmonella prevalence in the post-harvest environment. However, more work remains to be done to understand the mechanism of action.
RESUMO
Reducing Salmonella in commercial chickens is vital to decreasing human salmonellosis infections resulting from contact with contaminated poultry and poultry products. As the intestinal microbiota plays an important role in preventing pathogen colonization, we sought to understand the relationship between Salmonella infection and the cecal microbiota and the host immune system. Day-of-hatch broiler chicks were assigned to three treatments: control, artificial (SA), and natural (SN) Salmonella infection. At seven days of age, control and SA birds were inoculated with PBS or Salmonella Typhimurium, respectively. Five SA birds were transferred to SN cages to facilitate natural infection. Cecal content and blood samples were collected at 0, 8, 14, and 21 days of age for microbiota and leukocyte analysis, respectively. A significant change in microbiota composition was observed in both groups as noted by a decrease in Lactobacillus and Escherichia and an increase in Bacteroides. Leukocyte analysis revealed a decrease in the percentage of circulating monocytes at 7 days post-infection while a decrease in thrombocyte and an increase in heterophil percentages were seen at 14 days post-infection. Taken together, these results demonstrate the ability of Salmonella to modulate the intestinal microbiota to facilitate colonization. Additionally, results indicated an early role of monocytes and thrombocytes during colonization, followed by heterophils.
RESUMO
Citrus limonoids are unique secondary metabolites, characterized by a triterpenoid skeleton with a furan ring. Studies have demonstrated beneficial health properties of limonoids. In addition, certain citrus limonoids play a role in plant defence against insect pests. In the present study, five limonoids were purified from sour orange and evaluated for their ability to inhibit cell-cell signalling. The purified limonoids were tested for their ability to interfere with cell-cell signalling and biofilm formation in Vibrio harveyi. Isolimonic acid, deacetylnomilinic acid glucoside and ichangin demonstrated significant inhibition of autoinducer-mediated cell-cell signalling and biofilm formation. Furthermore, isolimonic acid and ichangin treatment resulted in induced expression of the response regulator gene luxO. In addition, luxR promoter activity was not affected by isolimonic acid or ichangin. Therefore, the ability of isolimonic acid and ichangin to interfere with cell-cell signalling and biofilm formation seems to stem from the modulation of luxO expression. The results suggest that isolimonic acid and ichangin are potent modulators of bacterial cell-cell signalling.
Assuntos
Proteínas de Bactérias/biossíntese , Biofilmes/efeitos dos fármacos , Citrus/química , Limoninas/metabolismo , Percepção de Quorum/efeitos dos fármacos , Proteínas Repressoras/biossíntese , Vibrio/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Expressão Gênica , Limoninas/isolamento & purificação , Regulação para Cima , Vibrio/crescimento & desenvolvimento , Vibrio/fisiologiaRESUMO
Consumption or handling of poultry and poultry products contaminated with Campylobacter species are a leading cause of foodborne illness in humans. Current strategies employed to reduce Campylobacter in live chickens provide inconsistent results indicating the need for an alternative approach. This study investigated the efficacy of phytochemicals, namely, turmeric, curcumin, allyl sulfide, garlic oil, and ginger oil, to reduce Campylobacter jejuni in postharvest poultry and sought to delineate the underlying mechanisms of action. Two experiments were conducted on the thigh skin of the chicken, and each experiment was repeated twice. Samples were inoculated with 50 µl (â¼107 CFU/sample) of C. jejuni strain S-8 and allowed to adhere for 30 min. Skin samples were dipped into their respective prechilled treatment solutions (0.25 and 0.5% in experiments 1 and 2, respectively) at 4°C for an hour to simulate chilling tank treatment, followed by plating to enumerate C. jejuni (n = 3 samples/treatment/trial). The mechanisms of action(s) were investigated using subinhibitory concentration (SIC) in adhesion, quorum sensing, and gene expression analyses. Adhesion assay was conducted on the monolayers of ATCC CRL-1590 chicken embryo cells challenged with C. jejuni and incubated in the presence or absence of phytochemicals for 1.5 h, followed by plating to enumerate adhered C. jejuni. The effects of phytochemicals on quorum sensing and cell viability were investigated using Vibrio harveyi bioluminescence and LIVE/Dead BacLightTM bacterial viability assays, respectively. In addition, droplet digital PCR determined the gene expression analyses of C. jejuni exposed to phytochemicals. Data were analyzed by GraphPad Prism version 9. C. jejuni counts were reduced by 1.0-1.5 Log CFU/sample with garlic oil or ginger oil at 0.25 and 0.5% (p < 0.05). The selected phytochemicals (except curcumin) reduced the adhesion of C. jejuni to chicken embryo cells (p < 0.05). In addition, all the phytochemicals at SIC reduced quorum sensing of C. jejuni (p < 0.05). The cell viability test revealed that cells treated with 0.25% of phytochemicals had compromised cell membranes indicating this as a mechanism that phytochemicals use to damage/kill C. jejuni. This study supports that the application of phytochemicals in postharvest poultry would significantly reduce C. jejuni in poultry meat.
RESUMO
This study investigates the microbiological and immunological basis underlying the efficacy of electron beam-inactivated immune modulators. The underlying hypothesis is that exposure to eBeam-based ionization reactions inactivate microorganisms without modifying their antigenic properties and thereby creating immune modulators. The immunological correlates of protection induced by such eBeam based Salmonella Typhimurium (EBST) immune modulators in dendritic cell (DC) (in vitro) and mice (in vivo) models were assessed. The EBST stimulated innate pro inflammatory response (TNFα) and maturation (MHC-II, CD40, CD80 and CD86) of DC. Immuno-stimulatory potential of EBST was on par with both a commercial Salmonella vaccine, and live Salmonella cells. The EBST cells did not multiply under permissive in vitro and in vivo conditions. However, EBST cells remained metabolically active. EBST immunized mice developed Salmonella-specific CD4+ T-cells that produced the Th1 cytokine IFNγ at a level similar to that induced by the live attenuated vaccine (AroA- ST) formulation. The EBST retained stable immunogenic properties for several months at room temperature, 4°C, and -20°C as well as after lyophilization. Therefore, such eBeam-based immune modulators have potential as vaccine candidates since they offer the safety of a "killed" vaccine, while retaining the immunogenicity of an "attenuated" vaccine. The ability to store eBeam based immune modulators at room temperature without loss of potency is also noteworthy.
Assuntos
Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Vacinas Atenuadas/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Citocinas/imunologia , Células Dendríticas/imunologia , Elétrons , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Vacinas de Produtos Inativados/imunologiaRESUMO
Clostridium perfringens (Cp) is a Gram-positive anaerobe that is one of the causative agents of necrotic enteritis (NE) in chickens, which leads to high mortality. Owing to the ban of administering antibiotics in feed to chickens, there has been an increase in the number of NE outbreaks all over the world, and the estimated loss is approximately 6 billion U.S. dollars. The best alternative method to control NE without antibiotics could be vaccination. In this study, we exposed three different strains of Cp to electron beam (eBeam) irradiation to inactivate them and then used them as a killed vaccine to control the colonization of Cp in broiler chickens. The vaccine was delivered to 18-day old embryos in ovo and the chickens were challenged with the respective vaccine strain at two different time points (early and late) to test the protective efficacy of the vaccine. The results indicate that an effective eBeam dose of 10 kGy inactivated all three strains of Cp, did not affect the cell membrane or epitopes, induced significant levels of IgY in the vaccinated birds, and further reduced the colonization of Cp strains significantly (p < 0.0001) in late challenge (JGS4064: 4 out of 10; JGS1473: 0 out of 10; JGS4104: 3 out of 10). Further studies are necessary to enhance the efficacy of the vaccine and to understand the mechanism of vaccine protection.
RESUMO
The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.
Assuntos
Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Homosserina/análogos & derivados , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Biofilmes , Bioensaio , Transporte Biológico/genética , Liases de Carbono-Enxofre/genética , Ciclo Celular , Quimiotaxia/genética , Fímbrias Bacterianas/genética , Flagelos/genética , Glucose/administração & dosagem , Homosserina/análise , Homosserina/fisiologia , Lactonas/análise , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/fisiologia , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Listeria monocytogenes is a foodborne pathogen capable of employing stress adaptive responses to evade a variety of stressors including temperature stress. We employed two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight analysis to study the differential expression of L. monocytogenes (ATCC 43256) soluble proteins at heat shock (60 degrees C) conditions, prolonged heat shock (60 degrees C for 9 minutes) conditions, and thermotolerance-inducing (48 degrees C for 30 minutes followed by 60 degrees C for 9 minutes) conditions. We compared the proteome of L. monocytogenes under these conditions to the proteome at 37 degrees C. Eighteen proteins were differentially expressed at 60 degrees C (6 up-regulated and 12 down-regulated), 21 proteins were differentially expressed (12 up-regulated and 9 down-regulated) when the cells were exposed to 60 degrees C for 9 minutes, and 20 proteins were differentially expressed (10 up-regulated and 10 down-regulated) when cells were initially exposed to 48 degrees C for 30 minutes before 60 degrees C for 9 minutes. There was one unidentifiable protein with observed molecular weight of 50 kDa which was differentially expressed across the three temperature treatments. Thermotolerance-inducing conditions caused the up-regulation of a protein by as much as 12-fold. DnaN, a previously identified stress protein, was up-regulated almost threefold at 60 degrees C. TcsA, a lipoprotein (CD4 T cell-stimulating antigen), and Gap (glyceraldehyde-3-phosphate-dehydrogenase) were selectively expressed under prolonged heat shock conditions suggesting their potential as candidate marker proteins targets for identifying temperature-stressed L. monocytogenes cells.
Assuntos
Proteínas de Bactérias/metabolismo , Temperatura Alta/efeitos adversos , Listeria monocytogenes/metabolismo , Proteoma/metabolismo , Estresse Fisiológico , Regulação para Baixo , Eletroforese em Gel Bidimensional , Listeria monocytogenes/genética , Proteômica/métodos , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para CimaRESUMO
Salmonella Typhimurium is known to exhibit LuxS/AI-2-mediated cell signaling. We investigated the role of LuxS/AI-2 system on Salmonella Typhimurium protein expression using a proteomics approach based on two-dimensional gel electrophoresis (2DGE)-MALDI-MS. The global protein expression profiles of the wild-type, a luxS mutant, and a luxS mutant strain supplemented with AI-2 were compared. Seven proteins were differentially expressed when comparing the wild-type and luxS mutant strains, whereas 13 proteins were differentially expressed when comparisons were made between luxS mutant strains with and without AI-2 supplementation. The seven proteins that were differentially expressed between the wild-type and the luxS mutant strain were also differentially expressed in the luxS mutant strain supplemented with AI-2. The level of PhoP, a virulence determinant, was higher in the presence of AI-2. Proteins associated with the carbohydrate metabolism (pfkA, gpmI, and talB) and ATP synthesis (Pta gene product) were up-regulated by the presence of AI-2 molecules. These results provide experimental evidence that AI-2 molecules regulate a variety of cellular processes in Salmonella Typhimurium.
Assuntos
Proteínas de Bactérias/análise , Liases de Carbono-Enxofre/fisiologia , Homosserina/análogos & derivados , Proteômica , Salmonella typhimurium/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Bioensaio , Metabolismo dos Carboidratos , Liases de Carbono-Enxofre/genética , Eletroforese em Gel Bidimensional , Regulação Bacteriana da Expressão Gênica , Homosserina/biossíntese , Homosserina/genética , Homosserina/fisiologia , Lactonas , Mutação , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transcrição Gênica , Regulação para Cima , Virulência/genéticaRESUMO
RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.
Assuntos
Bactérias/genética , Infecções por Enterovirus/patologia , Poliovirus/genética , Recombinação Genética/genética , Animais , Bactérias/metabolismo , Bactérias/virologia , Linhagem Celular Tumoral , Coinfecção , Infecções por Enterovirus/virologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliovirus/patogenicidadeRESUMO
The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high.IMPORTANCE One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations.
Assuntos
Variação Genética , Poliovirus/classificação , Poliovirus/isolamento & purificação , Ensaio de Placa Viral , Replicação Viral , Citometria de Fluxo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Mutagênese , Poliovirus/genética , Poliovirus/fisiologiaRESUMO
Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways.