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INTRODUCTION: Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity. OBJECTIVES: For instance, the C. jejuni 11168 strain can utilize both L-fucose and L-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of L-fucose and L-glutamate in the growth medium. METHODS: To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h. RESULTS: This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon L-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy. CONCLUSIONS: Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon L-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.
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Campylobacter jejuni/metabolismo , Fucose/farmacologia , Ácido Glutâmico/farmacologia , Metaboloma , Campylobacter jejuni/efeitos dos fármacos , Fucose/metabolismo , Ácido Glutâmico/metabolismoRESUMO
Bloodstream infection remains a major cause of morbidity and death worldwide. Timely and appropriate treatment can reduce mortality among critically ill patients. Current diagnostic methods are too slow to inform precise antibiotic choice, leading to the prescription of empirical antibiotics, which may fail to cover the resistance profile of the pathogen, risking poor patient outcomes. Additionally, overuse of broad-spectrum antibiotics may lead to more resistant organisms, putting further pressure on the dwindling pipeline of antibiotics, and risk transmission of these resistant organisms in the health care environment. Therefore, rapid diagnostics are urgently required to better inform antibiotic choice early in the course of treatment. Sequencing offers great promise in reducing time to microbiological diagnosis; however, the amount of host DNA compared with the pathogen in patient samples presents a significant obstacle. To address this, various host-depletion and bacterial-enrichment strategies have been used in samples, such as saliva, urine, or tissue. However, these methods have yet to be collectively integrated and/or extensively explored for rapid bloodstream infection diagnosis. Although most of these workflows possess individual strengths, their lack of analytical/clinical sensitivity and/or comprehensiveness demands additional improvements or synergistic application. Therefore, this review provides a distinctive classification system for these methods based on their working principles to guide future research, discusses their strengths and limitations, and explores potential avenues for improvement.
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Salmonella enterica has two pathogenicity islands encoding separate type three secretion systems (T3SS). Proteins secreted through these systems facilitate invasion and survival. After entry, Salmonella reside within a membrane bound vacuole, the Salmonella containing vacuole (SCV), where translocation of a second set of effectors by the Salmonella pathogenicity island 2 (SPI-2) T3SS is initiated. SPI-2 secretion in vitro can be induced by conditions that mimic the Salmonella containing vacuole. Utilising high-throughput mass spectrometry, we mapped the surface-attached proteome of S. Typhimurium SL1344 grown in vitro under SPI-2-inducing conditions and identified 108 proteins; using secretion signal prediction software, 43% of proteins identified contained a signal sequence. Of these proteins, 13 were known secreted effector proteins including SPI-2 effector proteins SseB, SseC, SseD, SseL, PipB2 and SteC, although surprisingly five were SPI-1 proteins, SipA, SipB, SipC, SipD and SopD, while 2 proteins SteA and SlrP are secreted by both T3SSs. This is the first in vitro study to demonstrate dual secretion of SPI-1 and SPI-2 proteins by S. Typhimurium and demonstrates the potential of high-throughput LC-ESI/MS/MS sequencing for the identification of novel proteins, providing a platform for subsequent comparative proteomic analysis, which should greatly assist understanding of the pathogenesis and inherent variation between serovars of Salmonella and ultimately help towards development of novel control strategies.
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Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Western Blotting , Cromatografia Líquida , Infecções por Salmonella/microbiologia , Transdução de SinaisRESUMO
Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.
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Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Intestino Grosso/microbiologia , Intestino Delgado/microbiologia , Aderência Bacteriana , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
Deletion of the lipooligosaccharide biosynthesis region (Cj1132c to Cj1152c) from the genome of Campylobacter jejuni NCTC11168 shows that the core is not required for viability. The mutant was attenuated for growth and has increased sensitivity to antibiotics and detergents. Natural transformation and invasion of cultured host cells was abolished.
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Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Deleção de Genes , Lipopolissacarídeos/biossíntese , Viabilidade Microbiana , Família Multigênica , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Campylobacter jejuni/química , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/metabolismo , Lipopolissacarídeos/química , MutaçãoRESUMO
Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact.
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Campylobacter jejuni/genética , Campylobacter jejuni/isolamento & purificação , Variação Genética , Genoma Bacteriano , DNA Bacteriano/genética , FenótipoRESUMO
To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.
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Infecções por Campylobacter/veterinária , Campylobacter jejuni/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Animais , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/patogenicidade , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Trato Gastrointestinal/microbiologia , Vida Livre de Germes , Humanos , Mutagênese Insercional , Suínos , Doenças dos Suínos/microbiologia , Virulência/genéticaRESUMO
Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and -poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.
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Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/veterinária , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/genética , Aptidão Genética , Genoma Bacteriano , Animais , Linhagem Celular , Galinhas , Meios de Cultura/química , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Moscas Domésticas , Humanos , Viabilidade Microbiana , Mutagênese Insercional , TemperaturaRESUMO
One of the most serious complications of typhoid fever is intestinal perforation. Of 27 patients admitted to a provincial hospital in the Mekong Delta region of Vietnam who had gastrointestinal perforation secondary to suspected typhoid fever, 67% were male, with a median age of 23 years and a median duration of illness of 10 days. Salmonella enterica subspecies enterica serotype Typhi (S. Typhi) was isolated from 11 (41%) of 27 patients; of 27 patients, only 4 (15%) had positive cultures from gut biopsies. S. Typhi DNA was detected by polymerase chain reaction for all perforation biopsy samples. Detailed histological examination of the gastrointestinal mucosa at the site of perforation in all cases showed a combination of discrete acute and chronic inflammation. Acute inflammation at the serosal surface indicated additional tissue damage after perforation. Immunohistochemical results showed that the predominant infiltrating cell types at the site of perforation were CD68+ leukocytes (macrophages) or CD3+ leukocytes (T lymphocytes).
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Perfuração Intestinal/etiologia , Salmonella typhi/isolamento & purificação , Febre Tifoide/complicações , Adulto , Técnicas de Tipagem Bacteriana , Técnicas de Laboratório Clínico , Feminino , Humanos , Imuno-Histoquímica , Perfuração Intestinal/microbiologia , Masculino , Reação em Cadeia da Polimerase , Salmonella enterica/isolamento & purificação , Salmonella typhi/genética , Testes Sorológicos , Febre Tifoide/microbiologia , VietnãRESUMO
Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC-ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith-Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.
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Campylobacter jejuni causes symptoms of acute inflammatory diarrhoea in man. C. jejuni interaction with epithelial cells elicits interleukin-8 (IL-8) production, and IL-8 recruits neutrophils to sites of infection. Cell culture models of bacterial interaction with epithelium are useful to define bacteria-host interaction and are used because it is thought they mimic the same bacteria-host cell interaction in the natural disease. This study looks at the ability of C. jejuni strains to elicit IL-8 production from a variety of cell lines previously used for investigating the interaction of C. jejuni with host cells. A spectrum of IL-8 responses was observed, with minimal IL-8 elicited from Caco-2 cells and more marked responses elicited from HeLa and T84 cells. These in vitro-infected cell line responses were compared to IL-8 production from in vitro C. jejuni-infected human colonic and ileal tissue. The in vitro-infected tissue elicited the highest IL-8 responses and the cytokine was manifested earlier compared to the infected cell lines.
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Campylobacter jejuni/imunologia , Células Epiteliais/microbiologia , Interleucina-8/biossíntese , Mucosa Intestinal/microbiologia , Células CACO-2 , Linhagem Celular Tumoral , Células Cultivadas , Células Epiteliais/imunologia , Células HT29 , Células HeLa , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologiaRESUMO
Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in 'domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting ('doming') Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.
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Translocação Bacteriana , Campylobacter jejuni/fisiologia , Junções Íntimas/microbiologia , Células CACO-2 , Permeabilidade da Membrana Celular/fisiologia , Impedância Elétrica , Humanos , Junções Íntimas/fisiologiaRESUMO
The mitogen-activated protein kinases (MAPKs) play a central role in many host signalling pathways. These signalling proteins are known to be involved in host responses against invasive bacteria including generation of chemotactic and inflammatory cytokines. It was hypothesized that Campylobacter jejuni may activate MAPKs, as intestinal infection may induce a clinical and pathological picture of acute colonic inflammation. Infection of Caco-2 cell monolayers (human colonic epithelial cell line) and human colonic tissue with C. jejuni in vitro demonstrated increased MAPK activity for ERK 1/2 (p44/42 MAPK), JNK and p38 MAPKs. Kinase activity and phosphorylated forms were increased in infected Caco-2 cells and human colonic explants, suggesting that these pathways are important in inflammatory responses induced by C. jejuni in man.
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Células CACO-2/microbiologia , Campylobacter jejuni/fisiologia , Colo/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Campylobacter jejuni/imunologia , Colo/citologia , Colo/microbiologia , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 Delta aroC Delta ssaV, designated ZH9) and S. enterica serovar Typhimurium (TML Delta aroC Delta ssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 10(7), 10(8), or 10(9) CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 10(8) and two of three receiving 10(9) CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 10(8) and 10(9) CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 10(9) CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi.
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Proteínas de Bactérias/imunologia , Proteínas de Membrana/imunologia , Fósforo-Oxigênio Liases/imunologia , Infecções por Salmonella/prevenção & controle , Salmonella typhi/imunologia , Salmonella typhimurium/imunologia , Proteínas de Bactérias/genética , Nível de Saúde , Voluntários Saudáveis , Humanos , Proteínas de Membrana/genética , Mutagênese , Fósforo-Oxigênio Liases/genética , Salmonella typhi/genética , Salmonella typhimurium/genética , Febre Tifoide/imunologia , Febre Tifoide/prevenção & controle , VacinaçãoRESUMO
To identify the major outer surface proteins of Streptococcus agalactiae (group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as S. agalactiae specific, 27 have been identified. Six of these proteins, previously unidentified in S. agalactiae, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.