scholarly journals Signature-Tagged Transposon Mutagenesis Studies Demonstrate the Dynamic Nature of Cecal Colonization of 2-Week-Old Chickens by Campylobacter jejuni

2005 ◽  
Vol 71 (12) ◽  
pp. 8031-8041 ◽  
Author(s):  
Andrew J. Grant ◽  
Christopher Coward ◽  
Michael A. Jones ◽  
Claire A. Woodall ◽  
Paul A. Barrow ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Hot Spots ◽  
Dynamic Nature ◽  
Transposon Insertion ◽  
Random Loss ◽  
Colonization Process ◽  
Insertion Sites ◽  
Different Strains ◽  
Constructed Plasmids

ABSTRACT We have constructed plasmids to be used for in vitro signature-tagged mutagenesis (STM) of Campylobacter jejuni and used these to generate STM libraries in three different strains. Statistical analysis of the transposon insertion sites in the C. jejuni NCTC 11168 chromosome and the plasmids of strain 81-176 indicated that their distribution was not uniform. Visual inspection of the distribution suggested that deviation from uniformity was not due to preferential integration of the transposon into a limited number of hot spots but rather that there was a bias towards insertions around the origin. We screened pools of mutants from the STM libraries for their ability to colonize the ceca of 2-week-old chickens harboring a standardized gut flora. We observed high-frequency random loss of colonization proficient mutants. When cohoused birds were individually inoculated with different tagged mutants, random loss of colonization-proficient mutants was similarly observed, as was extensive bird-to-bird transmission of mutants. This indicates that the nature of campylobacter colonization in chickens is complex and dynamic, and we hypothesize that bottlenecks in the colonization process and between-bird transmission account for these observations.

scholarly journals Pyruvate is required for catecholamine-stimulated growth of different strains of Campylobacter jejuni

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10011
Author(s):  
Meicen Liu ◽  
Mark Lyte
Keyword(s):  
Escherichia Coli ◽  
Campylobacter Jejuni ◽  
Pathogenic Bacteria ◽  
Intestinal Tract ◽  
Nerve Terminals ◽  
In Vitro Growth ◽  
Gastrointestinal Infections ◽  
Different Strains ◽  
Iron Delivery

Humans and food-producing animals are constantly exposed to and affected by stress. As a consequence of stress, the release of stress-related catecholamines, such as norepinephrine (NE) and dopamine (DA), from nerve terminals in the gastrointestinal tract potentiates both the growth and the virulence of pathogenic bacteria. This may lead to the enhancement of gastrointestinal infections in humans or food-producing animals. Compared with foodborne bacterial pathogens such as Escherichia coli and Salmonella spp., less is known about the effect of stress catecholamines on Campylobacter jejuni subsp. jejuni. The present study focuses on the effect(s) of stress catecholamines DA and NE in iron-restricted media and how they affect the growth of different C. jejuni strains NCTC 11168, 81–176, and ML2126. Results demonstrated that DA- and NE-enhanced growth of C. jejuni in iron-restricted media may involve different mechanisms that cannot be explained by current understanding which relies on catecholamine-mediated iron delivery. Specifically, we found that DA-enhanced growth requires pyruvate, whereas NE-enhanced growth does not. We further report significant strain-specific dependence of C. jejuni growth on various catecholamines in the presence or absence of pyruvate. These data provide novel insights into the effect(s) of stress catecholamines on the in vitro growth of C. jejuni in iron-restricted environments, such as the intestinal tract. They suggest a mechanism by which stress-related catecholamines affect the growth of C. jejuni in the intestinal tract of food-producing animals, which in turn may influence colonization and transmission to humans.

scholarly journals Heterogeneity of a Campylobacter jejuni Protein That Is Secreted through the Flagellar Filament

2007 ◽  
Vol 75 (8) ◽  
pp. 3859-3867 ◽  
Author(s):  
Frédéric Poly ◽  
Cheryl Ewing ◽  
Scarlett Goon ◽  
Thomas E. Hickey ◽  
David Rockabrand ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Virulence Factor ◽  
Promoter Analysis ◽  
Acidic Protein ◽  
Rapid Induction ◽  
Virulence Potential ◽  
Flagellar Filament ◽  
Induction Of Apoptosis ◽  
Different Strains

ABSTRACTCj0859c, or FspA, is a small, acidic protein ofCampylobacter jejunithat is expressed by a σ28promoter. Analysis of thefspAgene in 41 isolates ofC. jejunirevealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novelC. jejunivirulence factor, and the observed heterogeneity amongfspAalleles suggests alternate virulence potential among different strains.

scholarly journals Attraction of Brachyspira pilosicoli to mucin

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 191-197 ◽  
Author(s):  
Ram Naresh ◽  
David J. Hampson
Keyword(s):  
Capillary Tube ◽  
Experimental Studies ◽  
Mucus Layer ◽  
Brachyspira Pilosicoli ◽  
Brachyspira Hyodysenteriae ◽  
Potential Marker ◽  
Colonization Process ◽  
Different Strains

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1 % mucin, but some strains started to enter a 2 % solution, and attraction then increased with increasing concentrations to peak at around 6–8 % mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6 % mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6–8 % mucin solutions were around 7–12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of d-serine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.

scholarly journals Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1134-1143 ◽  
Author(s):  
Muhammad Afzal Javed ◽  
Andrew J. Grant ◽  
Mary. C. Bagnall ◽  
Duncan J. Maskell ◽  
Diane G. Newell ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Campylobacter Jejuni ◽  
Transposon Mutagenesis ◽  
Genomic Variation ◽  
Host Cells ◽  
Transposon Insertion ◽  
Strain Nctc ◽  
Transposon Insertions

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (∼14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

scholarly journals Identification of Motility and Autoagglutination Campylobacter jejuni Mutants by Random Transposon Mutagenesis

2002 ◽  
Vol 70 (4) ◽  
pp. 1761-1771 ◽  
Author(s):  
Neal J. Golden ◽  
David W. K. Acheson
Keyword(s):  
Campylobacter Jejuni ◽  
The United States ◽  
Transposon Mutagenesis ◽  
Intestinal Epithelial ◽  
Transposon Insertion ◽  
Gentamicin Treatment ◽  
Strong Homology ◽  
Transposon Mutants ◽  
Insertion Sites

ABSTRACT Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.

scholarly journals Development of Genomic Array Footprinting for Identification of Conditionally Essential Genes in Streptococcus pneumoniae

2007 ◽  
Vol 73 (5) ◽  
pp. 1514-1524 ◽  
Author(s):  
Jetta J. E. Bijlsma ◽  
Peter Burghout ◽  
Tomas G. Kloosterman ◽  
Hester J. Bootsma ◽  
Anne de Jong ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
High Throughput ◽  
Insertion Site ◽  
Essential Genes ◽  
Degree Of Saturation ◽  
Transposon Insertion ◽  
High Zinc ◽  
Genomic Array ◽  
Insertion Sites

ABSTRACT Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput, genome-wide technique, genomic array footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in nonreproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1 showed that both have an equal degree of saturation but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for surviving zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high-zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.

Hot spots of retroviral integration in human CD34+ hematopoietic cells

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 1770-1778 ◽  
Author(s):  
Claudia Cattoglio ◽  
Giulia Facchini ◽  
Daniela Sartori ◽  
Antonella Antonelli ◽  
Annarita Miccio ◽  
...  
Keyword(s):  
Human Genome ◽  
Hot Spots ◽  
Clonal Selection ◽  
System Development ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Immune System Development ◽  
Insertion Sites

Abstract Insertional oncogenesis is a possible consequence of the integration of gamma-retroviral (RV) or lentiviral (LV) vectors into the human genome. RV common insertion sites (CISs) have been identified in hematopoietic malignancies and in the nonmalignant progeny of transduced hematopoietic stem/progenitor cells (HSCs), possibly as a consequence of clonal selection in vivo. We have mapped a large number of RV and LV integrations in human CD34+ HSCs, transduced in vitro and analyzed without selection. Recurrent insertion sites (hot spots) account for more than 21% of the RV integration events, while they are significantly less frequent in the case of LV vectors. RV but not LV hot spots are highly enriched in proto-oncogenes, cancer-associated CISs, and growth-controlling genes, indicating that at least part of the biases observed in the HSC progeny in vivo are characteristics of RV integration, already present in nontransplanted cells. Genes involved in hematopoietic and immune system development are targeted at high frequency and enriched in hot spots, suggesting that the CD34+ gene expression program is instrumental in directing RV integration. The lower propensity of LV vectors for integrating in potentially dangerous regions of the human genome may be a factor determining a better safety profile for gene therapy applications.

scholarly journals The glycosyltransferase ST3GAL2 is regulated by miR-615-3p in the intestinal tract of Campylobacter jejuni infected mice

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
De Xi ◽  
Lukas Hofmann ◽  
Thomas Alter ◽  
Ralf Einspanier ◽  
Stefan Bereswill ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Regulatory Networks ◽  
In Silico Analysis ◽  
Luciferase Reporter ◽  
Colonic Tissue ◽  
Tissue Samples ◽  
Vitro Model ◽  
Pathway Analyses

Abstract Background Campylobacter jejuni (C. jejuni) infections are of increasing importance worldwide. As a typical mucosal pathogen, the interaction of C. jejuni with mucins is a prominent step in the colonisation of mucosal surfaces. Despite recent advances in understanding the interaction between bacterial pathogens and host mucins, the mechanisms of mucin glycosylation during intestinal C. jejuni infection remain largely unclear. This prompted us to identify relevant regulatory networks that are concerted by miRNAs and could play a role in the mucin modification and interaction. Results We firstly used a human intestinal in vitro model, in which we observed altered transcription of MUC2 and TFF3 upon C. jejuni NCTC 11168 infection. Using a combined approach consisting of in silico analysis together with in vitro expression analysis, we identified the conserved miRNAs miR-125a-5p and miR-615-3p associated with MUC2 and TFF3. Further pathway analyses showed that both miRNAs appear to regulate glycosyltransferases, which are related to the KEGG pathway ‘Mucin type O-glycan biosynthesis’. To validate the proposed interactions, we applied an in vivo approach utilising a well-established secondary abiotic IL-10−/− mouse model for infection with C. jejuni 81-176. In colonic tissue samples, we confirmed infection-dependent aberrant transcription of MUC2 and TFF3. Moreover, two predicted glycosyltransferases, the sialyltransferases ST3GAL1 and ST3GAL2, exhibited inversely correlated transcriptional levels compared to the expression of the identified miRNAs miR-125a-5p and miR-615-3p, respectively. In this study, we mainly focused on the interaction between miR-615-3p and ST3GAL2 and were able to demonstrate their molecular interaction using luciferase reporter assays and RNAi. Detection of ST3GAL2 in murine colonic tissue by immunofluorescence demonstrated reduced intensity after C. jejuni 81-176 infection and was thus consistent with the observations made above. Conclusions We report here for the first time the regulation of glycosyltransferases by miRNAs during murine infection with C. jejuni 81-176. Our data suggest that mucin type O-glycan biosynthesis is concerted by the interplay of miRNAs and glycosyltransferases, which could determine the shape of intestinal glycosylated proteins during infection.

scholarly journals Mutations That Affect the Tropism of DA and GDVII Strains of Theiler's Virus In Vitro Influence Sialic Acid Binding and Pathogenicity

2002 ◽  
Vol 76 (16) ◽  
pp. 8138-8147 ◽  
Author(s):  
Karima Jnaoui ◽  
Muriel Minet ◽  
Thomas Michiels
Keyword(s):  
Sialic Acid ◽  
Demyelinating Disease ◽  
Binding Capacity ◽  
Single Amino Acid ◽  
Sialic Acid Binding ◽  
Encephalomyelitis Virus ◽  
The Central Nervous System ◽  
Different Strains ◽  
Single Amino Acid Mutation

ABSTRACT Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse. The different strains of TMEV are divided into two subgroups according to the pathology they provoke. The neurovirulent strains GDVII and FA induce an acute fatal encephalitis, while persistent strains, like DA and BeAn, cause a chronic demyelinating disease associated with viral persistence in the central nervous system. Different receptor usage was proposed to account for most of the phenotype difference between neurovirulent and persistent strains. Persistent but not neurovirulent strains were shown to bind sialic acid. We characterized DA and GDVII derivatives adapted to grow on CHO-K1 cells. Expression of glycosaminoglycans did not influence infection of CHO-K1 cells by parental and adapted viruses. Mutations resulting from adaptation of DA and GDVII to CHO-K1 cells notably mapped to the well-characterized VP1 CD and VP2 EF loops of the capsid. Adaptation of the DA virus to CHO-K1 cells correlated with decreased sialic acid usage for entry. In contrast, adaptation of the GDVII virus to CHO-K1 cells correlated with the appearance of a weak sialic acid usage for entry. The sialic acid binding capacity of the GDVII variant resulted from a single amino acid mutation (VP1-51, Asn→Ser) located out of the sialic acid binding region defined for virus DA. Mutations affecting tropism in vitro and sialic acid binding dramatically affected the persistence and neurovirulence of the viruses.

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