CARPETA DE RODAMIENTO DE CONCRETO ASFALTICO EN CALIENTE TIPO CAC D-19 CON CA - 30
XII. 2.5.2.- Texturómetro láser (tramo)
Poultry intestinal mucus modulates Campylobacter jejuni gene expression Looft Torey, Casey Thomas
United States Department of Agriculture, Agriculture Research Service, Food Safety Enteric Pathogens Research Unit, National Animal Disease Center,
Ames, Iowa, USA
Campylobacter jejuni is an important human pathogen, causing up to 400 million infections a year
world-wide. Poultry is a natural reservoir of C. jejuni, colonizing and residing within the intestinal mucus without causing disease. Mucus colonization is an essential step in C. jejuni colonization and previous studies suggest that host intestinal mucus impacts Campylobacter function. In this study we characterize the global transcriptome of C. jejuni grown on host mucus isolated from avian (chicken or turkey) and mammalian (cow, pig, or sheep) sources. C. jejuni NCTC 11168 was grown for 24 hours on defined media supplemented with or without 0.5 % wt/vol of each host mucus. Following RNA isolation, directional RNA libraries were sequenced, and mapped to the reference genome. Avian and mammalian mucus sources differentially impacted gene expression in ways that may reflect Campylobacter’s ability to colonize different animal intestinal tracts. Non- coding antisense RNAs were associated with differentially expressed genes between avian and mammalian mucus, and may be in response to environmental cues. These data suggest that C.
jejuni alters its gene expression in the presence of avian mucus in such a way that promotes
intestinal colonization. Understanding how C. jejuni interacts within the host-intestinal environment will provide insights into how C. jejuni has adapted to colonizing different intestinal environments.
RNAseq reveals complex response of Campylobacter jejuni to bile and the ovine gallbladder environment
Amanda J. Kreuder1,2, Micheal J. Yaeger3, Jennfier A. Schleining1, Brandon Ruddell2, Qijing Zhang2, Paul J. Plummer1,2
1
Departments of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, United States
2
Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, United States
3
Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA, United States
Recent advances in the use of high throughput deep sequencing of RNA (RNAseq) have revolutionized the study of gene expression and have allowed unprecedented examination of the whole transcriptome of bacterial pathogens. The highly virulent Campylobacter jejuni sheep abortion (SA) clone, represented by the isolate IA3902, has recently emerged as the dominant cause for sheep abortion in the United States. The SA clone has also been increasingly identified from human outbreaks of foodborne gastroenteritis, making further understanding of the molecular mechanisms that allow for disease and persistence within the animal host of this particular strain is especially important. Survival of C. jejuni within the intestinal tract is known to require adaptation to various levels of bile salts. In addition, abattoir studies have frequently identified the gallbladder as a site of positive culture for C. jejuni in sheep despite the assumed inhospitable nature of this environment. To study the survival of C. jejuni IA3902 in bile and the ovine gallbladder, both in vitro and in vivo studies were performed to collect high quality total RNA following up to 24 hours exposure to these environments. High throughput deep sequencing of strand specific rRNA-depleted total RNA was then performed on the Illumina Hi-Seq platform to characterize the transcriptome of IA3902 and Rockhopper was used to analyze differences in gene expression. Our results indicated a large number of protein coding genes differentially expressed in ovine bile, the ovine gallbladder, and the ovine gallbladder mucosal layer along with differential expression of several previously identified small non-coding RNAs. This research provides valuable insight into the mechanisms that may be utilized by C. jejuni to survive and develop a carrier state within the inhospitable host gallbladder environment.
Contributions of Phase Variation to Phage-Escape by Campylobacter jejuni
Sawant Prachi 1, Sorensen Martine 2, Cayrou Caroline1, Aidley Jack1, Verma Amit 1, Morozov Andrey 3, Brondsted Lone 2, Bayliss Christopher D. 1
1
Department of Genetics, University of Leicester
2
Department of Veterinary Disease Biology, University of Copenhagen
3
Department of Mathematics, University of Leicester
Background: Multiple surface-exposed structures of Campylobacter jejuni are subject to phase
variation (PV) due to mutations in polyG/C tracts. Emerging structural PV-mediated differences in capsular polysaccharide (CPS) composition are linked to phage and serum resistance of C. jejuni. Specifically, ON/OFF switches in expression of CPS genes cj1421, cj1422 and cj1426 were shown to modulate sensitivity of NCTC11168 to phage F336 (Sorensen et al. 2011 J. Bact. 193:6742). Cj1421 and Cj1422 transfer O-methyl-phosphoramidate groups to alternate positions in CPS while Cj1426 attaches a 6-O-methyl group to the heptose. We hypothesize that this combination of hypermutable sequences and multiple phase-variable genes provides a fitness advantage to C.
jejuni for survival of attacks by phage.
Method: We constructed deletion mutations of one, two or three genes for cj1421, cj1422 and cj1426 and complementation strains carrying cj1421 with varying poly G tract lengths in C. jejuni
strain NCTC11168 . Single strain and competition experiments of construct versus wild-type were performed in the presence and absence of phage. PolyG tract lengths were determined for multiple output colonies using a PCR-based-GeneScan method.
Results: The wild-type strain exhibited escape of phage due primarily to mutations in the polyG
tracts of either cj1421 or cj1422. Deletion of either of these genes did not produce changes in competitiveness but an increase in polyG tract length from G9 to G12 resulted in higher switching rates, enhanced escape of phage and increased competitiveness relative to the wild-type strain.
Discussion: Our study indicates that pre-existing variants generated by PV are key determinants of
adaptation to selection pressures by C. jejuni and that the rate of switching can enhance competitiveness during selection. This study provides a framework for development of combined phage therapy and immunisation protocols targeting alternate phase-variable expression states of CPS epitopes as a strategy for reducing C. jejuni loads in poultry.
The phospholipidome of C.jejuni
Marc Wösten1, Linda Heijmen van Dijk1, Craig Parker3, Steven Huynh3, Jos Brouwers2, Jos van Putten1
1
Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands
2
Department of Biochemie & Celbiologie, Utrecht University, Utrecht, The Netherlands
3
Produce Safety and Microbiology Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Albany, California, USA
To survive, bacteria need to change not only their protein repertoire, but also their lipid composition in response to changes in their environment. Bacterial membranes are composed of phospholipids, glycolipids and proteins. In most cases, the phospholipids are composed of two fatty acids, a glycerol moiety, a phosphate group and a variable head group. Bacteria have evolved mechanisms to control the formation of fatty acids and modify the structure of existing fatty acids. These modifications allow bacteria to adjust their membrane viscosity to match environmental requirements. Campylobacter jejuni is a highly motile spiral shaped bacterium that is capable of changing its cell shape to a coccoid immotile bacterium. Knowledge of the composition of the phospholipids in the spiral or coccoid form of C. jejuni as well as the gene regulation and biosynthesis of the phospholipids is largely lacking. Moreover, changes in phospholipid composition in bacteria due to environmental conditions or age has hardly been studied in bacteria.
We followed the composition of the C. jejuni phospholipidome in cultures growing for 4 days on different carbon sources as well as oxygen availability by high performance liquid chromatography (LC-MS/MS) and studied the transcription of the genes involved in this process by RNA-seq. The phospholipidome of C. jejuni compromises more than a hundred different phospholipids and it displays a high variation dependent on the oxygen availability or age of the Campylobacter culture. Abnormal amounts (30-50%) of the phospholipids of C. jejuni are lysophospholipids. As in several pathogenic bacteria, accumulation of lysolipids is crucial to cause disease or to survive after phagocytosis, the role of the C. jejuni phospholipids in Campylobacteriosis might be an underestimated factor.
Membrane protein complexome of C. jejuni using 2D blue native/SDS-PAGE
Alizée Guérin1, Sheiam Sulaeman1, Lucile Bugros1, Armelle Ménard2, Emmanuelle Dé3, Odile Tresse1
1
SECALIM, UMR 1014 INRA, Oniris, Route de Gachet, 44307 Nantes, France
2
Université de Bordeaux, Laboratoire de Bactériologie, Centre National de Référence des Helicobacters et Campylobacters,
33076 Bordeaux, France
3
Université de Rouen, Laboratoire Polymères Biopolymères Surfaces, UMR 6270 and FR 3038 CNRS, IFRMP23,
76130 Mont-Saint-Aignan, France
Campylobacter has emerged as the leading cause of bacterial foodborne infections in developed
countries with a significant increase in the prevalence of campylobacteriosis cases for a decade. The perception cues from biotic or abiotic environments by the bacteria are often related to bacterial surface and membrane proteins. These proteins mediate the cellular response for the adaptation of C. jejuni to the environment. These proteins function rarely as a unique entity, they are often organized in functional complexes. In C. jejuni, these complexes are not fully identified and some of them remain unknown. To identify functional multi-subunit entities at the membrane subproteome level of C. jejuni, holistic non a priori method was addressed using two-dimensional (2-D) blue native (BN)/Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Couples of acrylamide gradient/migration-time, membrane detergent concentration and hand- made strips were optimized to obtain reproducible extraction and separation of intact membrane protein complexes (MCPs). MCPs were subsequently denatured using SDS-PAGE and each spot from each MCP was identified by mass spectrometry (nanoLC-MS/MS). All together 20 MPCs could be identified including multihomooligomeric and multiheteroligomeric complexes. These MPCs are distributed in both inner and outer membranes. The maximum number of subunits detected in a MCP for C. jejuni using 2-D BN SDS-PAGE approach was three. Function and conservation of MPCs across C. jejuni strains were inspected by functional and genomic comparison analyses. The MPCs identified in C. jejuni membrane are involved in protein folding, molecules trafficking, oxidative phosphorylation, membrane structuration, peptidoglycan biosynthesis, motility and chemotaxis, stress signaling, efflux pumps and virulence. This is the first time that such a holistic non a priori method is applied to C. jejuni to detect MPCs.
Global control of Campylobacter jejuni biology by protein lysine acetylation
Thomas Puttick1, J.A. Butler1, R.J. Dixon1, M.O. Collins2, D.J. Kelly1
1
Molecular Biology and Biotechnology, The University of Sheffield, UK
2
Department of Biomedical Science, The University of Sheffield, UK
The small genome size and regulator complement of C. jejuni suggests post-transcriptional adaptation mechanisms might be crucial in its life-cycle, but the control of protein activity by post- translational modifications (PTMs) of specific amino acids has not been widely studied. Here, we show for the first time that protein lysine acetylation is widespread in C. jejuni and that it impacts multiple aspects of its biology. Immunoblots using a commercially available anti-AcK antibody detected a wide range of acetylated proteins in strain NCTC 11168. While the acetylation profile was similar in both wild-type and pta mutants, deletion of ackA clearly increased protein acetylation, which returned to wild-type levels in a complemented strain. This pattern strongly suggests (i) a dominant role for chemical acetylation from acetyl-P, (ii) acetyl-CoA dependent acetylation via acetyl-transferases in the pta mutant. We identified a Sirtuin homologue, CobB which we show is a lysine deacetylase that is important for in vitro growth and in vivo colonisation of the Galleria larval model. Using a state-of-the-art proteomic workflow with antibody enrichment coupled to LC-MS/MS analysis, we have identified 7,322 acetylation sites in over 1,200 proteins in wild-type cells. Therefore, over 70% of the C. jejuni proteome is acetylated, a greater proportion than in any other bacterium to date. The cobB mutation caused significant alterations in the C. jejuni acetylome; we identified 566 lysines that are controlled by CobB and show that many fundamental cellular processes in C. jejuni are impacted by this reversible acetylation, including chemotaxis, motility, nitrogen and carbon metabolism. Target candidate proteins with cobB sensitive lysines were selected for further characterisation, to determine the effects of acetylation on their activity. Our results have revealed a previously unsuspected but extensive landscape of protein regulation by acetylation in C. jejuni, which we are now investigating in detail.