Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum.

Infected pork meat is an important source of non-typhoidal human salmonellosis. Understanding of molecular mechanisms involved in disease pathogenesis is important for the development of therapeutic and preventive strategies. Thus, hereby we study the transcriptional profiles along the porcine intestine during infection with Salmonella Typhimurium, as well as post-transcriptional gene modulation by microRNAs (miRNA). Sixteen piglets were orally challenged with S. Typhimurium. Samples from jejunum, ileum and colon, collected 1, 2 and 6 days post infection (dpi) were hybridized to mRNA and miRNA expression microarrays and analyzed. Jejunum showed a reduced transcriptional response indicating mild inflammation only at 2 dpi. In ileum inflammatory genes were overexpressed (e.g., IL-1B, IL-6, IL-8, IL1RAP, TNFα), indicating a strong immune response at all times of infection. Infection also down-regulated genes of the FXR pathway (e.g., NR1H4, FABP6, APOA1, SLC10A2), indicating disruption of the bile acid absorption in ileum. This result was confirmed by decreased high-density lipoprotein cholesterol in serum of infected pigs. Ileal inflammatory gene expression changes peaked at 2 dpi and tended to resolve at 6 dpi. Furthermore, miRNA analysis of ileum at 2 dpi revealed 62 miRNAs potentially regulating target genes involved in this inflammatory process (e.g., miR-374 and miR-451). In colon, genes involved in epithelial adherence, proliferation and cellular reorganization were down-regulated at 2 and 6 dpi. In summary, here we show the transcriptional changes occurring at the intestine at different time points of the infection, which are mainly related to inflammation and disruption of the bile acid metabolism.

Quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to Salmonella Typhimurium.

The enteropathogen Salmonella Typhimurium (S. Typhimurium) is the most commonly non-typhoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon) along a time course of 1, 2, and 6 days post infection (dpi) with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signaling, free radical scavengers or antimicrobial peptides (AMP) expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signaling pathway in ileum has been established for the first time in pigs. Taken together, our results provide a better understanding of the porcine response to Salmonella infection and the molecular mechanisms underlying Salmonella-host interactions.

Effect of 7H(ch) Hordeum chilense chromosome introgressions on the wheat endosperm proteomic profile.

Hordeum chilense is an excellent genetic resource for wheat breeding due to its potential to improve breadmaking quality and nutritional value and provide resistance to some biotic and abiotic stresses. Hexaploid wheat lines carrying chromosome 7H(ch) introgressions, namely, chromosome additions of the whole chromosome 7H(ch) or the 7H(ch)α or the 7H(ch)ß chromosome arms, and chromosome substitutions of the homeologous chromosomes 7A, 7B, or 7D by chromosome 7H(ch) were compared by 2D-PAGE analysis to study the effect of these alien introgressions on the wheat endosperm proteome. The addition of the 7H(ch)α chromosome arm did not alter the profile of most glutenins and gliadins, but showed higher quantities of puroindolines and lower xylanase inhibitors, which might improve also resistance to plant pathogens. On the other hand, (7A)7H(ch) or (7D)7H(ch) substitution lines showed enhanced avenin-like b proteins and triticin levels but reduced puroindolines, which could be desirable to improve dough properties and nutritional value and increase kernel hardness in wheat.

Unravelling the proteomic profile of rice meiocytes during early meiosis.

Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier.

Exploring the immune response of porcine mesenteric lymph nodes to Salmonella enterica serovar Typhimurium: an analysis of transcriptional changes, morphological alterations and pathogen burden.

Infections caused by Salmonella enterica serovar Typhimurium (S. typhimurium) cause important economic problems in the swine industry and threaten the integrity of a safe and healthy food supply. Controlling the prevalence of Salmonella in pig production requires a thorough knowledge of the response processes that occurs in the gut associated immune tissues. To explore the in vivo porcine response to S. typhimurium, MLN samples from four control pigs and twelve infected animals at 1, 2 and 6 days post infection (dpi) were collected to quantify the mRNA expression of gene coding for 42 innate immune-related molecules. In addition, the presence of S. typhimurium in MLN was examined and its effect on tissue micro-anatomy. Higher S. typhimurium loads were observed at 2dpi, triggering an innate immune response, marked by a substantial infiltration of phagocytes and up-regulation of pro-inflammatory genes. Such response resulted in a significant decrease in pathogen burden in MLN at 6dpi, although Salmonella could not be completely eliminated from tissue. Furthermore, our results suggest that in porcine infections, S. typhimurium might interferes with dendritic cell-T cell interactions and this strategy could be involved in the conversion of Salmonella infected pigs to a carrier state.

Proteomic analysis of porcine mesenteric lymph-nodes after Salmonella typhimurium infection.

In this study we employed for the first time an in vivo approach coupled to DIGE-based proteomics to explore the response of porcine mesenteric lymph nodes (MLN) to Salmonella typhimurium infection. MLN samples were collected from four control and twelve infected pigs (at 1, 2 and 6 days post infection) for histological analysis, protein and RNA purification. Afterwards, expressed proteins were screened by differential in gel analysis and data were analyzed by bioinformatic tools to generate interaction networks, and identify enriched signaling pathways and biological annotations. S. typhimurium labeling in tissue and phagocyte infiltration were analyzed by immunohistochemistry and RNA was employed to determine the relative expression of immune-related genes by quantitative RNA analysis. The proteome response of porcine MLN to infection was associated to the induction of processes such as phagocyte infiltration, cytoskeleton remodeling and pyroptosis. Moreover, our results suggest that S. typhimurium antigens are cross-presented via MHC-I in a proteasome-dependent manner in porcine MLN. Since pathogen burden in tissue was noticeably reduced at the end of the time course, we infer that host innate and adaptive immunity act in association in MLN to control S. typhimurium dissemination in swine infections.

An in vivo proteomic study of the interaction between Salmonella Typhimurium and porcine ileum mucosa.

The enteropathogen Salmonella Typhimurium is one of the main causes of porcine and human enterocolitis. We have used a 2-DE, MALDI-TOF/TOF-based approach to characterize in vivo proteome changes in porcine ileum mucosa after pathogen interaction. Ileum samples from non-infected and orally infected animals were collected at 2 days post infection and S. Typhimurium presence was confirmed by immunohistochemistry. Fifty one proteins, involved in immune response (acute phase response, inflammation and immune response regulation), apoptosis and pathogen-mediated cell invasion, were identified as being differentially expressed after pathogen challenge. Overall, anti-inflammatory signals and a possible down-regulation of dendritic cell maturation were observed. According to this, we identified the up-regulation of FK506-binding protein 4 (FKBP4), a negative regulator of the transcription factor IRF4 (interferon regulatory factor 4), implicated in Th2 and Th17 response. Transcriptional analysis using RT-qPCR indicated a general trend toward down-regulation of Th2 and Th17 cytokines genes, which would be in agreement with an IRF4 reduced transactivation activity. On the other hand, proteins that could be involved in maturation of Salmonella-containing vacuole and intracellular pathogen survival were up-regulated. Results derived from this study would be valuable to better characterize a possible pathogen led modulation of host responses in vivo.

DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae.

The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), ß-tubulin (ß-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.

Quantitative analysis of the immune response upon Salmonella typhimurium infection along the porcine intestinal gut.

Salmonella enterica serovar Typhimurium causes enteric disease and compromises food safety. In pigs, the molecular response of the intestine to S. typhimurium has been traditionally characterized by in vitro models that do not reflect the actual immunological competence of the intestinal mucosa. In this work, we performed an oral S. typhimurium infection study to obtain insight into the in vitro response in three different sections (jejunum, ileum and colon) of the porcine intestine. For this, samples from one-month-old infected piglets were collected during a time course comprising 1, 2, and 6 days post inoculation to evaluate the intestinal response by quantifying the mRNA expression of gene coding for 28 innate immune system molecules using quantitative real-time PCR assays. In addition, samples from non-infected control animals were also employed to establish differences in the steady state gene expression between intestinal sections. The panel of quantified molecules included an assortment of cytokines, chemokines, pattern-recognition receptors, intracellular signaling molecules, transcription factors and antimicrobial molecules. Changes in gene expression occurred in the three different parts of the intestine and during the course of the S. typhimurium infection. Moreover, the high variation observed in expression patterns of genes coding for inflammatory mediators could indicate that each intestinal section responds differently to the infection. Thus, on the contrary to findings in the jejunum and colon, a down-regulation and lack of induction of some proinflammatory cytokine transcripts was observed in the ileum. Nevertheless, all chemoattractant cytokines assayed were up-regulated in the ileum and jejunum whereas only interleukin-8 and MIP-1alpha mRNA were over expressed in the colon. In conclusion, our results reveal regional differences in gene expression profiles along the porcine intestinal gut as well as regional differences in the inflammatory response to S. typhimurium infection. Taken together, these data should provide a basis for a complete understanding of the porcine intestinal response to bacterial infection.

Biological pathway analysis by ArrayUnlock and Ingenuity Pathway Analysis.

BACKGROUND: Once a list of differentially expressed genes has been identified from a microarray experiment, a subsequent post-analysis task is required in order to find the main biological processes associated to the experimental system. This paper describes two pathways analysis tools, ArrayUnlock and Ingenuity Pathways Analysis (IPA) to deal with the post-analyses of microarray data, in the context of the EADGENE and SABRE post-analysis workshop. Dataset employed in this study proceeded from an experimental chicken infection performed to study the host reactions after a homologous or heterologous secondary challenge with two species of Eimeria. RESULTS: Analysis of the same microarray data source employing both commercial pathway analysis tools in parallel let to identify several biological and/or molecular functions altered in the chicken Eimeria maxima infection model, including several immune system related pathways. Biological functions differentially altered in the homologous and heterologous second infection were identified. Similarly, the effect of the timing in a homologous second infection was characterized by several biological functions. CONCLUSION: Functional analysis with ArrayUnlock and IPA provided information related to functional differences with the three comparisons of the chicken infection leading to similar conclusions. ArrayUnlock let an improvement of the annotations of the chicken genome adding InterPro annotations to the data set file. IPA provides two powerful tools to understand the pathway analysis results: the networks and canonical pathways that showed several pathways related to an adaptative immune response.

Methods for interpreting lists of affected genes obtained in a DNA microarray experiment.

BACKGROUND: The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. RESULTS: Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached. CONCLUSION: It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experiment.

Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers.

ABSTRACT A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study.

Genetic and Virulence Diversity in Verticillium dahliae Populations Infecting Artichoke in Eastern-Central Spain.

ABSTRACT Severe Verticillium dahliae attacks have occurred in artichoke crops in the Comunidad Valenciana region of eastern-central Spain since the late 1990s. Knowledge of genetic and virulence diversity in the pathogen population is a key factor for the management of the disease through disease risk assessment as well as development and use of resistant cultivars. V. dahliae isolates from artichoke (109 isolates) and cotton (three isolates) in that region were characterized by vegetative compatibility grouping (VCG), and specific polymerase chain reaction assays using three sets of primer pairs that differentiate the cotton-defoliating (D) and -nondefoliating (ND) V. dahliae pathotypes. In all, 35 and 39 V. dahliae isolates representative of the identified VCGs and geographic origins were tested for virulence to artichoke cvs. Nun 6374 and Nun 9444, and cotton cv. Acala SJ-2, respectively. Four VCGs were identified among 107 artichoke isolates, and 2 isolates were heterokaryon self-incompatible: VCG1A (one isolate), VCG2A (31 isolates), VCG2B (72 isolates), and VCG4B (three isolates). The three cotton isolates were VCG1A. Isolates in VCG2B were distributed across the region and were the most prevalent isolates in the northern part. Conversely, 83.9% of isolates in VCG2A were recovered from the southern part of the region. Two subgroups of isolates were identified in VCG2B based on heterokaryon compatibility with either international or local tester isolates, which further showed diversity in the amplification of 334- and 824-bp DNA fragments which are markers of the D and ND pathotypes, respectively. Virulence of isolates to artichoke and cotton correlated with VCG but the pattern of correlation varied with the host. VCG1A isolates from artichoke and cotton induced defoliation in cotton but not in artichoke. Collectively, isolates of VCG2B and VCG4B were the most virulent and isolates of VCG1A or HSI were the least virulent to artichoke; but isolates of VCG1A were more virulent to cotton than those of any other VCG. Also, molecular subgrouping in VCG2B determined by amplification of the 334- and 824-bp markers correlated with virulence of isolates to the two hosts tested.