Tamara Gull

CsgA is a pathogen‐associated molecular pattern of Salmonella enterica serotype Typhimurium that is recognized by Toll‐like receptor 2

Knowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen‐associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant (csgBA) was attenuated in its ability to elicit fluid accumulation and GROα mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL‐8 production than the wild type in human macrophage‐like cells. Purified thin curled fimbriae induced IL‐8 expression in human embryonic kidney (HEK293) cells transfected with Toll‐like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione‐S‐transferase (GST) elicited IL‐8 production in HEK293 cells transfected with TLR2/CD14. Proteinase K treatment abrogated IL‐8 production elicited in these cells by GST–CsgA, but not by synthetic lipoprotein. GST–CsgA elicited more IL‐6 production than GST in bone marrow‐derived macrophages from TLR2+/+ mice, while there was no difference in IL‐6 secretion between GST–CsgA and GST in macrophages from TLR2–/– mice. These data suggested that CsgA is a PAMP that is recognized by TLR2.

Early Phase Morphological Lesions and Transcriptional Responses of Bovine Ileum Infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johnes disease) and a possible etiopathologic agent in human Crohns disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5–12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric β-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohns disease.

Role of spi-1 secreted effectors in acute bovine response to salmonella enterica serovar typhimurium: A systems biology analysis approach

Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway.

Systems biology analysis of gene expression during in vivo Mycobacterium avium paratuberculosis enteric colonization reveals role for immune tolerance.

Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyers patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways and specifically modified mechanistic genes contributing to the colonization of Peyers patch.

Morphologic and Cytokine Profile Characterization of Salmonella enterica Serovar Typhimurium Infection in Calves With Bovine Leukocyte Adhesion Deficiency

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium–induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18–), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18– calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18– calves—including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration—were similar to the ones reported in the literature for CD18– calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18– calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene α in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1β and tumor necrosis factor α were poorly correlated with the presence or absence of tissue neutrophils.

Systems Biology Analysis of Brucella Infected Peyer's Patch Reveals Rapid Invasion with Modest Transient Perturbations of the Host Transcriptome

Brucella melitensis causes the most severe and acute symptoms of all Brucella species in human beings and infects hosts primarily through the oral route. The epithelium covering domed villi of jejunal-ileal Peyers patches is an important site of entry for several pathogens, including Brucella. Here, we use the calf ligated ileal loop model to study temporal in vivo Brucella-infected host molecular and morphological responses. Our results document Brucella bacteremia occurring within 30 min after intraluminal inoculation of the ileum without histopathologic traces of lesions. Based on a system biology Dynamic Bayesian Network modeling approach (DBN) of microarray data, a very early transient perturbation of the host enteric transcriptome was associated with the initial host response to Brucella contact that is rapidly averted allowing invasion and dissemination. A detailed analysis revealed active expression of Syndecan 2, Integrin alpha L and Integrin beta 2 genes, which may favor initial Brucella adhesion. Also, two intestinal barrier-related pathways (Tight Junction and Trefoil Factors Initiated Mucosal Healing) were significantly repressed in the early stage of infection, suggesting subversion of mucosal epithelial barrier function to facilitate Brucella transepithelial migration. Simultaneously, the strong activation of the innate immune response pathways would suggest that the host mounts an appropriate protective immune response; however, the expression of the two key genes that encode innate immunity anti-Brucella cytokines such as TNF-α and IL12p40 were not significantly changed throughout the study. Furthermore, the defective expression of Toll-Like Receptor Signaling pathways may partially explain the lack of proinflammatory cytokine production and consequently the absence of morphologically detectable inflammation at the site of infection. Cumulatively, our results indicate that the in vivo pathogenesis of the early infectious process of Brucella is primarily accomplished by compromising the mucosal immune barrier and subverting critical immune response mechanisms.

Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer’s patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum, inhibition of Ca signaling associated with decreased phagosome-lysosome fusion as well as phagocytosis inhibition, bias toward Th2 cell immune response accompanied by cell recruitment, cell proliferation and cell differentiation; leading to persistent infection. Contrarily, MAA infection was related to cellular responses associated with activation of molecular pathways that release chemicals and cytokines involved with containment of infection and a strong bias toward Th1 immune response, resulting in a transient infection.

Models of Contagious Bovine Pleuropneumonia: Evaluation of Two Novel Strains

The clinical course of infection following endobronchial inoculation of three strains of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), the causative agent of Contagious Bovine Pleuropneumonia (CBPP), was compared in 15 mixed-breed Bos taurus steers. Two of these strains have not previously been investigated under experimental conditions. The cattle were inoculated endobronchially with MmmSC strain Gladysdale, Ondangwa or Shawawa and monitored for 42 days. Four of five Gladysdale-inoculated cattle developed acute clinical CBPP. Four of five Ondangwa and 3/5 Shawawa-inoculated cattle developed subacute to chronic CBPP without overt clinical signs but had characteristic pathological lesions. MmmSC was isolated from cattle both during the course of infection and at necropsy. Clinical signs and necropsy lesions were consistent within groups. Ondangwa and Shawawa strains of MmmSC may be useful for study of subacute and chronic CBPP, which is more common in natural infection than peracute CBPP. Use of recent strains will enhance study of CBPP host response, pathogen detection and vaccine development.

Systems Biology Analysis of Temporal In vivo Brucella melitensis and Bovine Transcriptomes Predicts host:Pathogen Protein–Protein Interactions

To date, fewer than 200 gene-products have been identified as Brucella virulence factors, and most were characterized individually without considering how they are temporally and coordinately expressed or secreted during the infection process. Here, we describe and analyze the in vivo temporal transcriptional profile of Brucella melitensis during the initial 4 h interaction with cattle. Pathway analysis revealed an activation of the “Two component system” providing evidence that the in vivo Brucella sense and actively regulate their metabolism through the transition to an intracellular lifestyle. Contrarily, other Brucella pathways involved in virulence such as “ABC transporters” and “T4SS system” were repressed suggesting a silencing strategy to avoid stimulation of the host innate immune response very early in the infection process. Also, three flagellum-encoded loci (BMEII0150-0168, BMEII1080-1089, and BMEII1105-1114), the “flagellar assembly” pathway and the cell components “bacterial-type flagellum hook” and “bacterial-type flagellum” were repressed in the tissue-associated B. melitensis, while RopE1 sigma factor, a flagellar repressor, was activated throughout the experiment. These results support the idea that Brucella employ a stealthy strategy at the onset of the infection of susceptible hosts. Further, through systems-level in silico host:pathogen protein–protein interactions simulation and correlation of pathogen gene expression with the host gene perturbations, we identified unanticipated interactions such as VirB11::MAPK8IP1; BtaE::NFKBIA, and 22 kDa OMP precursor::BAD and MAP2K3. These findings are suggestive of new virulence factors and mechanisms responsible for Brucella evasion of the hosts protective immune response and the capability to maintain a dormant state. The predicted protein–protein interactions and the points of disruption provide novel insights that will stimulate advanced hypothesis-driven approaches toward revealing a clearer understanding of new virulence factors and mechanisms influencing the pathogenesis of brucellosis.