Association of an IRF3 putative functional uORF variant with resistance to Brucella infection: A candidate gene based analysis of InDel polymorphisms in goats.

Brucellosis is an important zoonotic disease caused by infection with Brucella spp. It generates major economic losses in livestock production worldwide. Goats are the principal hosts of B. melitensis, the main infection agent of caprine and human brucellosis. The selection of resistance-related genes is considered one of the best long-term means to improve control to bacterial infection in domestic ruminants. We performed a candidate gene association study to test if six short insertion/deletion polymorphisms (InDels) at bacterial-infection related genes influence the resistance to Brucella infection in female creole goats. InDels (IRF3-540: rs660531540, FKBP5-294: rs448529294, TIRAP-561: rs657494561, PTPRT-588: rs667380588, KALRN-989: rs667660989 and RAB5a-016: rs661537016) were resolved by PCR-capillary electrophoresis in samples from 64 cases and 64 controls for brucellosis. Allelic frequencies were significantly different between cases and controls at IRF3-540 and KALRN-989 (p = 0.001 and 0.005). Indeed, the minor alleles (a and k) at InDels IRF3-540 and KALRN-989 were more frequent among controls than cases, providing evidence that these alleles confer protection against Brucella infection. Moreover, IRF3-540 a-containing genotypes (Aa and aa) were associated with absence of Brucella-specific antibodies in goats (p = 0.003; OR = 3.52; 95% CI = 1.55-7.96), and more specifically, a-allele was associated with resistance to Brucella infection in a dose-dependent manner. Also, we observed that the IRF3-540 deletion (a-allele) extends a conserved upstream ORF by 75 nucleotides to the main ORF, and thus it may decrease gene expression by reducing translation efficiency from the main ORF. These results suggest a potential functional role of IRF3-540 deletion in genetic resistance to Brucella infection in goats.

Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions.

This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics.

Cytokine expression profile of B. melitensis-infected goat monocyte-derived macrophages.

Brucella parasitize the macrophage where is able to replicate and modulate the immune response in order to establish a chronic infection. The most adequate response to control and eliminate Brucella infection is a type 1 (Th1) cell-mediated effector immunity. Research in immune response of B. melitensis-infected goats is relatively scarce. In this study, we first evaluated changes in the gene expression of cytokines, a chemokine (CCL2) and the inducible nitric oxide synthase (iNOS) of goat macrophage cultures derived from monocytes (MDMs) infected for 4 and 24 h with Brucella melitensis strain 16 M. TNFα, IL-1ß and iNOS, and IL-12p40, IFNγ and also iNOS were significantly expressed (p < 0.05) at 4 and 24 h respectively, in infected compared to non-infected MDMs. Therefore, the in vitro challenge of goat MDMs with B. melitensis promoted a transcriptional profile consistent with a type 1 response. However, when the immune response to B. melitensis infection was contrasted between MDM cultures phenotypically restrictive or permissive to intracellular multiplication of B. melitensis 16 M, it was observed that the relative IL-4 mRNA expression was significantly higher in permissive macrophage cultures with respect to restrictive cultures (p < 0.05), independently of the time p.i. A similar trend, although non-statistical, was recorded for IL-10, but not for pro-inflammatory cytokines. Thus, the up-expression profile of inhibitory instead of pro-inflammatory cytokines could explain, in part, the difference observed in the ability to restrict intracellular replication of Brucella. In this sense, the present results make a significant contribution to the knowledge of the immune response induced by B. melitensis in macrophages of its preferential host species.

Deletion in KARLN intron 5 and predictive relationship with bovine tuberculosis and brucellosis infection phenotype.

Kalirin (gene: KALRN) is a Rho-GEF kinase linked to neurodegenerative diseases in humans. Unexpectedly, various polymorphisms in KALRN gene were previously associated with resistance to bacterial infections in ruminants. In this study, we evaluated the effect of the rs384223075 (RS-075) deletion in KALRN intron 5 on the occurrence of Mycobacterium bovis and Brucella abortus infections in cattle. We performed two separate case-control association analyses: one for bovine tuberculosis (bTB) using 308 Holstein and Jersey cows from three herds with prevalence between 5 and 15% for this infection; and another for brucellosis using 140 Holstein and beef crossbred cows from two herds with high prevalence for brucellosis (> 30%). In the bTB analysis, the RS-075 deletion frequency was higher among cases than controls (p = 0.0001), and the absence of the RS-075 deletion allele was associated with negative PPD-skin test results (p = 0.0009) at genotype level. On the contrary, RS-075 was not associated with Brucella spp. serological status (p = 0.72) but, unexpectedly, the deletion allele was more frequent among controls than cases in the beef crossbred herd (0.31 vs. 0.14, p = 0.02). In concordance with this observation, in vitro assays showed that the RS-075 deletion could be linked to an enhanced cellular response to bacterial antigens and unspecific stimulation in mononuclear cells derived from beef crossbred cows, specifically the reactive nitrogen species production (p = 0.008) and proliferation capacity (p = 0.018). This study is consistent with other reports that support an important role of the KALRN gene and its polymorphisms in the host response to intracellular pathogens.

Evaluation of B. melitensis whole-cell lysate antigen-based indirect ELISA for the serodiagnosis of caprine brucellosis.

Different fractions of Brucella (B) abortus or Brucella melitensis have been used as antigens for the detection of anti-Brucella antibodies in goat sera, being their accomplishment cumbersome and time consuming. In an attempt to achieve a simpler enzyme-linked immunosorbent assay (ELISA) antigen preparation method for serodiagnosis of caprine brucellosis, we developed and evaluated a B. melitensis whole-cell lysate antigen-based indirect ELISA (Bm-WCL iELISA). A total of 162 serum samples from female crossbred goats collected from non-vaccinated herds against brucellosis were classified according to the buffered plate antigen (BPA) screening test and the complement fixation (CF) test and used for the indirect ELISA (iELISA) evaluation. The Bm-WCL iELISA showed a high Se and Sp [95.7% (CI 88.1% - 98.8%), and 92.4% (CI 83.4% - 96.7%), respectively] to detect the serological response against Brucella in commercial goat herds, and an almost perfect agreement with combined official tests results (κ = 0.88), when goat sera with concordant results in both official serological tests (BPA and CF; n = 136) were used. However, the agreement dropped to substantial (k > 0.73) when 26 goat serum samples with BPA and CF not concordant results were incorporated for the iELISA performance evaluation and the comparison was made for each test independently. Comparison of the Bm-WCL iELISA results with Brucella abortus sLPS iELISA showed almost perfect agreement (κ > 0.83). Even when a larger number of samples are needed to validate this test, these preliminary results encourage the optimization of the Brucella melitensis whole cell lysate antigen-based iELISA.

Transcriptional profile of the intracellular pathogen Brucella melitensis following HeLa cells infection.

Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces; however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. Using cDNA microarray analysis, we characterized the transcriptional profile of the intracellular pathogen Brucella melitensis at 4 h (adaptational period) and 12 h (replicative phase) following HeLa cells infection. The intracellular pathogen transcriptome was determined using initially enriched and then amplified B. melitensis RNA from total RNA of B. melitensis-infected HeLa cells. Analysis of microarray results identified 161 and 115 pathogen genes differentially expressed at 4 and 12 h p.i., respectively. In concordance with phenotypic studies, most of the genes expressed were involved in pathogen growth and metabolism, and were down-regulated at the earliest time point (78%), but up-regulated at 12 h p.i. (75%). Further characterization of specific genes identified in this study will elucidate biological processes and pathways to help understand how both host and Brucella interact during the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve host.

Evaluation of the safety profile of the vaccine candidate Brucella melitensis 16MΔvjbR strain in goats.

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x109 CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host.

Brucella melitensis global gene expression study provides novel information on growth phase-specific gene regulation with potential insights for understanding Brucella:host initial interactions.

BACKGROUND: Brucella spp. are the etiological agents of brucellosis, a zoonotic infectious disease that causes abortion in animals and chronic debilitating illness in humans. Natural Brucella infections occur primarily through an incompletely defined mechanism of adhesion to and penetration of mucosal epithelium. In this study, we characterized changes in genome-wide transcript abundance of the most and the least invasive growth phases of B. melitensis cultures to HeLa cells, as a preliminary approach for identifying candidate pathogen genes involved in invasion of epithelial cells. RESULTS: B. melitensis at the late logarithmic phase of growth are more invasive to HeLa cells than mid-logarithmic or stationary growth phases. Microarray analysis of B. melitensis gene expression identified 414 up- and 40 down-regulated genes in late-log growth phase (the most invasive culture) compared to the stationary growth phase (the least invasive culture). As expected, the majority of up-regulated genes in late-log phase cultures were those associated with growth, including DNA replication, transcription, translation, intermediate metabolism, energy production and conversion, membrane transport, and biogenesis of the cell envelope and outer membrane; while the down-regulated genes were distributed among several functional categories. CONCLUSION: This Brucella global expression profile study provides novel information on growth phase-specific gene expression. Further characterization of some genes found differentially expressed in the most invasive culture will likely bring new insights into the initial molecular interactions between Brucella and its host.

Caprine brucellosis: A historically neglected disease with significant impact on public health.

Caprine brucellosis is a chronic infectious disease caused by the gram-negative cocci-bacillus Brucella melitensis. Middle- to late-term abortion, stillbirths, and the delivery of weak offspring are the characteristic clinical signs of the disease that is associated with an extensive negative impact in a flock's productivity. B. melitensis is also the most virulent Brucella species for humans, responsible for a severely debilitating and disabling illness that results in high morbidity with intermittent fever, chills, sweats, weakness, myalgia, abortion, osteoarticular complications, endocarditis, depression, anorexia, and low mortality. Historical observations indicate that goats have been the hosts of B. melitensis for centuries; but around 1905, the Greek physician Themistokles Zammit was able to build the epidemiological link between "Malta fever" and the consumption of goat milk. While the disease has been successfully managed in most industrialized countries, it remains a significant burden on goat and human health in the Mediterranean region, the Middle East, Central and Southeast Asia (including India and China), sub-Saharan Africa, and certain areas in Latin America, where approximately 3.5 billion people live at risk. In this review, we describe a historical evolution of the disease, highlight the current worldwide distribution, and estimate (by simple formula) the approximate costs of brucellosis outbreaks to meat- and milk-producing farms and the economic losses associated with the disease in humans. Successful control leading to eradication of caprine brucellosis in the developing world will require a coordinated Global One Health approach involving active involvement of human and animal health efforts to enhance public health and improve livestock productivity.

A haplotype at intron 8 of PTPRT gene is associated with resistance to Brucella infection in Argentinian creole goats.

Brucellosis is the leading zoonosis on a worldwide scale and constitutes a major public health threat in many regions of the world. Several molecular markers associated with natural resistance to intracellular bacterial infection have been identified. Recently seven single-nucleotide polymorphisms (SNPs) located in the PTPRT gene were associated with resistance to Mycobacterium bovis infection in cattle. Here, we perform a case-control study to test if polymorphisms at PTPRT intron 8 might influence the resistance or susceptibility to Brucella infection in goats. DNA samples from 22 seropositive (cases) and 22 seronegative (controls) for brucellosis, unrelated female creole goats, were included in the present study. Four previously reported polymorphisms (SNP1: rs643551276, SNP2: rs651618967, SNP3: rs662137815 and SNP4: rs657542977) and a new SNP (SNP5: chr13: 691695526) were detected by PCR-DNA sequencing method. Genotypic and allelic frequencies differed significantly between cases and controls at SNPs 1, 2, 4 and 5 (p≤0.001). Indeed, the SNP1 TT, SNP2 TT, SNP4 CC and SNP5 TT genotypes were associated with absence of Brucella-specific antibodies (ORs=0.019 to 0.045). Moreover, haplotype association analysis revealed a significant association of the TTCCT haplotype with protection to Brucella infection (p≤1×10-4; OR=18), including the major allelic variants associated with resistance. These results represent the first evidence of genetic association between polymorphisms in the PTPRT gene and absence of brucellosis in goats.

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 host's 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.

Human Brucellosis and Adverse Pregnancy Outcomes.

PURPOSE OF REVIEW: Brucellosis is a neglected, zoonotic disease of nearly worldwide distribution. Despite brucellosis being recognized as a reproductive disease in animals, it has been historically known as a flu-like illness in humans with little or no significant role in maternal or newborn health. This review focuses on what is currently known relative to the epidemiology of brucellosis in human pregnancy as well as new insights of placental immunology. RECENT FINDINGS: New evidence suggests that maternal infection poses a significant risk factor for adverse pregnancy outcomes including increased risk for miscarriage during the first and second trimester of gestation, preterm delivery, and vertical transmission to the fetus. Adverse pregnancy outcomes were not associated with any specific clinical sign. However, prompt diagnosis and treatment significantly decreased the risk of miscarriage or any other adverse effect. SUMMARY: Brucellosis during pregnancy should be considered a significant risk factor for adverse pregnancy outcomes in humans. The identification of the mechanism behind bacterial tropism should prove powerful for the development of new countermeasures to prevent these detrimental effects. Increased awareness concerning brucellosis in pregnant women, its transmission, and prevention measures should be considered as a pressing need.

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.

Buenos Aires, a new Leptospira serovar of serogroup Djasiman, isolated from an aborted dog fetus in Argentina.

This study describes the isolation of a new leptospiral serovar from the Djasiman group from an Argentinean aborted fetus of a dog. The strain was isolated from a culture of mixed liver and kidney tissue from one aborted dog fetus. Bitch's serum showed a titre of 1:800 against the new serovar and 1:400 or less against other serovars tested. Microscopic Agglutination test (MAT) with a panel of 38 rabbit anti-Leptospira sera representative for all pathogenic serogroups was performed to establish the putative serogroup of the isolated strain. Serovar identification was done by Cross-Agglutination Absorption Test (CAAT). Species determination was carried out on basis of sequence identity of primer pair G1/G2 generated PCR products from the isolate with those from reference strains belonging to the various pathogenic Leptospira species. Results showed that the new strain named, Baires, belongs to species L. interrogans sensu stricto and the serogroup Djasiman. However, the isolate could not be identified as any of the known serovars within this serogroup. These results allow us to conclude that the strain Baires represents a new serovar (Buenos Aires) of Djasiman serogroup.

Polymorphisms at the 3' untranslated region of SLC11A1 gene are associated with protection to Brucella infection in goats.

Goats are susceptible to brucellosis and the detection of Brucella-infected animals is carried out by serological tests. In other ruminant species, polymorphisms in microsatellites (Ms) of 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to Brucella abortus infection. Goats present two polymorphic Ms at the 3'UTR end of SLC11A1 gene, called regions A and B. Here, we evaluated if polymorphisms in regions A and/or B are associated with Brucella infection in goats. Serum (for the detection of Brucella-specific antibodies) and hair samples (for DNA isolation and structure analysis of the SLC11A1 gene) were randomly collected from 229 adult native goats from the northwest of Argentina. Serological status was evaluated by buffer plate antigen test (BPAT) complemented by the fluorescent polarization assay (FPA), and the genotype of the 3'UTR of the SLC11A1 gene was determined by capillary electrophoresis and confirmed by sequence analysis. Polymorphisms in regions A and B of the 3'UTR SLC11A1 gene were found statistically significant associated with protection to Brucella infection. Specifically, the association study indicates statistical significance of the allele A15 and B7/B7 genotype with absence of Brucella-specific antibodies (p=0.0003 and 0.0088, respectively). These data open a promising opportunity for limiting goat brucellosis through selective breeding of animals based on genetic markers associated with natural resistance to B. melitensis infection.

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 Peyer's 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.

Transcriptome analysis of HeLa cells response to Brucella melitensis infection: a molecular approach to understand the role of the mucosal epithelium in the onset of the Brucella pathogenesis.

Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces, however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. We hypothesized that characterizing the epithelial-like human HeLa cell line molecular response to wild type Brucella melitensis infection would help to understand the role of the mucosal epithelium at the onset of the Brucella pathogenesis. RNA samples from B. melitensis-infected HeLa cells were taken at 4 and 12 h of infection and hybridized in a cDNA microarray. The analysis using a dynamic Bayesian network modeling approach (DBN) identified several pathways, biological processes, cellular components and molecular functions altered due to infection at 4 h p.i., but almost none at 12 h p.i. The in silico modeling results were experimentally tested by knocking down the expression of MAPK1 by siRNA technology. MAPK1-siRNA transfected cell cultures decreased the internalization and impaired the intracellular replication of the pathogen in HeLa cells after 4 h p.i. DBN analysis provides important insights into the role of the epithelial cells response to Brucella infection and guide research to novel mechanisms identification.

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 Peyer's 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 Peyer's patch.

Enhancing the role of veterinary vaccines reducing zoonotic diseases of humans: linking systems biology with vaccine development.

The aim of research on infectious diseases is their prevention, and brucellosis and salmonellosis as such are classic examples of worldwide zoonoses for application of a systems biology approach for enhanced rational vaccine development. When used optimally, vaccines prevent disease manifestations, reduce transmission of disease, decrease the need for pharmaceutical intervention, and improve the health and welfare of animals, as well as indirectly protecting against zoonotic diseases of people. Advances in the last decade or so using comprehensive systems biology approaches linking genomics, proteomics, bioinformatics, and biotechnology with immunology, pathogenesis and vaccine formulation and delivery are expected to enable enhanced approaches to vaccine development. The goal of this paper is to evaluate the role of computational systems biology analysis of host:pathogen interactions (the interactome) as a tool for enhanced rational design of vaccines. Systems biology is bringing a new, more robust approach to veterinary vaccine design based upon a deeper understanding of the host-pathogen interactions and its impact on the host's molecular network of the immune system. A computational systems biology method was utilized to create interactome models of the host responses to Brucella melitensis (BMEL), Mycobacterium avium paratuberculosis (MAP), Salmonella enterica Typhimurium (STM), and a Salmonella mutant (isogenic ΔsipA, sopABDE2) and linked to the basis for rational development of vaccines for brucellosis and salmonellosis as reviewed by Adams et al. and Ficht et al. [1,2]. A bovine ligated ileal loop biological model was established to capture the host gene expression response at multiple time points post infection. New methods based on Dynamic Bayesian Network (DBN) machine learning were employed to conduct a comparative pathogenicity analysis of 219 signaling and metabolic pathways and 1620 gene ontology (GO) categories that defined the host's biosignatures to each infectious condition. Through this DBN computational approach, the method identified significantly perturbed pathways and GO category groups of genes that define the pathogenicity signatures of the infectious agent. Our preliminary results provide deeper understanding of the overall complexity of host innate immune response as well as the identification of host gene perturbations that defines a unique host temporal biosignature response to each pathogen. The application of advanced computational methods for developing interactome models based on DBNs has proven to be instrumental in elucidating novel host responses and improved functional biological insight into the host defensive mechanisms. Evaluating the unique differences in pathway and GO perturbations across pathogen conditions allowed the identification of plausible host-pathogen interaction mechanisms. Accordingly, a systems biology approach to study molecular pathway gene expression profiles of host cellular responses to microbial pathogens holds great promise as a methodology to identify, model and predict the overall dynamics of the host-pathogen interactome. Thus, we propose that such an approach has immediate application to the rational design of brucellosis and salmonellosis vaccines.

Multi-comparative systems biology analysis reveals time-course biosignatures of in vivo bovine pathway responses to B.melitensis, S.enterica Typhimurium and M.avium paratuberculosis.

BACKGROUND: To decipher the complexity and improve the understanding of host-pathogen interactions, biologists must adopt new system level approaches in which the hierarchy of biological interactions and dynamics can be studied. This paper presents the application of systems biology for the cross-comparative analysis and interactome modeling of three different infectious agents, leading to the identification of novel, unique and common molecular host responses (biosignatures). METHODS: A computational systems biology method was utilized to create interactome models of the host responses to Brucella melitensis (BMEL), Salmonella enterica Typhimurium (STM) and Mycobacterium avium paratuberculosis (MAP). A bovine ligated ileal loop biological model was employed to capture the host gene expression response at four time points post infection. New methods based on Dynamic Bayesian Network (DBN) machine learning were employed to conduct a systematic comparative analysis of pathway and Gene Ontology category perturbations. RESULTS: A cross-comparative assessment of 219 pathways and 1620 gene ontology (GO) categories was performed on each pathogen-host condition. Both unique and common pathway and GO perturbations indicated remarkable temporal differences in pathogen-host response profiles. Highly discriminatory pathways were selected from each pathogen condition to create a common system level interactome model comprised of 622 genes. This model was trained with data from each pathogen condition to capture unique and common gene expression features and relationships leading to the identification of candidate host-pathogen points of interactions and discriminatory biosignatures. CONCLUSIONS: Our results provide deeper understanding of the overall complexity of host defensive and pathogen invasion processes as well as the identification of novel host-pathogen interactions. The application of advanced computational methods for developing interactome models based on DBN has proven to be instrumental in conducting multi-conditional cross-comparative analyses. Further, this approach generates a fully simulateable model with capabilities for predictive analysis as well as for diagnostic pattern recognition. The resulting biosignatures may represent future targets for identification of emerging pathogens as well as for development of antimicrobial drugs, immunotherapeutics, or vaccines for prevention and treatment of diseases caused by known, emerging/re-emerging infectious agents.