The Skin Microbiome in Healthy and Allergic Dogs

Changes in the microbial populations on the skin of animals have traditionally been evaluated using conventional microbiology techniques. The sequencing of bacterial 16S rRNA genes has revealed that the human skin is inhabited by a highly diverse and variable microbiome that had previously not been demonstrated by culture-based methods. The goals of this study were to describe the microbiome inhabiting different areas of the canine skin, and to compare the skin microbiome of healthy and allergic dogs. DNA extracted from superficial skin swabs from healthy (n = 12) and allergic dogs (n = 6) from different regions of haired skin and mucosal surfaces were used for 454-pyrosequencing of the 16S rRNA gene. Principal coordinates analysis revealed clustering for the different skin sites across all dogs, with some mucosal sites and the perianal regions clustering separately from the haired skin sites. The rarefaction analysis revealed high individual variability between samples collected from healthy dogs and between the different skin sites. Higher species richness and microbial diversity were observed in the samples from haired skin when compared to mucosal surfaces or mucocutaneous junctions. In all examined regions, the most abundant phylum and family identified in the different regions of skin and mucosal surfaces were Proteobacteria and Oxalobacteriaceae. The skin of allergic dogs had lower species richness when compared to the healthy dogs. The allergic dogs had lower proportions of the Betaproteobacteria Ralstonia spp. when compared to the healthy dogs. The study demonstrates that the skin of dogs is inhabited by much more rich and diverse microbial communities than previously thought using culture-based methods. Our sequence data reveal high individual variability between samples collected from different patients. Differences in species richness was also seen between healthy and allergic dogs, with allergic dogs having lower species richness when compared to healthy dogs.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Bayesian Approach to Model CD137 Signaling in Human M.tuberculosis in vitro Responses

Abstract Immune responses are qualitatively and quantitatively influenced by a complex network of receptor-ligand interactions. Among them, the CD137:CD137L pathway is known to modulate innate and adaptive human responses against Mycobacterium tuberculosis. However, the underlying mechanisms of this regulation remain unclear. In this work, we developed a Bayesian Computational Model (BCM) of in vitro CD137 signaling, devised to fit previously gathered experimental data. The BCM is fed with the data and the prior distribution of the model parameters and it returns theirposterior distribution and the model evidence, which allows comparing alternative signaling mechanisms. The BCM uses a coupled system of non-linear differential equations to describe the dynamics of Antigen Presenting Cells, Natural Killer and T Cells together with the interpheron (IFN)-c and tumor necrosis factor (TNF)-a levels in the media culture. Fast and complete mixing of the media is assumed. The prior distribution of the parameters that describe the dynamics of the immunological response was obtained from the literature and theoretical considerations Our BCM applies successively the Levenberg-Marquardt algorithm to find the maximum a posteriori likelihood (MAP); the Metropolis Markov Chain Monte Carlo method to approximate the posterior distribution of the parameters and Thermodynamic Integration to calculate the evidence of alternative hypothesis. Bayes factors provided decisive evidence favoring direct CD137 signaling on T cells. Moreover, the posterior distribution of the parameters that describe the CD137 signaling showed that the regulation of IFNc levels is based more on T cells survival than on direct induction. Furthermore, the mechanisms that account for the effect of CD137 signaling on TNF-a production were based on a decrease of TNF-a production by APC and, perhaps, on the increase in APC apoptosis. BCM proved to be a useful tool to gain insight on the mechanisms of CD137 signaling during human response against Mycobacterium tuberculosis.Fil: Darío A Fernández Do Porto. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Jerónimo Auzmendi. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Delfina Peña. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. DTO.DE QUIMICA BIOLOGICA.Fil: Veronica E Garcia. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES.Fil: Luciano Moffatt. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG

Variable Carbon Catabolism among Salmonella enterica Serovar Typhi Isolates

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever) and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study. METHODOLOGY/PRINCIPAL FINDINGS: To obtain insight into the intracellular lifestyle of S. Typhi, a high-throughput phenotypic microarray was employed to characterise the catabolic capacity of 190 carbon sources in S. Typhi strains. The success of this study lies in the carefully selected library of S. Typhi strains, including strains from two geographically distinct areas of typhoid endemicity, an asymptomatic human carrier, clinical stools and blood samples and sewage-contaminated rivers. An extremely low carbon catabolic capacity (27% of 190 carbon substrates) was observed among the strains. The carbon catabolic profiles appeared to suggest that S. Typhi strains survived well on carbon subtrates that are found abundantly in the human body but not in others. The strains could not utilise plant-associated carbon substrates. In addition, α-glycerolphosphate, glycerol, L-serine, pyruvate and lactate served as better carbon sources to monosaccharides in the S. Typhi strains tested. CONCLUSION: The carbon catabolic profiles suggest that S. Typhi could survive and persist well in the nutrient depleted metabolic niches in the human host but not in the environment outside of the host. These findings serve as caveats for future studies to understand how carbon catabolism relates to the pathogenesis and transmission of this pathogen

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5′-UTR, 3′-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5’-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3’-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37°C versus 28°C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49% and 51% similarity, and 16% and 17% identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species

Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors

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

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species