Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host

A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.IMPORTANCE Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi Our results suggest that H. ducreyi survives in an abscess by utilizing l-ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection

Characterization and sequence analysis of the lsg (LOS synthesis genes) locus from Haemophilus influenzae type b

Analysis of the lsg (LOS synthesis genes) cluster in Escherichia coli strain K12 and mutations in the lsg locus in Haemophilus influenzae type b indicated the presence of 3 regions responsible for sequential modifications of E. coli lipopolysaccharide (LPS). Sequencing of the lsg region yielded 7,435 bp that encompassed 7 complete and 1 partial open reading frames (ORFs 1-8). The predicted product of ORF1 had homology to the consensus sequence of cytochrome b proteins (21% identity, 51% similarity) and to other transmembrane proteins. The products of ORF5 and ORF6 share overall 23% identity and 49% similarity with each other. The ORF6 protein had high homology with the product of ORF275 of the E. coli rfb gene cluster (40% identity, 58% similarity), whose function is not known. Multiple sequence alignment of the ORF5 and ORF6 proteins with the RfbB, RfbJ and RfbX proteins revealed conserved motifs over the N-terminal half region of all these proteins. The products of ORF7 and ORF8 are homologous with Azotobacter vinelandii MolA protein (30% identity, 51% similarity) and MolB protein (26% identity, 48% similarity), respectively. The promoter regions of ORF1, 7 and 8 were determined by primer extension analysis and found to be similar to bacterial σ70-dependent promoters. ORF7 and ORF8 are transcribed into diverse orientation. At least 5 of the encoded proteins have been identified using coupled E. coli transcription/translation system and labeling with [35S]-methionine. We conclude that the genetic organization of the lsg biosynthesis pathway involves multiple operons that lead to the assembly of an H. influenzae LOS structure.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67077/2/10.1177_096805199400100305.pd

Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions

Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi

Providing adaptive traffic routing based on user and network context

Providing real-time traffic guarantees and fairness based on the availability of network resources has been a major issue presented in the literature. However, due the convergent nature of digital architectures, the increasing demand of upcoming real-time sensitive traffic, such as VoIP, and a higher user´s adaptability (devices, global positioning, content quality, etc.), solutions based on Quality of Service (QoS) turned out to be insufficient in order to meet user´s requirements. Indeed, QoS metrics are network-centered, and mostly related to the dynamic nature of the traffic (such as throughput, delay, jitter, among others). In order to meet the need for a user-centered network, this paper proposes a context-aware solution where the concepts of Quality of Service, Quality of Experience and Adaptive Routing are integrated in order to provide a more dynamic and pro-active approach for the delivery of context-oriented time-sensitive traffic.info:eu-repo/semantics/publishedVersio

Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains

BACKGROUND: Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin? METHODOLOGY/PRINCIPAL FINDINGS: To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin. CONCLUSIONS/SIGNIFICANCE: These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions

Astabiotics: Antimicrobial Signal Transduction Activators

poster abstractThe increasing prevalence of multi-drug resistant Gram-negative bacteria is a major public health concern. All available antibiotics are inhibitors of targets essential for virulence or growth. CpxRA is a highly conserved bacterial signal transduction system that responds to extracytoplasmic membrane stress. CpxA is a sensor with kinase and phosphatase activity; upon activation, CpxA donates a phosphate group to CpxR, activating a transcriptional response. Activation of CpxRA reduces the flow of protein traffic through the cytoplasmic membrane, dramatically reducing the expression of virulence determinants. Activation of CpxRA abolishes the virulence of Salmonella Typhimurium in mice. We found that activation of CpxRA crippled the ability of Haemophilus ducreyi to cause disease in experimentally infected human volunteers. Using an Escherichia coli reporter strain, we developed a high throughput screen to detect compounds that activate CpxRA. In a pilot screen of 36,000 compounds, we identified 1 class of compounds that shifts the equilibrium of CpxA to kinase activity, activating CpxR. Based on its potency, the calculated effective dose of the lead compound (a nitroindole) was 10 mg/kg. Female mice tolerated 100 mg/kg of the nitroindole given twice a day for 3 days. A CpxRA activating mutant constructed in uropathogenic E. coli (UPEC) was severely impaired in a murine urinary tract infection model; thus, activation of CpxRA is a valid treatment strategy for UPEC. However, when female mice were challenged with UPEC and treated with 100 mg/kg of the nitroindole using the schedule above, there were no differences in the recovered CFU in the urine, bladder, and kidney of sham and compound-treated mice. Future studies will include medicinal optimization of the nitroindole and identification and optimization other leads that activate CpxRA. Although our pilot test of efficacy was negative, astabiotics have great potential as broad-spectrum, adjunctive therapies to existing antimicrobials for treatment of Gram-negative infections

Outer Membrane Protein DsrA Is the Major Fibronectin-Binding Determinant of Haemophilus ducreyi

The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains

Joint association of polymorphism of the FGFR4 gene and mutation TP53 gene with bladder cancer prognosis

The impact of the fibroblast growth factor receptor 4 (FGFR4) Gly388Arg polymorphism on bladder cancer is unknown. We found no clear correlations between the FGFR4 genotype and risk of bladder cancer or pathological parameters. Neither the polymorphism nor TP53 mutation status was an independent predictor of prognosis, but they might act jointly on the disease-specific survival of patients

Naming the pain in requirements engineering : Contemporary problems, causes, and effects in practice

Requirements Engineering (RE) has received much attention in research and practice due to its importance to software project success. Its interdisciplinary nature, the dependency to the customer, and its inherent uncertainty still render the discipline difficult to investigate. This results in a lack of empirical data. These are necessary, however, to demonstrate which practically relevant RE problems exist and to what extent they matter. Motivated by this situation, we initiated the Naming the Pain in Requirements Engineering (NaPiRE) initiative which constitutes a globally distributed, bi-yearly replicated family of surveys on the status quo and problems in practical RE. In this article, we report on the qualitative analysis of data obtained from 228 companies working in 10 countries in various domains and we reveal which contemporary problems practitioners encounter. To this end, we analyse 21 problems derived from the literature with respect to their relevance and criticality in dependency to their context, and we complement this picture with a cause-effect analysis showing the causes and effects surrounding the most critical problems. Our results give us a better understanding of which problems exist and how they manifest themselves in practical environments. Thus, we provide a first step to ground contributions to RE on empirical observations which, until now, were dominated by conventional wisdom only.Peer reviewe

First-generation structure-activity relationship studies of 2,3,4,9-tetrahydro-1H-carbazol-1-amines as CpxA phosphatase inhibitors

Genetic activation of the bacterial two-component signal transduction system, CpxRA, abolishes the virulence of a number of pathogens in human and murine infection models. Recently, 2,3,4,9-tetrahydro-1H-carbazol-1-amines were shown to activate the CpxRA system by inhibiting the phosphatase activity of CpxA. Herein we report the initial structure-activity relationships of this scaffold by focusing on three approaches 1) A-ring substitution, 2) B-ring deconstruction to provide N-arylated amino acid derivatives, and 3) C-ring elimination to give 2-ethylamino substituted indoles. These studies demonstrate that the A-ring is amenable to functionalization and provides a promising avenue for continued optimization of this chemotype. Further investigations revealed that the C-ring is not necessary for activity, although it likely provides conformational constraint that is beneficial to potency, and that the (R) stereochemistry is required at the primary amine. Simplification of the scaffold through deconstruction of the B-ring led to inactive compounds, highlighting the importance of the indole core. A new lead compound 26 was identified, which manifests a ∼30-fold improvement in CpxA phosphatase inhibition over the initial hit. Comparison of amino and des-amino derivatives in bacterial strains differing in membrane permeability and efflux capabilities demonstrate that the amine is required not only for target engagement but also for permeation and accumulation in Escherichia coli