Gene regulatory network inference and analysis of multidrug-resistant Pseudomonas aeruginosa

BACKGROUND Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.

Helper T-cell identity and evolution of differential transcriptomes and epigenomes.

CD4(+) T cells are critical for the elimination of an immense array of microbial pathogens. Among the ways they accomplish this task is to generate progeny with specialized, characteristic patterns of gene expression. From this perspective, helper cells can be viewed as pluripotent precursors that adopt distinct cell fates. Although there are aspects of helper cell differentiation that can be modeled as a classic cell fate commitment, CD4(+) T cells also maintain considerable flexibility in their transcriptional program. This makes sense in terms of host defense, but raises the question of how these remarkable cells balance both these requirements, a high degree of specific gene expression and the capacity for plasticity. In this review, we discuss recent advances in our understanding of CD4(+) T-cell specification, focusing on how genomic perspectives have influenced our views of these processes. The relative contributions of sensors of the cytokine milieu, especially the signal transducer and activator of transcription family transcription factors, 'master regulators', and other transcription factors are considered as they relate to the helper cell transcriptome and epigenome.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy: known and novel aspects of the syndrome.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a monogenic autosomal recessive disease caused by mutations in the autoimmune regulator (AIRE) gene and, as a syndrome, is characterized by chronic mucocutaneous candidiasis and the presentation of various autoimmune diseases. During the last decade, research on APECED and AIRE has provided immunologists with several invaluable lessons regarding tolerance and autoimmunity. This review describes the clinical and immunological features of APECED and discusses emerging alternative models to explain the pathogenesis of the disease.

Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis.

We have identified a transcriptional regulator, named Ers (for enterococcal regulator of survival), of Enterococcus faecalis, an important opportunistic bacterium commonly recovered from hospitalized patients. Ers is a member of the Crp/Fnr family and is 69% similar to Srv, a PrfA-like regulator of Streptococcus pyogenes implicated in virulence, and is the E. faecalis protein most closely related to PrfA, a positive regulator of virulence genes in Listeria monocytogenes. In an in vivo-in vitro macrophage infection model, the survival of an ers mutant was highly significantly decreased compared with that of the parental strain JH2-2. This mutant was more than 10-fold more sensitive to oxidative challenge by hydrogen peroxide. In order to identify genes whose expression was under Ers control, the RNA levels of 31 likely candidates were measured by real-time quantitative PCR. The results indicate that ers may be autoregulated and that the locus ef0082 appears to be positively regulated by Ers. Nevertheless, mutation of ef0082 did not result in any detectable changes in the survival of the bacterium within murine macrophages.

Signaling and transcriptional control of Fas ligand gene expression.

Fas ligand (FasL), a member of the tumor necrosis factor family, initiates apoptosis by binding to its surface receptor Fas. As a consequence, there is sequential activation of caspases and the release of cytochrome c from the mitochondria, with additional caspase activation followed by cellular degradation and death. Recent studies have shed important insight into the molecular mechanisms controlling FasL gene expression at the level of transcription. Nuclear factors such as nuclear factor in activated T cells, nuclear factor-kappa B, specificity protein-1, early growth response factor, interferon regulatory factor, c-Myc and the forkhead transcriptional regulator, alone or cooperatively, activate FasL expression. These factors are often coexpressed with FasL in pathophysiologic settings including human atherosclerotic lesions. Here, we review these important advances in our understanding of the signaling and transcriptional mechanisms controlling FasL gene expression.

The role of Ras in T lymphocyte activation.

Ras plays an important role in T cell signal transduction through multiple pathways. Here, we demonstrate that, upon stimulation, increasing Ras activity partially substitutes for calcium-mediated signals leading to IL-2 induction. The increase of Ras activity renders Jurkat cells the resistant to cyclosporin A (CsA) through increasing calcineurin activity. Coincidentally, the inducible binding of NIL-2 to a negative-regulatory element in the IL-2 promoter becomes less sensitive to CsA in the cells with increasing Ras activity. The dose of CsA required for inhibition of IL-2 induction in the cells with increased Ras activity remains similar to the concentration of CsA needed for the suppression of NFAT activation in control cells. The results suggest that Ras regulates calcium/calcineurin signalling during T cell activation and the existence of new immune-related target(s) for CsA action at the posttranscriptional level.

Human natural killer cells in asymptomatic human immunodeficiency virus-1 infection.

OBJECTIVES: We evaluated whether DNA immunization with HIV-1 regulatory genes change natural killer (NK) effector cell activity. NK cells are the most important cells for the immediate host defense against virus-infected and tumor cells. We analyzed the NK activities of HIV-1-infected individuals against K562 cells (the classical assay) as well as against a CD4+ cell line with and without HIV-1 infection. CD4+ T lymphocytes are the main target cells for HIV-1 infection in vivo. Various proportions of the CD4+ T lymphocyte population carry the HIV-1 genome, produce virus and contribute to the systemic spread of HIV-1. METHODS: CD4+ cell lines were established through HTLV-1 transformation which made the cells susceptible to NK lysis. NK activity was then tested in a (51)Cr release assay. RESULTS: NK cells of asymptomatic HIV-infected individuals mediated considerable lytic activity against K562 cells as well as against the uninfected and HIV-1-infected CD4+ cell line, and so did the NK cells of HIV-1-infected patients on highly active antiretroviral treatment. CONCLUSION: DNA immunization with HIV-1-regulatory genes did not significantly change the NK effector cell activity.

Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients.

BACKGROUND: DNA vaccination is known to generate immune responses against HIV-1 in animal models. We aimed to assess the efficacy of DNA vaccination in induction of immune responses in HIV-1-infected human beings. METHODS: Nine symptom-free HIV-1-infected patients were immunised with DNA constructs encoding the nef, rev, or tat regulatory genes of HIV-1. The patients were selected for having no or low antibody reactivities to these antigens. HIV-1-specific cytotoxic T-lymphocytes (CTLs), precursor frequencies, and antigen-specific proliferative responses were measured before, during, and after three immunisations over 6 months. FINDINGS: Cellular immune reactivities against the HIV-1 regulatory proteins were absent or low before DNA immunisation. DNA vaccination induced detectable memory cells in all patients and specific cytotoxicity in eight patients. CTLs were MHC-class-I restricted and mainly of CD8+ origin. In three patients the cellular activity was transient, decreasing after an initial response. INTERPRETATION: DNA immunisation with HIV-1 genes can induce specific cellular responses in human beings with no apparent side-effects. It is theoretically possible that HIV-1-specific cytotoxic responses to regulatory proteins could lead to infected cells being eliminated before they have released new viral particles. However, it is possible that the patients we selected responded less than would non-selected or non-infected individuals. The small number of patients presented here does not allow generalisation of our findings.

Nucleic acid vaccination with HIV regulatory genes: a combination of HIV-1 genes in separate plasmids induces strong immune responses.

The concept of combining several genes in order to immunize against a microbial agent has been tested. We selected human immunodeficiency virus (HIV) genes that individually have been shown to mediate immune responses against HIV proteins. These proteins were the regulating genes/proteins of HIV-1 rev, tat and nef as well as structural genes for gp160 under the control of rev, and the capsid p24 represented by the larger precursor gene p37. Two findings were of particular interest. The combination of these five gene constructs gave strong reactivity to all of them, compared with previous results using each one in single injections. The intranasal immunization route gave good mucosal reactivity by inducing IgG, IgA and T-cell proliferative responses.

No association or linkage to IDDM of an interferon regulatory factor-1 gene polymorphism in a Danish population. The Danish Study Group for Diabetes in Childhood.

Nitric oxide is a potent mediator of the cytokine-induced cytotoxic effect on pancreatic beta cells. It has been shown that the inducible nitric oxide synthase (iNOS) is induced in islets of Langerhans by interleukin-1 beta (IL-1 beta). Interferon regulatory factor-1 (IRF-1), a transcriptional factor known to play an essential role in the induction of the inducible nitric oxide synthase, has also been shown to be induced by IL-1 beta in isolated islets of Langerhans. In the present study we analysed a GT nucleotide repeat polymorphism in the intron 7 of the IRF-1 gene. We typed 123 Danish Caucasian insulin-dependent diabetes mellitus (IDDM) multiplex families (550 individuals including 271 diabetic patients) and 108 control subjects of Danish Caucasian origin. In total, seven alleles were identified. No significant differences in either allele or genotype distribution were found comparing IDDM patients with control subjects (P = 0.7 and P = 0.5, respectively). An extended transmission disequilibrium test (ETDT) did not reveal transmission disequilibrium in an allele-wise manner. A 16-nucleotide deletion was found when sequencing the region containing the polymorphic GT repeat. This new deletion was in linkage disequilibrium with the GT-repeat polymorphism, as it was only seen with alleles of more than 13 GT tandem repeats. No association with IDDM for the deletion was observed. Furthermore, three single base substitutions linked to the 16 nucleotide deletion were identified. Even though we could not associate the GT-repeat polymorphism to IDDM in this study, additional mutation screening is warranted, as we still think the IRF-1 gene is a potential candidate gene for IDDM.

Identification and characterization of a human CD4 silencer.

Using transgenic mice, we have identified a human CD4 silencer contained within a 484-bp fragment in the first intron of the CD4 gene. Further experiments have mapped a lineage-specific silencing activity to a region of 190 bp. This region contains two protein-binding sites detected by deoxyribonuclease I footprinting analyses. Tested in transient transfection assays, these two DNA elements showed significant silencing activity restricted to the CD8 phenotype. In CD4 cells, either no clear effect (FP I) or strong enhancing activity (FP II) was observed by transient transfection assays. Despite the lineage-specific activity of these two elements, electrophoretic mobility shift assays (EMSA) showed similar levels of protein binding to the silencer element FP I in CD4 and CD8 T cells. Base substitutions in the FP I fragment abolished the silencing activity in transfected CD8 cells as well as the protein binding in EMSA, suggesting an important role of this protein-DNA interaction in CD4 gene regulation.

[The phenotypic expression of the genes controlling the yield of a factor suppressing macrophage migration to Candida albicans antigens and tuberculin in mice]. / Fenotipicheskaia ékspressiia genov, kontroliruiushchikh vyrabotku faktora, ugnetaiushchego migratsiiu makrofagov na antigeny Candida albicans i tuberkulin u myshei.

The regulation of the synthesis of factor inhibiting the migration of macrophages in response to C. albicans antigen in CBA (H-2k) and C57BL/6 (H-2b) mice has been studied. The low level of macrophage migration inhibition factor in response to this antigen is due to the existence of cyclophosphamide-inhibited specific suppressors. Differences between various strains of mice ensue from different activity of suppressors of thymic origin, whose nature has been revealed as the result of the transfer of marrow cells treated with anti-Thy-1 serum.