Structural and mechanistic insights into Helicobacter pylori NikR activation

NikR is a transcriptional metalloregulator central in the mandatory response to acidity of Helicobacter pylori that controls the expression of numerous genes by binding to specific promoter regions. NikR/DNA interactions were proposed to rely on protein activation by Ni(II) binding to high-affinity (HA) and possibly secondary external (X) sites. We describe a biochemical characterization of HpNikR mutants that shows that the HA sites are essential but not sufficient for DNA binding, while the secondary external (X) sites and residues from the HpNikR dimer–dimer interface are important for DNA binding. We show that a second metal is necessary for HpNikR/DNA binding, but only to some promoters. Small-angle X-ray scattering shows that HpNikR adopts a defined conformation in solution, resembling the cis-conformation and suggests that nickel does not trigger large conformational changes in HpNikR. The crystal structures of selected mutants identify the effects of each mutation on HpNikR structure. This study unravels key structural features from which we derive a model for HpNikR activation where: (i) HA sites and an hydrogen bond network are required for DNA binding and (ii) metallation of a unique secondary external site (X) modulates HpNikR DNA binding to low-affinity promoters by disruption of a salt bridge

Opposite Effects of HIV-1 p17 Variants on PTEN Activation and Cell Growth in B Cells

The HIV-1 matrix protein p17 is a structural protein that can act in the extracellular environment to deregulate several functions of immune cells, through the interaction of its NH2-terminal region with a cellular surface receptor (p17R). The intracellular events triggered by p17/p17R interaction have been not completely characterized yet. In this study we analyze the signal transduction pathways induced by p17/p17R interaction and show that in Raji cells, a human B cell line stably expressing p17R on its surface, p17 induces a transient activation of the transcriptional factor AP-1. Moreover, it was found to upregulate pERK1/2 and downregulate pAkt, which are the major intracellular signalling components involved in AP-1 activation. These effects are mediated by the COOH-terminal region of p17, which displays the capability of keeping PTEN, a phosphatase that regulates the PI3K/Akt pathway, in an active state through the serin/threonin (Ser/Thr) kinase ROCK. Indeed, the COOH-terminal truncated form of p17 (p17Δ36) induced activation of the PI3K/Akt pathway by maintaining PTEN in an inactive phosphorylated form. Interestingly, we show that among different p17s, a variant derived from a Ugandan HIV-1 strain, named S75X, triggers an activation of PI3K/Akt signalling pathway, and leads to an increased B cell proliferation and malignant transformation. In summary, this study shows the role of the COOH-terminal region in modulating the p17 signalling pathways so highlighting the complexity of p17 binding to and signalling through its receptor(s). Moreover, it provides the first evidence on the presence of a p17 natural variant mimicking the p17Δ36-induced signalling in B cells and displaying the capacity of promoting B cell growth and tumorigenesis

Differential Effects of Antibiotic Therapy on the Structure and Function of Human Gut Microbiota

The human intestinal microbiota performs many essential functions for the host. Antimicrobial agents, such as antibiotics (AB), are also known to disturb microbial community equilibrium, thereby having an impact on human physiology. While an increasing number of studies investigate the effects of AB usage on changes in human gut microbiota biodiversity, its functional effects are still poorly understood. We performed a follow-up study to explore the effect of ABs with different modes of action on human gut microbiota composition and function. Four individuals were treated with different antibiotics and samples were taken before, during and after the AB course for all of them. Changes in the total and in the active (growing) microbiota as well as the functional changes were addressed by 16S rRNA gene and metagenomic 454-based pyrosequencing approaches. We have found that the class of antibiotic, particularly its antimicrobial effect and mode of action, played an important role in modulating the gut microbiota composition and function. Furthermore, analysis of the resistome suggested that oscillatory dynamics are not only due to antibiotic-target resistance, but also to fluctuations in the surviving bacterial community. Our results indicated that the effect of AB on the human gut microbiota relates to the interaction of several factors, principally the properties of the antimicrobial agent, and the structure, functions and resistance genes of the microbial community

Trans-Translation in Helicobacter pylori: Essentiality of Ribosome Rescue and Requirement of Protein Tagging for Stress Resistance and Competence

BACKGROUND: The ubiquitous bacterial trans-translation is one of the most studied quality control mechanisms. Trans-translation requires two specific factors, a small RNA SsrA (tmRNA) and a protein co-factor SmpB, to promote the release of ribosomes stalled on defective mRNAs and to add a specific tag sequence to aberrant polypeptides to direct them to degradation pathways. Helicobacter pylori is a pathogen persistently colonizing a hostile niche, the stomach of humans. PRINCIPAL FINDINGS: We investigated the role of trans-translation in this bacterium well fitted to resist stressful conditions and found that both smpB and ssrA were essential genes. Five mutant versions of ssrA were generated in H. pylori in order to investigate the function of trans-translation in this organism. Mutation of the resume codon that allows the switch of template of the ribosome required for its release was essential in vivo, however a mutant in which this codon was followed by stop codons interrupting the tag sequence was viable. Therefore one round of translation is sufficient to promote the rescue of stalled ribosomes. A mutant expressing a truncated SsrA tag was viable in H. pylori, but affected in competence and tolerance to both oxidative and antibiotic stresses. This demonstrates that control of protein degradation through trans-translation is by itself central in the management of stress conditions and of competence and supports a regulatory role of trans-translation-dependent protein tagging. In addition, the expression of smpB and ssrA was found to be induced upon acid exposure of H. pylori. CONCLUSIONS: We conclude to a central role of trans-translation in H. pylori both for ribosome rescue possibly due to more severe stalling and for protein degradation to recover from stress conditions frequently encountered in the gastric environment. Finally, the essential trans-translation machinery of H. pylori is an excellent specific target for the development of novel antibiotics

Small Molecule Control of Virulence Gene Expression in Francisella tularensis

In Francisella tularensis, the SspA protein family members MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes critical for the intramacrophage growth and survival of the organism. Although the association of the MglA-SspA complex with RNAP is evidently central to its role in controlling gene expression, the molecular details of how MglA and SspA exert their effects are not known. Here we show that in the live vaccine strain of F. tularensis (LVS), the MglA-SspA complex works in concert with a putative DNA-binding protein we have called PigR, together with the alarmone guanosine tetraphosphate (ppGpp), to regulate the expression of target genes. In particular, we present evidence that MglA, SspA, PigR and ppGpp regulate expression of the same set of genes, and show that mglA, sspA, pigR and ppGpp null mutants exhibit similar intramacrophage growth defects and are strongly attenuated for virulence in mice. We show further that PigR interacts directly with the MglA-SspA complex, suggesting that the central role of the MglA and SspA proteins in the control of virulence gene expression is to serve as a target for a transcription activator. Finally, we present evidence that ppGpp exerts its effects by promoting the interaction between PigR and the RNAP-associated MglA-SspA complex. Through its responsiveness to ppGpp, the contact between PigR and the MglA-SspA complex allows the integration of nutritional cues into the regulatory network governing virulence gene expression

Achieving a cure for HIV infection: do we have reasons to be optimistic?

The introduction of highly active antiretroviral therapy (HAART) in 1996 has transformed a lethal disease to a chronic pathology with a dramatic decrease in mortality and morbidity of AIDS-related symptoms in infected patients. However, HAART has not allowed the cure of HIV infection, the main obstacle to HIV eradication being the existence of quiescent reservoirs. Several other problems have been encountered with HAART (such as side effects, adherence to medication, emergence of resistance and cost of treatment), and these motivate the search for new ways to treat these patients. Recent advances hold promise for the ultimate cure of HIV infection, which is the topic of this review. Besides these new strategies aiming to eliminate the virus, efforts must be made to improve current HAART. We believe that the cure of HIV infection will not be attained in the short term and that a strategy based on purging the reservoirs has to be associated with an aggressive HAART strategy

Built Shallow to Maintain Homeostasis and Persistent Infection: Insight into the Transcriptional Regulatory Network of the Gastric Human Pathogen Helicobacter pylori

Transcriptional regulatory networks (TRNs) transduce environmental signals into coordinated output expression of the genome. Accordingly, they are central for the adaptation of bacteria to their living environments and in host–pathogen interactions. Few attempts have been made to describe a TRN for a human pathogen, because even in model organisms, such as Escherichia coli, the analysis is hindered by the large number of transcription factors involved. In light of the paucity of regulators, the gastric human pathogen Helicobacter pylori represents a very appealing system for understanding how bacterial TRNs are wired up to support infection in the host. Herein, we review and analyze the available molecular and “-omic” data in a coherent ensemble, including protein–DNA and protein–protein interactions relevant for transcriptional control of pathogenic responses. The analysis covers ∼80% of the annotated H. pylori regulators, and provides to our knowledge the first in-depth description of a TRN for an important pathogen. The emerging picture indicates a shallow TRN, made of four main modules (origons) that process the physiological responses needed to colonize the gastric niche. Specific network motifs confer distinct transcriptional response dynamics to the TRN, while long regulatory cascades are absent. Rather than having a plethora of specialized regulators, the TRN of H. pylori appears to transduce separate environmental inputs by using different combinations of a small set of regulators

The therapeutic potential of epigenetic manipulation during infectious diseases.

Epigenetic modifications are increasingly recognized as playing an important role in the pathogenesis of infectious diseases. They represent a critical mechanism regulating transcriptional profiles in the immune system that contributes to the cell-type and stimulus specificity of the transcriptional response. Recent data highlight how epigenetic changes impact macrophage functional responses and polarization, influencing the innate immune system through macrophage tolerance and training. In this review we will explore how post-translational modifications of histone tails influence immune function to specific infectious diseases. We will describe how these may influence outcome, highlighting examples derived from responses to acute bacterial pathogens, models of sepsis, maintenance of viral latency and HIV infection. We will discuss how emerging classes of pharmacological agents, developed for use in oncology and other settings, have been applied to models of infectious diseases and their potential to modulate key aspects of the immune response to bacterial infection and HIV therapy