The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein

The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators

Targeting the Essential Transcription Factor HP1043 of Helicobacter pylori: A Drug Repositioning Study

Antibiotic-resistant bacterial pathogens are a very challenging problem nowadays. Helicobacter pylori is one of the most widespread and successful human pathogens since it colonizes half of the world population causing chronic and atrophic gastritis, peptic ulcer, mucosa-associated lymphoid tissue-lymphoma, and even gastric adenocarcinoma. Moreover, it displays resistance to numerous antibiotics. One of the H. pylori pivotal transcription factors, HP1043, plays a fundamental role in regulating essential cellular processes. Like other bacterial transcription factors, HP1043 does not display a eukaryote homolog. These characteristics make HP1043 a promising candidate to develop novel antibacterial strategies. Drug repositioning is a relatively recent strategy employed in drug development; testing approved drugs on new targets considerably reduces the time and cost of this process. The combined computational and in vitro approach further reduces the number of compounds to be tested in vivo. Our aim was to identify a subset of known drugs able to prevent HP1043 binding to DNA promoters. This result was reached through evaluation by molecular docking the binding capacity of about 14,350 molecules on the HP1043 dimer in both conformations, bound and unbound to the DNA. Employing an ad hoc pipeline including MMGBSA molecular dynamics, a selection of seven drugs was obtained. These were tested in vitro by electrophoretic mobility shift assay to evaluate the HP1043–DNA interaction. Among these, three returned promising results showing an appreciable reduction of the DNA-binding activity of HP1043. Overall, we applied a computational methodology coupled with experimental validation of the results to screen a large number of known drugs on one of the H. pylori essential transcription factors. This methodology allowed a rapid reduction of the number of drugs to be tested, and the drug repositioning approach considerably reduced the drug design costs. Identified drugs do not belong to the same pharmaceutical category and, by computational analysis, bound different cavities, but all display a reduction of HP1043 binding activity on the DNA

Helicobacter pylori Stress-Response: Definition of the HrcA Regulon

Bacteria respond to different environmental stresses by reprogramming the transcription of specific genes whose proper expression is critical for their survival. In this regard, the heat-shock response, a widespread protective mechanism, triggers a sudden increase in the cellular concentration of different proteins, including molecular chaperones and proteases, to preserve protein folding and maintain cellular homeostasis. In the medically important gastric pathogen Helicobacter pylori the regulation of the principal heat-shock genes is under the transcriptional control of two repressor proteins named HspR and HrcA. To define the HrcA regulon, we carried out whole transcriptome analysis through RNA-sequencing, comparing the transcriptome of the H. pylori G27 wild type strain to that of the isogenic hrcA-knockout strain. Overall, differential gene expression analysis outlined 49 genes to be deregulated upon hrcA gene inactivation. Interestingly, besides controlling the transcription of genes coding for molecular chaperones and stress-related mediators, HrcA is involved in regulating the expression of proteins whose function is linked to several cellular processes crucial for bacterial survival and virulence. These include cell motility, membrane transporters, Lipopolysaccharide modifiers and adhesins. The role of HrcA as a central regulator of H. pylori transcriptome, as well as its interconnections with the HspR regulon are here analyzed and discussed. As the HrcA protein acts as a pleiotropic regulator, influencing the expression of several stress-unrelated genes, it may be considered a promising target for the design of new antimicrobial strategies

Analisi della trasmissione di SARS-CoV-2: influenza delle condizioni termoigrometriche rispetto al rischio di diffusione del contagio

In questo articolo vengono analizzati i principali meccanismi di diffusione del virus SARS-CoV-2, fornendo elementi utili per la corretta gestione per il contrasto alla diffusione del virus stesso. Lo studio prende le mosse dalla constatazione che, allo stato dell’arte, manca una terminologia rigorosa e univocamente accettata nella letteratura tecnico-scientifica con riferimento alle modalità con cui può avvenire la trasmissione del contagio di SARS-CoV-2 e, in particolare, alle modalità di trasporto cosiddette “droplet” e “airborne”. La corretta analisi dei meccanismi che governano la diffusione del virus non può prescindere da una rigorosa definizione dei termini che entrano in gioco nella descrizione dei fenomeni, da cui discendono importanti indicazioni volte a determinare soluzioni tecniche finalizzate alla minimizzazione del rischio di contagio. Partendo dalla definizione di una terminologia rigorosa ed univoca, attraverso l’applicazione un nuovo modello per lo studio della propagazione del contagio, lo studio, sulla base di valutazioni fisico-matematiche, dà ragione di come il contagio avvenga solo mediante goccioline, sole a poter veicolare efficacemente il virus. Sulla base di queste valutazioni, lo studio analizza il trasporto del virus, incluso nelle goccioline, sulla base dei principi della termo-fluidodinamica, ovvero accoppiando alle equazioni del moto le equazioni che descrivono la variazione di massa per evaporazione. Infatti, la gittata di una goccia emessa da un soggetto infetto, ovvero la distanza di sicurezza da mantenere per minimizzare il rischio di contagio, dipende da numerosi parametri fra i quali, di notevole importanza, il grado igrometrico dell’ambiente in cui questa, la goccia, si trova: da esso, infatti, dipende la velocità di evaporazione e, quindi, la sussistenza della goccia. Pertanto, lo studio dimostra che il controllo del trasporto del contagio non può essere affrontato in assenza di controllo del grado igrometrico dell’ambiente in cui la goccia si muove: infatti, la definizione stessa di distanza di sicurezza perderebbe di significato in quanto la gocciolina potrebbe “sopravvivere” nell’ambiente con elevata umidità, anche per tempi lunghi e realizzando percorsi casuali, qualora le goccioline fossero al di sotto di certe dimensioni. A conclusione dello studio, è riportato un caso di studio che dimostra non solo la correlazione fra grado igrometrico e distanza di sicurezza ma anche che la trasmissione del contagio di tipo airborne, ovvero tramite virioni rilasciati a seguito di evaporazione della goccia, in virtù della loro bassissima concentrazione e del moto di tipo browniano che li caratterizza, abbia una probabilità del tutto trascurabile rispetto alla trasmissione dell’infezione da SARS-CoV-2

The Helicobacter pylori Heat-Shock Repressor HspR: Definition of Its Direct Regulon and Characterization of the Cooperative DNA-Binding Mechanism on Its Own Promoter

The ability of pathogens to perceive environmental conditions and modulate gene expression accordingly is a crucial feature for bacterial survival. In this respect, the heat-shock response, a universal cellular response, allows cells to adapt to hostile environmental conditions and to survive during stress. In the major human pathogen Helicobacter pylori the expression of chaperone-encoding operons is under control of two auto-regulated transcriptional repressors, HrcA and HspR, with the latter acting as the master regulator of the regulatory circuit. To further characterize the HspR regulon in H. pylori, we used global transcriptome analysis (RNA-sequencing) in combination with Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-sequencing) of HspR genomic binding sites. Intriguingly, these analyses showed that HspR is involved in the regulation of different crucial cellular functions through a limited number of genomic binding sites. Moreover, we further characterized HspR-DNA interactions through hydroxyl-radical footprinting assays. This analysis in combination with a nucleotide sequence alignment of HspR binding sites, revealed a peculiar pattern of DNA protection and highlighted sequence conservation with the HAIR motif (an HspR-associated inverted repeat of Streptomyces spp.). Site-directed mutagenesis demonstrated that the HAIR motif is fundamental for HspR binding and that additional nucleotide determinants flanking the HAIR motif are required for complete binding of HspR to its operator sequence spanning over 70 bp of DNA. This finding is compatible with a model in which possibly a dimer of HspR recognizes the HAIR motif overlapping its promoter for binding and in turn cooperatively recruits two additional dimers on both sides of the HAIR motif

One ligand, two regulators and three binding sites: How KDPG controls primary carbon metabolism in Pseudomonas

Effective regulation of primary carbon metabolism is critically important for bacteria to successfully adapt to different environments. We have identified an uncharacterised transcriptional regulator; RccR, that controls this process in response to carbon source availability. Disruption of rccR in the plant-associated microbe Pseudomonas fluorescens inhibits growth in defined media, and compromises its ability to colonise the wheat rhizosphere. Structurally, RccR is almost identical to the Entner-Doudoroff (ED) pathway regulator HexR, and both proteins are controlled by the same ED-intermediate; 2-keto-3-deoxy-6-phosphogluconate (KDPG). Despite these similarities, HexR and RccR control entirely different aspects of primary metabolism, with RccR regulating pyruvate metabolism (aceEF), the glyoxylate shunt (aceA, glcB, pntAA) and gluconeogenesis (pckA, gap). RccR displays complex and unusual regulatory behaviour; switching repression between the pyruvate metabolism and glyoxylate shunt/gluconeogenesis loci depending on the available carbon source. This regulatory complexity is enabled by two distinct pseudo-palindromic binding sites, differing only in the length of their linker regions, with KDPG binding increasing affinity for the 28 bp aceA binding site but decreasing affinity for the 15 bp aceE site. Thus, RccR is able to simultaneously suppress and activate gene expression in response to carbon source availability. Together, the RccR and HexR regulators enable the rapid coordination of multiple aspects of primary carbon metabolism, in response to levels of a single key intermediate

A Novel Phase Variation Mechanism in the Meningococcus Driven by a Ligand-Responsive Repressor and Differential Spacing of Distal Promoter Elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract and contributes to the differential expression levels of phase variant promoters with different numbers of repeats likely due to different spacing between operators. We show that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces NadA expression by inhibiting the DNA binding activity of the repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants and are likely due to differential RNA polymerase contacts leading to altered promoter activity. Our results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, both mediated by the NadR repressor, and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals

The Interplay between Two Transcriptional Repressors and Chaperones Orchestrates Helicobacter pylori Heat-Shock Response

The ability to gauge the surroundings and modulate gene expression accordingly is a crucial feature for the survival bacterial pathogens. In this respect, the heat-shock response, a universally conserved mechanism of protection, allows bacterial cells to adapt rapidly to hostile conditions and to survive during environmental stresses. The important and widespread human pathogen Helicobacter pylori enrolls a collection of highly conserved heat-shock proteins to preserve cellular proteins and to maintain their homeostasis, allowing the pathogen to adapt and survive in the hostile niche of the human stomach. Moreover, various evidences suggest that some chaperones of H. pylori may play also non-canonical roles as, for example, in the interaction with the extracellular environment. In H. pylori, two dedicated transcriptional repressors, named HspR and HrcA, homologues to well-characterized regulators found in many other bacterial species, orchestrate the regulation of heat-shock proteins expression. Following twenty years of intense research, characterized by molecular, as well as genome-wide, approaches, it is nowadays possible to appreciate the complex picture representing the heat-shock regulation in H. pylori. Specifically, the HspR and HrcA repressors combine to control the transcription of target genes in a way that the HrcA regulon results embedded within the HspR regulon. Moreover, an additional level of control of heat-shock genes’ expression is exerted by a posttranscriptional feedback regulatory circuit in which chaperones interact and modulate HspR and HrcA DNA-binding activity. This review recapitulates our understanding of the roles and regulation of the most important heat-shock proteins of H. pylori, which represent a crucial virulence factor for bacterial infection and persistence in the human host