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Cefiderocol (FDC), a siderophore-cephalosporin conjugate, is the newest option for treating infection with carbapenem-resistant gram-negative bacteria. We identified a novel mechanism contributing to decreased FDC susceptibility in Klebsiella pneumoniae clinical isolates. The mechanism involves 2 coresident plasmids: pKpQIL, carrying variants of bla KPC carbapenemase gene, and pKPN, carrying the ferric citrate transport (FEC) system. We observed increasing FDC MICs in an Escherichia coli model system carrying different natural pKpQIL plasmids, encoding different K. pneumoniae carbapenemase (KPC) variants, in combination with a conjugative low copy number vector carrying the fec gene cluster from pKPN. We observed transcriptional repression of fiu, cirA, fepA, and fhuA siderophore receptor genes in bla KPC -fec-E. coli cells treated with ferric citrate. Screening of 27,793 K. pneumoniae whole-genome sequences revealed that the fec cluster occurs frequently in some globally distributed different KPC-producing K. pneumoniae clones (sequence types 258, 14, 45, and 512), contributing to reduced FDC susceptibility.

Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC 50 ) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC 50 : 12.6-305 µM, MIC: 0.5-128 µg ml -1 ). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity., (© 2024. The Author(s).)

Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Shigella, the aetiological agent of human bacillary dysentery, controls the expression of its virulence determinants through an environmentally stimulated cascade of transcriptional activators. VirF is the leading activator and is essential for proper virulence expression. In this work, we report on in vitro and in vivo experiments showing that two autoinducers of the DSF family, XcDSF and BDSF interact with the jelly roll module of VirF causing its inhibition and affecting the expression of the entire virulence system of Shigella, including its ability to invade epithelial cells. We propose a molecular model explaining how the binding of XcDSF and BDSF causes inhibition of VirF by preventing its dimerization. Overall, our experimental results suggest that XcDSF and BDSF may contribute to "colonisation resistance" in the human gut or, alternatively, may be exploited for the fine-tuning of Shigella virulence expression as the bacterium migrates from the lumen to approach the intestinal mucosa. Our findings also stress how a detailed understanding of the interaction of DSF ligands with VirF may contribute to the rational development of innovative antivirulence drugs to treat shigellosis., (© 2023. Springer Nature Limited.)

The pathogenicity of Shigella , the intracellular pathogen responsible for human bacillary dysentery, depends on a coordinated and tightly regulated expression of its virulence determinants. This is the result of a cascade organization of its positive regulators, with VirF, a transcriptional activator belonging to the AraC-XylS family, in a pivotal position. VirF itself is submitted to several well-known regulations at the transcriptional level. In this work, we present evidence for a novel posttranslational regulatory mechanism of VirF mediated by the inhibitory interaction with specific fatty acids. By homology modeling and molecular docking analyses, we identify a jelly roll motif in the structure of ViF capable of interacting with medium-chain saturated and long-chain unsaturated fatty acids. In vitro and in vivo assays show that capric, lauric, myristoleic, palmitoleic, and sapienic acids interact effectively with the VirF protein, abolishing its transcription-promoting activity. This silences the virulence system of Shigella , leading to a drastic reduction in its ability to invade epithelial cells and proliferate in their cytoplasm. IMPORTANCE In the absence of a valid vaccine, the main therapeutic approach currently used to treat shigellosis is based on the use of antibiotics. The emergence of antibiotic resistance jeopardizes the future effectiveness of this approach. The importance of the present work resides both in the identification of a new level of posttranslational regulation of the Shigella virulence system and in the characterization of a mechanism offering new opportunities for the design of antivirulence compounds, which may change the treatment paradigm of Shigella infections by limiting the emergence of antibiotic-resistant bacteria., Competing Interests: The authors declare no conflict of interest.

The tripartite complex AcrAB-TolC is the major RND pump in Escherichia coli and other Enterobacteriaceae, including Shigella , the etiological agent of bacillary dysentery. In addition to conferring resistance to many classes of antibiotics, AcrAB plays a role in the pathogenesis and virulence of several bacterial pathogens. Here, we report data demonstrating that AcrAB specifically contributes to Shigella flexneri invasion of epithelial cells. We found that deletion of both acrA and acrB genes causes reduced survival of S. flexneri M90T strain within Caco-2 epithelial cells and prevents cell-to-cell spread of the bacteria. Infections with single deletion mutant strains indicate that both AcrA and AcrB favor the viability of the intracellular bacteria. Finally, we were able to further confirm the requirement of the AcrB transporter activity for intraepithelial survival by using a specific EP inhibitor. Overall, the data from the present study expand the role of the AcrAB pump to an important human pathogen, such as Shigella , and add insights into the mechanism governing the Shigella infection process., Competing Interests: The authors declare no conflict of interest.

The essential oils from the Centaurea genus are well known for their pharmacological properties. The most abundant and dominant chemical components in Centaurea essential oils are ß-caryophyllene, hexadecanoic acid, spathulenol, pentacosane, caryophyllene oxide, and phytol. However, whether these dominant components are the key drivers for observed antimicrobial activity remains unclear. Thus, the aim of this study was dual. Here we provide comprehensive, literature-based data to correlate the chemical compounds in Centaurea essential oils with the tested antimicrobial activity. Secondly, we characterized the essential oil of Centaurea triumfettii All. squarrose knapweed using coupled system gas chromatography-mass spectrometry and tested its phytochemicals for antimicrobial activity against E. coli and S. epidermis using disc diffusion assay and monitoring their growth in Muller Hinton broth. The most abundant compounds in C. triumfettii essential oil were hexadecanoic acid (11.1%), spathulenol (10.8%), longifolene (8.8%), germacrene D (8.4%), aromadendrene oxide (6.0%) and linoleic acid (5.3%). Based on our analysis of literature data from other Centaurea essential oils, they were positively correlated with antimicrobial activity. Using an agar disk diffusion method, tested chemical constituents did not show experimental evidence to support this positive correlation to antimicrobial activity when we tested them as pure components. The antibacterial effect of essential oil constituents may be related to a complex synergistic, rather than a single component as suggested by performed network pharmacology analysis, underlying the theoretical interactions between the essential oil phytochemicals listed as potentially responsible for antimicrobial activity and should be confirmed in further in-depth studies. This is the first report on the comparative analysis of Centaurea essential oils with good antimicrobial activity, as well as the first analysis of chemical components of the essential oil from C. triumfettii and the first report of antimicrobial activity of the representative, pure components: aromadendrene, germacrene D, spathulenol, longifolene, and the mixture of selected chemical compounds. This work contributes to the body of knowledge on the genus Centaurea and C. triumfettii species., (© 2023. The Author(s).)

Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella , an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.

Shigella spp, the etiological agents of bacillary dysentery in humans, have evolved an intricate regulatory strategy to ensure fine-tuned expression of virulence genes in response to environmental stimuli. A key component in this regulation is VirF, an AraC-like transcription factor, which at the host temperature (37°C) triggers, directly or indirectly, the expression of > 30 virulence genes important for invasion of the intestinal epithelium. Previous work identified two different forms of VirF with distinct functions: VirF30 activates virulence gene expression, while VirF21 appears to negatively regulate virF itself. Moreover, VirF21 originates from either differential translation of the virF mRNA or from a shorter leaderless mRNA (llmRNA). Here we report that both expression of the virF21 llmRNA and the VirF21:VirF30 protein ratio are higher at 30°C than at 37°C, suggesting a possible involvement of VirF21 in minimizing virulence gene expression outside the host (30°C). Ectopic elevation of VirF21 levels at 37°C indeed suppresses Shigella´s ability to infect epithelial cells. Finally, we find that the VirF21 C-terminal portion, predicted to contain a Helix-Turn-Helix motif (HTH2), is required for the functionality of this negative virulence regulator., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Bacterial pathogens are able to survive within diverse habitats. The dynamic adaptation to the surroundings depends on their ability to sense environmental variations and to respond in an appropriate manner. This involves, among others, the activation of various cell-to-cell communication strategies. The capability of the bacterial cells to rapidly and co-ordinately set up an interplay with the host cells and/or with other bacteria facilitates their survival in the new niche. Efflux pumps are ubiquitous transmembrane transporters, able to extrude a large set of different molecules. They are strongly implicated in antibiotic resistance since they are able to efficiently expel most of the clinically relevant antibiotics from the bacterial cytoplasm. Besides antibiotic resistance, multidrug efflux pumps take part in several important processes of bacterial cell physiology, including cell to cell communication, and contribute to increase the virulence potential of several bacterial pathogens. Here, we focus on the structural and functional role of multidrug efflux pumps belonging to the Major Facilitator Superfamily (MFS), the largest family of transporters, highlighting their involvement in the colonization of host cells, in virulence and in biofilm formation. We will offer an overview on how MFS multidrug transporters contribute to bacterial survival, adaptation and pathogenicity through the export of diverse molecules. This will be done by presenting the functions of several relevant MFS multidrug efflux pumps in human life-threatening bacterial pathogens as Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumoniae , Shigella/E. coli, Acinetobacter baumannii., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pasqua, Bonaccorsi di Patti, Fanelli, Utsumi, Eguchi, Trirocco, Prosseda, Grossi and Colonna.)

Outer membrane vesicles (OMVs) are nanostructures mostly produced by blebbing of the outer membrane in Gram negative bacteria. They contain biologically active proteins and perform a variety of processes. OMV production is also a typical response to events inducing stress in the bacterial envelope. In these cases, hypervesiculation is regarded as a strategy to avoid the dangerous accumulation of undesired products within the periplasm. Several housekeeping genes influence the biogenesis of OMVs, including those correlated with peptidoglycan and cell wall dynamics. In this work, we have investigated the relationship between OMV production and the lysis module of the E. coli DLP12 cryptic prophage. This module is an operon encoding a holin, an endolysin and two spannins, and is known to be involved in cell wall maintenance. We find that deleting the lysis module increases OMV production, suggesting that during evolution this operon has been domesticated to regulate vesiculation, likely through the elimination of non-recyclable peptidoglycan fragments. We also show that the expression of the lysis module is negatively regulated by environmental stress stimuli as high osmolarity, low pH and low temperature. Our data further highlight how defective prophages finely contribute to bacterial host fitness.

Efflux pumps (EPs) are present in all living cells and represent a large and important group of transmembrane proteins involved in transport processes. In bacteria, multidrug resistance efflux pumps (MDR EPs) confer resistance to antibiotics at different levels and are deeply implicated in the fast and dramatic emergence of antibiotic resistance. Recently, several reports have outlined the great versatility of MDR EPs in exporting a large variety of compounds other than antibiotics, thus promoting bacterial adaptation to a wide range of habitats. In several bacterial pathogens, MDR EPs contribute to increase the virulence potential and are directly involved in the crosstalk with host cells. In this work, we have investigated the possible role of MDR EPs in the infectious process of the adherent-invasive Escherichia coli (AIEC), a group of pathogenic E. coli that colonize the ileal mucosa of Crohn disease (CD) patients causing a strong intestinal inflammation. The results we have obtained indicate that, with the exception of mdtM , all MDR-EPs encoding genes present in E.coli K12 are conserved in the AIEC prototype strain LF82. The analysis of MDR EP expression during LF82 infection of macrophages and epithelial cells reveals that their transcription is highly modulated during the bacterial intracellular life. Notably, some EP genes are regulated in a cell-type specific manner, strongly suggesting that their function is required for LF82 successful infection. AIEC are able to adhere to and invade intestinal epithelial cells and, importantly, to survive and multiply within macrophages. Thus, we further investigated the role of EPs specifically induced by macrophage environment. We present evidence indicating that deletion of mdtEF genes, encoding an MDR EP belonging to the resistance nodulation division (RND) family, significantly impairs survival of LF82 in macrophages and that the wild type phenotype can be restored by trans-complementation with functional MdtEF pump. Altogether, our results indicate a strong involvement of MDR EPs in host pathogen interaction also in AIEC and highlight the contribution of MdtEF to the fitness of LF82 in the macrophage environment., (Copyright © 2020 Fanelli, Pasqua, Colonna, Prosseda and Grossi.)

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness through the same pathogenic mechanism used by Shigella spp. The latter species can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification within E. coli species. Recently the first appearance of a highly pathogenic EIEC O96:H19 was described in Europe as the causative agent of two large outbreaks that occurred in Italy and in the United Kingdom. In contrast to Shigella spp and to the majority of EIEC strains, EIEC O96:H19 fermented lactose, lacked pathoadaptive mutations, and showed good fitness in extracellular environment, similarly to non-pathogenic E. coli , suggesting they have emerged following acquisition of the invasion plasmid by a non-pathogenic E. coli . Here we describe the whole genome comparison of two EIEC O96:H19 strains isolated from severe cases of diarrhea in Uruguay in 2014 with the sequences of EIEC O96:H19 available in the public domain. The phylogenetic comparison grouped all the O96:H19 strains in a single cluster, while reference EIEC strains branched into different clades with Shigella strains occupying apical positions. The comparison of the virulence plasmids showed the presence of a complete conjugation region in at least one O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed in this strain the expression of the pilin-encoding gene and conjugation experiments suggested its ability to mobilize an accessory plasmid in a recipient strain. Noteworthy, the tra region was comprised between two reversely oriented IS 600 elements, which were also found as remnants in another EIEC O96:H19 plasmid lacking the tra locus . We hypothesize that an IS-mediated recombination mechanism may have caused the loss of the conjugation region commonly observed in EIEC and Shigella virulence plasmids. The results of this study support the hypothesis of EIEC originating from non-pathogenic E. coli through the acquisition of the virulence plasmid via conjugation. Remarkably, this study showed the ability of a circulating EIEC strain to mobilize plasmids through conjugation, suggesting a mechanism for the emergence of novel EIEC clones., (Copyright © 2020 Michelacci, Tozzoli, Arancia, D'Angelo, Boni, Knijn, Prosseda, Greig, Jenkins, Camou, Sirok, Navarro, Schelotto, Varela and Morabito.)

Efflux pumps represent an important and large group of transporter proteins found in all organisms. The importance of efflux pumps resides in their ability to extrude a wide range of antibiotics, resulting in the emergence of multidrug resistance in many bacteria. Besides antibiotics, multidrug efflux pumps can also extrude a large variety of compounds: Bacterial metabolites, plant-produced compounds, quorum-sensing molecules, and virulence factors. This versatility makes efflux pumps relevant players in interactions not only with other bacteria, but also with plant or animal cells. The multidrug efflux pumps belonging to the major facilitator superfamily (MFS) are widely distributed in microbial genomes and exhibit a large spectrum of substrate specificities. Multidrug MFS efflux pumps are present either as single-component transporters or as tripartite complexes. In this review, we will summarize how the multidrug MFS efflux pumps contribute to the interplay between bacteria and targeted host cells, with emphasis on their role in bacterial virulence, in the colonization of plant and animal host cells and in biofilm formation. We will also address the complexity of these interactions in the light of the underlying regulatory networks required for the effective activation of efflux pump genes., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Efflux pumps are membrane protein complexes conserved in all living organisms. Beyond being involved in antibiotic extrusion in several bacteria, efflux pumps are emerging as relevant players in pathogen-host interactions. We have investigated on the possible role of the efflux pump network in Shigella flexneri, the etiological agent of bacillary dysentery. We have found that S. flexneri has retained 14 of the 20 pumps characterized in Escherichia coli and that their expression is differentially modulated during the intracellular life of Shigella. In particular, the emrKY operon, encoding an efflux pump of the Major Facilitator Superfamily, is specifically and highly induced in Shigella-infected U937 macrophage-like cells and is activated in response to a combination of high K + and acidic pH, which are sensed by the EvgS/EvgA two-component system. Notably, we show that following S. flexneri infection, macrophage cytosol undergoes a mild reduction of intracellular pH, permitting EvgA to trigger the emrKY activation. Finally, we present data suggesting that EmrKY is required for the survival of Shigella in the harsh macrophage environment, highlighting for the first time the key role of an efflux pump during the Shigella invasive process.

Among the intestinal pathogenic Escherichia coli , enteroinvasive E. coli (EIEC) are a group of intracellular pathogens able to enter epithelial cells of colon, multiplicate within them, and move between adjacent cells with a mechanism similar to Shigella , the ethiological agent of bacillary dysentery. Despite EIEC belong to the same pathotype of Shigella , they neither have the full set of traits that define Shigella nor have undergone the extensive gene decay observed in Shigella . Molecular analysis confirms that EIEC are widely distributed among E. coli phylogenetic groups and correspond to bioserotypes found in many E. coli serogroups. Like Shigella , also in EIEC the critical event toward a pathogenic life-style consisted in the acquisition by horizontal gene transfer of a large F-type plasmid (pINV) containing the genes required for invasion, intracellular survival, and spreading through the intestinal mucosa. In Shigella , the ample gain in virulence determinants has been counteracted by a substantial loss of functions that, although important for the survival in the environment, are redundant or deleterious for the life inside the host. The pathoadaptation process that has led Shigella to modify its metabolic profile and increase its pathogenic potential is still in infancy in EIEC, although maintenance of some features typical of E. coli might favor their emerging relevance as intestinal pathogens worldwide, as documented by recent outbreaks in industrialized countries. In this review, we will discuss the evolution of EIEC toward Shigella -like invasive forms going through the epidemiology, including the emergence of new virulent strains, their genome organization, and the complex interactions they establish with the host.

Shigella, the etiological agent of bacillary dysentery (shigellosis), is a highly adapted human pathogen. It evolved from an innocuous ancestor resembling the Escherichia coli strain by gain and loss of genes and functions. While the gain process concerns the acquisition of the genetic determinants of virulence, the loss is related to the adaptation of the genome to the new pathogenic status and occurs by pathoadaptive mutation of antivirulence genes. In this study, we highlight that the SRRz/Rz 1 lambdoid lysis cassette, even though stably adopted in E. coli K12 by virtue of its beneficial effect on cell physiology, has undergone a significant decay in Shigella. Moreover, we show the antivirulence nature of the SRRz/Rz 1 lysis cassette in Shigella. In fact, by restoring the SRRz/Rz 1 expression in this pathogen, we observe an increased release of peptidoglycan fragments, causing an unbalance in the fine control exerted by Shigella on host innate immunity and a mitigation of its virulence. This strongly affects the virulence of Shigella and allows to consider the loss of SRRz/Rz 1 lysis cassette as another pathoadaptive event in the life of Shigella., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF 30 (30 kDa), and the shorter VirF 21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF 30 and VirF 21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF 21 is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF 21 The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF 30 is responsible for activation of the virulence system, VirF 21 negatively autoregulates virF expression itself. Since VirF 21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA., Importance: Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF 30 and VirF 21 , that are functionally distinct. The major form, VirF 30 , activates the genes necessary for virulence, whereas the minor VirF 21 , which shares the C-terminal two-thirds of VirF 30 , negatively autoregulates virF expression itself. VirF 21 is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis., (Copyright © 2016 Di Martino et al.)

Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella . In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella . We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

The polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation.

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host-bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic⁻ phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic⁻ phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella., (Copyright © 2013. Published by Elsevier GmbH.)

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.

Stenotrophomonas maltophilia is a microorganism of environmental and clinical importance as well as a frequent airway colonizer of cystic fibrosis (CF) individuals. We combined 2-DE and MALDI-TOF MS to profile the protein expression in S. maltophilia K279a, a completely sequenced clinical isolate, grown at 37 °C with respect to the strain grown at 26 °C. Among the proteins up-regulated at 37 °C, we identified GroEL, a molecular chaperone that mainly assist the folding and unfolding of proteins under both normal and stress conditions. A 2.4-kb groESL mRNA was detected independently by Northern blot analyses with a groES- and a groEL-specific probe, indicating that S. maltophilia groES and groEL form an operon. Primer extension analysis of S. maltophilia groESL done in Escherichia coli showed that 2 promoters, Pσ(32) and Pσ(70), were utilized under the heat-shock and normal condition, respectively, whereas S. maltophilia groEL was shown to act as a heat-shock gene at 37 °C, 42 °C, and, to a lesser extent, at 50 °C by real-time RT-PCR analyses. Finally, immunoblot analyses revealed that S. maltophilia GroEL strongly reacted with sera from CF patients chronically infected by the microorganism, but did not with sera from CF patients with sporadic infection or uninfected., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.