The Multifaceted Activity of the VirF Regulatory Protein in the Shigella Lifestyle

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

Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose negative Escherichia coli strains of serotype O4 isolated from children with dhiarrea

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin

The Varied Role of Efflux Pumps of the MFS Family in the Interplay of Bacteria with Animal and Plant Cells

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

Fatty Acids Abolish Shigella Virulence by Inhibiting Its Master Regulator, VirF

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

Role of the MDR Efflux Pump AcrAB in Epithelial Cell Invasion by Shigella flexneri

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

Data for action: The Family and Community Safety for Aboriginal and Torres Strait Islander peoples (FaCtS) Study

Purpose: The purpose of this paper is to describe the development, methodology, methods and final data resource for the Family and Community Safety for Aboriginal and Torres Strait Islander Peoples (FaCtS) Study. Improving family and community safety is a priority for Aboriginal and Torres Strait Islander communities and organisations, and governments, but to date there has been insufficient appropriate evidence to underpin action. The FaCtS Study aims to improve understanding of family and community safety and violence in Aboriginal and Torres Strait Islander communities, using mixed methods, Community-based Action Research. Methods: The FaCtS Study is an Aboriginal and Torres Strait Islander-led and governed study, founded on principles of participation and collaboration. Key components included self-nomination of partner communities, establishment of study governance structures, co development of data collection tools, locally-led data collection and collaborative decision-making around data ownership and use. The FaCtS Study is a mixed-method study in which Community Researchers were trained to undertake quantitative and qualitative research in communities, supported by the Study team. Primary data collection components included quantitative surveys and qualitative interviews and focus groups with community members and service provider staff, and identification (mapping) of services relevant to family and community violence operating in the communities. Results: Eighteen communities covering very remote, remote, regional, and urban areas in Australia self-nominated to take part in the Study. A training resource was developed, and over 30 Community Researchers were trained across participating communities. A total of 1584 eligible community member surveys, 98 service provider surveys, 56 community focus groups, 49 community member interviews, and 41 service provider interviews are available for analysis. Community-specific data were provided to each of the 18 communities and are being, or can be, used to inform local planning and/or advocacy. Conclusions: The FaCtS Study data resource provides valuable insight to inform effective community, policy, and service responses to support family and community safety and to improve service provision for those exposed to or involved in violence. It also serves as an exemplar of ethical research, demonstrating the application of Community-based Action Research principles, reciprocity, and local data ownership. To maximise the benefit that can come from the Study, confidentialised data will be available to communities, academics, services, and government agencies for approved research purposes under Indigenous data governance arrangements.The FaCtS Study was funded by the Australian Government Department of Social Services

A novel antisense RNA regulates at transcriptional level the virulence gene icsA of Shigella flexneri

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

The Triggering Receptor Expressed on Myeloid Cells 2 Inhibits Complement Component 1q Effector Mechanisms and Exerts Detrimental Effects during Pneumococcal Pneumonia

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2(-/-) AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2(-/-) mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs

Changing geographical patterns and trends in cancer incidence in children and adolescents in Europe, 1991–2010 (Automated Childhood Cancer Information System): a population-based study

Background: A deceleration in the increase in cancer incidence in children and adolescents has been reported in several national and regional studies in Europe. Based on a large database representing 1·3 billion person-years over the period 1991–2010, we provide a consolidated report on cancer incidence trends at ages 0–19 years. Methods: We invited all population-based cancer registries operating in European countries to participate in this population-based registry study. We requested a listing of individual records of cancer cases, including sex, age, date of birth, date of cancer diagnosis, tumour sequence number, primary site, morphology, behaviour, and the most valid basis of diagnosis. We also requested population counts in each calendar year by sex and age for the registration area, from official national sources, and specific information about the covered area and registration practices. An eligible registry could become a contributor if it provided quality data for all complete calendar years in the period 1991–2010. Incidence rates and the average annual percentage change with 95% CIs were reported for all cancers and major diagnostic groups, by region and overall, separately for children (age 0–14 years) and adolescents (age 15–19 years). We examined and quantified the stability of the trends with joinpoint analyses. Findings: For the years 1991–2010, 53 registries in 19 countries contributed a total of 180 335 unique cases. We excluded 15 162 (8·4%) of 180 335 cases due to differing practices of registration, and considered the quality indicators for the 165 173 cases included to be satisfactory. The average annual age-standardised incidence was 137·5 (95% CI 136·7–138·3) per million person-years and incidence increased significantly by 0·54% (0·44–0·65) per year in children (age 0–14 years) with no change in trend. In adolescents, the combined European incidence was 176·2 (174·4–178·0) per million person-years based on all 35 138 eligible cases and increased significantly by 0·96% (0·73–1·19) per year, although recent changes in rates among adolescents suggest a deceleration in this increasing trend. We observed temporal variations in trends by age group, geographical region, and diagnostic group. The combined age-standardised incidence of leukaemia based on 48 458 cases in children was 46·9 (46·5–47·3) per million person-years and increased significantly by 0·66% (0·48–0·84) per year. The average overall incidence of leukaemia in adolescents was 23·6 (22·9–24·3) per million person-years, based on 4702 cases, and the average annual change was 0·93% (0·49–1·37). We also observed increasing incidence of lymphoma in adolescents (average annual change 1·04% [0·65–1·44], malignant CNS tumours in children (average annual change 0·49% [0·20–0·77]), and other tumours in both children (average annual change 0·56 [0·40–0·72]) and adolescents (average annual change 1·17 [0·82–1·53]). Interpretation: Improvements in the diagnosis and registration of cancers over time could partly explain the observed increase in incidence, although some changes in underlying putative risk factors cannot be excluded. Cancer incidence trends in this young population require continued monitoring at an international level. Funding: Federal Ministry of Health of the Federal German Government, the European Union's Seventh Framework Programme, and International Agency for Research on Cancer