Group B Streptococcus transcriptome when interacting with brain endothelial cells.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY, encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text]codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators. IMPORTANCE: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin.

BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization.

BACKGROUND: Lipoprotein(a) [Lp(a)] level variability, related to atherothrombotic risk increase, is mainly attributed to LPA gene, encoding apolipoprotein(a), with kringle IV type 2 (KIV2) copy number variation (CNV) acting as the primary genetic determinant. Genetic characterization of Lp(a) is in continuous growth; nevertheless, the peculiar structural characteristics of this variant constitute a significant challenge to the development of effective detection methods. The aim of the study was to compare quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR) in the evaluation of KIV2 repeat polymorphism. METHODS: We analysed 100 subjects tested for cardiovascular risk in which Lp(a) plasma levels were assessed. RESULTS: Correlation analysis between CNV values obtained with the two methods was slightly significant (R = 0.413, p = 0.00002), because of the wider data dispersion in qPCR compared with ddPCR. Internal controls C1, C2 and C3 measurements throughout different experimental sessions revealed the superior stability of ddPCR, which was supported by a reduced intra/inter-assay coefficient of variation determined in this method compared to qPCR. A significant inverse correlation between Lp(a) levels and CNV values was confirmed for both techniques, but it was higher when evaluated by ddPCR than qPCR (R = -0.393, p = 0.000053 vs R = -0.220, p = 0.028, respectively). When dividing subjects into two groups according to 500 mg/L Lp(a) cut-off value, a significantly lower number of KIV2 repeats emerged among subjects with greater Lp(a) levels, with stronger evidence in ddPCR than in qPCR (p = 0.000013 and p = 0.001, respectively). CONCLUSIONS: Data obtained support a better performance of ddPCR in the evaluation of KIV2 repeat polymorphism.

Associations Between Dietary Patterns and Kidney Health Assessed in the Population-Based CHRIS Study Using Reduced Rank Regression.

OBJECTIVE: While diet plays a key role in chronic kidney disease (CKD) management, the potential for diet to impact CKD prevention in the general population is less clear. Using a priori knowledge, we derived disease-related dietary patterns (DPs) through reduced rank regression (RRR) and investigated associations with kidney function, separately focusing on generally healthy individuals and those with self-reported kidney diseases, hypertension, or diabetes mellitus. METHODS: Eight thousand six hundred eighty-six participants from the population-based Cooperative Health Research in South Tyrol study were split into a group free of kidney disease, hypertension and diabetes (n = 6,133) and a group with any of the 3 conditions (n = 2,553). Diet was assessed through the self-administered Global Allergy and Asthma Network of Excellence food frequency questionnaire and DPs were derived through RRR selecting food frequency questionnaire-derived sodium, potassium, phosphorus, and protein intake as mediators. Outcomes were creatinine-based estimated glomerular filtration rate, urinary albumin-to-creatinine ratio, CKD and microalbuminuria. Multiple linear and logistic models were used to assess associations between RRR-based DPs and kidney outcomes separately in the 2 analytic groups. RESULTS: We identified 3 DPs, where high adherence reflected high levels of all nutrients (DP1), high potassium-phosphorus and low protein-sodium levels (DP2), and low potassium-sodium and high protein-phosphorus levels (DP3), respectively. We observed heterogeneous associations with kidney outcomes, varying by analytic group and sex. Kidney outcomes were much more strongly associated with DPs than with single nutrients. CONCLUSION: RRR is a feasible approach to estimate disease-related DPs and explore the combined effects of nutrients on kidney health. Heterogeneous associations across kidney outcomes suggest possible specificity to kidney function or damage. In individuals reporting kidney disease, hypertension or diabetes, specific dietary habits were associated with better kidney health, indicating that disease-specific dietary interventions can be effective for disease control.

Perfluoroalkyl substances and thyroid stimulating hormone levels in a highly exposed population in the Veneto Region.

BACKGROUND: Per- and poly-fluoroalkyl substances (PFAS) are persistent and widespread environmental pollutants. People living in Veneto Region (Italy) have been exposed from the late 1970s to 2013 to elevated concentrations of PFAS through drinking water. The effect of PFAS on thyroid function is still controversial and studies focusing on thyroid stimulating hormone (TSH) have shown inconsistent results. The aim of this study was to evaluate the association between serum PFAS and TSH levels and its dose-response relationship in a large population of highly exposed individuals. METHODS: A cross-sectional study was conducted on 21,424 individuals aged 14-39 living in the contaminated area. In the main analysis, participants with prevalent thyroid disease and pregnant women were excluded. Serum levels of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) were measured. Generalized Additive Models were used to evaluate the association between TSH levels and serum PFAS, using thin plate spline smooth terms to model the potential non-linear relationship. Models were stratified by sex and age group and adjusted for potential confounders. A secondary analysis was conducted to evaluate the association between PFAS with prevalent self-reported thyroid disorders. RESULTS: We found no association between TSH and any type of PFAS among adolescents or women. A decrease in TSH concentration was observed in association with an IQR increase in PFHxS and a mild decrease in TSH at low levels of PFOA, PFOS and PFHxS among male adults. Self-reported thyroid disease was more common among women with higher levels of PFNA concentrations, whereas all other PFAS were not associated with thyroid diseases regardless of sex or age. CONCLUSIONS: Overall there is no evidence of an association between TSH and PFAS. However, some results are suggestive of a possible inverse association of TSH with PFOA, PFOS and PFHxS among adult males.

Methodological tools to study species of the genus Burkholderia.

Bacteria belonging to the Burkholderia genus are extremely versatile and diverse. They can be environmental isolates, opportunistic pathogens in cystic fibrosis, immunocompromised or chronic granulomatous disease patients, or cause disease in healthy people (e.g., Burkholderia pseudomallei) or animals (as in the case of Burkholderia mallei). Since the genus was separated from the Pseudomonas one in the 1990s, the methodological tools to study and characterize these bacteria are evolving fast. Here we reviewed the techniques used in the last few years to update the taxonomy of the genus, to study gene functions and regulations, to deepen the knowledge on the drug resistance which characterizes these bacteria, and to elucidate their mechanisms to establish infections. The availability of these tools significantly impacts the quality of research on Burkholderia and the choice of the most appropriated is fundamental for a precise characterization of the species of interest.Key points• Updated techniques to study the genus Burkholderia were reviewed.• Taxonomy, genomics, assays, and animal models were described.• A comprehensive overview on recent advances in Burkholderia studies was made.

The association between perfluoroalkyl substances and lipid profile in exposed pregnant women in the Veneto region, Italy.

BACKGROUND: Residents of a large area of North-Eastern Italy were exposed for decades to high concentrations of perfluoroalkyl and polyfluoroalkyl substances (PFAS) via drinking water. Serum PFAS levels have been consistently associated with elevated serum lipids, but few studies have been conducted among pregnant women, and none has stratified analyses by trimester of gestation. Elevated serum lipid levels during pregnancy can have both immediate and long-lasting effects on pregnant women and the developing fetus. We evaluated the association between perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and perfluoro-hexanesulfonate (PFHxS) levels in relation to lipid profiles in highly-exposed pregnant women. METHODS: A cross-sectional analysis was conducted in 319 pregnant women (age 14-48 years) enrolled in the Regional health surveillance program. Non-fasting blood samples were obtained in any trimester of pregnancy and analyzed for PFOA, PFOS and PFHxS, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C). Low-density lipoprotein cholesterol (LDL-C) was calculated. The associations between ln-transformed PFAS (and categorized into quartiles) and lipids were assessed using generalized additive models. Analyses were adjusted for potential confounders and stratified according to pregnancy trimester. RESULTS: The geometric means of PFOA, PFOS and PFHxS were 14.78 ng/mL, 2.67 ng/mL and 1.89 ng/mL, respectively. The plasma levels of TC, HDL-C and LDL-C increased steadily throughout the trimesters. In the 1st trimester, PFOS was positively associated with TC and PFHxS with HDL-C. In the 3rd trimester, instead, an inverse relationship was seen between PFOA and PFHxS and both TC and LDL-C. CONCLUSIONS: Results suggest the associations between PFAS concentrations and lipid profiles in pregnant women might differ by trimesters of pregnancy. In the first trimester, patterns are similar to those of non-pregnant women, while they differ late in pregnancy. Different independent behavior of PFAS and lipid levels throughout the pregnancy might explain our observations. These findings support the ubiquitous exposure to PFAS and possible influence on lipid metabolisms during pregnancy and suggest a careful evaluation of the timing of PFAS measurement, when examining effects of PFAS during pregnancy on gestational outcomes related to serum lipids amounts.

Perfluoroalkyl substances are associated with elevated blood pressure and hypertension in highly exposed young adults.

BACKGROUND: Residents in a large area of North-Eastern Italy were exposed to perfluoroalkyl substances (PFAS) via drinking water. Studies on the association between PFAS and blood pressure levels are limited, and results are inconsistent. Using cross-sectional data from the Regional health surveillance program, we aimed to quantify the associations between PFAS serum concentrations and blood pressure and hypertension prevalence. METHODS: The study comprised 16,224 individuals aged 20-39 years. Pregnant women (n = 327), or individuals with missing information on the selected covariates (n = 111) were excluded, leaving 15,786 subjects for the analyses. Hypertension was defined as any self-reported diagnosis, use of antihypertensive drugs, or elevated systolic blood pressure (SBP ≥ 140 mmHg)/diastolic blood pressure (DBP ≥ 90 mmHg). Generalized additive models were used to investigate the relation between perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), and perfluorononanoic acid (PFNA)) natural log (ln) transformed and by decile, and SBP, DBP, hypertension, adjusted for potential confounders. RESULTS: Both SBP and DBP increased significantly with an increase in the ln-transformed serum PFAS concentrations in a monotonic way. The predicted increase in SBP and DBP were 1.54 mmHg (95%CI 0.61-2.47), 1.60 mmHg (95%CI 0.92-2.27) from lowest to highest decile of PFOA. The associations were stronger for SBP in men and for DBP in women. One unit increase in each In-transformed PFAS was positively associated with an increased odd of hypertension in men: PFOA OR = 1.06 (1.01-1.11), PFOS OR = 1.13 (1.03-1.23), PFHxS OR = 1.08 (1.02-1.15), PFNA OR = 1.20 (1.02-1.40). CONCLUSIONS: Our findings suggest that serum PFAS concentrations were associated with increased systolic and diastolic blood pressure in a large highly exposed young adult population. Although the magnitude of the observed effect was relatively small, if confirmed it would be of public health relevance since even small increases in blood pressure levels at the population level may be associated to a raised risk of adverse outcomes such as cardiovascular disease and target organ damage.

Avoidable hospitalisation for diabetes mellitus among immigrants and natives: Results from the Italian Network for Longitudinal Metropolitan Studies.

BACKGROUND AND AIMS: Italy has experienced a relevant increase in migration inflow over the last 20 years. Although the Italian Health Service is widely accessible, immigrants can face many barriers that limit their use of health services. Diabetes mellitus (DM) has a different prevalence across ethnic groups, but studies focusing on DM care among immigrants in Europe are scarce. This study aimed to compare the rates of avoidable hospitalisation (AH) between native and immigrant adults in Italy. METHODS AND RESULTS: A multi-centre open cohort study including all 18- to 64-year-old residents in Turin, Venice, Reggio-Emilia, Modena, Bologna and Rome between 01/01/2001 and 31/12/2013-14 was conducted. Italian citizens were compared with immigrants from high migratory pressure countries who were further divided by their area of origin. We calculated age-, sex- and calendar year-adjusted rate ratios (RRs) and 95% confidence intervals (95% CIs) of AH for DM by citizenship using negative binomial regression models. The RRs were summarized using a random effects meta-analysis. The results showed higher AH rates among immigrant males (RR: 1.63, 95% CI: 1.16-2.23), whereas no significant difference was found for females (RR: 1.14, 95% CI: 0.65-1.99). Immigrants from Asia and Africa showed a higher risk than Italians, whereas those from Central-Eastern Europe and Central-Southern America did not show any increased risk. CONCLUSION: Adult male immigrants were at higher risk of experiencing AH for DM than Italians, with differences by area of origin, suggesting that they may experience lower access to and lower quality of primary care for DM. These services should be improved to reduce disparities.

Potentially avoidable hospitalization for asthma in children and adolescents by migrant status: results from the Italian Network for Longitudinal Metropolitan Studies.

BACKGROUND: Global migration toward Europe is increasing. Providing health assistance to migrants is challenging because numerous barriers limit their accessibility to health services. Migrants may be at a greater risk of developing asthma and receiving lower quality healthcare assistance than non-migrants. We aim to investigate whether immigrants as children and adolescents have higher rates of potentially avoidable hospitalization (PAH) for asthma compared to Italians. METHODS: We performed a retrospective longitudinal study using six cohorts of 2-17-year-old residents in North and Central Italy from 01/01/2001 to 31/12/2014 (N = 1,256,826). We linked asthma hospital discharges to individuals using anonymized keys. We estimated cohort-specific age and calendar-year-adjusted asthma PAH rate ratios (HRRs) and 95% confidence intervals (95%CIs) among immigrants compared to Italians. We applied a two-stage random effect model to estimate asthma PAH meta-analytic rate ratios (MHRRs). We analyzed data by gender and geographical area of origin countries. RESULTS: Three thousand three hundred four and 471 discharges for asthma PAH occurred among Italians and immigrants, respectively. Compared to Italians, the asthma PAH cohort-specific rate was higher for immigrant males in Bologna (HRR:2.42; 95%CI:1.53-3.81) and Roma (1.22; 1.02-1.45), and for females in Torino (1.56; 1.10-2.20) and Roma (1.82; 1.50-2.20). Asthma PAH MHRRs were higher only among immigrant females (MHRRs:1.48; 95%CI:1.18-1.87). MHRRs by area of origin were 63 to 113% higher among immigrants, except for Central-Eastern Europeans (0.80; 0.65-0.98). CONCLUSION: The asthma PAH meta-analytic rate was higher among female children and adolescent immigrants compared to Italians, with heterogeneity among cohorts showing higher cohort-specific PAH also among males, with some differences by origin country. Access to primary care for children and adolescent immigrants should be improved and immigrants should be considered at risk of severe asthma outcomes and consequently targeted by clinicians.

Quorum Sensing as Antivirulence Target in Cystic Fibrosis Pathogens.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder which leads to the secretion of a viscous mucus layer on the respiratory epithelium that facilitates colonization by various bacterial pathogens. The problem of drug resistance has been reported for all the species able to colonize the lung of CF patients, so alternative treatments are urgently needed. In this context, a valid approach is to investigate new natural and synthetic molecules for their ability to counteract alternative pathways, such as virulence regulating quorum sensing (QS). In this review we describe the pathogens most commonly associated with CF lung infections: Staphylococcus aureus, Pseudomonas aeruginosa, species of the Burkholderia cepacia complex and the emerging pathogens Stenotrophomonas maltophilia, Haemophilus influenzae and non-tuberculous Mycobacteria. For each bacterium, the QS system(s) and the molecules targeting the different components of this pathway are described. The amount of investigations published in the last five years clearly indicate the interest and the expectations on antivirulence therapy as an alternative to classical antibiotics.

[Immigrants' health and socioeconomic inequalities of overall population residing in Italy evaluated through the Italian network of Longitudinal Metropolitan Studies]. / Salute degli immigrati e disuguaglianze socioeconomiche nella popolazione residente in Italia valutate attraverso la rete degli Studi Longitudinali Metropolitani.

INTRODUCTION: Describing and monitoring socioeconomic inequalities in health are the prerequisite for planning equity policies. In Italy, some cities have integrated personal information from the municipal registries with Census data and with data from healthcare information systems to set up Longitudinal Metropolitan Studies (LMS). Under the coordination of the Italian National Institute for Health, Migration, and Poverty (NIHMP), six cities in the LMS network have contributed to the present monograph: Turin, Venice, Reggio Emilia, Modena, Bologna, and Rome. MORTALITY RESULTS. Significant socioeconomic differences by level of education were seen in all the participating centres. People who live alone or in single-parent households are more likely to die, as are those living in a substandard dwelling. Immigrants resident in the six cities included in the study showed lower all-cause mortality than Italians (males: MRR 0.83; 95%CI 0.78-0.90 - females: MRR 0.70; 95%CI 0.64-0.77). Sub-Saharan Africans experienced a significant higher mortality than Italians (males: MRR 1.33; 95%CI 1.12-1.59 - females: MMR 1.69; 95%CI 1.31-2.17). Immigrants had a neonatal and post-neonatal mortality risk about 1.5 times higher than Italians (neonatal: OR 1.71; 95%CI 1.22-2.39 - post-neonatal: OR 1.63; 95%CI 1.03-2.57). A difference between Italians and immigrants was also observed for mortality in children aged 1-4 years, though less marked (OR 1.24; 95%CI 0.73-2.11). Excesses concerned particularly immigrants from North Africa and from sub-Saharan Africa as well as those residing in Italy for >5 years. HOSPITALISATION RESULTS. Hospitalisation rates are lower for immigrants than for Italians, except when due to infectious diseases, blood disorders, and, among women, for reasons linked to pregnancy and childbirth. Avoidable hospitalisation rates of adults from low migratory pressure Countries are lower than or equal to those of Italians. On the contrary, adults from low migratory pressure Countries show higher avoidable hospitalisation rates compared to Italians in every cohort, with the exception of Rome (RR 0.81; 95%CI 0.78-0.85), with RR ranging from 1.08 (95%CI 0.96-1.22) in Venice to 1.64 (95%CI 1.47-1.83) in Modena. CONCLUSIONS: Maternal and child health is the most critical area of health for immigrant population. Considering the importance that the issue of health equity has taken on in the political agenda, the data presented in this volume are a great asset, particularly in light of the long recession and the social crisis that have impacted the Country.

Interplay of CodY and ScoC in the Regulation of Major Extracellular Protease Genes of Bacillus subtilis.

UNLABELLED: AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC, little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC. Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB, allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients. IMPORTANCE: The major Bacillus subtilis exoproteases, AprE and NprE, are important metabolic enzymes whose genes are subject to complex regulation by multiple transcription factors. We show here that expression of the aprE and nprE genes is also controlled, both directly and indirectly, by CodY, a global transcriptional regulator that responds to the intracellular pools of amino acids. Direct CodY-mediated repression explains a long-standing puzzle, that is, why exoproteases are not produced when cells are growing exponentially in a medium containing abundant quantities of proteins or their degradation products. Indirect regulation of aprE and nprE through CodY-mediated repression of the scoC gene, encoding another pleiotropic repressor, serves to maintain a significant level of repression of exoprotease genes when CodY loses activity.

Interactive regulation by the Bacillus subtilis global regulators CodY and ScoC.

CodY and ScoC are Bacillus subtilis transcriptional regulators that control the expression of dozens of genes and operons. Using scoC-lacZ fusions and DNA-binding experiments, we show here that scoC is directly repressed by CodY. This effect creates multiple forms of cascade regulation. For instance, expression of the dtpT gene, which is directly and negatively controlled by ScoC and encodes a putative oligopeptide permease, was activated indirectly by CodY due to CodY-mediated repression of scoC. The opp operon, which encodes an oligopeptide permease that is essential for sporulation and genetic competence development, proved to be a direct target of repression by both ScoC and CodY but was not significantly affected in codY or scoC single mutants. The combined actions of CodY and ScoC maintain opp repression when either one of the regulators loses activity but limit the level of repression to that provided by one of the regulators acting alone. Under conditions of nitrogen limitation, repression by ScoC of dtpT and opp was partly prevented by TnrA. Thus, the functioning of ScoC is determined by other transcription factors via modulation of its expression or DNA binding.

CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr.

UNLABELLED: CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE: CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein utilization pathway.

New Antimicrobial Strategies to Treat Multi-Drug Resistant Infections Caused by Gram-Negatives in Cystic Fibrosis.

People with cystic fibrosis (CF) suffer from recurrent bacterial infections which induce inflammation, lung tissue damage and failure of the respiratory system. Prolonged exposure to combinatorial antibiotic therapies triggers the appearance of multi-drug resistant (MDR) bacteria. The development of alternative antimicrobial strategies may provide a way to mitigate antimicrobial resistance. Here we discuss different alternative approaches to the use of classic antibiotics: anti-virulence and anti-biofilm compounds which exert a low selective pressure; phage therapies that represent an alternative strategy with a high therapeutic potential; new methods helping antibiotics activity such as adjuvants; and antimicrobial peptides and nanoparticle formulations. Their mechanisms and in vitro and in vivo efficacy are described, in order to figure out a complete landscape of new alternative approaches to fight MDR Gram-negative CF pathogens.

Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

BACKGROUND: The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. METHODS: We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. RESULTS: All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. CONCLUSIONS: Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Genetic determinants of complement activation in the general population.

Complement is a fundamental innate immune response component. Its alterations are associated with severe systemic diseases. To illuminate the complement's genetic underpinnings, we conduct genome-wide association studies of the functional activity of the classical (CP), lectin (LP), and alternative (AP) complement pathways in the Cooperative Health Research in South Tyrol study (n = 4,990). We identify seven loci, encompassing 13 independent, pathway-specific variants located in or near complement genes (CFHR4, C7, C2, MBL2) and non-complement genes (PDE3A, TNXB, ABO), explaining up to 74% of complement pathways' genetic heritability and implicating long-range haplotypes associated with LP at MBL2. Two-sample Mendelian randomization analyses, supported by transcriptome- and proteome-wide colocalization, confirm known causal pathways, establish within-complement feedback loops, and implicate causality of ABO on LP and of CFHR2 and C7 on AP. LP causally influences collectin-11 and KAAG1 levels and the risk of mouth ulcers. These results build a comprehensive resource to investigate the role of complement in human health.

von Willebrand factor-binding protein (vWbp)-activated factor XIII and transglutaminase 2 (TG2) promote cross-linking between FnBPA from Staphylococcus aureus and fibrinogen.

The secreted von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus interacts with the coagulation factors prothrombin and fibrinogen (Fbg), leading to the non-proteolytic transglutaminase activation of Factor XIII (FXIII). In this study we found that vWbp-activated FXIII catalyses the incorporation of amino-donor dansylcadaverine into region A of fibronectin-binding protein A (FnBPA). Incubation of Fbg with recombinant region A of S. aureus Fbg-binding proteins FnBPA, FnBPB, ClfA or ClfB in presence of vWbp-activated FXIII resulted in the formation of high molecular heteropolymers with FnBPA only, suggesting a specificity of the cross-linking reaction between fibrin(ogen) and the staphylococcal surface. As previously observed, cross-linking sites were mapped to the α-chain and the N1 subdomain of fibrin(ogen) and region A of FnBPA, respectively. Comparable results were obtained when tissue tranglutaminase-2 (TG2) was tested for cross-linking of FnBPA and Fbg. Of note, FnBPA-mediated covalent cross-linking promoted by vWbp-activated FXIII was also observed when bacteria were allowed to attach to fibrin(ogen). Together these findings suggest a novel pathogenetic mechanism by which the transglutaminase action of FXIII and/or TG2 contributes to entrapment and persistence of S. aureus in blood and host tissues.