Non-coding regulatory sRNAs from bacteria of the Burkholderia cepacia complex.

Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Hundreds of sRNAs have been found using in silico genome analysis and experimentally based approaches in bacteria of the Burkholderia cepacia complex (Bcc). However, and despite the hundreds of sRNAs identified so far, the number of functionally characterized sRNAs from these bacteria remains very limited. In this mini-review, we describe the general characteristics of sRNAs and the main mechanisms involved in their action as regulators of post-transcriptional gene expression, as well as the work done so far in the identification and characterization of sRNAs from Bcc. The number of functionally characterized sRNAs from Bcc is expected to increase and to add new knowledge on the biology of these bacteria, leading to novel therapeutic approaches to tackle the infections caused by these opportunistic pathogens, particularly severe among cystic fibrosis patients. KEY POINTS: •Hundreds of sRNAs have been identified in Burkholderia cepacia complex bacteria (Bcc). •A few sRNAs have been functionally characterized in Bcc. •Functionally characterized Bcc sRNAs play major roles in metabolism, biofilm formation, and virulence.

Identification of Burkholderia cenocepacia non-coding RNAs expressed during Caenorhabditis elegans infection.

Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their roles on bacterial physiology and virulence remain largely unknown, as is the case of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of opportunistic pathogens with relatively large genomes that can cause lethal lung infections amongst cystic fibrosis (CF) patients. To characterise sRNAs expressed by Bcc bacteria when infecting a host, the nematode Caenorhabditis elegans was used as an infection model by the epidemic CF strain B. cenocepacia J2315. A total of 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified, most of them located on chromosome 1. RIT11b, a sRNA downregulated under C. elegans infection conditions, was shown to directly affect B. cenocepacia virulence, biofilm formation, and swimming motility. RIT11b overexpression reduced the expression of the direct targets dusA and pyrC, involved in biofilm formation, epithelial cell adherence, and chronic infections in other organisms. The in vitro direct interaction of RIT11b with the dusA and pyrC messengers was demonstrated by electrophoretic mobility shift assays. To the best of our knowledge this is the first report on the functional characterization of a sRNA directly involved in B. cenocepacia virulence. KEY POINTS: • 139 sRNAs expressed by B. cenocepacia during C. elegans infection were identified • The sRNA RIT11b affects B. cenocepacia virulence, biofilm formation, and motility • RIT11b directly binds to and regulates dusA and pyrC mRNAs.

Comparison of Virulence-Factor-Encoding Genes and Genotype Distribution amongst Clinical Pseudomonas aeruginosa Strains.

Pseudomonas aeruginosa is an opportunistic pathogen encoding several virulence factors in its genome, which is well-known for its ability to cause severe and life-threatening infections, particularly among cystic fibrosis patients. The organism is also a major cause of nosocomial infections, mainly affecting patients with immune deficiencies and burn wounds, ventilator-assisted patients, and patients affected by other malignancies. The extensively reported emergence of multidrug-resistant (MDR) P. aeruginosa strains poses additional challenges to the management of infections. The aim of this study was to compare the incidence rates of selected virulence-factor-encoding genes and the genotype distribution amongst clinical multidrug-sensitive (MDS) and MDR P. aeruginosa strains. The study involved 74 MDS and 57 MDR P. aeruginosa strains and the following virulence-factor-encoding genes: lasB, plC H, plC N, exoU, nan1, pilA, and pilB. The genotype distribution, with respect to the antimicrobial susceptibility profiles of the strains, was also analyzed. The lasB and plC N genes were present amongst several P. aeruginosa strains, including all the MDR P. aeruginosa, suggesting that their presence might be used as a marker for diagnostic purposes. A wide variety of genotype distributions were observed among the investigated isolates, with the MDS and MDR strains exhibiting, respectively, 18 and 9 distinct profiles. A higher prevalence of genes determining the virulence factors in the MDR strains was observed in this study, but more research is needed on the prevalence and expression levels of these genes in additional MDR strains.

LipNanoCar Technology - A Versatile and Scalable Technology for the Production of Lipid Nanoparticles.

The extensive knowledge in the miniemulsion technique used in biocatalysis applications by the authors allowed the development of drug delivery systems that constitutes the LipNanoCar technology core for the production of lipid nanoemulsions and solid lipid nanoparticles. The LipNanoCar technology, together with adequate formulations of different oils, fatty acids, surfactants, and temperature, allows the entrapment of several bioactive and therapeutic compounds in lipid nanoparticles for cosmetic, nutrition, and pharmaceutical applications.The LIpNanoCar technology allowed lipid nanoparticles production with average sizes ranging from 100 to 300 nm and Zeta Potentials between -55 and -20 mV. Concomitantly, high entrapment or encapsulation efficiencies (%EE) were achieved, as illustrated in this work for ß-carotene and vitamins derivatives (>85%) for cosmetic application, and for antibiotics currently used in chemotherapy, like rifampicin (69-85%) and pyrazinamide (14-29%) against Mycobacterium tuberculosis (TB), and ciprofloxacin (>65%) and tobramycin (~100%) in Cystic Fibrosis (CF) respiratory infections therapy. Ciprofloxacin presented, for example, a quick-release from the lipid nanoparticles using a dialysis tubing (96% in the first 7 h), but slower than the free antibiotic (95% in the first 3 h). This result suggests that ciprofloxacin is loaded near the external surface of the lipid nanoparticles.The toxicity and validation of entrapment of antibiotics in lipid nanoparticles for Cystic Fibrosis therapy were assessed using Caenorhabditis elegans as an animal model of bacterial infection. Fluorescence microscopy of an entrapped fluorescent dye (DiOC) confirmed the uptake of the lipid nanoparticles by ingestion, and their efficacy was successfully tested in C. elegans. Burkholderia contaminans IST408 and Burkholderia cenocepacia K56-2 infections were tested as model bacterial pathogens difficult to eradicate in Cystic Fibrosis respiratory diseases.

Broad Spectrum Functional Activity of Structurally Related Monoanionic Au(III) Bis(Dithiolene) Complexes.

The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ diselenolene) complexes were evaluated. The complexes differ in the nature of the heteroatom connected to the gold atom (AuS for dithiolene, AuSe for diselenolene), the substituent on the nitrogen atom of the thiazoline ring (Me, Et, Pr, iPr and Bu), the nature of the exocyclic atom or group of atoms (O, S, Se, C(CN)2) and the counter-ion (Ph4P+ or Et4N+). The anticancer and antimicrobial activities of all the complexes were investigated, while the anti-HIV activity was evaluated only for selected complexes. Most complexes showed relevant anticancer activities against Cisplatin-sensitive and Cisplatin-resistant ovarian cancer cells A2780 and OVCAR8, respectively. After 48 h of incubation, the IC50 values ranged from 0.1-8 µM (A2780) and 0.8-29 µM (OVCAR8). The complexes with the Ph4P+ ([P]) counter-ion are in general more active than their Et4N+ ([N]) analogues, presenting IC50 values in the same order of magnitude or even lower than Auranofin. Studies in the zebrafish embryo model further showed that, despite their marked anticancer effect, the complexes with [P] counter-ion exhibited low in vivo toxicity. In general, the exocyclic exchange of sulfur by oxygen or ylidenemalononitrile (C(CN)2) enhanced the compounds toxicity. Most complexes containing the [P] counter ion exhibited exceptional antiplasmodial activity against the Plasmodium berghei parasite liver stages, with submicromolar IC50 values ranging from 400-700 nM. In contrast, antibacterial/fungi activities were highest for most complexes with the [N] counter-ion. Auranofin and two selected complexes [P][AuSBu(=S)] and [P][AuSEt(=S)] did not present anti-HIV activity in TZM-bl cells. Mechanistic studies for selected complexes support the idea that thioredoxin reductase, but not DNA, is a possible target for some of these complexes. The complexes [P] [AuSBu(=S)], [P] [AuSEt(=S)], [P] [AuSEt(=Se)] and [P] [AuSeiPr(=S)] displayed a strong quenching of the fluorescence intensity of human serum albumin (HSA), which indicates a strong interaction with this protein. Overall, the results highlight the promising biological activities of these complexes, warranting their further evaluation as future drug candidates with clinical applicability.

Prevalence and risk factors of fatty liver in Portuguese adults.

BACKGROUND: Prevalence of fatty liver (FL) and nonalcoholic fatty liver disease (NAFLD) depends mainly on obesity, diabetes and genetic factors. FL and NAFLD prevalence was evaluated in Portuguese adult population and correlated with several risk factors and related mortality data, within the same period. MATERIALS AND METHODS: A cross-sectional, population-based multicenter study, voluntary and randomly selected in 834 Portuguese adults (18-79 years). Participants were evaluated after 12-hour fasting. Anthropometric data, past history including alcohol consumption, and associated diseases were registered. Blood samples were collected for biochemical testing. Dietary intake was evaluated using a semi-quantitative food frequency questionnaire. Presence of FL was evaluated using ultrasound, and NAFLD was diagnosed after exclusion of other causes for liver disease. RESULTS: Adjusted prevalence of FL and NAFLD was 37.8% and 17.0%, respectively. FL individuals were older, more frequently males, with increased probability of having obesity, diabetes or harmful alcohol consumption (HAC). NAFLD individuals were also older, but had a similar sex distribution and an increased probability of obesity and diabetes. In both groups, no differences were found regarding dietary pattern or physical activity. During the same time period, nonalcoholic steatohepatitis (NASH) liver-related deaths in Portugal were 0.105/100 000, while alcohol-related liver disease mortality was 6.790/100 000. CONCLUSION: The large spectrum of FL was present in more than one third of the population, although only less than half could be classified as NAFLD. Other significant risk factors, such as HAC, are probably implicated in FL, explaining the low NASH-related mortality compared with the high alcohol-related mortality during the same time period.

Microbial Virulence Factors.

Microbial virulence factors encompass a wide range of molecules produced by pathogenic microorganisms, enhancing their ability to evade their host defenses and cause disease [...].

Extracellular RNAs in Bacterial Infections: From Emerging Key Players on Host-Pathogen Interactions to Exploitable Biomarkers and Therapeutic Targets.

Non-coding RNAs (ncRNAs) are key regulators of post-transcriptional gene expression in prokaryotic and eukaryotic organisms. These molecules can interact with mRNAs or proteins, affecting a variety of cellular functions. Emerging evidence shows that intra/inter-species and trans-kingdom regulation can also be achieved with exogenous RNAs, which are exported to the extracellular medium, mainly through vesicles. In bacteria, membrane vesicles (MVs) seem to be the more common way of extracellular communication. In several bacterial pathogens, MVs have been described as a delivery system of ncRNAs that upon entry into the host cell, regulate their immune response. The aim of the present work is to review this recently described mode of host-pathogen communication and to foster further research on this topic envisaging their exploitation in the design of novel therapeutic and diagnostic strategies to fight bacterial infections.

Determination of estrone and 17α-ethinylestradiol in digested sludge by ultrasonic liquid extraction and high-performance liquid chromatography with fluorescence detection.

Estrone, 17ß-estradiol and 17α-ethinylestradiol are increasingly recognised as important micropollutants to be monitored in wastewater treatment plants. These estrogens are retained onto sludge due to their high adsorption and since they are largely used in land applications, the monitoring of these chemicals in sludge samples is of great importance. This study describes a method for the determination of estrone and 17α-ethinylestradiol in fresh sludge samples. After spiking fresh digested sludge with estrone and 17α-ethinylestradiol and maintaining in contact during 5, 30 and 60 min, the freeze-dried samples were subjected to ultrasonic liquid extraction, with methanol and acetone, and analysed by high-performance liquid chromatography with fluorescence detection. The average recoveries obtained for estrone and 17α-ethinylestradiol using the different contact times were 103 ± 3 and 97 ± 4%, respectively. Fresh sludge samples from one waste water treatment plant located in Portugal were analysed and estrone was detected in primary fresh sludge, anaerobic digested sludge and dehydrated sludge at a concentration in the range of 1-4.8 µg/g. The method here developed does not require any sample clean-up, being fast and simple, reliable and inexpensive, making possible its application for monitoring the contamination of sludge with these estrogens.

Taming chaos to sample rare events: The effect of weak chaos.

Rare events in nonlinear dynamical systems are difficult to sample because of the sensitivity to perturbations of initial conditions and of complex landscapes in phase space. Here, we discuss strategies to control these difficulties and succeed in obtaining an efficient sampling within a Metropolis-Hastings Monte Carlo framework. After reviewing previous successes in the case of strongly chaotic systems, we discuss the case of weakly chaotic systems. We show how different types of nonhyperbolicities limit the efficiency of previously designed sampling methods, and we discuss strategies on how to account for them. We focus on paradigmatic low-dimensional chaotic systems such as the logistic map, the Pomeau-Maneville map, and area-preserving maps with mixed phase space.

Differential effects of Th17 cytokines during the response of neutrophils to Burkholderia cenocepacia outer membrane protein A.

T helper 17 cells are involved in the immunopathology of cystic fibrosis. They play a key role in recruitment of neutrophils, which is the first line of defence against bacteria. Additionally, Burkholderia cenocepacia outer membrane protein A (OmpA) BCAL2958 is considered a potential protective epitope for vaccine development. The present study aimed to investigate the neutrophil response to OmpA in the presence of Th17 cytokines, IL-17 and IL-22 at different times of activation. Neutrophils were isolated from whole blood of healthy volunteers and activated with OmpA in the presence of IL-17, IL-22 or both cytokines together. Supernatant was collected after 1 h, 2 h, 4 h, 8 h, and 12 h. Neutrophil activation was assessed by measuring MPO, TNF-α, elastase, hydrogen peroxide, catalase and NO. The results revealed that the combination of IL-17 and IL-22 cytokines induced the release of NE, catalase, H2O2 and TNF-α from neutrophils activated with Burkholderia OmpA at late stages of activation. However, IL-22 alone or IL-17 alone decreased the myeloperoxidase (MPO), catalase and NE levels at early stages of neutrophil activation. The presence of IL-17 alone led to a significant increase in TNF-α level after 1 h and 12 h. However, the presence of IL-22 alone led to a significant increase in TNF-α level after only 1 h but a significant decrease after 8 h of activation was observed as compared to OmpA stimulated neutrophils. In conclusion, Th17 cytokines IL-17 and IL-22, have differential effects during the neutrophil response to Burkholderia OmpA.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils.

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and ß-galactosidase activities.

No Evidence for Lower Levels of Serum Vitamin D in the Presence of Hepatic Steatosis. A Study on the Portuguese General Population.

Introduction and aims: Nonalcoholic fatty liver disease (NAFLD) has become highly prevalent, paralleling the pandemic of obesity and diabetes, and represents an important burden. Nutrition knowledge is fundamental, in prevention, evolution and treatment of NAFLD. Association of low serum levels of vitamin D (VD) with several diseases, including NAFLD, has been emphasized in the last decade. We evaluated how serum levels of VD correlate with the presence of hepatic steatosis, and VD intake, in a random sample of the Portuguese adult population. Methods: Participants underwent a dietary intake inquiry, using a semi-quantitative food frequency questionnaire representative of the usual intake over the previous year. Anthropometric measures, blood tests and ultrasound were done. Hepatic steatosis was quantified according to Hamaguchi's ultrasonographic score (steatosis defined by a score ≥ 2). Results: We recruited 789 adult individuals, 416 males (52.7%), mean age of 49.9 ± 17.0 years (18-79). Prevalence of hepatic steatosis was 35.5%, and after exclusion of excessive alcohol consumption, 28.0%. Mean VD serum levels were 26.0 ± 9.8 ng/ml and 68.4% participants had serum VD levels below 30 ng/ml. Mean serum levels of VD were not significantly different between participants with steatosis vs. no steatosis: 25.2±8.7 vs. 26.4±10.3 ng/ml, respectively (p=0.071). There was no correlation between VD serum levels and VD intake, measured by the FFQ, r=0.075 (p= 0.383). Conclusions: In spite of a high prevalence rate, there was no evidence that decreased VD serum levels were associated with hepatic steatosis. No significant correlation was found between VD dietary ingestion and VD serum levels.

Small Noncoding Regulatory RNAs from Pseudomonas aeruginosa and Burkholderia cepacia Complex.

Cystic fibrosis (CF) is the most life-limiting autosomal recessive disorder in Caucasians. CF is characterized by abnormal viscous secretions that impair the function of several tissues, with chronic bacterial airway infections representing the major cause of early decease of these patients. Pseudomonas aeruginosa and bacteria from the Burkholderia cepacia complex (Bcc) are the leading pathogens of CF patients' airways. A wide array of virulence factors is responsible for the success of infections caused by these bacteria, which have tightly regulated responses to the host environment. Small noncoding RNAs (sRNAs) are major regulatory molecules in these bacteria. Several approaches have been developed to study P. aeruginosa sRNAs, many of which were characterized as being involved in the virulence. On the other hand, the knowledge on Bcc sRNAs remains far behind. The purpose of this review is to update the knowledge on characterized sRNAs involved in P. aeruginosa virulence, as well as to compile data so far achieved on sRNAs from the Bcc and their possible roles on bacteria virulence.

Sampling Motif-Constrained Ensembles of Networks.

The statistical significance of network properties is conditioned on null models which satisfy specified properties but that are otherwise random. Exponential random graph models are a principled theoretical framework to generate such constrained ensembles, but which often fail in practice, either due to model inconsistency or due to the impossibility to sample networks from them. These problems affect the important case of networks with prescribed clustering coefficient or number of small connected subgraphs (motifs). In this Letter we use the Wang-Landau method to obtain a multicanonical sampling that overcomes both these problems. We sample, in polynomial time, networks with arbitrary degree sequences from ensembles with imposed motifs counts. Applying this method to social networks, we investigate the relation between transitivity and homophily, and we quantify the correlation between different types of motifs, finding that single motifs can explain up to 60% of the variation of motif profiles.

Chest pain in the emergency department: risk stratification with Manchester triage system and HEART score.

BACKGROUND: Fast and accurate chest pain risk stratification in the emergency department (ED) is critical. The HEART score predicts the short-term incidence of major adverse cardiac events (MACE) in this population, dividing it in three risk categories. We aimed to describe the population with chest pain, to characterize the subgroup of patients with acute coronary syndrome (ACS) and to assess the prognostic value of Manchester triage system and of HEART score. METHODS: Retrospective observational study including patients admitted to the ED of a tertiary hospital with chest pain as the presenting symptom. The primary outcome was a composite of all-cause mortality, myocardial infarction or unscheduled revascularization at 6 weeks. RESULTS: We enrolled 233 patients (age 58 ± 19; 55.4 % males). The most common final diagnosis was non-specific chest pain (n = 86, 36.9 %), followed by ACS (n = 22, 9.4 %). Male gender, smoking and chronic kidney disease were associated with higher risk of ACS. According to Manchester triage system, chest pain patients stratified with red or orange priority had a higher incidence of ACS (16.5 % vs. 3.8 %, p = 0.006). The application of HEART score showed that most patients were in low risk category (56.3 %). The six-week incidence of MACE in each category was 2 %, 15.6 % and 76.9 % (p < 0.001). HEART score accurately predicted the short-term incidence of MACE in chest pain patients (c-statistic 0.880; 95 % CI, 0.807-0.950, p < 0.001). CONCLUSIONS: Chest pain patients have very different levels of severity and the discriminatory power of Manchester triage system should be used in the assessment of this population. The HEART score seems to be an effective tool for risk stratification in the ED.

Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia.

Volume 195, no. 16, p. 3514­3523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitão, J. Bacteriol. 193:1515­1526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitão, Microbiology 156:896­908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitão, Microb. Pathog. 48:168­177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

How good is controlled attenuation parameter and fatty liver index for assessing liver steatosis in general population: correlation with ultrasound.

BACKGROUND & AIMS: Liver steatosis measurement by controlled attenuation parameter (CAP) is a non-invasive method for diagnosing steatosis, based on transient elastography. Its usefulness as screening procedure for hepatic steatosis in general population has not been previously evaluated. The aim of this study was to evaluate the diagnostic accuracy of CAP and fatty liver index (FLI) for detection and quantification of steatosis in general population. METHODS: Recruitment was done from a prospective epidemiological study of the general adult population. Steatosis was evaluated using CAP, FLI and ultrasound (US). Steatosis scored according to Hamaguchi's US scoring, from 0 (S0) to 6 (S6) points. Hepatic steatosis defined by score ≥2 (S≥2) and moderate/severe steatosis by score ≥4 (S≥4). Performance of CAP and FLI for diagnosing steatosis compared with US was assessed using areas under receiver operating characteristic curves (AUROC). RESULTS: From 219 consecutive individuals studied, 13 (5.9%) excluded because of failure/unreliable liver stiffness measurements. Steatosis prevalence: S≥2 38.4% and S≥4 12.1%. CAP significantly correlated with steatosis (ρ = 0.73, P < 0.0001), steatosis score (ρ = 0.76; P < 0.0001), FLI (ρ = 0.69), waist circumference (ρ = 0.62), body mass index (ρ = 0.55), triglyceride (ρ = 0.49), HOMA-IR (ρ = 0.26), alcohol consumption (ρ = 0.24) and cholesterol (ρ = 0.19), not with liver stiffness measurements. Using CAP and FLI, AUROC's were 0.94 (95% CI 0.91-0.97, P < 0.001) and 0.91 for S≥2; 0.95 (95% CI 0.90-0.99, P < 0.001) and 0.93 for S≥4 respectively. Optimal cut-off value of CAP and FLI were 243 dB/m and 48 for S≥2; 303.5 dB/m and 62 for S≥4 respectively. CONCLUSION: Controlled attenuation parameter and FLI seem promising tools for screening and steatosis quantification in the general population. Larger studies are needed for validation.

Experimental identification of small non-coding regulatory RNAs in the opportunistic human pathogen Burkholderia cenocepacia J2315.

Small non-coding regulatory RNAs (sRNAs) play important roles in regulating gene expression at the post-transcriptional level and often require the RNA chaperone Hfq. The human opportunistic pathogen Burkholderia cenocepacia J2315 encodes two distinct RNA chaperones, Hfq and Hfq2. The present work describes the experimental identification and validation of 24 sRNAs from B. cenocepacia J2315, based on the co-purification of sRNAs with the bacterium Hfq protein, followed by conversion into cDNA, cloning, computational analysis of sequences and validation by Northern blot analysis. The sRNAs here reported escaped identification by previous studies based on transcriptomics or bioinformatic analyses. Results presented indicate that 3 sRNAs are exclusive to bacteria of the Burkholderia cepacia complex and have no homologues in other bacteria, while the other 21 share homology, at different extents, to sRNAs of other bacterial species.

Surface-Exposed Protein Moieties of Burkholderia cenocepacia J2315 in Microaerophilic and Aerobic Conditions.

Burkholderia cepacia complex infections remain life-threatening to cystic fibrosis patients, and due to the limited eradication efficiency of current treatments, novel antimicrobial therapies are urgently needed. Surface proteins are among the best targets to develop new therapeutic strategies since they are exposed to the host's immune system. A surface-shaving approach was performed using Burkholderia cenocepacia J2315 to quantitatively compare the relative abundance of surface-exposed proteins (SEPs) expressed by the bacterium when grown under aerobic and microaerophilic conditions. After trypsin incubation of live bacteria and identification of resulting peptides by liquid chromatography coupled with mass spectrometry, a total of 461 proteins with ≥2 unique peptides were identified. Bioinformatics analyses revealed a total of 53 proteins predicted as localized at the outer membrane (OM) or extracellularly (E). Additionally, 37 proteins were predicted as moonlight proteins with OM or E secondary localization. B-cell linear epitope bioinformatics analysis of the proteins predicted to be OM and E-localized revealed 71 SEP moieties with predicted immunogenic epitopes. The protegenicity higher scores of proteins BCAM2761, BCAS0104, BCAL0151, and BCAL0849 point out these proteins as the best antigens for vaccine development. Additionally, 10 of the OM proteins also presented a high probability of playing important roles in adhesion to host cells, making them potential targets for passive immunotherapeutic approaches. The immunoreactivity of three of the OM proteins identified was experimentally demonstrated using serum samples from cystic fibrosis patients, validating our strategy for identifying immunoreactive moieties from surface-exposed proteins of potential interest for future immunotherapies development.