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.

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.

Advances in Human Pathogen Control-A 21st Century Challenge.

The emergence of new pathogens, coupled with the reemergence of old pathogens and the steep worldwide increase in multiple resistances to available antimicrobials, poses major challenges to human health at the global scale [...].

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.

Tuning the Biological Activity of Camphorimine Complexes through Metal Selection.

The cytotoxic activity of four sets of camphorimine complexes based on the Cu(I), Cu(II), Ag(I), and Au(I) metal sites were assessed against the cisplatin-sensitive A2780 and OVCAR3 ovarian cancer cells. The results showed that the gold complexes were ca. one order of magnitude more active than the silver complexes, which in turn were ca. one order of magnitude more active than the copper complexes. An important finding was that the cytotoxic activity of the Ag(I) and Au(I) camphorimine complexes was higher than that of cisplatin. Another relevant aspect was that the camphorimine complexes did not interact significantly with DNA, in contrast with cisplatin. The cytotoxic activity of the camphorimine complexes displayed a direct relationship with the cellular uptake by OVCAR3 cells, as ascertained by PIXE (particle-induced X-ray emission). The levels of ROS (reactive oxygen species) formation exhibited an inverse relationship with the reduction potentials for the complexes with the same metal, as assessed by cyclic voltammetry. In order to gain insight into the toxicity of the complexes, their cytotoxicity toward nontumoral cells (HDF and V79 fibroblasts) was evaluated. The in vivo cytotoxicity of complex 5 using the nematode Caenorhabditis elegans was also assessed. The silver camphorimine complexes displayed the highest selectivity coefficients (activity vs. toxicity).

A Polyclonal Antibody Raised against the Burkholderia cenocepacia OmpA-like Protein BCAL2645 Impairs the Bacterium Adhesion and Invasion of Human Epithelial Cells In Vitro.

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain a life threat to cystic fibrosis (CF) patients, due to the faster lung function decline and the absence of effective eradication strategies. Immunotherapies are regarded as an attractive alternative to control and reduce the damages caused by these infections. In this work, we report the cloning and functional characterization of the OmpA-like BCAL2645 protein, previously identified and found to be immunoreactive against sera from CF patients with a record of Bcc infections. The BCAL2645 protein is shown to play a role in biofilm formation, adherence to mucins and invasion of human lung epithelial cells. The expression of the BCAL2645 protein was found to be increased in culture medium, mimicking the lungs of CF patients and microaerophilic conditions characteristic of the CF lung. Moreover, a polyclonal antibody raised against BCAL2645 was found to inhibit, by about 75 and 85%, the ability of B. cenocepacia K56-2 to bind and invade in vitro CFBE41o- human bronchial epithelial cells. These results highlight the potential of anti-BCAL2645 antibodies for the development of passive immunization therapies to protect CF patients against Bcc infections.

Bacterial Nosocomial Infections: Multidrug Resistance as a Trigger for the Development of Novel Antimicrobials.

Nosocomial bacterial infections are associated with high morbidity and mortality, posing a huge burden to healthcare systems worldwide. The ongoing COVID-19 pandemic, with the raised hospitalization of patients and the increased use of antimicrobial agents, boosted the emergence of difficult-to-treat multidrug-resistant (MDR) bacteria in hospital settings. Therefore, current available antibiotic treatments often have limited or no efficacy against nosocomial bacterial infections, and novel therapeutic approaches need to be considered. In this review, we analyze current antibacterial alternatives under investigation, focusing on metal-based complexes, antimicrobial peptides, and antisense antimicrobial therapeutics. The association of new compounds with older, commercially available antibiotics and the repurposing of existing drugs are also revised in this work.

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.

Comparative Genomics and Evolutionary Analysis of RNA-Binding Proteins of Burkholderia cenocepacia J2315 and Other Members of the B. cepacia Complex.

RNA-binding proteins (RBPs) are important regulators of cellular functions, playing critical roles on the survival of bacteria and in the case of pathogens, on their interaction with the host. RBPs are involved in transcriptional, post-transcriptional, and translational processes. However, except for model organisms like Escherichia coli, there is little information about the identification or characterization of RBPs in other bacteria, namely in members of the Burkholderia cepacia complex (Bcc). Bcc is a group of bacterial species associated with a poor clinical prognosis in cystic fibrosis patients. These species have some of the largest bacterial genomes, and except for the presence of two-distinct Hfq-like proteins, their RBP repertoire has not been analyzed so far. Using in silico approaches, we identified 186 conventional putative RBPs in Burkholderia cenocepacia J2315, an epidemic and multidrug resistant pathogen of cystic fibrosis patients. Here we describe the comparative genomics and phylogenetic analysis of RBPs present in multiple copies and predicted to play a role in transcription, protein synthesis, and RNA decay in Bcc bacteria. In addition to the two different Hfq chaperones, five cold shock proteins phylogenetically close to E. coli CspD protein and three distinct RhlE-like helicases could be found in the B. cenocepacia J2315 genome. No RhlB, SrmB, or DeaD helicases could be found in the genomes of these bacteria. These results, together with the multiple copies of other proteins generally involved in RNA degradation, suggest the existence, in B. cenocepacia and in other Bcc bacteria, of some extra and unexplored functions for the mentioned RBPs, as well as of alternative mechanisms involved in RNA regulation and metabolism in these bacteria.

New insights into the immunoproteome of B. cenocepacia J2315 using serum samples from cystic fibrosis patients.

Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous multidrug resistant organisms and opportunistic pathogens capable of causing life threatening lung infections among cystic fibrosis (CF) patients. No effective therapies are currently available to eradicate Bcc bacteria from CF patients, as these organisms are inherently resistant to the majority of clinically available antimicrobials. An immunoproteomics approach was used to identify Bcc proteins that stimulate the humoral immune response of the CF host, using bacterial cells grown under conditions mimicking the CF lung environment and serum samples from CF patients with a clinical record of Bcc infection. 24 proteins of the Bcc strain B. cenocepacia J2315 were identified as immunoreactive, 19 here reported as immunogenic for the first time. Ten proteins were predicted as extracytoplasmic, 9 of them being conserved in Bcc genomes. The immunogenic Bcc extracytoplasmic proteins are potential targets for development of novel therapeutic strategies and diagnostic tools to protect patients against the onset of chronic Bcc lung infections.

The afc antifungal activity cluster, which is under tight regulatory control of ShvR, is essential for transition from intracellular persistence of Burkholderia cenocepacia to acute pro-inflammatory infection.

The opportunistic pathogen Burkholderia cenocepacia is particularly life-threatening for cystic fibrosis (CF) patients. Chronic lung infections with these bacteria can rapidly develop into fatal pulmonary necrosis and septicaemia. We have recently shown that macrophages are a critical site for replication of B. cenocepacia K56-2 and the induction of fatal pro-inflammatory responses using a zebrafish infection model. Here, we show that ShvR, a LysR-type transcriptional regulator that is important for biofilm formation, rough colony morphotype and inflammation in a rat lung infection model, is also required for the induction of fatal pro-inflammatory responses in zebrafish larvae. ShvR was not essential, however, for bacterial survival and replication in macrophages. Temporal, rhamnose-induced restoration of shvR expression in the shvR mutant during intramacrophage stages unequivocally demonstrated a key role for ShvR in transition from intracellular persistence to acute fatal pro-inflammatory disease. ShvR has been previously shown to tightly control the expression of the adjacent afc gene cluster, which specifies the synthesis of a lipopeptide with antifungal activity. Mutation of afcE, encoding an acyl-CoA dehydrogenase, has been shown to give similar phenotypes as the shvR mutant. We found that, like shvR, afcE is also critical for the switch from intracellular persistence to fatal infection in zebrafish. The closely related B. cenocepacia H111 has been shown to be less virulent than K56-2 in several infection models, including Galleria mellonella and rats. Interestingly, constitutive expression of shvR in H111 increased virulence in zebrafish larvae to almost K56-2 levels in a manner that absolutely required afc. These data confirm a critical role for afc in acute virulence caused by B. cenocepacia that depends on strain-specific regulatory control by ShvR. We propose that ShvR and AFC are important virulence factors of the more virulent Bcc species, either through pro-inflammatory effects of the lipopeptide AFC, or through AFC-dependent membrane properties.

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.

Correction: Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.

[This corrects the article DOI: 10.1371/journal.ppat.1006437.].

Macrophages, but not neutrophils, are critical for proliferation of Burkholderia cenocepacia and ensuing host-damaging inflammation.

Bacteria of the Burkholderia cepacia complex (Bcc) can cause devastating pulmonary infections in cystic fibrosis (CF) patients, yet the precise mechanisms underlying inflammation, recurrent exacerbations and transition from chronic stages to acute infection and septicemia are not known. Bcc bacteria are generally believed to have a predominant extracellular biofilm life style in infected CF lungs, similar to Pseudomonas aeruginosa, but this has been challenged by clinical observations which show Bcc bacteria predominantly in macrophages. More recently, Bcc bacteria have emerged in nosocomial infections of patients hospitalized for reasons unrelated to CF. Research has abundantly shown that Bcc bacteria can survive and replicate in mammalian cells in vitro, yet the importance of an intracellular life style during infection in humans is unknown. Here we studied the contribution of innate immune cell types to fatal pro-inflammatory infection caused by B. cenocepacia using zebrafish larvae. In strong contrast to the usual protective role for macrophages against microbes, our results show that these phagocytes significantly worsen disease outcome. We provide new insight that macrophages are critical for multiplication of B. cenocepacia in the host and for development of a fatal, pro-inflammatory response that partially depends on Il1-signalling. In contrast, neutrophils did not significantly contribute to disease outcome. In subcutaneous infections that are dominated by neutrophil-driven phagocytosis, the absence of a functional NADPH oxidase complex resulted in a small but measurably higher increase in bacterial growth suggesting the oxidative burst helps limit bacterial multiplication; however, neutrophils were unable to clear the bacteria. We suggest that paradigm-changing approaches are needed for development of novel antimicrobials to efficiently disarm intracellular bacteria of this group of highly persistent, opportunistic pathogens.

Synthesis, antimicrobial activity and toxicity to nematodes of cyclam derivatives.

The antimicrobial activity and toxicity to nematodes of the cyclam salt [H2{H2(4-CF3PhCH2)2Cyclam}](CH3COO)2⋅(CH3COOH)2 were evaluated. Estimated minimum inhibitory concentrations (MICs) of 9, 261 and 15 µg/mL were obtained for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. For selected Candida spp., the estimated MICs obtained ranged from 32 µg/mL to 63 µg/mL. Bactericidal activity was demonstrated but the compound was not reliably fungicidal. Concentrations of the cyclam salt up to 32 µg/mL did not significantly affect survival of the nematode Caenorhabditis elegans; however, concentrations equal or above this value significantly affected nematode survival in a dose-dependent manner.

Burkholderia cepacia Complex Regulation of Virulence Gene Expression: A Review.

Burkholderia cepacia complex (Bcc) bacteria emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Their eradication is very difficult due to the high level of intrinsic resistance to clinically relevant antibiotics. Bcc bacteria have large and complex genomes, composed of two to four replicons, with variable numbers of insertion sequences. The complexity of Bcc genomes confers a high genomic plasticity to these bacteria, allowing their adaptation and survival to diverse habitats, including the human host. In this work, we review results from recent studies using omics approaches to elucidate in vivo adaptive strategies and virulence gene regulation expression of Bcc bacteria when infecting the human host or subject to conditions mimicking the stressful environment of the cystic fibrosis lung.

The Burkholderia cenocepacia OmpA-like protein BCAL2958: identification, characterization, and detection of anti-BCAL2958 antibodies in serum from B. cepacia complex-infected Cystic Fibrosis patients.

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among cystic fibrosis patients, highlighting the need for novel therapeutic strategies. In the present work we have studied the B. cenocepacia protein BCAL2958, a member of the OmpA-like family of proteins, demonstrated as highly immunogenic in other pathogens and capable of eliciting strong host immune responses. The encoding gene was cloned and the protein, produced as a 6× His-tagged derivative, was used to produce polyclonal antibodies. Bioinformatics analyses led to the identification of sequences encoding proteins with a similarity higher than 96 % to BCAL2958 in all the publicly available Bcc genomes. Furthermore, using the antibody it was experimentally demonstrated that this protein is produced by all the 12 analyzed strains from 7 Bcc species. In addition, results are also presented showing the presence of anti-BCAL2958 antibodies in sera from cystic fibrosis patients with a clinical record of respiratory infection by Bcc, and the ability of the purified protein to in vitro stimulate neutrophils. The widespread production of the protein by Bcc members, together with its ability to stimulate the immune system and the detection of circulating antibodies in patients with a documented record of Bcc infection strongly suggest that the protein is a potential candidate for usage in preventive therapies of infections by Bcc.

Hfq: a multifaceted RNA chaperone involved in virulence.

Hfq has emerged in recent years as a master regulator of gene expression in bacteria, mainly due to its ability to mediate the interaction of small noncoding RNAs with their mRNA targets, including those related to virulence in Gram-negative bacteria. In this work, we review current knowledge on the involvement of Hfq in the regulation of virulence traits related to secretion systems, alternative sigma factors, outer membrane proteins, polysaccharides and iron metabolism. Recent data from transcriptomics and proteomics studies performed for major pathogens are included. We also summarize and correlate current knowledge on how Hfq protein impacts pathogenicity of bacterial pathogens.

Suitability of a Saccharomyces cerevisiae-based assay to assess the toxicity of pyrimethanil sprayed soils via surface runoff: comparison with standard aquatic and soil toxicity assays.

The present study is aimed at evaluating whether a gene expression assay with the microbial eukaryotic model Saccharomyces cerevisiae could be used as a suitable warning tool for the rapid preliminary screening of potential toxic effects on organisms due to scenarios of soil and water contamination with pyrimethanil. The assay consisted of measuring changes in the expression of the selected pyrimethanil-responsive genes ARG3 and ARG5,6 in a standardized yeast population. Evaluation was held by assessing the toxicity of surface runoff, a major route of pesticide exposure in aquatic systems due to non-point-source pollution, which was simulated with a pyrimethanil formulation at a semifield scale mimicking worst-case scenarios of soil contamination (e.g. accident or improper disposal). Yeast cells 2-h exposure to the runoff samples led to a significant 2-fold increase in the expression of both indicator genes. These results were compared with those from assays with organisms relevant for the aquatic and soil compartments, namely the nematode Caenorhabditis elegans (reproduction), the freshwater cladoceran Daphnia magna (survival and reproduction), the benthic midge Chironomus riparius (growth), and the soil invertebrates Folsomia candida and Enchytraeus crypticus (survival and reproduction). Under the experimental conditions used to simulate accidental discharges into soil, runoff waters were highly toxic to the standard test organisms, except for C. elegans. Overall, results point out the usefulness of the yeast assay to provide a rapid preview of the toxicity level in preliminary screenings of environmental samples in situations of inadvertent high pesticide contamination. Advantages and limitations of this novel method are discussed.

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.