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.

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.

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.

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.