RNA-Seq reveals that Pseudomonas aeruginosa mounts growth medium-dependent competitive responses when sensing diffusible cues from Burkholderia cenocepacia.

Most habitats host diverse bacterial communities, offering opportunities for inter-species interactions. While competition might often dominate such interactions, little is known about whether bacteria can sense competitors and mount adequate responses. The competition sensing hypothesis proposes that bacteria can use cues such as nutrient stress and cell damage to prepare for battle. Here, we tested this hypothesis by measuring transcriptome changes in Pseudomonas aeruginosa exposed to the supernatant of its competitor Burkholderia cenocepacia. We found that P. aeruginosa exhibited significant growth-medium-dependent transcriptome changes in response to competition. In an iron-rich medium, P. aeruginosa upregulated genes encoding the type-VI secretion system and the siderophore pyoverdine, whereas genes encoding phenazine toxins and hydrogen cyanide were upregulated under iron-limited conditions. Moreover, general stress response and quorum sensing regulators were upregulated upon supernatant exposure. Altogether, our results reveal nuanced competitive responses of P. aeruginosa when confronted with B. cenocepacia supernatant, integrating both environmental and social cues.

Glyphosate resistance and biodegradation by Burkholderia cenocepacia CEIB S5-2.

Glyphosate is a broad spectrum and non-selective herbicide employed to control different weeds in agricultural and urban zones and to facilitate the harvest of various crops. Currently, glyphosate-based formulations are the most employed herbicides in agriculture worldwide. Extensive use of glyphosate has been related to environmental pollution events and adverse effects on non-target organisms, including humans. Reducing the presence of glyphosate in the environment and its potential adverse effects requires the development of remediation and treatment alternatives. Bioremediation with microorganisms has been proposed as a feasible alternative for treating glyphosate pollution. The present study reports the glyphosate resistance profile and degradation capacity of the bacterial strain Burkholderia cenocepacia CEIB S5-2, isolated from an agricultural field in Morelos-México. According to the agar plates and the liquid media inhibition assays, the bacterial strain can resist glyphosate exposure at high concentrations, 2000 mg·L-1. In the degradation assays, the bacterial strain was capable of fast degrading glyphosate (50 mg·L-1) and the primary degradation metabolite aminomethylphosphonic acid (AMPA) in just eight hours. The analysis of the genomic data of B. cenocepacia CEIB S5-2 revealed the presence of genes that encode enzymes implicated in glyphosate biodegradation through the two metabolic pathways reported, sarcosine and AMPA. This investigation provides novel information about the potential of species of the genus Burkholderia in the degradation of the herbicide glyphosate and its main degradation metabolite (AMPA). Furthermore, the analysis of genomic information allowed us to propose for the first time a metabolic route related to the degradation of glyphosate in this bacterial group. According to the findings of this study, B. cenocepacia CEIB S5-2 displays a great glyphosate biodegradation capability and has the potential to be implemented in glyphosate bioremediation approaches.

Mutation of hmgA, encoding homogentisate 1,2-dioxygenase, is responsible for pyomelanin production but does not impact the virulence of Burkholderia cenocepacia in a chronic granulomatous disease mouse lung infection.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

Glycoproteomic and proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility.

Across the Burkholderia genus O-linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in Burkholderia cepacia complex species, such as Burkholderia cenocepacia, little is known about how specific glycosylation sites impact protein functionality. Within this study, we sought to improve our understanding of the breadth, dynamics, and requirement for glycosylation across the B. cenocepacia O-glycoproteome. Assessing the B. cenocepacia glycoproteome across different culture media using complementary glycoproteomic approaches, we increase the known glycoproteome to 141 glycoproteins. Leveraging this repertoire of glycoproteins, we quantitively assessed the glycoproteome of B. cenocepacia using Data-Independent Acquisition (DIA) revealing the B. cenocepacia glycoproteome is largely stable across conditions with most glycoproteins constitutively expressed. Examination of how the absence of glycosylation impacts the glycoproteome reveals that the protein abundance of only five glycoproteins (BCAL1086, BCAL2974, BCAL0525, BCAM0505, and BCAL0127) are altered by the loss of glycosylation. Assessing ΔfliF (ΔBCAL0525), ΔmotB (ΔBCAL0127), and ΔBCAM0505 strains, we demonstrate the loss of FliF, and to a lesser extent MotB, mirror the proteomic effects observed in the absence of glycosylation in ΔpglL. While both MotB and FliF are essential for motility, we find loss of glycosylation sites in MotB or FliF does not impact motility supporting these sites are dispensable for function. Combined this work broadens our understanding of the B. cenocepacia glycoproteome supporting that the loss of glycoproteins in the absence of glycosylation is not an indicator of the requirement for glycosylation for protein function. IMPORTANCE: Burkholderia cenocepacia is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of B. cenocepacia gained over the last 20 years a complete understanding of the proteome and especially the O-glycoproteome, is lacking. In this study, we utilize systems biology approaches to expand the known B. cenocepacia glycoproteome as well as track the dynamics of glycoproteins across growth phases, culturing media and in response to the loss of glycosylation. We show that the glycoproteome of B. cenocepacia is largely stable across conditions and that the loss of glycosylation only impacts five glycoproteins including the motility associated proteins FliF and MotB. Examination of MotB and FliF shows, while these proteins are essential for motility, glycosylation is dispensable. Combined this work supports that B. cenocepacia glycosylation can be dispensable for protein function and may influence protein properties beyond stability.

Regulation of Burkholderia cenocepacia virulence by the fatty acyl-CoA ligase DsfR as a response regulator of quorum sensing signal.

Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia cenocepacia virulence as a cis-2-dodecenoic acid (BDSF) QS signal receptor. Here, we report that the fatty acyl-CoA ligase DsfR (BCAM2136), which efficiently catalyzes in vitro synthesis of lauryl-CoA and oleoyl-CoA from lauric acid and oleic acid, respectively, acts as a global transcriptional regulator to control B. cenocepacia virulence by sensing BDSF. We show that BDSF binds to DsfR with high affinity and enhances the binding of DsfR to the promoter DNA regions of target genes. Furthermore, we demonstrate that the homolog of DsfR in B. lata, RS02960, binds to the target gene promoter, and perception of BDSF enhances the binding activity of RS02960. Together, these results provide insights into the evolved unusual functions of DsfR that control bacterial virulence as a response regulator of QS signal.

Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device.

DNA is a component of biofilms, but the triggers of DNA release during biofilm formation and how DNA contributes to biofilm development are poorly investigated. One key mechanism involved in DNA release is explosive cell lysis, which is a consequence of prophage induction. In this article, the role of explosive cell lysis in biofilm formation was investigated in the opportunistic human pathogen Burkholderia cenocepacia H111 (H111). Biofilm streamers, flow-suspended biofilm filaments, were used as a biofilm model in this study, as DNA is an essential component of their matrix. H111 contains three prophages on chromosome 1 of its genome, and the involvement of each prophage in causing explosive cell lysis of the host and subsequent DNA and membrane vesicle (MV) release, as well as their contribution to streamer formation, were studied in the presence and absence of genotoxic stress. The results show that two of the three prophages of H111 encode functional lytic prophages that can be induced by genotoxic stress and their activation causes DNA and MVs release by explosive cell lysis. Furthermore, it is shown that the released DNA enables the strain to develop biofilm streamers, and streamer formation can be enhanced by genotoxic stress. Overall, this study demonstrates the involvement of prophages in streamer formation and uncovers an often-overlooked problem with the use of antibiotics that trigger the bacterial SOS response for the treatment of bacterial infections.

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.

A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: Implications for chronic infection in cystic fibrosis.

Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low-oxygen activated (lxa) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis (CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa-encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56-2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild-type and complement strains but not the Δusp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.

The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB.

The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the Burkholderia diffusible signaling factor (BDSF) quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia. An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators like berA and berB restored the wild-type smooth colony morphology. We found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of a diguanylate cyclase or a c-di-GMP phosphodiesterase, respectively. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Except for rpfR, deletion mutants of all 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia showed no change to berA and bep gene expression. Moreover, bacterial two-hybrid assays provided evidence that RpfR and BerB physically interact and give specificity to the regulation of the bep genes. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA, and the BerA protein subsequently activates transcription of the bep genes.

Targeting a Multidrug-Resistant Pathogen: First Generation Antagonists of Burkholderia cenocepacia's BC2L-C Lectin.

Multidrug-resistant pathogens such as Burkholderia cenocepacia have become a hazard in the context of healthcare-associated infections, especially for patients admitted with cystic fibrosis or immuno-compromising conditions. Like other opportunistic Gram-negative bacteria, this pathogen establishes virulence and biofilms through lectin-mediated adhesion. In particular, the superlectin BC2L-C is believed to cross-link human epithelial cells to B. cenocepacia during pulmonary infections. We aimed to obtain glycomimetic antagonists able to inhibit the interaction between the N-terminal domain of BC2L-C (BC2L-C-Nt) and its target fucosylated human oligosaccharides. In a previous study, we identified by fragment virtual screening and validated a small set of molecular fragments that bind BC2L-C-Nt in the vicinity of the fucose binding site. Here, we report the rational design and synthesis of bifunctional C- or N-fucosides, generated by connecting these fragments to a fucoside core using a panel of rationally selected linkers. A modular route starting from two key fucoside intermediates was implemented for the synthesis, followed by evaluation of the new compounds as BC2L-C-Nt ligands with a range of techniques (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR, differential scanning calorimetry, and X-ray crystallography). This study resulted in a hit molecule with an order of magnitude gain over the starting methyl fucoside and in two crystal structures of antagonist/lectin complexes.

Oridonin Attenuates Burkholderia cenocepacia Virulence by Suppressing Quorum-Sensing Signaling.

Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and pathogenicity: the cis-2-dodecenoic acid (BDSF) system and the N-acyl homoserine lactone (AHL) system. In this study, we reported that oridonin, which was screened from a collection of natural products, disrupted important B. cenocepacia phenotypes, including motility, biofilm formation, protease production, and virulence. Genetic and biochemical analyses showed that oridonin inhibited the production of BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Furthermore, we revealed that oridonin directly binds to the regulator RqpR of the two-component system RqpSR that dominates the above-mentioned QS systems to inhibit the expression of the BDSF and AHL signal synthase-encoding genes. Oridonin also binds to the transcriptional regulator CepR of the cep AHL system to inhibit its binding to the promoter of bclACB. These findings suggest that oridonin could potentially be developed as a new QS inhibitor against pathogenic B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important human opportunistic pathogen that can cause life-threatening infections in susceptible individuals. It employs quorum-sensing (QS) systems to regulate biological functions and virulence. In this study, we have identified a lead compound, oridonin, that is capable of interfering with B. cenocepacia QS signaling and physiology. We demonstrate that oridonin suppressed cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) signal production and attenuated virulence in B. cenocepacia. Oridonin also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that oridonin could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.

In Silico and In Vitro Analysis of Helicobacter pullorum Type Six Secretory Protein Hcp and Its Role in Bacterial Invasion and Pathogenesis.

Helicobacter pullorum is a human zoonotic pathogen transmitted through poultry where it is associated with vibrionic hepatitis and colitis. Hemolysin co-regulated protein (Hcp) is an important structural as well as effector protein of type six secretory system; however, its role in H. pullorum invasion and pathogenesis has not been elucidated. In this study, we predicted the Helicobacter pullorum Hcp (HpuHcp) structure and identified Campylobacter jejuni Hcp (CjHcp) as its nearest homologue. Analysis of the predicted structure shows several common bacterial Hcp motifs like Protein kinase C phosphorylation site, Casein kinase II phosphorylation site, N-myristoylation site, cAMP-and cCGMP-dependent protein kinase phosphorylation site, N-glycosylation site. The presence of unique microbodies C-terminal targeting signal domain was present in HpuHcp which was seen for the first time in CjHcp. This could indicate that Hcp is a structural protein as well as a secretory protein. Moreover, the presence of a deamidase domain, similar to the tecA of Burkholderia cenocepacia an opportunistic pathogen, may help in bacterial internalization as it depolymerises the membranous actin by deamidation of the host cell Rho GTPases cdc42 and Rac1, which was supported by increased invasion of hepatocytes by Hcp-positive isolates.

A Bacterial Mannose Binding Lectin as a Tool for the Enrichment of C- and O-Mannosylated Peptides.

Mass spectrometry (MS) easily detects C-mannosylated peptides from purified proteins but not from complex biological samples. Enrichment of specific glycopeptides by lectin affinity prior to MS analysis has been widely applied to support glycopeptide identification but was until now not available for C-mannosylated peptides. Here, we used the α-mannose-specific Burkholderia cenocepacia lectin A (BC2L-A) and show that, in addition to its previously demonstrated high-mannose N-glycan binding capability, this lectin is able to retain C- and O-mannosylated peptides. Besides testing binding abilities to standard peptides, we applied BC2L-A affinity to enrich C-mannosylated peptides from complex samples of tryptic digests of HEK293 and MCF10A whole cell extracts, which led to the identification of novel C-mannosylation sites. In conclusion, BC2L-A enabled specific enrichment of C- and O-mannosylated peptides and might have superior properties over other mannose binding lectins for this purpose.

Pellicle Biofilm Formation in Burkholderia cenocepacia J2315 is Epigenetically Regulated through WspH, a Hybrid Two-Component System Kinase-Response Regulator.

The chemosensory signal transduction system Wsp regulates biofilm formation and related phenotypes by influencing cyclic-di-GMP (c-di-GMP) levels in bacterial cells. This is typically achieved by activation of the diguanylate cyclase WspR, through phosphorylation of its phosphoreceiver domain. The Wsp system of Burkholderia cenocepacia J2315 is in one operon with the hybrid response regulator/histidine kinase wspH, but lacks the diguanylate cyclase wspR which is located in a different operon. The expression of wspH, the first gene in the B. cenocepacia Wsp operon as well as pellicle biofilm formation are epigenetically regulated in B. cenocepacia J2315. To investigate whether WspH regulates pellicle biofilm formation, several mutants with altered expression of wspH were constructed. Mutants with increased expression of wspH showed accelerated pellicle biofilm formation, reduced swimming motility and increased c-di-GMP levels. This was independent of WspR phosphorylation, showing that WspR is not the cognate response receiver for histidine kinase WspH. IMPORTANCE Biofilms are surface-attached or suspended aggregates of cells, that are problematic in the context of bacterial infections, as they provide protection from antibiotic treatment. Burkholderia cenocepacia can colonize the lung of immunocompromised patients and forms biofilms that increase its recalcitrance to antibiotic treatment. Pellicles are biofilms which form at an air-liquid interface to take advantage of the higher oxygen concentrations in this environment. How quickly pellicles are formed is crucial for the fitness of obligate aerobic bacteria such as B. cenocepacia. Cyclic-di-GMP (c-di-GMP) levels determine the transition between planktonic and biofilm lifestyle, and WspH controls c-di-GMP production. WspH is therefore important for the fitness of B. cenocepacia in environments with gradients in oxygen concentration, such as the human lung.

The anti-virulence activity of the non-mevalonate pathway inhibitor FR900098 towards Burkholderia cenocepacia is maintained during experimental evolution.

Burkholderia cenocepacia infections are difficult to treat and there is an urgent need for alternative (combination) treatments. The use of anti-virulence therapies in combination with antibiotics is a possible strategy to increase the antimicrobial susceptibility of the pathogen and to slow down the development of resistance. In the present study we evaluated the ß-lactam and colistin-potentiating activity, and anti-virulence effect of the non-mevalonate pathway inhibitor FR900098 against B. cenocepacia in various in vitro and in vivo models. In addition, we evaluated whether repeated exposure to FR900098 alone or when combined with ceftazidime leads to increased resistance. FR900098 potentiated the activity of colistin and several ß-lactam antibiotics (aztreonam, cefepime, cefotaxime, ceftazidime, mecillinam and piperacillin) but not of imipenem and meropenem. When used alone or in combination with ceftazidime, FR900098 increased the survival of infected Galleria mellonella and Caenorhabditis elegans. Furthermore, combining ceftazidime with FR900098 resulted in a significant inhibition of the biofilm formation of B. cenocepacia. Repeated exposure to FR900098 in the C. elegans infection model did not lead to decreased activity, and the susceptibility of the evolved B. cenocepacia HI2424 lineages to ceftazidime, FR900098 and the combination of both remained unchanged. In conclusion, FR900098 reduces B. cenocepacia virulence and potentiates ceftazidime in an in vivo C. elegans model, and this activity is not lost during the experimental evolution experiment carried out in the present study.

CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

The cis-2-Dodecenoic Acid (BDSF) Quorum Sensing System in Burkholderia cenocepacia.

It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, Burkholderiadiffusible signal factor (BDSF) (cis-2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research on this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.

Characterization of glyphosate-resistant Burkholderia anthina and Burkholderia cenocepacia isolates from a commercial Roundup® solution.

Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate-resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate-sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia.

Identification of genes required for gold and silver tolerance in Burkholderia cenocepacia H111 by transposon sequencing.

Members of the genus Burkholderia show remarkable abilities to adapt to a wide range of environmental conditions and is frequently isolated from soils contaminated with heavy metals. In this study, we used a transposon sequencing approach to identify 138 and 164 genes that provide a benefit for growth of the opportunistic pathogen Burkholderia cenocepacia H111 in the presence of silver and gold ions respectively. The data suggest that arginine metabolism and citrate biosynthesis are important for silver tolerance, while components of an ABC transporter (BCAL0307-BCAL0308) and de novo cysteine biosynthesis are required for tolerance to gold ions. We show that determinants that affect tolerance to both metal ions include the two-component systems BCAL0497/99 and BCAL2830/31 and genes that are involved in maintaining the integrity of the cell envelope, suggesting that membrane proteins represent important targets of silver and gold ions. Furthermore, we show that that the P-type ATPase CadA (BCAL0055), which confers tolerance to cadmium contributes to silver but not gold tolerance. Our results may be useful for improving the antibacterial effect of silver and gold ions to combat drug-resistant pathogens.

Metabolomic profiling of Burkholderia cenocepacia in synthetic cystic fibrosis sputum medium reveals nutrient environment-specific production of virulence factors.

Infections by Burkholderia cenocepacia lead to life-threatening disease in immunocompromised individuals, including those living with cystic fibrosis (CF). While genetic variation in various B. cenocepacia strains has been reported, it remains unclear how the chemical environment of CF lung influences the production of small molecule virulence factors by these strains. Here we compare metabolomes of three clinical B. cenocepacia strains in synthetic CF sputum medium (SCFM2) and in a routine laboratory medium (LB), in the presence and absence of the antibiotic trimethoprim. Using a mass spectrometry-based untargeted metabolomics approach, we identify several compound classes which are differentially produced in SCFM2 compared to LB media, including siderophores, antimicrobials, quorum sensing signals, and various lipids. Furthermore, we describe that specific metabolites are induced in the presence of the antibiotic trimethoprim only in SCFM2 when compared to LB. Herein, C13-acyl-homoserine lactone, a quorum sensing signal previously not known to be produced by B. cenocepacia as well as pyochelin-type siderophores were exclusively detected during growth in SCFM2 in the presence of trimethoprim. The comparative metabolomics approach described in this study provides insight into environment-dependent production of secondary metabolites by B. cenocepacia strains and suggests future work which could identify personalized strain-specific regulatory mechanisms involved in production of secondary metabolites. Investigations into whether antibiotics with different mechanisms of action induce similar metabolic alterations will inform development of combination treatments aimed at effective clearance of Burkholderia spp. pathogens.