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Applied and Environmental Microbiology Feb 2016Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis....
Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate p-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated in vivo and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.
Topics: Animals; Bacterial Proteins; Biofilms; Burkholderia cenocepacia; Gene Deletion; Gene Expression Regulation, Bacterial; Humans; Larva; Macrophages; Mice; Moths; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; RAW 264.7 Cells; Virulence
PubMed: 26590274
DOI: 10.1128/AEM.03513-15 -
Microbial Cell (Graz, Austria) Sep 2017Opportunistic pathogens are a worldwide cause of mortality and morbidity, and infections with intrinsically antibiotic-resistant pathogens have a large clinical, social...
Opportunistic pathogens are a worldwide cause of mortality and morbidity, and infections with intrinsically antibiotic-resistant pathogens have a large clinical, social and economic impact. Bacteria belonging to the complex (Bcc), ubiquitous in natural and industrial environments, are notorious pathogens for individuals with cystic fibrosis (CF). In addition, is emerging as the culprit of non-CF related, sometimes fatal infections. Knowledge of the underlying infection mechanism of these pathogens is important for efficient treatment, however, to date not much is known about the lifestyle of Bcc bacteria during infection. In our recent study published in PLoS Pathogens, we provide experimental evidence that macrophages are critically important for proliferation of , and are major drivers of fatal pro-inflammatory infections in zebrafish larvae. This is in agreement with recent clinical information showing that is mainly localised in phagocytes in infected CF lungs. A predominant intramacrophage stage and a host-detrimental role for macrophages have major implications for treatment strategies of both CF and non-CF infections. Intracellular survival of bacteria traditionally classified as extracellular, including and , is an emerging theme. Our finding that macrophages are essential for proliferation of in the host suggests a new paradigm for Bcc infections and urges the development of novel anti-infectious therapies to efficiently disarm these intrinsically antibiotic resistant facultative intracellular pathogens.
PubMed: 29082233
DOI: 10.15698/mic2017.10.595 -
Carbohydrate Research Jan 2021Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic...
Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex, a group of 22 closely related species both of clinical and environmental origin, infecting cystic fibrosis patients. B. cenocepacia accounts for the majority of the clinical isolates, comprising the most virulent and transmissible strains. The capacity to form biofilms is among the many virulence determinants of B. cenocepacia, a characteristic that confers enhanced tolerance to some antibiotics, desiccation, oxidizing agents, and host defenses. Exopolysaccharides are a major component of biofilm matrices, particularly providing mechanical stability to biofilms. Recently, a water-insoluble exopolysaccharide produced by B. cenocepacia H111 in biofilm was characterized. In the present study, a water-soluble exopolysaccharide was extracted from B. cenocepacia H111 biofilm, and its structure was determined by GLC-MS, NMR and ESI-MS. The repeating unit is a linear rhamno-tetrasaccharide with 50% replacement of a 3-α-L-Rha with a α-3-L-Man. [2)-α-L-Rhap-(1→3)-α-L-[Rhap or Manp]-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→] Molecular modelling was used to obtain information about local structural motifs which could give information about the polysaccharide conformation.
Topics: Biofilms; Burkholderia cenocepacia; Carbohydrate Conformation; Mannose; Models, Molecular; Polysaccharides, Bacterial; Rhamnose
PubMed: 33440288
DOI: 10.1016/j.carres.2020.108231 -
MSphere Jul 2020Respiratory tract infections by the opportunistic pathogen often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these...
Respiratory tract infections by the opportunistic pathogen often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in strains J2315 and K56-2 and their role in regulating gene expression. predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of , including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of genes involved in biofilm formation, cell aggregation, and motility. CF patients diagnosed with infections often experience rapid deterioration of lung function, known as cepacia syndrome. has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle infections in CF patients.
Topics: Animals; Bacterial Proteins; Biofilms; Burkholderia cenocepacia; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Bacterial; Larva; Methyltransferases; Moths; Movement; Virulence
PubMed: 32669472
DOI: 10.1128/mSphere.00455-20 -
Microbiology Spectrum May 2024Across the Burkholderia genus -linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in...
UNLABELLED
Across the Burkholderia genus -linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in complex species, such as , 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 glycoproteome. Assessing the 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 using Data-Independent Acquisition (DIA) revealing the 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 Δ (ΔBCAL0525), Δ (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 Δ. 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 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
is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of 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 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 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 glycosylation can be dispensable for protein function and may influence protein properties beyond stability.
PubMed: 38709084
DOI: 10.1128/spectrum.00346-24 -
BMC Microbiology Mar 2017Genomic islands (GIs) are genomic regions that reveal evidence of horizontal DNA transfer. They can code for many functions and may augment a bacterium's adaptation to...
BACKGROUND
Genomic islands (GIs) are genomic regions that reveal evidence of horizontal DNA transfer. They can code for many functions and may augment a bacterium's adaptation to its host or environment. GIs have been identified in strain J2315 of Burkholderia cenocepacia, whereas in strain AU 1054 there has been no published works on such regions according to our text mining and keyword search in Medline.
RESULTS
In this study, we identified 21 GIs in AU 1054 by combining two computational tools. Feature analyses suggested that the predictions are highly reliable and hence illustrated the advantage of joint predictions by two independent methods. Based on putative virulence factors, four GIs were further identified as pathogenicity islands (PAIs). Through experiments of gene deletion mutants in live bacteria, two putative PAIs were confirmed, and the virulence factors involved were identified as lipA and copR. The importance of the genes lipA (from PAI 1) and copR (from PAI 2) for bacterial invasion and replication indicates that they are required for the invasive properties of B. cenocepacia and may function as virulence determinants for bacterial pathogenesis and host infection.
CONCLUSIONS
This approach of in silico prediction of GIs and subsequent identification of potential virulence factors in the putative island regions with final validation using wet experiments could be used as an effective strategy to rapidly discover novel virulence factors in other bacterial species and strains.
Topics: A549 Cells; Bacterial Adhesion; Bacterial Proteins; Base Composition; Burkholderia Infections; Burkholderia cenocepacia; Cell Culture Techniques; Colony Count, Microbial; Computational Biology; DNA, Bacterial; Gene Deletion; Gene Transfer, Horizontal; Genes, Bacterial; Genome, Bacterial; Genomic Islands; Genomics; Humans; Virulence Factors
PubMed: 28347342
DOI: 10.1186/s12866-017-0986-6 -
Microbiology (Reading, England) Mar 2021Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a...
Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in , an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGC sequences. Methylation motif GCGGCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in pG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when strains J2315 and K56-2 were compared, which suggests that GGC methylation patterns in are strain-specific. It seems likely that 4mC methylation of GGC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of .
Topics: Burkholderia Infections; Burkholderia cenocepacia; Cytosine; DNA Methylation; DNA, Bacterial; Genome, Bacterial; Humans; Whole Genome Sequencing
PubMed: 33565960
DOI: 10.1099/mic.0.001027 -
Applied and Environmental Microbiology Apr 2019Quorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities....
Quorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities. employs a molecular mechanism in which the -2-dodecenoic acid (named iffusible ignal actor [BDSF]) QS system regulates -acyl homoserine lactone (AHL) signal production and virulence by modulating intracellular levels of cyclic diguanosine monophosphate (c-di-GMP). Thus, inhibition of BDSF signaling may offer a non-antibiotic-based therapeutic strategy against BDSF-regulated bacterial infections. In this study, we report the synthesis of small-molecule mimics of the BDSF signal and evaluate their ability to inhibit BDSF QS signaling in A novel structural analogue of BDSF, 14-Me-C (-14-methylpentadec-2-enoic acid), was observed to inhibit BDSF production and impair BDSF-regulated phenotypes in , including motility, biofilm formation, and virulence, while it did not inhibit the growth rate of this pathogen. 14-Me-C also reduced AHL signal production. Genetic and biochemical analyses showed that 14-Me-C inhibited the production of the BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Notably, 14-Me-C attenuated BDSF-regulated phenotypes in various species. These findings suggest that 14-Me-C could potentially be developed as a new therapeutic agent against pathogenic species by interfering with their QS signaling. is an important opportunistic pathogen which can cause life-threatening infections in susceptible individuals, particularly in cystic fibrosis and immunocompromised patients. It usually employs two types of quorum sensing (QS) systems, including the -2-dodecenoic acid (BDSF) system and -acyl homoserine lactone (AHL) system, to regulate virulence. In this study, we have designed and identified an unsaturated fatty acid compound (-14-methylpentadec-2-enoic acid [14-Me-C]) that is capable of interfering with QS signaling and virulence. We demonstrate that 14-Me-C reduced BDSF and AHL signal production in It also impaired QS-regulated phenotypes in various species. These results suggest that 14-Me-C could interfere with QS signaling in many species and might be developed as a new antibacterial agent.
Topics: Acyl-Butyrolactones; Bacterial Proteins; Biofilms; Burkholderia Infections; Burkholderia cenocepacia; Cyclic GMP; Fatty Acids, Monounsaturated; Gene Expression Regulation, Bacterial; Microbial Sensitivity Tests; Phenotype; Quorum Sensing; Signal Transduction; Virulence
PubMed: 30770405
DOI: 10.1128/AEM.00105-19 -
BioMed Research International 2018is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. survives in macrophages...
is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. survives in macrophages in membrane-bound vacuoles; however, the mechanism by which gains entry into macrophages remains unknown. After macrophage internalization, survival of within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of to enter macrophages through the induction of macropinocytosis.
Topics: Animals; Bacterial Secretion Systems; Burkholderia cenocepacia; Female; Macrophages; Mice, Inbred BALB C; Pinocytosis
PubMed: 29850514
DOI: 10.1155/2018/4271560 -
Frontiers in Microbiology 2020There is an urgent need for new antimicrobials to treat the opportunistic Gram-negative , which represents a problematic challenge for cystic fibrosis patients....
There is an urgent need for new antimicrobials to treat the opportunistic Gram-negative , which represents a problematic challenge for cystic fibrosis patients. Recently, a benzothiadiazole derivative, C109, was shown to be effective against the infections caused by and other Gram-negative and-positive bacteria. C109 has a promising cellular target, the cell division protein FtsZ, and a recently developed PEGylated formulation make it an attractive molecule to counteract infections. However, the ability of efflux pumps to extrude it out of the cell represents a limitation for its use. Here, more than 50 derivatives of C109 were synthesized and tested against Gram-negative species and the Gram-positive . In addition, their activity was evaluated on the purified FtsZ protein. The chemical, metabolic and cellular stability of C109 has been assayed using different biological systems, including quantitative single-cell imaging. However, no further improvement on C109 was achieved, and the role of efflux in resistance was further confirmed. Also, a novel nitroreductase that can inactivate the compound was characterized, but it does not appear to play a role in natural resistance. All these data allowed a deep characterization of the compound, which will contribute to a further improvement of its properties.
PubMed: 32318042
DOI: 10.3389/fmicb.2020.00562