scholarly journals Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Cameron C. Oppy ◽  
Leila Jebeli ◽  
Miku Kuba ◽  
Clare V. Oates ◽  
Richard Strugnell ◽  
...  

ABSTRACT O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins. IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.

2019 ◽  
Author(s):  
Cameron C. Oppy ◽  
Leila Jebeli ◽  
Miku Kuba ◽  
Clare V. Oates ◽  
Richard Strugnell ◽  
...  

AbstractO-linked protein glycosylation is a conserved feature of the Burkholderia genus. For Burkholderia cenocepacia, the addition of the trisaccharide β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc to membrane exported proteins is required for virulence and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. This study demonstrates that the global B. cenocepacia proteome undergoes dramatic changes consistent with alterations in global transcriptional regulation in the absence of glycosylation. Using luciferase reporter assays and DNA cross-linking analysis, we confirm the repression of the master quorum sensing regulon CepR/I in response to the loss of glycosylation, which leads to the abolition of biofilm formation, defects in siderophore production, and reduced virulence. The abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants except for BCAL1086 and BCAL2974, which were found in reduced amount, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, as well as for reduced virulence and siderophore production. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the CepR/I regulon, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins.IMPORTANCEProtein glycosylation is increasingly recognised as a common protein modification in bacterial species. Despite this commonality our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that virulence attenuation observed within glycosylation-null B. cenocepacia strains are consistent with alteration of the master virulence regulator CepR. The repression of CepR transcription and its associated phenotypes support a model in which the virulence defects observed in glycosylation-null strains are at least in part due to transcriptional alteration and not the direct result of the loss of glycosylation per-se. This research unravels the pleotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.


2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Reed M. Stubbendieck ◽  
Paul D. Straight

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.


2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Erin R. Murphy ◽  
Johanna Roßmanith ◽  
Jacob Sieg ◽  
Megan E. Fris ◽  
Hebaallaha Hussein ◽  
...  

ABSTRACT RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae. First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.


2016 ◽  
Vol 198 (19) ◽  
pp. 2643-2650 ◽  
Author(s):  
Boo Shan Tseng ◽  
Charlotte D. Majerczyk ◽  
Daniel Passos da Silva ◽  
Josephine R. Chandler ◽  
E. Peter Greenberg ◽  
...  

ABSTRACTMembers of the genusBurkholderiaare known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterizedBurkholderia thailandensisbiofilm development under flow conditions and sought to determine whether QS contributes to this process.B. thailandensisbiofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by “dome” structures filled with biofilm matrix material. We showed that this process was dependent on QS.B. thailandensishas three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the threeB. thailandensisQS systems, we show that QS-1 is required for proper biofilm development, since abtaR1mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. ThebtaR1mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions.IMPORTANCEThe saprophyteBurkholderia thailandensisis a close relative of the pathogenic bacteriumBurkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms,B. thailandensisis an ideal model organism for investigating questions inBurkholderiaphysiology. In this study, we characterizedB. thailandensisbiofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows thatB. thailandensisproduces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience ofB. thailandensisbiofilms against changes in the nutritional environment.


2005 ◽  
Vol 71 (9) ◽  
pp. 5208-5218 ◽  
Author(s):  
Kerry L. Tomlin ◽  
Rebecca J. Malott ◽  
Gordon Ramage ◽  
Douglas G. Storey ◽  
Pamela A. Sokol ◽  
...  

ABSTRACT Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.


2017 ◽  
Vol 83 (24) ◽  
Author(s):  
M. Slany ◽  
J. Oppelt ◽  
L. Cincarova

ABSTRACT Staphylococcus aureus is a common biofilm-forming pathogen. Low doses of disinfectants have previously been reported to promote biofilm formation and to increase virulence. The aim of this study was to use transcriptome sequencing (RNA-seq) analysis to investigate global transcriptional changes in S. aureus in response to sublethal concentrations of the commonly used food industry disinfectants ethanol (EtOH) and chloramine T (ChT) and their combination (EtOH_ChT) in order to better understand the effects of these agents on biofilm formation. Treatment with EtOH and EtOH_ChT resulted in more significantly altered expression profiles than treatment with ChT. Our results revealed that EtOH and EtOH_ChT treatments enhanced the expression of genes responsible for regulation of gene expression (sigB), cell surface factors (clfAB), adhesins (sdrDE), and capsular polysaccharides (cap8EFGL), resulting in more intact biofilm. In addition, in this study we were able to identify the pathways involved in the adaptation of S. aureus to the stress of ChT treatment. Further, EtOH suppressed the effect of ChT on gene expression when these agents were used together at sublethal concentrations. These data show that in the presence of sublethal concentrations of tested disinfectants, S. aureus cells trigger protective mechanisms and try to cope with them. IMPORTANCE So far, the effect of disinfectants is not satisfactorily explained. The presented data will allow a better understanding of the mode of disinfectant action with regard to biofilm formation and the ability of bacteria to survive the treatment. Such an understanding could contribute to the effort to eliminate possible sources of bacteria, making disinfectant application as efficient as possible. Biofilm formation plays significant role in the spread and pathogenesis of bacterial species.


2012 ◽  
Vol 80 (5) ◽  
pp. 1681-1689 ◽  
Author(s):  
Ane Mohn Bjelland ◽  
Henning Sørum ◽  
Daget Ayana Tegegne ◽  
Hanne C. Winther-Larsen ◽  
Nils Peder Willassen ◽  
...  

ABSTRACTVibrio(Aliivibrio)salmonicidais the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth ofV. salmonicidain the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome ofV. salmonicidaLFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS inV. fischeri, was deleted. Compared to the parental strain, thelitRmutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, thelitRmutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with thelitRmutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.


2015 ◽  
Vol 197 (13) ◽  
pp. 2104-2111 ◽  
Author(s):  
Izumi Mashima ◽  
Futoshi Nakazawa

Dental plaque is a multispecies oral biofilm, the development of which is initiated by adherence of the pioneerStreptococcusspp. OralVeillonellaspp., includingV. atypica,V. denticariosi,V. dispar,V. parvula,V. rogosae, andV. tobetsuensis, are known as early colonizers in oral biofilm formation. These species have been reported to coaggregate withStreptococcusspp. in a metabolic cooperation-dependent manner to form biofilms in human oral cavities, especially in the early stages of biofilm formation. However, in our previous study,Streptococcus gordoniishowed biofilm formation to the greatest extent in the presence ofV. tobetsuensis, without coaggregation between species. These results suggest thatV. tobetsuensisproduces signaling molecules that promote the proliferation ofS. gordoniiin biofilm formation. It is well known in many bacterial species that the quorum-sensing (QS) system regulates diverse functions such as biofilm formation. However, little is known about the QS system with autoinducers (AIs) with respect toVeillonella and Streptococcusspp. Recently, autoinducer 1 (AI-1) and AI-2 were detected and identified in the culture supernatants ofV. tobetsuensisas strong signaling molecules in biofilm formation withS. gordonii. In particular, the supernatant fromV. tobetsuensisshowed the highest AI-2 activity among 6 oralVeillonellaspecies, indicating that AIs, mainly AI-2, produced byV. tobetsuensismay be important factors and may facilitate biofilm formation ofS. gordonii. Clarifying the mechanism that underlies the QS system betweenS. gordoniiandV. tobetsuensismay lead to the development of novel methods for the prevention of oral infectious diseases caused by oral biofilms.


mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Christopher L. Hager ◽  
Nancy Isham ◽  
Kory P. Schrom ◽  
Jyotsna Chandra ◽  
Thomas McCormick ◽  
...  

ABSTRACTDysbiosis of the gut microbiome has been implicated in inflammatory bowel diseases. We have shown that levels ofCandida tropicalis, along with those ofEscherichia coliandSerratia marcescens, are significantly elevated in Crohn’s disease (CD) patients. Here, we evaluated the ability of a novel probiotic to prevent and treat polymicrobial biofilms (PMB) formed byC. tropicaliswithE. coliandS. marcescens. SinceCandida albicanshas been reported to be elevated in CD patients, we investigated the interactions ofC. albicanswith these bacterial species in biofilm formation. We determined whether the interaction betweenCandidaspp. and bacteria is specific by usingTrichosporon inkinandSaccharomyces fibuligeraas comparators. Additionally, the effects of probiotics onC. albicansgermination and biofilm formation were determined. To determine the ability of the probiotic to prevent or treat mature biofilms, probiotic filtrate was added to the PMB at early (prevention) and mature (treatment) phases. Biofilm thickness and architecture were assessed by confocal scanning laser microscopy. The effects of the probiotic on germination were evaluated in the presence of serum. Exposure ofC. tropicalisPMB to probiotic filtrate reduced biofilm matrix, decreased thickness, and inhibited hyphal formation. We showed thatC. albicansorC. tropicalisformed significantly thicker PMB than control biofilms, indicating that this interaction isCandidaspecific. Treatment with probiotic filtrate inhibitedC. albicansgermination and prevented/treatedC. albicansPMB. The designed probiotic may have utility in the management of biofilm-associated gastrointestinal diseases such as Crohn’s and colorectal cancer.IMPORTANCEThe effects of diversity of the gut microbiome on inflammation have centered mainly on bacterial flora. Recent research has implicated fungal species and their interactions with other organisms in the inflammatory process. New ways to restore microbial balance in the gut are being explored. Our goal was to identify beneficial probiotic strains that would antagonize these fungal and bacterial pathogens that are elevated in the inflamed gut, and which also have antibiofilm activity. Fungus-bacterium correlation analysis allowed us to identify candidate probiotic species that can antagonize microbial pathogens, which we subsequently incorporated into a novel probiotic formulation. Amylase, which is known to have some antibiofilm activity, was also added to the probiotic mixture. This novel probiotic may have utility for the management of inflammatory bowel diseases by disrupting polymicrobial biofilm formation.


2016 ◽  
Vol 63 (6) ◽  
pp. 477-489 ◽  
Author(s):  
R.P. George ◽  
U. Kamachi Mudali ◽  
Baldev Raj

Purpose The purpose of this paper is to study the metal-Microbe interaction playing a crucial role in microbiologically influenced corrosion (MIC) and biofouling of materials in cooling water systems. Treatment regimens should be planned based on this understanding. Design/methodology/approach Attempts were made in the past decades to characterize and understand biofilm formation on important power plant structural materials such as carbon steel (CS), stainless steel (SS) and titanium in fresh water and in seawater to achieve better control of biofouling and minimize MIC problems. Findings This report presents the results of detailed studies on tuberculation-formed CS because of the action of iron-oxidizing bacteria and the effects of algae- and bacteria-dominated biofilms on the passivity of SS. The preferential adhesion of different bacterial species on SS under the influence of inclusions and sensitization was studied in the context of preferential corrosion of SS weldments due to microbial action. Detailed characterization of biofilms formed on titanium (the likely condenser material for fast breeder reactors) after exposure for two years in Kalpakkam coastal waters revealed intense biofouling and biomineralization of manganese even in chlorinated seawater. Studies on the effectiveness of conventional fouling control strategies were also evaluated. Originality/value The detailed studies of different metal/biofilm/microbe interactions demonstrated the physiological diversity of microbes in the biofilms that were formed on different materials, coupling their cooperative metabolic activities with consequent corrosion behaviour. These interactions could enhance either anodic or cathodic reactions and exploit metallurgical features that enhance biofilm formation and/or the capacity of microbes to mutate and overcome mitigation measures.


Sign in / Sign up

Export Citation Format

Share Document