Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei

Chatruthai Meethai; Muthita Vanaporn; Narin Intarak; Varintip Lerdsittikul; Patoo Withatanung; Sujintana Janesomboon; Paiboon Vattanaviboon; Sorujsiri Chareonsudjai; Toby Wilkinson; Mark P. Stevens; Joanne M. Stevens; Sunee Korbsrisate

doi:10.1371/journal.pone.0282098

Abstract

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se.

Citation: Meethai C, Vanaporn M, Intarak N, Lerdsittikul V, Withatanung P, Janesomboon S, et al. (2023) Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei. PLoS ONE 18(2): e0282098. https://doi.org/10.1371/journal.pone.0282098

Editor: Christopher Rao, University of Illinois at Urbana-Champaign, UNITED STATES

Received: April 4, 2022; Accepted: February 7, 2023; Published: February 23, 2023

Copyright: © 2023 Meethai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Meethai C. was supported by the Royal Golden Jubilee (RGJ) Ph.D. Programme (PHD/0070/2559), Thailand. Korbsrisate S., Withatanung P. and Janesomboon S. were supported by the Siriraj Grant for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University. Stevens M., Stevens J. and Wilkinson T. were supported by strategic funding from the Biotechnology & Biological Sciences Research Council of the United Kingdom (BBS/E/D/20002173). No additional external funding was received for this study.

Competing interests: The authors declare that no competing interests exist.

Introduction

Burkholderia pseudomallei is a motile Gram-negative pathogen that causes melioidosis, a severe invasive human disease endemic in Southeast Asia and Northern Australia [1]. It has been estimated that 165,000 cases of meliodiosis occur globally each year, causing 89,000 deaths [2]. B. pseudomallei is prevalent in soil and water in endemic areas, and inhalation and injury are believed to be important routes of infection. Clinical presentations vary, but frequently include acute pneumonia, septicaemia and abscess formation [3]. No vaccine is available for melioidosis and antibiotic treatment is hindered by intrinsic and transmissible drug resistances. Targeted and genome-wide mutagenesis has identified numerous virulence factors of B. pseudomallei [4, 5]. Among the key factors required for invasion, escape from endosomes and intracellular net replication of B. pseudomallei is a Type III secretion system encoded by the bsa locus [6].

Two-component sensory systems play a key role in regulation of virulence genes in Gram-negative bacteria, including pathogenic Burkholderia [7]. These comprise a histidine kinase which senses an environmental cue, and on receipt of this, phosphorylates a cognate transcriptional regulator. This typically activates the regulator, altering the transcription of genes under its control. Among the two-component systems so far implicated in the virulence of B. pseudomallei in murine models are the BprRS system [8], BPSL0127-BPSL0128 system [9] and the VirAG system that regulates a virulence-associated Type VI secretion system in response to cytosolic glutathione [10]. Here, we identified homologues of the QseBC system in B. pseudomallei, which has been implicated in virulence in diverse Gram-negative bacterial pathogens.

The QseBC system (quorum sensing E. coli regulators B and C) was first described as a regulator of motility and flagellar gene expression in enterohaemorrhagic Escherichia coli O157:H7 in response to quorum sensing [11]. It was later reported that in addition to sensing the bacterial autoinducer AI-3, QseC senses the host stress-related catecholamine hormones epinephrine and norepinephrine [12]. In addition to activating motility, epinephrine and norepinephrine were reported to enhance expression of virulence-associated genes, including a Type III secretion system associated with intestinal colonisation [12, 13]. Epinephrine and norepinephrine have further been reported to stimulate biofilm formation in E. coli in a manner that requires QseC [14] and the QseBC system has been associated with biofilm formation by mastitis-associated E. coli [15].

QseBC homologues have been identified in diverse Gram-negative pathogens, but predicting the role of these in virulence is complicated by significant disparities in mutant phenotypes observed by different laboratories. For example, in contrast to the findings of Sperandio et al. [11], Sharma et al. [16] found no impact of a qseBC deletion on motility of E. coli O157:H7 or transcription of fliC, and the mutant colonized the bovine intestines at a higher level than the parent strain in co-infection studies. Independently, Hamed et al. [17] reported that a qseC deletion did not significantly alter motility of E. coli O157:H7 in the presence or absence of epinephrine or norepinephrine. Similarly, while QseC has been reported to be required for norepinephrine-induced motility in Salmonella enterica serovar Typhimurium [18] and for epinephrine-dependent regulation of Type III secretion systems, intra-macrophage survival and virulence in this organism [19, 20], Pullinger et al. [21] did not observe such impacts. The latter authors found no defect in motility or transcription of Type III secretion in a S. Typhimurium qseC mutant, and while norepinephrine induced bacterial growth and augmented inflammatory and secretory responses in a bovine ligated ileal loop model, this occurred at a similar level without qseC. Consistent with these studies, Hamed et al. [17] reported that motility of an S. Typhimurium qseC mutant was comparable to the wild-type in the presence or absence of epinephrine, norepinephrine or dopamine.

We previously reported that epinephrine activates motility and flagellar gene expression in B. pseudomallei [22]. Given literature on the role of QseBC in this phenotype in other pathogenic bacteria, we identified homologues of QseBC, constructed an unmarked qseBC deletion mutant and analysed the impact of this on epinephrine-induced motility, biofilm formation and intracellular net replication.

Materials and methods

Bacterial strains and culture conditions

We used the prototype genome-sequenced B. pseudomallei strain K96243 [23], which has been extensively studied in our laboratories. K96243 wild-type, ΔqseBC mutant and ΔqseBC trans-complemented strains were grown in Luria-Bertani broth or agar (LB; Titan Biotech Ltd., Delhi, India) at 37°C for 24–48 h. A previously described bsaZ mutant strain was used as a control in intracellular survival assays [6]. Chloramphenicol (40 μg/mL), kanamycin (1 mg/mL) or zeocin (2 mg/mL) were used for selection where required. E. coli K-12 strains DH5α and RHO3 [24] were cultured in LB agar or broth at 37°C for 18–24 h. To support growth of E. coli RHO3, 2,6 diaminopimelic acid (DAP; Sigma, St. Louis, MO) was added to the culture medium. Chloramphenicol (30 μg/mL) and kanamycin (35 μg/mL) were added when needed for E. coli. Strain genotypes and sources are described in S1 Table.

Construction of a B. pseudomallei F044qseBC mutant

An unmarked deletion mutant of B. pseudomallei strain K96423 lacking qseBC was constructed essentially as described [24]. We used PCR to amplify a region 201-bp upstream of the gene encoding putative the QseB response regulator (bpsl0806) and 393-bp downstream of the gene encoding putative the QseC sensor kinase (bpsl0807) and then joined these by overlap extension PCR using primers described in S2 Table as previously described [24]. The resulting PCR product was cloned into the pGEM-T easy vector (Promega, USA) and the insert was then subcloned into the positive-selection suicide replicon pEXKm5 [24] by digestion with EcoRI, yielding plasmid pEXqseBC. E. coli RHO3 harboring pEXqseBC was used to introduce the plasmid into B. pseudomallei K96243 by conjugation. Merodiploids resulting from homologous recombination were selected by plating on LB agar containing 1 mg/mL kanamycin. To select for double recombinants in which the suicide replicon was lost, homing nuclease I-Sce-I-mediated gene replacement was performed. The pBADSce vector [24] was introduced into a merodiploid strain by electroporation. Clones were selected by plating on LB agar containing 2 mg/mL zeocin (Invivogen, California, USA) with 0.5% (w/v) L-arabinose and then incubated at 30°C for 36 h. After incubation, putative double recombinants were patched on LB with or without 1 mg/mL kanamycin. A kanamycin sensitive colony was grown in LB at 42°C to select for loss of the pBADSce vector. A double recombinant lacking qseBC mutant was initially identified by PCR using primers flanking the qseBC genes (S2 Table).

Whole-genome sequencing and bioinformatic analysis

Genomic DNA of the B. pseudomallei ΔqseBC mutant was extracted using a Genomic DNA mini kit (Geneaid Biotech, Taiwan). Whole-genome sequencing was performed by Illumina Miseq sequencing (San Diego, CA, USA) at the ‘Omics Sciences and Bioinformatic Center at Chulalongkorn University, Bangkok, Thailand. Quality of the sequence reads was checked using FASTQC software [25]. The average coverage of sequence reads was 100X. Sequence reads were aligned to the K96423 reference genome [23] (accession number NC_006350 and NC_006351) using BWA mem v0.7.12. Alignments to the K96243 reference genome were also used to identify single nucleotide polymorphisms (SNPs), insertions or deletions using Pilon (v1.22) [26]. Integrative Genomics Viewer was used to visualise the site of the deletion on chromosome 1. The nucleotide sequence of the K96243 ΔqseBC strain has been deposited in GenBank (accession number JAJOLO000000000). Raw sequence reads were deposited in the Short Read Archive (accession number PRJNA785800).

Homology of qseBC genes in B. pseudomallei relative to those in E. coli and other Burkholderia species was assessed by the UniProt Tool, Ident and Sim software and Simple Modular Architecture Research tool. Accession numbers of the query and target sequences used are listed in the corresponding figure legend.

Cloning of the B. pseudomallei qseBC genes for trans-complementation

The qseBC genes were amplified by with primers Com_BPSL0806-F and Com_BPSL0807-R (S2 Table), including the predicted ribosome-binding sequence 5’ of qseB to enable translation. The amplicon was digested with KpnI and XbaI restriction enzymes and cloned in the pBBR1MCS-1 broad host-range vector [27]. The sequence of the insert was confirmed to be as expected by Sanger sequencing (S1 Fig). The resulting plasmid (pBBRqseBC) was introduced into the B. pseudomallei ΔqseBC mutant by conjugation from E. coli strain RHO3 with selection for chloramphenicol resistance. Transcription of the qseBC genes in wild-type, mutant and trans-complemented strains was analysed by reverse-transcriptase PCR as described below.

Motility assays

Swimming and swarming motility assays were performed by measuring the diameter of motility zone on motility plates according to Déziel et al. [28] with some modifications. B. pseudomallei strains were grown overnight and adjusted to an optical density at 600nm (OD₆₀₀) of 0.5. Then, 3 μl of the adjusted suspension was carefully spotted onto 0.3% (w/v) or 0.5% agar plates to respectively investigate swimming and swarming, that contained yeast extract (3 g/L; Titan Biotech Ltd., Delhi, India), tryptone (5 g/L; Hardy Diagnostics, Ohio, USA), and glucose (5 g/L; Ajax Finechem, Australia), with or without 50 μM epinephrine hydrochloride (Sigma, St. Louis, MO). After inoculation, plates were incubated at 37°C for 18 h. The diameter of the zone of bacterial swimming or swarming motility was measured on the following day. Results represented the mean diameter of the motility zones. The experiment was performed with three technical replicates each performed on three separate occasions.

Biofilm formation assay

Biofilm formation was assayed essentially as described [29] with the following modifications. Overnight bacterial cultures were adjusted to a concentration of 1x10⁸ colony-forming units (CFU)/mL and then inoculated into 96-well plates with or without 50 μM epinephrine. The plates were then incubated statically at 37°C for 72 h. After incubation, non-adherent cells were removed by washing twice in phosphate-buffered saline (PBS; Sigma, St. Louis, MO). The biofilm was then fixed using 99% (v/v) methanol for 15 minutes and stained with 0.1% (w/v) crystal violet (Merck, New Jersey, USA) for 15 minutes. Finally, crystal violet was solubilized using 33% (v/v) glacial acetic acid (VWR international, Pennsylvania, USA). Optical density of the solubilized crystal violet suspension was then measured at 550 nm using a microplate reader (TECAN, Switzerland). Assays were performed with eight technical replicates on three separate occasions.

RNA extraction and real-time reverse transcriptase-PCR

B. pseudomallei strains were subcultured in 10 mL of LB broth with or without 50 μM epinephrine and grown at 37°C. Logarithmic phase cells were harvested at OD₆₀₀ of 0.5 and total RNA was extracted using a Total RNA-mini kit (Geneaid Biotech, Taipei, TW). Total RNA was treated with RNase-free DNase I (Thermo Scientific, MA, USA). We confirmed the absence of DNA by PCR using primers specific to 16S rRNA genes (S2 Table). RNA was then reverse transcribed to cDNA by using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA), according to the manufacturers instructions.

Analysis of the transcription of the flagellin gene (fliC) and qseBC gene was performed by real-time PCR using the cDNA samples obtained above and gene-specific primers (S2 Table) using a LightCycler 480 instrument (Roche diagnostics, Penzburgh, Germany). Relative gene expression was calculated using the 2^-ΔΔCt method [30] with normalization relative to 16S ribosomal RNA. To detect qseBC transcripts in wild-type, mutant or trans-complemented strains, conventional RT-PCR was performed with gene-specific primers (S2 Table) and amplicons were resolved by agarose gel electrophoresis.

Analysis of bacterial growth kinetics

B. pseudomallei K96243 wild-type, ΔqseBC mutant and ΔqseBC/pBBRqseBC trans-complemented strains were grown overnight and adjusted to reach OD₆₀₀ value of 0.17. Then, 300 μl of the adjusted bacterial suspension was then added to 15 mL of fresh LB broth. At 0, 2, 4, 8, 12 and 24 h, OD₆₀₀ measurements were taken to generate bacterial growth curves. At these same intervals, ten-fold serial dilutions of samples of the cultures were prepared and spotted onto LB agar plates. The plates were incubated at 37°C for 24–48 h and then viable bacteria were enumerated and expressed as CFU/mL. Experiments were performed with three technical replicates on three separate occasions.

Intracellular survival assay

J774A.1 murine macrophage cells were used to analyse net intracellular survival of the strains. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (HyClone, South Logan, UT) at 37°C in a humidified 5% CO₂ atmosphere. J774A.1 cells were seeded at 2x10⁵ per well in 24-well plates. They were then inoculated with B. pseudomallei wild-type or mutant strains at a multiplicity of infection of 0.1. Plates were centrifuged at 13x g for 5 minutes to drive bacterial into contact with the macrophages and obviate any effects on flagella-mediated motility. After 1 h post-inoculation, the infected cells were overlaid with DMEM supplemented with 10% (v/v) heat-inactivated foetal bovine serum containing 250 μg/mL kanamycin to eliminate extracellular bacteria. At 2, 4, 6, 8 and 24 h post-infection, the infected cells were washed with pre-warmed PBS and lysed with 0.1% (v/v) Triton X-100 (Sigma, St. Louis, MO) in PBS for 5 minutes. The viable intracellular bacteria released were enumerated following plating of serial ten-fold dilutions to LB agar plates and incubation for 24–28 h.

Statistical analysis

Statistical analysis was performed by using a student’s t-test using GraphPad Prism8 software. Differences were considered significant at P values of ≤ 0.05.

Results and discussion

B. pseudomallei BPSL0806 and BPSL0807 are homologues of E. coli QseB and QseC, respectively

BLAST analysis of the complete genome sequence of B. pseudomallei K96243 [23] using E. coli O157:H7 qseB and qseC genes as query sequences identified bpsl0806 and bpsl0807 on chromosome 1 as the closest homologues, respectively. BPSL0806 is predicted to be the response regulator and to be a 220 amino acid protein that is 50.91% identical and 66.36% similar to E. coli O157:H7 QseB over 220 amino acids. BPSL0807 is predicted to be the sensor kinase and 438 amino acids in length, sharing 28.88% identity and 48.49% similarity with E. coli O157:H7 QseC over 449 amino acids. We analysed the conservation of 8 residues that have been reported to be conserved in the predicted periplasmic domains of QseC homologues from 12 bacteria [31]. This revealed that 6 of these amino residues were shared in B. pseudomallei QseC (S2 Fig). Protein sequences of B. pseudomallei QseBC shared 99–100% identity with the corresponding proteins Burkholderia mallei ATCC23344 and 90–97% identity with those from Burkholderia thailandensis E264 (S3 Table). Across representatives of the B. cepacia complex and phytopathogenic Burkholderia, QseB exhibited at least 83% identity and 85% similarity with the B. pseudomallei protein, and QseC was at least 80% identical and 85% similar (S3 Table).

Analysis of the transcriptome of B. pseudomallei strain K96243 under 82 different conditions using a tiling whole-genome microarray indicated that the bpsl0806-0807 are transcribed [32]. In particular, the authors demonstrated that bpsl0806 is up-regulated within 1 h of nutrient-deprivation. We confirmed that a transcript spanning the qseBC genes could be detected in K96243 during growth in LB medium. This finding indicated that qseB and qseC are co-transcribed as an operon. The abundance of the transcript was unchanged in the presence of 50 μM epinephrine (S3 Fig).

Generation of B. pseudomallei qseBC mutant and trans-complemented strains

An unmarked ΔqseBC deletion mutant was constructed using a suicide replicon containing a fusion of amplicons spanning 201-bp 5’ of bpsl0806 and 393-bp 3’ of bpsl0807, as described in Materials and Methods. A putative double recombinant was confirmed to harbor the qseBC deletion by PCR with flanking primers (S4 Fig) and was then sequenced using the Illimina MiSeq platform. Sequence reads were aligned to the original K96243 reference genome, which confirmed the location of the expected deletion on chromosome 1 (S5 Fig). Importantly, we observed no unwanted rearrangements of the genome, including proximal to the deletion site, as has been reported following the use of suicide vectors for allelic exchange [33]. Sequence analysis identified a number of candidate polymorphisms and small insertions or deletions (indels) relative to the original published sequence for K96243 (S4 Table). Specifically, 44 SNPs and 9 indels were identified across chromosomes 1 and 2. The vast majority of these SNPs and indels have been previously reported following resequencing of four K96243 stocks from different laboratories [34, 35]. They affect a total of 10 genes while 12 are in intergenic regions. Just two SNPs associated with missense mutations in bpsl1036 (a hypothetical gene with homology to ompR) and bpss1755 (proximal to the C-terminus of the RpoD sigma factor), and a single nucleotide insertion predicted to cause a frameshift mutation in bpss1194, were unique relative to K96243 strains sequenced to date (S4 Table). A large number of other SNPs were detected in bpss1194 that are shared with other isolates. The extent to which polymorphisms could be due to sequencing errors in our analysis or with the reference strain is unclear.

As we cannot preclude the possibility that minor variants detected in the K96243 ΔqseBC genome sequence may contribute to its phenotypes, we cloned the qseBC genes under the control of an inducible promoter in the broad-host range vector pBBR1MCS-1 (S6A Fig). This was introduced into the mutant strain and we confirmed by reverse-transcriptase PCR that the qseBC transcript was present in the wild-type and trans-complemented strains, but absent in the ΔqseBC mutant (S6B Fig). We analysed the growth kinetics of the wild-type K96243 strain, ΔqseBC mutant and ΔqseBC/pBBRqseBC trans-complemented strain in LB broth by measuring both optical density and viable bacteria over time. Growth of the wild-type and ΔqseBC mutant strains was almost identical, indicating that polymorphisms detected by genome sequencing have not grossly affected bacterial fitness. However, we observed that growth of the ΔqseBC/pBBRqseBC strain was significantly suppressed at all the time intervals studied (S6C and S6D Fig). This is consistent with the phenotype of an E. coli O157:H7 qseBC mutant upon plasmid-mediated expression of the qseBC genes [16]. It is possible that this may be explained by recent data indicating that QseBC controls the timing of initiation of chromosomal replication in E. coli, likely by regulating expression of dnaA [36].

The B. pseudomallei qseBC genes influence motility and fliC transcription

Motility of the B. pseudomallei K96243 wild-type and ΔqseBC mutant strains was analysed on plates containing 0.3% (w/v) or 0.5% (w/v) agar to measure swimming and swarming motility, respectively. In soft agar (0.3% w/v), bacterial cells swim through the relatively fluid agar suspension while bacteria swarm over the surface when agar is present a 0.5% (w/v) [37]. We observed a significant reduction in both swimming motility (Fig 1A; c. 2.8-fold P <0.0001) and swarming motility (Fig 1B; c. 4.1-fold P = <0.0001) between the wild-type strain and ΔqseBC mutant. Moreover, we observed a reduction in transcription of the fliC gene encoding flagellin in the ΔqseBC mutant (Fig 1C; c. 132.1-fold P = <0.0001). We were unable to rescue the motility defect of the mutant strain by introduction of pBBRqseBC, likely as a consequence of markedly suppressed growth (S6C and S6D Fig). We also attempted to repair the qseBC mutant by reintroduction of the intact qseBC genes using a positive-selection suicide replicon, but were unsuccessful.

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Fig 1. QseBC homologues are required for B. pseudomallei motility.

B. pseudomallei wild-type K96243 and ΔqseBC mutant were grown on motility plates in the presence or absence 50 μM epinephrine. Motility zones after 18 h of incubation were determined. (A) 0.3% (w/v) nutrient agar was used to assess for swimming motility. (B) Swarming motility was assayed using 0.5% (w/v) nutrient agar. (C) Transcription of fliC gene was investigated by real-time reverse transcriptase PCR using B. pseudomallei wild-type K96243 and ΔqseBC mutant cultured to exponential phase in LB medium in the presence or absence 50 μM epinephrine. Three independent experiments were performed. Error bars represent standard errors of the means following students t-test. Asterisks indicate significant differences (P ≤ 0.05, t-test).

https://doi.org/10.1371/journal.pone.0282098.g001

Consistent with our previous research [22], 50 μM epinephrine significantly enhanced swimming motility Fig 1A; c. 1.3 fold P = 0.0311), swarming motility of B. pseudomallei K96243 (Fig 1B; c. 1.2-fold P = 0.0385) and transcription of fliC (Fig 1C; c. 1.7- fold P = 0.0464). Swimming motility, swarming motility and fliC transcription in the ΔqseBC mutant were near-identical in the presence and absence of 50 μM epinephrine. Thus, QseBC is required for full motility but epinephrine does not have to be present for it to regulate it. While QseBC have been reported to act as adrenergic sensors controlling motility in E. coli O157:H7 [12] and S. Typhimurium [18], other laboratories have found no defects in motility for qseBC mutants of these pathogens [16, 17, 21]. The extent to which this reflects differences in the strains, types of mutation and assay conditions remains unclear.

The B. pseudomallei qseBC genes influence biofilm production

Biofilms can contribute to bacterial pathogenesis and aid resistance to antibiotics and host defences [38]. We observed that biofilm formation by strain K96243 was significantly reduced by the ΔqseBC mutation (Fig 2; c. 4-fold P = 0.0034). It is plausible that this is a consequence of the impact of the ΔqseBC mutation on flagella expression, as a genome-wide screen of transposon mutants for defects in biofilm formation identified mutations in multiple flagellum-related genes [39]. In the presence of 50 μM epinephrine, a slight but non-significant increase in biofilm formation was detected for both the wild-type and ΔqseBC mutant strains (Fig 2). The impact of ΔqseBC mutation on biofilm formation by B. pseudomallei is consistent with reports using qseC mutants of Haemophilus parasuis [40]. Aggregatibacter actinomycetemcomitans [41], E. coli strains [14, 15], and a qseB mutant of Salmonella Typhi [42]. We consider it unlikely that the missense mutation detected in bpsl1036 accounts for the phenotype of the ΔqseBC mutant, as deletion of this gene in B. pseudomallei has been reported to increase biofilm formation [43].

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Fig 2. Effect of qseBC mutation and epinephrine on biofilm formation by B. pseudomallei.

Biofilm formations by B. pseudomallei wild-type K96243 and its ΔqseBC mutant was measured after incubation in LB broth supplemented with or without 50 μM epinephrine for 72 h. Three independent experiments were performed. Error bars represent standard errors of the means. Asterisks indicate significant differences following students t-test (P ≤ 0.05, t-test).

https://doi.org/10.1371/journal.pone.0282098.g002

The B. pseudomallei qseBC genes influence net intracellular survival in macrophages

B. pseudomallei is a facultative intracellular pathogen and factors that influence its survival in macrophages are known to be important for virulence in murine models of melioidosis [4, 44]. We studied uptake and net intracellular survival of B. pseudomallei K96243 and the ΔqseBC mutant in J774A.1 murine macrophage-like cells over time. A bsaZ mutant known to be attenuated in this model was included as a control [6]. After inoculation, bacteria were driven into contact with the macrophages by centrifugation with the aim of avoiding indirect effects due to impaired motility. At 2 h post-infection (1 h to allow uptake and 1 h for kanamycin to kill extracellular bacteria), the number of intracellular (kanamycin-protected) ΔqseBC mutant bacteria was not significantly different than the wild-type implying comparable uptake (Fig 3). However, at 4, 6, 8 and 24 h post-infection the numbers of intracellular ΔqseBC mutant were significantly reduced compared to the wild-type strain (P = 0.0120, P = 0.0306, P = 0.0099 and P = 0.0060, respectively). A significant difference was also detected for the mutant lacking the function of the Bsa Type III secretion system as expected [6]. Sequence analysis of the ΔqseBC mutant revealed a missense mutation resulting in a predicted a single amino acid substitution in BapA, an effector protein injected by the Bsa system (S4 Table). However, it is considered unlikely that this affects the phenotype of the ΔqseBC mutant as a bapA mutant exhibited no defect in invasion or net survival in epithelial or macrophage cell lines [45].

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Fig 3. Effect of qseBC mutation on the intracellular survival of B. pseudomallei in J774A.1 macrophage-like cells.

Murine J774A.1 macrophages were infected with either B. pseudomallei wild-type K96243 or its ΔqseBC mutant at an MOI 0.1. At 2, 4, 6, 8 and 24 h post-infection, infected cells were lysed with 0.1% Triton X-100 and the numbers of viable intracellular bacteria were enumerated. A B. pseudomallei bsaZ mutant was included as positive control. Two independent experiments were performed. Error bars represent standard errors of the means. Asterisks indicate significant differences following students t-test (P ≤ 0.05, t-test) compared with the B. pseudomallei wild-type strain.

https://doi.org/10.1371/journal.pone.0282098.g003

A role for the QseBC system in intracellular life of B. pseudomallei is consistent with the phenotype in J774 macrophages of qseC mutants of Edwardsiella tarda [46] and S. Typhimurium [20]. Introduction of pBBRqseBC into the ΔqseBC mutant strain did not rescue the ability of the mutant strain to survive in J774A.1 cells to wild-type levels likely owing to suppression of growth as evident from S6C and S6D Fig.

Conclusions

We identified homologues of the E. coli QseBC proteins in B. pseudomallei (BPSL0806 and BPSL0807). A ΔqseBC non-polar deletion mutant was constructed and verified to be near-identical at the whole genome sequence level to sequenced K96243 strains. Consistent with observations in other pathogenic bacteria, deletion of B. pseudomallei qseBC impaired motility, fliC transcription, biofilm formation and intra-macrophage survival. While QseBC controls similar phenotypes in B. pseudomallei to those described in other bacteria, epinephrine does not need to be present for it to do so.

Supporting information

S1 Fig. Confirmation of the sequence of the qseBC genes inserted in pBBR1MCS-1 by Sanger sequencing.

Nucleotide sequences were aligned using Bioedit version 7.2.5. The B. pseudomallei K96243 qseBC nucleotide sequence (accession number: NC_006350.1) was compared with the sequence obtained for the insert in pBBRqseBC. The predicted ribosome-binding site sequence is highlighted in yellow.

https://doi.org/10.1371/journal.pone.0282098.s001

(PDF)

S2 Fig. Multiple protein sequence alignments of B. pseudomallei QseB and QseC relative to homologous proteins from other Burkholderia species and E. coli O157:H7.

QseB and QseC amino acid sequences from B. pseudomallei K96243 (UniProt accession numbers: Q63WT5, Q63WT4) was compared with those from B. mallei ATCC 23344 (UniProt accession numbers: A0A0H2WJH0, A0A0H2WIM0), B. thailandensis E264 (UniProt accession numbers: Q2T0S0, Q2T0R9), B. cepacia ATCC25416 (UniProt accession numbers: A0A806UVQ1, A0A806V178), B. cenocepacia ATCCJ2315 (UniProt accession numbers: B4EA15, B4EA14), B. multivorans ATCC17616 (UniProt accession numbers: A0A0H3KLT5, A0A0H3KH15), B. gladioli (UniProt accession numbers: A0A095FGQ9, A0A095FGJ4), B. dolosa AU0158 (UniProt accession numbers: A2W7S9, A2W7T0), B. glumae BGR1 (UniProt accession numbers: C5ACR6, C5ACR5) and E. coli O157:H7 (UniProt accession numbers: Q8XBS3, Q8X524). Residues in the periplasmic domain of E. coli O157:H7 QseC that are conserved across QseC homologues from diverse bacteria are highlighted in yellow. The symbols demonstrate the scale of conservation, whereby asterisk (*) indicates a fully conserved residue, colon (:) indicates conservation between groups of strongly similar properties and period (.) indicates conservation between groups of weakly similar properties. The functional domains were predicted using a Simple Modular Architecture Research Tool or SMART (http://smart.embl.de). The functional regions were highlighted in different colors (green, receiver domain; blue, transcriptional regulatory domain; grey, transmembrane region; orange, HAMP domain; pink, histidine kinase domain; brown, histidine kinase-like ATPase).

https://doi.org/10.1371/journal.pone.0282098.s002

(PDF)

S3 Fig. Transcription of the B. pseudomallei qseBC genes is not affected by epinephrine.

B. pseudomallei wild-type K96243 was grown in LB medium with or without 50μM epinephrine. The transcription of qseBC was analysed by real-time reverse transcriptase PCR. Three independent experiments were performed. Error bars represent the standard error of the mean. NS indicates no significant difference following a student’s t-test.

https://doi.org/10.1371/journal.pone.0282098.s003

(TIF)

S4 Fig. Verification of B. pseudomallei ΔqseBC mutation.

PCR analysis of B. pseudomallei wild-type K96243 compared with the ΔqseBC mutant using primers BPSL0806-F and BPSL0807-R. The amplicon predicted to be generated from B. pseudomallei wild-type is 2639-bp (lane 1) whereas for the ΔqseBC mutant it is 574-bp (lane 2). Lane M and N represent 1 kb DNA ladder and negative control, respectively.

https://doi.org/10.1371/journal.pone.0282098.s004

(TIFF)

S5 Fig. Whole genome alignment between B. pseudomallei K96243 and ΔqseBC mutant using Integrative Genomics Viewer (IGV).

The sequence genomes of K96243 and ΔqseBC mutant were compared with each other in chromosome 1. Deletions between bpsl0806 and bpsl0807 in B. pseudomallei ΔqseBC mutant were shown. There were no effects on adjacent genes.

https://doi.org/10.1371/journal.pone.0282098.s005

(TIFF)

S6 Fig. Construction of a B. pseudomallei qseBC-complemented strain.

(A) Schematic representation of generation of plasmid pBBRqseBC. (B) RT-PCR analysis of qseBC expression using B. pseudomallei wild-type K96243 cDNA (lane 2), ΔqseBC mutant cDNA (lane 3) and ΔqseBC/pBBRqseBC cDNA (lane 4). Positive control using gDNA wild-type is shown in lane 1. Lanes M and N represent 100-bp DNA ladder and negative control, respectively. B. pseudomallei wild-type K96243, ΔqseBC mutant and ΔqseBC/pBBRqseBC complemented strains were grown in LB medium and bacterial cells were collected at 2, 4, 8, 12 and 24 h. for OD measurement (C) and colony count (D). Data represents the mean of triplicate determinations.

https://doi.org/10.1371/journal.pone.0282098.s006

(TIF)

S1 Table. Bacterial strains and plasmids used in this study.

https://doi.org/10.1371/journal.pone.0282098.s007

(PDF)

S2 Table. List of primers used in this study.

https://doi.org/10.1371/journal.pone.0282098.s008

(PDF)

S3 Table. Identity and similarity of homologues of QseB and QseC across Burkholderia species.

https://doi.org/10.1371/journal.pone.0282098.s009

(PDF)

S4 Table. Analysis of nucleotide variation in the B. pseudomallei ΔqseBC mutant genome compared to the original reference K96243 genome and resequencing data for different laboratory stocks.

https://doi.org/10.1371/journal.pone.0282098.s010

(XLSX)

Acknowledgments

The authors gratefully acknowledge the Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University for the laboratory facilities. We would like to thank Prof. Tararaj Dharakul for providing B. pseudomallei K96243 strain and Prof. Herbert P. Schweizer for providing the plasmid used to construct qseBC mutant strain.

References

1. Wiersinga WJ, Virk HS, Torres AG, Currie BJ, Peacock SJ, Dance DAB, et al. Melioidosis. Nat Rev Dis Primers. 2018;4:17107. Epub 20180201. pmid:29388572; PubMed Central PMCID: PMC6456913.
- View Article
- PubMed/NCBI
- Google Scholar
2. Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, et al. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol. 2016;1(1). Epub 2016/02/16. pmid:26877885; PubMed Central PMCID: PMC4746747.
- View Article
- PubMed/NCBI
- Google Scholar
3. Gassiep I, Armstrong M, Norton R. Human melioidosis. Clin Microbiol Rev. 2020;33(2). Epub 20200311. pmid:32161067; PubMed Central PMCID: PMC7067580.
- View Article
- PubMed/NCBI
- Google Scholar
4. Galyov EE, Brett PJ, DeShazer D. Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis. Annu Rev Microbiol. 2010;64:495–517. Epub 2010/06/10. pmid:20528691.
- View Article
- PubMed/NCBI
- Google Scholar
5. Willcocks SJ, Denman CC, Atkins HS, Wren BW. Intracellular replication of the well-armed pathogen Burkholderia pseudomallei. Curr Opin Microbiol. 2016;29:94–103. Epub 20160122. pmid:26803404.
- View Article
- PubMed/NCBI
- Google Scholar
6. Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, Jones PW, et al. An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol. 2002;46(3):649–59. pmid:12410823.
- View Article
- PubMed/NCBI
- Google Scholar
7. Schaefers MM. Regulation of virulence by two-component systems in pathogenic Burkholderia. Infect Immun. 2020;88(7). Epub 2020/04/15. pmid:32284365; PubMed Central PMCID: PMC7309619.
- View Article
- PubMed/NCBI
- Google Scholar
8. Lazar Adler NR, Allwood EM, Deveson Lucas D, Harrison P, Watts S, Dimitropoulos A, et al. Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei. BMC Genomics. 2016;17:331. Epub 2016/05/06. pmid:27147217; PubMed Central PMCID: PMC4855414.
- View Article
- PubMed/NCBI
- Google Scholar
9. Hasselbring BM, Patel MK, Schell MA. Dictyostelium discoideum as a model system for identification of Burkholderia pseudomallei virulence factors. Infect Immun. 2011; 79(5): 2079–88. Epub 20110314. pmid:21402765; PubMed Central PMCID: PMC3088138.
- View Article
- PubMed/NCBI
- Google Scholar
10. Wong J, Chen Y, Gan YH. Host cytosolic glutathione sensing by a membrane histidine kinase activates the type VI secretion system in an intracellular bacterium. Cell Host Microbe. 2015;18(1):38–48. Epub 20150618. pmid:26094804.
- View Article
- PubMed/NCBI
- Google Scholar
11. Sperandio V, Torres AG, Kaper JB. Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol. 2002;43(3):809–21. Epub 2002/04/04. pmid:11929534.
- View Article
- PubMed/NCBI
- Google Scholar
12. Clarke MB, Hughes DT, Zhu C, Boedeker EC, Sperandio V. The QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad Sci U S A. 2006;103(27):10420–5. Epub 2006/06/29. pmid:16803956; PubMed Central PMCID: PMC1482837.
- View Article
- PubMed/NCBI
- Google Scholar
13. Hughes DT, Clarke MB, Yamamoto K, Rasko DA, Sperandio V. The QseC adrenergic signaling cascade in Enterohemorrhagic E. coli (EHEC). PLoS Pathog. 2009;5(8): e1000553. Epub 20090821. pmid:19696934; PubMed Central PMCID: PMC2726761.
- View Article
- PubMed/NCBI
- Google Scholar
14. Yang K, Meng J, Huang YC, Ye LH, Li GJ, Huang J, et al. The role of the qseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by Escherichia coli. Cell Biochem Biophys. 2014;70(1):391–8. pmid:24676679.
- View Article
- PubMed/NCBI
- Google Scholar
15. Li W, Xue M, Yu L, Qi K, Ni J, Chen X, et al. QseBC is involved in the biofilm formation and antibiotic resistance in Escherichia coli isolated from bovine mastitis. PeerJ. 2020;8:e8833. Epub 2020/04/08. pmid:32257646; PubMed Central PMCID: PMC7102498.
- View Article
- PubMed/NCBI
- Google Scholar
16. Sharma VK, Casey TA. Escherichia coli O157:H7 lacking the qseBC-encoded quorum-sensing system outcompetes the parental strain in colonization of cattle intestines. Appl Environ Microbiol. 2014;80(6):1882–92. Epub 20140110. pmid:24413602; PubMed Central PMCID: PMC3957642.
- View Article
- PubMed/NCBI
- Google Scholar
17. Hamed A, Pullinger G, Stevens M, Farveen F, Freestone P. Characterisation of the E. coli and Salmonella qseC and qseE mutants reveals a metabolic rather than adrenergic receptor role. FEMS Microbiol Lett. 2022. Epub 20220208. pmid:35137015.
- View Article
- PubMed/NCBI
- Google Scholar
18. Bearson BL, Bearson SM. The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium. Microb Pathog. 2008;44(4):271–8. Epub 20071012. pmid:17997077.
- View Article
- PubMed/NCBI
- Google Scholar
19. Moreira CG, Sperandio V. Interplay between the qseC and qseE bacterial adrenergic sensor kinases in Salmonella enterica serovar Typhimurium pathogenesis. Infect Immun. 2012;80(12):4344–53. Epub 20121001. pmid:23027532; PubMed Central PMCID: PMC3497441.
- View Article
- PubMed/NCBI
- Google Scholar
20. Moreira CG, Weinshenker D, Sperandio V. QseC mediates Salmonella enterica serovar Typhimurium virulence in vitro and in vivo. Infect Immun. 2010;78(3):914–26. Epub 2009/12/24. pmid:20028809; PubMed Central PMCID: PMC2825943.
- View Article
- PubMed/NCBI
- Google Scholar
21. Pullinger GD, Carnell SC, Sharaff FF, van Diemen PM, Dziva F, Morgan E, et al. Norepinephrine augments Salmonella enterica-induced enteritis in a manner associated with increased net replication but independent of the putative adrenergic sensor kinases qseC and qseE. Infect Immun. 2010;78(1):372–80. Epub 20091102. pmid:19884332; PubMed Central PMCID: PMC2798220.
- View Article
- PubMed/NCBI
- Google Scholar
22. Intarak N, Muangsombut V, Vattanaviboon P, Stevens MP, Korbsrisate S. Growth, motility and resistance to oxidative stress of the melioidosis pathogen Burkholderia pseudomallei are enhanced by epinephrine. Pathog Dis. 2014;72(1):24–31. Epub 2014/04/23. pmid:24753312.
- View Article
- PubMed/NCBI
- Google Scholar
23. Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, et al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A. 2004;101(39):14240–5. Epub 2004/09/21. 0403302101. pmid:15377794; PubMed Central PMCID: PMC521101.
- View Article
- PubMed/NCBI
- Google Scholar
24. Lopez CM, Rholl DA, Trunck LA, Schweizer HP. Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei. Appl Environ Microbiol. 2009;75(20):6496–503. Epub 2009/08/25. pmid:19700544; PubMed Central PMCID: PMC2765137.
- View Article
- PubMed/NCBI
- Google Scholar
25. Andrews S. FastQC: a quality control tool for high throughput 550 sequence data babraham bioinformatics. 2010. Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
- View Article
- Google Scholar
26. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014;9(11):e112963. Epub 20141119. pmid:25409509; PubMed Central PMCID: PMC4237348.
- View Article
- PubMed/NCBI
- Google Scholar
27. Obranic S, Babic F, Maravic-Vlahovicek G. Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors. Plasmid. 2013;70(2):263–7. Epub 2013/04/16. pmid:23583732.
- View Article
- PubMed/NCBI
- Google Scholar
28. Deziel E, Comeau Y, Villemur R. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J Bacteriol. 2001;183(4):1195–204. pmid:11157931; PubMed Central PMCID: PMC94992.
- View Article
- PubMed/NCBI
- Google Scholar
29. Vandecandelaere I, Van Acker H, Coenye T. A microplate-based system as in vitro model of biofilm growth and quantification. Methods Mol Biol. 2016;1333:53–66. Epub 2015/10/16. pmid:26468099.
- View Article
- PubMed/NCBI
- Google Scholar
30. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. Epub 2002/02/16. pmid:11846609.
- View Article
- PubMed/NCBI
- Google Scholar
31. Parker CT, Russell R, Njoroge JW, Jimenez AG, Taussig R, Sperandio V. Genetic and mechanistic analyses of the periplasmic domain of the enterohemorrhagic Escherichia coli qseC Histidine Sensor Kinase. J Bacteriol. 2017;199(8). Epub 20170328. pmid:28138098; PubMed Central PMCID: PMC5370426.
- View Article
- PubMed/NCBI
- Google Scholar
32. Ooi WF, Ong C, Nandi T, Kreisberg JF, Chua HH, Sun G, et al. The condition-dependent transcriptional landscape of Burkholderia pseudomallei. PLoS Genet. 2013;9(9): e1003795. Epub 20130912. pmid:24068961; PubMed Central PMCID: PMC3772027.
- View Article
- PubMed/NCBI
- Google Scholar
33. Johnson JR, Lockman HA, Owens K, Jelacic S, Tarr PI. High-frequency secondary mutations after suicide-driven allelic exchange mutagenesis in extraintestinal pathogenic Escherichia coli. J Bacteriol. 2003;185(17):5301–5. Epub 2003/08/19. pmid:12923106; PubMed Central PMCID: PMC180995.
- View Article
- PubMed/NCBI
- Google Scholar
34. Sahl JW, Allender CJ, Colman RE, Califf KJ, Schupp JM, Currie BJ, et al. Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence. PLoS One. 2015;10(3):e0121052. Epub 20150324. pmid:25803742; PubMed Central PMCID: PMC4372212.
- View Article
- PubMed/NCBI
- Google Scholar
35. Wagley S, Scott AE, Ireland PM, Prior JL, Atkins TP, Bancroft GJ, et al. Genome resequencing of laboratory stocks of Burkholderia pseudomallei K96243. Microbiol Resour Announc. 2019;8(9). Epub 20190228. pmid:30834386; PubMed Central PMCID: PMC6395871.
- View Article
- PubMed/NCBI
- Google Scholar
36. Wu D, Baigalmaa L, Yao Y, Li G, Su M, Fan L, et al. The Escherichia coli qseB/qseC signaling is required for correct timing of replication initiation and cell motility. Gene. 2021;773:145374. Epub 20201223. pmid:33359126.
- View Article
- PubMed/NCBI
- Google Scholar
37. Attmannspacher U, Scharf BE, Harshey RM. FliL is essential for swarming: motor rotation in absence of fliL fractures the flagellar rod in swarmer cells of Salmonella enterica. Mol Microbiol. 2008;68(2):328–41. Epub 20080219. pmid:18284590.
- View Article
- PubMed/NCBI
- Google Scholar
38. Vestby LK, Gronseth T, Simm R, Nesse LL. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics (Basel). 2020;9(2). Epub 2020/02/08. pmid:32028684; PubMed Central PMCID: PMC7167820.
- View Article
- PubMed/NCBI
- Google Scholar
39. Borlee GI, Plumley BA, Martin KH, Somprasong N, Mangalea MR, Islam MN, et al. Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster. PLoS Negl Trop Dis. 2017;11(6):e0005689. Epub 20170628. pmid:28658258; PubMed Central PMCID: PMC5507470.
- View Article
- PubMed/NCBI
- Google Scholar
40. He L, Dai K, Wen X, Ding L, Cao S, Huang X, et al. QseC mediates osmotic stress resistance and biofilm formation in Haemophilus parasuis. Front Microbiol. 2018;9:212. Epub 2018/03/01. pmid:29487590; PubMed Central PMCID: PMC5816903.
- View Article
- PubMed/NCBI
- Google Scholar
41. Novak EA, Shao H, Daep CA, Demuth DR. Autoinducer-2 and qseC control biofilm formation and in vivo virulence of Aggregatibacter actinomycetemcomitans. Infect Immun. 2010;78(7):2919–26. Epub 20100419. pmid:20404080; PubMed Central PMCID: PMC2897384.
- View Article
- PubMed/NCBI
- Google Scholar
42. Ji Y, Li W, Zhang Y, Chen L, Zhang Y, Zheng X, et al. QseB mediates biofilm formation and invasion in Salmonella enterica serovar Typhi. Microb Pathog. 2017;104:6–11. Epub 2017/01/08. pmid:28062286.
- View Article
- PubMed/NCBI
- Google Scholar
43. Alwis PA, Treerat P, Gong L, Deveson Lucas D, Allwood EM, Prescott M, et al. Disruption of the Burkholderia pseudomallei two-component signal transduction system BbeR-BbeS leads to increased extracellular DNA secretion and altered biofilm formation. Vet Microbiol. 2020;242:108603. Epub 20200208. pmid:32122607.
- View Article
- PubMed/NCBI
- Google Scholar
44. Wiersinga WJ, van der Poll T, White NJ, Day NP, Peacock SJ. Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nat Rev Microbiol. 2006;4(4):272–82. Epub 2006/03/17. pmid:16541135.
- View Article
- PubMed/NCBI
- Google Scholar
45. Treerat P, Alwis P, D’Cruze T, Cullinane M, Vadivelu J, Devenish RJ, et al. The Burkholderia pseudomallei proteins BapA and BapC are secreted TTSS3 effectors and BapB levels modulate expression of bopE. PLoS One. 2015;10(12):e0143916. Epub 20151201. pmid:26624293; PubMed Central PMCID: PMC4666416.
- View Article
- PubMed/NCBI
- Google Scholar
46. Wang X, Wang Q, Yang M, Xiao J, Liu Q, Wu H, et al. QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda. Fish Shellfish Immunol. 2011;30(3):944–53. Epub 2011/02/04. 01.019. pmid:21288493.
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Wiersinga WJ, Virk HS, Torres AG, Currie BJ, Peacock SJ, Dance DAB, et al. Melioidosis. Nat Rev Dis Primers. 2018;4:17107. Epub 20180201. pmid:29388572; PubMed Central PMCID: PMC6456913.
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, et al. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol. 2016;1(1). Epub 2016/02/16. pmid:26877885; PubMed Central PMCID: PMC4746747.
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Gassiep I, Armstrong M, Norton R. Human melioidosis. Clin Microbiol Rev. 2020;33(2). Epub 20200311. pmid:32161067; PubMed Central PMCID: PMC7067580.
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Galyov EE, Brett PJ, DeShazer D. Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis. Annu Rev Microbiol. 2010;64:495–517. Epub 2010/06/10. pmid:20528691.
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Willcocks SJ, Denman CC, Atkins HS, Wren BW. Intracellular replication of the well-armed pathogen Burkholderia pseudomallei. Curr Opin Microbiol. 2016;29:94–103. Epub 20160122. pmid:26803404.
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, Jones PW, et al. An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol. 2002;46(3):649–59. pmid:12410823.
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Schaefers MM. Regulation of virulence by two-component systems in pathogenic Burkholderia. Infect Immun. 2020;88(7). Epub 2020/04/15. pmid:32284365; PubMed Central PMCID: PMC7309619.
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Lazar Adler NR, Allwood EM, Deveson Lucas D, Harrison P, Watts S, Dimitropoulos A, et al. Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei. BMC Genomics. 2016;17:331. Epub 2016/05/06. pmid:27147217; PubMed Central PMCID: PMC4855414.
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Hasselbring BM, Patel MK, Schell MA. Dictyostelium discoideum as a model system for identification of Burkholderia pseudomallei virulence factors. Infect Immun. 2011; 79(5): 2079–88. Epub 20110314. pmid:21402765; PubMed Central PMCID: PMC3088138.
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Wong J, Chen Y, Gan YH. Host cytosolic glutathione sensing by a membrane histidine kinase activates the type VI secretion system in an intracellular bacterium. Cell Host Microbe. 2015;18(1):38–48. Epub 20150618. pmid:26094804.
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Sperandio V, Torres AG, Kaper JB. Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol. 2002;43(3):809–21. Epub 2002/04/04. pmid:11929534.
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Clarke MB, Hughes DT, Zhu C, Boedeker EC, Sperandio V. The QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad Sci U S A. 2006;103(27):10420–5. Epub 2006/06/29. pmid:16803956; PubMed Central PMCID: PMC1482837.
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Hughes DT, Clarke MB, Yamamoto K, Rasko DA, Sperandio V. The QseC adrenergic signaling cascade in Enterohemorrhagic E. coli (EHEC). PLoS Pathog. 2009;5(8): e1000553. Epub 20090821. pmid:19696934; PubMed Central PMCID: PMC2726761.
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Yang K, Meng J, Huang YC, Ye LH, Li GJ, Huang J, et al. The role of the qseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by Escherichia coli. Cell Biochem Biophys. 2014;70(1):391–8. pmid:24676679.
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Li W, Xue M, Yu L, Qi K, Ni J, Chen X, et al. QseBC is involved in the biofilm formation and antibiotic resistance in Escherichia coli isolated from bovine mastitis. PeerJ. 2020;8:e8833. Epub 2020/04/08. pmid:32257646; PubMed Central PMCID: PMC7102498.
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Sharma VK, Casey TA. Escherichia coli O157:H7 lacking the qseBC-encoded quorum-sensing system outcompetes the parental strain in colonization of cattle intestines. Appl Environ Microbiol. 2014;80(6):1882–92. Epub 20140110. pmid:24413602; PubMed Central PMCID: PMC3957642.
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Hamed A, Pullinger G, Stevens M, Farveen F, Freestone P. Characterisation of the E. coli and Salmonella qseC and qseE mutants reveals a metabolic rather than adrenergic receptor role. FEMS Microbiol Lett. 2022. Epub 20220208. pmid:35137015.
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Bearson BL, Bearson SM. The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium. Microb Pathog. 2008;44(4):271–8. Epub 20071012. pmid:17997077.
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Moreira CG, Sperandio V. Interplay between the qseC and qseE bacterial adrenergic sensor kinases in Salmonella enterica serovar Typhimurium pathogenesis. Infect Immun. 2012;80(12):4344–53. Epub 20121001. pmid:23027532; PubMed Central PMCID: PMC3497441.
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Moreira CG, Weinshenker D, Sperandio V. QseC mediates Salmonella enterica serovar Typhimurium virulence in vitro and in vivo. Infect Immun. 2010;78(3):914–26. Epub 2009/12/24. pmid:20028809; PubMed Central PMCID: PMC2825943.
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Pullinger GD, Carnell SC, Sharaff FF, van Diemen PM, Dziva F, Morgan E, et al. Norepinephrine augments Salmonella enterica-induced enteritis in a manner associated with increased net replication but independent of the putative adrenergic sensor kinases qseC and qseE. Infect Immun. 2010;78(1):372–80. Epub 20091102. pmid:19884332; PubMed Central PMCID: PMC2798220.
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Intarak N, Muangsombut V, Vattanaviboon P, Stevens MP, Korbsrisate S. Growth, motility and resistance to oxidative stress of the melioidosis pathogen Burkholderia pseudomallei are enhanced by epinephrine. Pathog Dis. 2014;72(1):24–31. Epub 2014/04/23. pmid:24753312.
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Holden MT, Titball RW, Peacock SJ, Cerdeno-Tarraga AM, Atkins T, Crossman LC, et al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci U S A. 2004;101(39):14240–5. Epub 2004/09/21. 0403302101. pmid:15377794; PubMed Central PMCID: PMC521101.
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Lopez CM, Rholl DA, Trunck LA, Schweizer HP. Versatile dual-technology system for markerless allele replacement in Burkholderia pseudomallei. Appl Environ Microbiol. 2009;75(20):6496–503. Epub 2009/08/25. pmid:19700544; PubMed Central PMCID: PMC2765137.
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Andrews S. FastQC: a quality control tool for high throughput 550 sequence data babraham bioinformatics. 2010. Available from: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref26] 26. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014;9(11):e112963. Epub 20141119. pmid:25409509; PubMed Central PMCID: PMC4237348.
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref27] 27. Obranic S, Babic F, Maravic-Vlahovicek G. Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors. Plasmid. 2013;70(2):263–7. Epub 2013/04/16. pmid:23583732.
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref28] 28. Deziel E, Comeau Y, Villemur R. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J Bacteriol. 2001;183(4):1195–204. pmid:11157931; PubMed Central PMCID: PMC94992.
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref29] 29. Vandecandelaere I, Van Acker H, Coenye T. A microplate-based system as in vitro model of biofilm growth and quantification. Methods Mol Biol. 2016;1333:53–66. Epub 2015/10/16. pmid:26468099.
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref30] 30. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. Epub 2002/02/16. pmid:11846609.
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref31] 31. Parker CT, Russell R, Njoroge JW, Jimenez AG, Taussig R, Sperandio V. Genetic and mechanistic analyses of the periplasmic domain of the enterohemorrhagic Escherichia coli qseC Histidine Sensor Kinase. J Bacteriol. 2017;199(8). Epub 20170328. pmid:28138098; PubMed Central PMCID: PMC5370426.
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref32] 32. Ooi WF, Ong C, Nandi T, Kreisberg JF, Chua HH, Sun G, et al. The condition-dependent transcriptional landscape of Burkholderia pseudomallei. PLoS Genet. 2013;9(9): e1003795. Epub 20130912. pmid:24068961; PubMed Central PMCID: PMC3772027.
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref33] 33. Johnson JR, Lockman HA, Owens K, Jelacic S, Tarr PI. High-frequency secondary mutations after suicide-driven allelic exchange mutagenesis in extraintestinal pathogenic Escherichia coli. J Bacteriol. 2003;185(17):5301–5. Epub 2003/08/19. pmid:12923106; PubMed Central PMCID: PMC180995.
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref34] 34. Sahl JW, Allender CJ, Colman RE, Califf KJ, Schupp JM, Currie BJ, et al. Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence. PLoS One. 2015;10(3):e0121052. Epub 20150324. pmid:25803742; PubMed Central PMCID: PMC4372212.
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref35] 35. Wagley S, Scott AE, Ireland PM, Prior JL, Atkins TP, Bancroft GJ, et al. Genome resequencing of laboratory stocks of Burkholderia pseudomallei K96243. Microbiol Resour Announc. 2019;8(9). Epub 20190228. pmid:30834386; PubMed Central PMCID: PMC6395871.
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref36] 36. Wu D, Baigalmaa L, Yao Y, Li G, Su M, Fan L, et al. The Escherichia coli qseB/qseC signaling is required for correct timing of replication initiation and cell motility. Gene. 2021;773:145374. Epub 20201223. pmid:33359126.
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref37] 37. Attmannspacher U, Scharf BE, Harshey RM. FliL is essential for swarming: motor rotation in absence of fliL fractures the flagellar rod in swarmer cells of Salmonella enterica. Mol Microbiol. 2008;68(2):328–41. Epub 20080219. pmid:18284590.
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref38] 38. Vestby LK, Gronseth T, Simm R, Nesse LL. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics (Basel). 2020;9(2). Epub 2020/02/08. pmid:32028684; PubMed Central PMCID: PMC7167820.
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref39] 39. Borlee GI, Plumley BA, Martin KH, Somprasong N, Mangalea MR, Islam MN, et al. Genome-scale analysis of the genes that contribute to Burkholderia pseudomallei biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster. PLoS Negl Trop Dis. 2017;11(6):e0005689. Epub 20170628. pmid:28658258; PubMed Central PMCID: PMC5507470.
View Article
PubMed/NCBI
Google Scholar

[153] View Article

[154] PubMed/NCBI

[155] Google Scholar

[ref40] 40. He L, Dai K, Wen X, Ding L, Cao S, Huang X, et al. QseC mediates osmotic stress resistance and biofilm formation in Haemophilus parasuis. Front Microbiol. 2018;9:212. Epub 2018/03/01. pmid:29487590; PubMed Central PMCID: PMC5816903.
View Article
PubMed/NCBI
Google Scholar

[157] View Article

[158] PubMed/NCBI

[159] Google Scholar

[ref41] 41. Novak EA, Shao H, Daep CA, Demuth DR. Autoinducer-2 and qseC control biofilm formation and in vivo virulence of Aggregatibacter actinomycetemcomitans. Infect Immun. 2010;78(7):2919–26. Epub 20100419. pmid:20404080; PubMed Central PMCID: PMC2897384.
View Article
PubMed/NCBI
Google Scholar

[161] View Article

[162] PubMed/NCBI

[163] Google Scholar

[ref42] 42. Ji Y, Li W, Zhang Y, Chen L, Zhang Y, Zheng X, et al. QseB mediates biofilm formation and invasion in Salmonella enterica serovar Typhi. Microb Pathog. 2017;104:6–11. Epub 2017/01/08. pmid:28062286.
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref43] 43. Alwis PA, Treerat P, Gong L, Deveson Lucas D, Allwood EM, Prescott M, et al. Disruption of the Burkholderia pseudomallei two-component signal transduction system BbeR-BbeS leads to increased extracellular DNA secretion and altered biofilm formation. Vet Microbiol. 2020;242:108603. Epub 20200208. pmid:32122607.
View Article
PubMed/NCBI
Google Scholar

[169] View Article

[170] PubMed/NCBI

[171] Google Scholar

[ref44] 44. Wiersinga WJ, van der Poll T, White NJ, Day NP, Peacock SJ. Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nat Rev Microbiol. 2006;4(4):272–82. Epub 2006/03/17. pmid:16541135.
View Article
PubMed/NCBI
Google Scholar

[173] View Article

[174] PubMed/NCBI

[175] Google Scholar

[ref45] 45. Treerat P, Alwis P, D’Cruze T, Cullinane M, Vadivelu J, Devenish RJ, et al. The Burkholderia pseudomallei proteins BapA and BapC are secreted TTSS3 effectors and BapB levels modulate expression of bopE. PLoS One. 2015;10(12):e0143916. Epub 20151201. pmid:26624293; PubMed Central PMCID: PMC4666416.
View Article
PubMed/NCBI
Google Scholar

[177] View Article

[178] PubMed/NCBI

[179] Google Scholar

[ref46] 46. Wang X, Wang Q, Yang M, Xiao J, Liu Q, Wu H, et al. QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda. Fish Shellfish Immunol. 2011;30(3):944–53. Epub 2011/02/04. 01.019. pmid:21288493.
View Article
PubMed/NCBI
Google Scholar

[181] View Article

[182] PubMed/NCBI

[183] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Bacterial strains and culture conditions

Construction of a B. pseudomallei F044qseBC mutant

Whole-genome sequencing and bioinformatic analysis

Cloning of the B. pseudomallei qseBC genes for trans-complementation

Motility assays

Biofilm formation assay

RNA extraction and real-time reverse transcriptase-PCR

Analysis of bacterial growth kinetics

Intracellular survival assay

Statistical analysis

Results and discussion

B. pseudomallei BPSL0806 and BPSL0807 are homologues of E. coli QseB and QseC, respectively

Generation of B. pseudomallei qseBC mutant and trans-complemented strains

The B. pseudomallei qseBC genes influence motility and fliC transcription

The B. pseudomallei qseBC genes influence biofilm production

The B. pseudomallei qseBC genes influence net intracellular survival in macrophages

Conclusions

Supporting information

S1 Fig. Confirmation of the sequence of the qseBC genes inserted in pBBR1MCS-1 by Sanger sequencing.

S2 Fig. Multiple protein sequence alignments of B. pseudomallei QseB and QseC relative to homologous proteins from other Burkholderia species and E. coli O157:H7.

S3 Fig. Transcription of the B. pseudomallei qseBC genes is not affected by epinephrine.

S4 Fig. Verification of B. pseudomallei ΔqseBC mutation.

S5 Fig. Whole genome alignment between B. pseudomallei K96243 and ΔqseBC mutant using Integrative Genomics Viewer (IGV).

S6 Fig. Construction of a B. pseudomallei qseBC-complemented strain.

S1 Table. Bacterial strains and plasmids used in this study.

S2 Table. List of primers used in this study.

S3 Table. Identity and similarity of homologues of QseB and QseC across Burkholderia species.

S4 Table. Analysis of nucleotide variation in the B. pseudomallei ΔqseBC mutant genome compared to the original reference K96243 genome and resequencing data for different laboratory stocks.

Acknowledgments

References