[Two-stage Inhibition Effects of Burkholderia sp. Y4 Application on Cadmium Uptake and Transport in Wheat].

In order to evaluate the feasibility of using Burkholderia sp. Y4 as a cadmium （Cd）-reducing bacterial agent in contaminated wheat fields， the changes in the rhizosphere soil microbial community and Cd available state， as well as the content and transport characteristics of Cd in the wheat root， basal node， internode， and grain under the treatment of strain Y4 were tested using microbial high-throughput sequencing， step-by-step extraction， subcellular distribution， and occurrence analyses. The results showed that root application of strain Y4 significantly reduced the root and grain Cd content of wheat by 7.7% and 30.3%， respectively， compared with that in the control treatment. The Cd content and Cd transfer factor results in wheat vegetative organs showed that strain Y4 reduced the Cd transfer factor from basal node to internode by 79.3%， and Cd content in the wheat internode stem also decreased by 50.9%. The study of Cd occurrence morphology showed that strain Y4 treatment increased the proportion of residual Cd in roots and basal ganglia， decreased the contents of inorganic and water-soluble Cd in roots， and increased the content of residual Cd in basal ganglia. Further examination of the subcellular distribution of Cd showed that the Cd content in root cell walls and basal ganglia cell fluid increased by 21.3% and 98.2%， respectively， indicating that the Cd fixation ability of root cell walls and basal ganglia cell fluid was improved by the strain Y4 treatment. In the rhizosphere soil， it was found that the microbial community structure was changed by strain Y4 application. Under the Y4 treatment， the relative abundance of Burkholderia increased from 9.6% to 11.5%， whereas that of Acidobacteriota decreased. Additionally， the relative abundance of Gemmatimonadales， Pseudomonadales， and Chitinophagales were also increased by strain Y4 treatment. At the same time， the application of strain Y4 increased the pH value of rhizosphere soil by 8.3%. The contents of exchangeable Cd， carbonate-bound Cd， and iron-manganese oxide-bound Cd in the soil decreased by 44.4%， 21.7%， and 15.9%， respectively， whereas the proportion of residual Cd reached 53.6%. Root application of strain Y4 increased the contents of nitrate nitrogen and ammonium nitrogen in the soil by 22.0% and 21.4%， respectively， and the contents of alkaline nitrogen also increased to a certain extent. In conclusion， the root application of strain Y4 not only improved soil nitrogen availability but also inhibited Cd transport and accumulation from contaminated soil to wheat grains in a "two-stage" manner by reducing Cd availability in rhizosphere soil and improving Cd interception and fixation capacity of wheat roots and basal nodes. Therefore， Burkholderia Y4 has application potential as a Cd-reducing and growth-promoting agent in wheat.

In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (ΔbimA) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the ΔbimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the ΔbimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease.

[Effects of Burkholderia sp. Y4 on Cadmium Damage and Uptake in Rice Seedlings].

In order to investigate the effects of Burkholderia sp. Y4 on rice seedlings under cadmium (Cd) stress, seed germination and vermiculite culture experiments were conducted using low Cd-accumulation xiangzaoxian 24 (X24) and high Cd-accumulation Tyou 705 (T705) varieties. The effects of Burkholderia sp. Y4 on rice growth, oxidative damage caused by Cd, and Cd accumulation were studied. Additionally, the Cd2+ flux rates in the elongation zone of rice roots under Burkholderia sp. Y4 application were detected using non-invasive micro-test technology. Burkholderia sp. Y4 alleviated the inhibition effect of Cd on rice seed germination by 13.8%. After inoculation with Burkholderia sp. Y4 for 7 d, the length of rice roots and buds increased by 83.3% and 12.2%, and their dry weight increased by 56.8% and 12.5%, respectively; those in the 10 d Y4 inoculation group increased by 28.6% and 20.0% in length and by 113.2% and 46.0% in dry weight, respectively. Burkholderia sp. Y4 inoculation also alleviated rice oxidative stress damage caused by Cd. The application of strain Y4 significantly reduced the content of the oxidative damage product malondialdehyde (MDA) in the shoots and roots of rice seedlings by 21.5% and 16.9%, respectively. Under Burkholderia sp. Y4 inoculation, the significant changes in antioxidant enzyme SOD and CAT activities caused by Cd stress disappeared in rice roots; those in shoots also decreased from 176.9% and 74.8% to 53.3% and 21.5%, respectively. Conversely, Burkholderia sp. Y4 inhibited Cd uptake by rice seedlings with different genotypes, including the low Cd-accumulation variety X24 and high Cd-accumulation variety T705. The root application of strain Y4 significantly reduced Cd accumulation in the shoots and roots of rice seedlings by 79.2% and 62.7% in T705 and by 57.3% and 24.1% in X24, respectively. The Cd2+ flux rate of high Cd-accumulation variety T705 was significantly higher than that of low Cd-accumulation variety X24. Under Burkholderia sp. Y4 inoculation, the yellow membrane was formed on the root surface of rice seedlings, and the Cd2+ flux rate in the elongation zone of T705 and X24 roots decreased by 36.0% and 35.0% in 3-day-old seedlings, as well as by 44.6% and 24.9% in 10-day-old seedlings, respectively. In conclusion, Burkholderia sp. Y4 inoculation inhibited the toxic effects of Cd on rice seedling growth through alleviating oxidative stress and damage caused by Cd. Furthermore, the root application of Burkholderia sp. Y4 effectively decreased the Cd2+ flux rate in the elongation zone of roots to inhibit the Cd uptake and accumulation in roots and shoots of rice seedlings. This study provides theoretical basis and data support for the application of Burkholderia sp. Y4 as a Cd-reducing and growth-promoting agent for rice in contaminated farmland.

A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans.

Burkholderia cepacia complex bacteria comprise opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream of the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. This insertion sequence (IS) element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pilus synthesis, type VI secretion, and chromosome-associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line and reduced surface hydrophobicity and forms smaller cellular aggregates but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of the Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, fulfilling a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein is one out of eight orthologs present in the B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.

Phosphate-solubilizing bacterium Burkholderia sp. strain N3 facilitates the regulation of gene expression and improves tomato seedling growth under cadmium stress.

Cadmium (Cd) is among the most toxic heavy metals in soils. The ways by which tomato plants inoculated with a phosphate-solubilizing bacterium (PSB) respond to Cd and regulate gene expression remain unclear. We investigated hormone metabolism and genes involved in Cd resistance in tomato seedlings inoculated with the PSB strain N3. Cd inhibited tomato plant growth and nutrient uptake and increase in dry weight. Compared with Cd treatment, N3 inoculation inhibited the accumulation of Cd in the shoots and roots, and the root dry weight significantly increased by 30.50% (P < 0.05). The nitrogen and potassium contents in the roots of seedlings treated with N3 increased, and the phosphorus levels were the same as those in the control. N3 decreased the rate of Zn2+ absorption but increased Fe3+ absorption in the roots, and the amount of accumulated Cd increased with Zn2+ uptake. The concentrations of hormones (indole-3-acetic acid, IAA; zeatin, ZEA; and jasmonic acid, JA) increased under Cd stress, whereas inoculation with N3 reduced IAA and ZEA levels. In the comparison between N3 + Cd and Cd treatments, the highest number of up- and downregulated genes was obtained. Pathways involved in signaling response, photosynthesis, phenylpropanoid biosynthesis, and DNA replication and the photosynthesis-antenna proteins pathway play important roles in the responses and adaptation of seedlings to Cd. Inoculation with N3 alleviates Cd stress in tomato seedlings. The present study provides new insights into the differentially expressed genes related to interaction between PSB and tomato exposed to Cd in soils.

Potential of cadmium resistant Burkholderia contaminans strain ZCC in promoting growth of soy beans in the presence of cadmium.

Bioremediation of Cd contaminated environments can be assisted by plant-growth-promoting bacteria (PGPB) enabling plant growth in these sites. Here a gram-negative Burkholderia contaminans ZCC was isolated from mining soil at a copper-gold mine. When exposed to Cd(II), ZCC displayed high Cd resistance and the minimal inhibitory concentration was 7 mM in LB medium. Complete genome analysis uncovered B. contaminans ZCC contained 3 chromosomes and 2 plasmids. One of these plasmids was shown to contain a multitude of heavy metal resistance determinants including genes encoding a putative Cd-translocating PIB-type ATPase and an RND-type related to the Czc-system. These additional heavy metal resistance determinants are likely responsible for the increased resistance to Cd(II) and other heavy metals in comparison to other strains of B. contaminans. B. contaminans ZCC also displayed PGPB traits such as 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore production, organic and inorganic phosphate solubilization and indole acetic acid production. Moreover, the properties and Cd(II) binding characteristics of extracellular polymeric substances was investigated. ZCC was able to induce extracellular polymeric substances production in response to Cd and was shown to be chemically coordinated to Cd(II). It could promote the growth of soybean in the presence of elevated concentrations of Cd(II). This work will help to better understand processes important in bioremediation of Cd-contaminated environment.

Dietary antioxidant seleno-L-methionine protects macrophages infected with Burkholderia thailandensis.

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a potentially life-threatening disease endemic in Southeast Asia and Northern Australia. Treatment of melioidosis is a long and costly process and the pathogen is inherently resistant to several classes of antibiotics, therefore there is a need for new treatments that can help combat the pathogen. Previous work has shown that the combination of interferon-gamma, an immune system activator, and the antibiotic ceftazidime synergistically reduced the bacterial burden of RAW 264.7 macrophages that had been infected with either B. pseudomallei or Burkholderia thailandensis. The mechanism of the interaction was found to be partially dependent on interferon-gamma-induced production of reactive oxygen species inside the macrophages. To further confirm the role of reactive oxygen species in the effectiveness of the combination treatment, we investigated the impact of the antioxidant and reactive oxygen species scavenger, seleno-L-methionine, on intracellular and extracellular bacterial burden of the infected macrophages. In a dose-dependent manner, high concentrations of seleno-L-methionine (1000 µM) were protective towards infected macrophages, resulting in a reduction of bacteria, on its own, that exceeded the reduction caused by the antibiotic alone and rivaled the effect of ceftazidime and interferon-gamma combined. Seleno-L-methionine treatment also resulted in improved viability of infected macrophages compared to untreated controls. We show that the protective effect of seleno-L-methionine was partly due to its inhibition of bacterial growth. In summary, our study shows a role for high dose seleno-L-methionine to protect and treat macrophages infected with B. thailandensis.

Regulation of Virulence by Two-Component Systems in Pathogenic Burkholderia.

The regulation and timely expression of bacterial genes during infection is critical for a pathogen to cause an infection. Bacteria have multiple mechanisms to regulate gene expression in response to their environment, one of which is two-component systems (TCS). TCS have two components. One component is a sensory histidine kinase (HK) that autophosphorylates when activated by a signal. The activated sensory histidine kinase then transfers the phosphoryl group to the second component, the response regulator, which activates transcription of target genes. The genus Burkholderia contains members that cause human disease and are often extensively resistant to many antibiotics. The Burkholderia cepacia complex (BCC) can cause severe lung infections in patients with cystic fibrosis (CF) or chronic granulomatous disease (CGD). BCC members have also recently been associated with several outbreaks of bacteremia from contaminated pharmaceutical products. Separate from the BCC is Burkholderia pseudomallei, which is the causative agent of melioidosis, a serious disease that occurs in the tropics, and a potential bioterrorism weapon. Bioinformatic analysis of sequenced Burkholderia isolates predicts that most strains have at least 40 TCS. The vast majority of these TCS are uncharacterized both in terms of the signals that activate them and the genes that are regulated by them. This review will highlight TCS that have been described to play a role in virulence in either the BCC or B. pseudomallei Since many of these TCS are involved in virulence, TCS are potential novel therapeutic targets, and elucidating their function is critical for understanding Burkholderia pathogenesis.

Burkholderia sp. Y4 inhibits cadmium accumulation in rice by increasing essential nutrient uptake and preferentially absorbing cadmium.

Microbial remediation of heavy metal-polluted soil is a commonly used method. Burkholderia sp. Y4, isolated from cadmium (Cd)-contaminated rice rhizosphere soil, was investigated for its direct and indirect effects on Cd accumulation in rice by SEM-EDS, FITR and sequencing analysis of the soil bacterial community. Burkholderia sp. Y4 inoculation reduced Cd accumulation in rice roots, rachises, and grains of the two rice varieties T705 and X24 and increased levels of essential elements, especially Fe and Mn, which competitively inhibited Cd transport through cationic channels. Living Burkholderia sp. Y4 cells, rather than non-living ones, could colonize the surface of rice roots and accumulated more Cd through direct biosorption associated with -CO and -NH/-CO bonds of amino acids and proteins. The results of soil microbial community showed that the colonization of externally added Burkholderia sp. Y4 could be maintained over some time to impact the total rhizospheric environment. Burkholderia sp. Y4 inoculation decreased the abundance of microbes involved in the iron cycle (Acidobacteria) as well as of those mediating the transformation of ammonium nitrogen to nitrate nitrogen (Nitrosomonadaceae and Nitrospira). So Burkholderia sp. Y4 inoculation may indirectly change the availability of micronutrients and Cd in rice rhizosphere soil through iron-nitrogen coupled cycles to increase essential nutrient uptake and inhibit Cd accumulation in rice by preferential Cd-biosorption. Therefore, Burkholderia sp. Y4 is potentially suitable for the bioremediation of Cd-contaminated paddy soil.

Ramachandran conformational energy maps for disaccharide linkages found in Burkholderia multivorans biofilm polysaccharides.

Ramachandran conformational energy maps have been prepared for all of the glycosidic linkages found in the C1576 exopolysaccharide that constitutes the biofilms of the bacterial species Burkholderia multivorans, a member of the Burkholderia cepacian complex that was isolated from a cystic fibrosis patient. This polysaccharide is a rhamnomannan with a tetrasaccharide repeat unit containing two mannose residues and two rhamnose residues, -[3-α-d-Man-(1→2)-α-d-Man-(1→2)-α-d-Rha-(1→3)-α-d-Rha-(1→]n-, where approximately 50% of the rhamnoses are randomly methylated on their O3 hydroxyl groups, further increasing the overall hydrophobicity of the chains. Because of the methylation, the tetrasaccharide repeat unit actually contains six possible linkages. The conformational energy maps are fully adiabatic relaxed maps in which the energy for each (ϕ,ψ) grid point on the map represents the lowest possible energy for the molecule in that conformation, considering all the combinations of the other degrees of freedom, such as hydroxyl orientations. Molecular dynamics simulations were used to verify that these maps indeed describe the conformational dynamics of these linkages. All six linkages were found to be quite restricted in possible ϕ angles, but to exhibit several possible low-energy ψ angles, suggesting that these chains could be quite flexible.

Unraveling the role of quorum sensing-dependent metabolic homeostasis of the activated methyl cycle in a cooperative population of Burkholderia glumae.

The activated methyl cycle (AMC) is responsible for the generation of S-adenosylmethionine (SAM), which is a substrate of N-acylhomoserine lactone (AHL) synthases. However, it is unknown whether AHL-mediated quorum sensing (QS) plays a role in the metabolic flux of the AMC to ensure cell density-dependent biosynthesis of AHL in cooperative populations. Here we show that QS controls metabolic homeostasis of the AMC critical for AHL biosynthesis and cellular methylation in Burkholderia glumae, the causal agent of rice panicle blight. Activation of genes encoding SAM-dependent methyltransferases, S-adenosylhomocysteine (SAH) hydrolase, and methionine synthases involved in the AMC by QS is essential for maintaining the optimal concentrations of methionine, SAM, and SAH required for bacterial cooperativity as cell density increases. Thus, the absence of QS perturbed metabolic homeostasis of the AMC and caused pleiotropic phenotypes in B. glumae. A null mutation in the SAH hydrolase gene negatively affected AHL and ATP biosynthesis and the activity of SAM-dependent methyltransferases including ToxA, which is responsible for the biosynthesis of a key virulence factor toxoflavin in B. glumae. These results indicate that QS controls metabolic flux of the AMC to secure the biosynthesis of AHL and cellular methylation in a cooperative population.

Environmental and cellular factors affecting the localization of T6SS proteins in Burkholderia thailandensis.

The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.

The polysaccharide extracted from the biofilm of Burkholderia multivorans strain C1576 binds hydrophobic species and exhibits a compact 3D-structure.

Microorganisms often grow in communities called biofilms where cells are imbedded in a complex self-produced biopolymeric matrix composed mainly of polysaccharides, proteins, and DNA. This matrix, together with cell proximity, confers many advantages to these microbial communities, but also constitutes a serious concern when biofilms develop in human tissues or on implanted prostheses. Although polysaccharides are considered the main constituents of the matrices, their specific role needs to be clarified. We have investigated the chemical and morphological properties of the polysaccharide extracted from biofilms produced by the C1576 reference strain of the opportunistic pathogen Burkholderia multivorans, which causes lung infections in cystic fibrosis patients. The aim of the present study is the definition of possible interactions of the polysaccharide and the three-dimensional conformation of its chain within the biofilm matrix. Surface plasmon resonance experiments confirmed the ability of the polysaccharide to bind hydrophobic molecules, due to the presence of rhamnose dimers in its primary structure. In addition, atomic force microscopy studies evidenced an extremely compact three-dimensional structure of the polysaccharide which may form aggregates, suggesting a novel view of its structural role into the biofilm matrix.

Gasdermin D Protects from Melioidosis through Pyroptosis and Direct Killing of Bacteria.

Gasdermin D (GSDMD) cleavage by caspase-1 or caspase-11 inflammasomes triggers pyroptosis, a lytic form of cell death protective against intracellular bacteria. In this study, we examine the role of GSDMD in a mouse model of melioidosis. Gsdmd-/- mice were more susceptible than wild-type mice to intranasal infection with Burkholderia thailandensis Production of IL-18, but not IL-1ß, was decreased in Gsdmd-/- infected mice. Despite lower IL-18, IFN-γ was produced in similar amounts in wild-type and Gsdmd-/- mice. In vitro, secretion of both IL-1ß and IL-18 by macrophages or dendritic cells infected with B. thailandensis was dependent on GSDMD. Surprisingly, wild-type or GSDMD-deficient neutrophils secreted similar amounts of IL-1ß, suggesting these cells may be the source of the GSDMD-independent IL-1ß detected in vivo. Recombinant GSDMD was able to directly kill B. thailandensis in vitro upon processing by active caspase-1. Moreover, bacteria harvested from wild-type, but not Gsdmd-/- , macrophages were more susceptible to the microbicidal effect of hydrogen peroxide or human ß-defensin-3. Finally, we provide evidence that pyroptosis of in vitro infected macrophages is directly microbicidal. Taken together, these results indicate that the protective action of GSDMD in melioidosis is primarily due to induction of pyroptosis and direct killing of bacteria rather than production of cytokines.

Natural killer cells kill Burkholderia cepacia complex via a contact-dependent and cytolytic mechanism.

Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection.

Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens.IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

[Multilocus sequence analysis, biofilm production, antibiotic susceptibility and synergy tests of Burkholderia species in patients with and without cystic fibrosis]. / Kistik fibrozisli ve kistik fibrozisi olmayan hasta grubunda Burkholderia türlerinin multilokus sekans analizi, biyofilm olusturma, antibiyotik duyarlilik ve sinerji testleri.

Burkholderia spp. emerged as important pathogens in the airways of immunocompromised humans, especially those with cystic fibrosis (CF). Failure of identification with conventional techniques, high intrinsic resistance to most antibiotics and biofilm formation can cause difficulties in the treatment of these infections. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with with routine microbiological methods, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multilocus sequence analysis (MLSA), to determine of the antibiotic susceptibility and synergies, and to evaluate biofilm formation of these isolates. A total of 38 Burkholderia spp. (25 CF, 13 non-CF) from 26 patients were identified by biochemical, phenotypical and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and sequence types were revealed by multilocus sequence analysis (MLSA). Sequence types of isolates were identified using the PubMLST database. Characteristics of biofilm formation of clinical isolates were evaluated by microplate method. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim-sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI (2017) guidelines. Synergy tests were performed by checkerboard method. Clinical isolates were identified as Burkholderia cenocepacia (n= 16), Burkholderia contaminans (n= 11), Burkholderia gladioli (n= 4), Burkholderia dolosa (n= 4), Burkholderia multivorans (n= 2) and Burkholderia seminalis (n= 1). Sequence types of these isolates were determined as ST19, ST72, ST102, ST180, ST482, ST602, ST629, ST740, ST839 and ST1392. The correct identification at the species-level with MALDI-TOF MS was 94-100% for all isolates except B.contaminans. Biofilm formation among the identified species in the study was determined as 53% (n= 20). There was no statistical difference when the biofilm production was evaluated separately among Burkholderia species and biofilm production rates between CF (56%, 14/25) and non-CF (46%, 6/13) Burkholderia isolates (p> 0.05). Overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin of the isolates were 35%, 66%, 50% and 40%, respectively. The antibiotic resistance against Burkholderia spp., isolates obtained from CF patients were more susceptible to ceftazidime, but no significant difference was found for other antibiotics. Synergy was determined between meropenem and TMP-SXT in two isolates. Antagonism was detected in 15 isolates, 12 of them were between meropenem and ceftazidime, three of them were between ceftazidime and TMP-SXT. Numerous resistance mechanisms may lead to higher resistance in this bacteria, whereas the antagonism between meropenem and ceftazidime in this study might be attributed to the expression of beta-lactamases. In this study, the distinctness of sequence types between Burkholderia spp. isolated from CF and non-CF patient, provided a better understanding about the importance of biofilm formation for the infections with these bacteria and emphasized that the management of therapy should be driven by the antibiotic test results.

Biological activities associated with the volatile compound 2,5-bis(1-methylethyl)-pyrazine.

Pyrazines are 1,4-diazabenzene-based volatile organic compounds and known for their broad-spectrum antimicrobial activity. In the present study, we assessed the antimicrobial activity of 2,5-bis(1-methylethyl)-pyrazine, produced by Paenibacillus sp. AD87 during co-culture with Burkholderia sp. AD24. In addition, we were using transcriptional reporter assays in E. coli and mammalian cells to decipher the possible mode of action. Bacterial and mammalian luciferase reporter strains were deployed to elucidate antimicrobial and toxicological effects of 2,5-bis(1-methylethyl)-pyrazine. At high levels of exposure, 2,5-bis(1-methylethyl)-pyrazine exerted strong DNA damage response. At lower concentrations, cell-wall damage response was observed. The activity was corroborated by a general toxicity reporter assay in E. coli ΔampD, defective in peptidoglycan turnover. The maximum E. coli cell-wall stress activity was measured at a concentration close to the onset of the mammalian cytotoxicity, while other adverse outcome pathways, such as the activation of aryl hydrocarbon and estrogenic receptor, the p53 tumour suppressor and the oxidative stress-related Nrf2 transcription factor, were induced at elevated concentrations compared to the response of mammalian cells. Because of its broad-spectrum antimicrobial activity at lower concentrations and the relatively low mammalian toxicity, 2,5-bis(1-methylethyl)-pyrazine is a potential bio-based fumigant with possible applications in food industry, agriculture or logistics.

An EmrB multidrug efflux pump in Burkholderia thailandensis with unexpected roles in antibiotic resistance.

The antibiotic trimethoprim is frequently used to manage Burkholderia infections, and members of the resistance-nodulation-division (RND) family of efflux pumps have been implicated in multidrug resistance of this species complex. We show here that a member of the distinct Escherichia coli multidrug resistance B (EmrB) family is a primary exporter of trimethoprim in Burkholderia thailandensis, as evidenced by increased trimethoprim sensitivity after inactivation of emrB, the gene that encodes EmrB. We also found that the emrB gene is up-regulated following the addition of gentamicin and that this up-regulation is due to repression of the gene encoding OstR, a member of the multiple antibiotic resistance regulator (MarR) family. The addition of the oxidants H2O2 and CuCl2 to B. thailandensis cultures resulted in OstR-dependent differential emrB expression, as determined by qRT-PCR analysis. Specifically, OstR functions as a rheostat that optimizes emrB expression under oxidizing conditions, and it senses oxidants by a unique mechanism involving two vicinal cysteines and one distant cysteine (Cys3, Cys4, and Cys169) per monomer. Paradoxically, emrB inactivation increased resistance of B. thailandensis to tetracycline, a phenomenon that correlated with up-regulation of an RND efflux pump. These observations highlight the intricate mechanisms by which expression of genes that encode efflux pumps is optimized depending on cellular concentrations of antibiotics and oxidants.

Caspase-11-dependent pyroptosis of lung epithelial cells protects from melioidosis while caspase-1 mediates macrophage pyroptosis and production of IL-18.

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1ß and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1ß and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1ß and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18's protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells.