RESUMO
INTRODUCTION: The global poultry industry produces millions of tons of waste feathers every year, which can be bio-degraded to make feed, fertilizer, and daily chemicals. However, feather bio-degradation is a complex process that is not yet fully understood. This results in low degradation efficiency and difficulty in industrial applications. Omics-driven system biology research offers an effective solution to quickly and comprehensively understand the molecularmechanisms involved in a metabolic pathway. METHODS: In the early stage of this process, feathers are hydrolyzed into water-soluble keratin monomers. In this study, we used high-throughput RNA-seq technology to analyze the genes involved in the internalization and degradation of keratin monomers in Stenotrophomonas maltophilia DHHJ strain cells. Moreover, we used Co-IP with LC-MS/MS technology to search for proteins that interact with recombinant keratin monomers. RESULTS: We discovered TonB transports and molecular chaperones associating with the keratin monomer, which may play a crucial role in the transmembrane transport of keratin. Meanwhile, multiple proteases belonging to distinct families were identified as binding partners of keratin monomers, among which ATPases associated with diverse cellular activity (AAA+) family proteases are overrepresented. Four genes, including JJL50_15620, JJL50_17955 (TonB-dependent receptors), JJL50_03260 (ABC transporter ATP-binding protein), and JJL50_20035 (ABC transporter substrate-binding protein), were selected as representatives for determining their expressions under different culture conditions using qRT-PCR, and they were found to be upregulated in response to keratin degradation consistent with the data from RNA-seq and Co-IP. CONCLUSION: This study highlights the complexity of keratin biodegradation in S. maltophilia DHHJ, in which multiple pathways are involved such as protein folding, protein transport, and several protease systems. Our findings provide new insights into the mechanism of feather degradation.
Assuntos
Proteínas de Bactérias , Biodegradação Ambiental , Queratinas , Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/metabolismo , Stenotrophomonas maltophilia/genética , Queratinas/metabolismo , Queratinas/genética , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Plumas/metabolismo , Plumas/microbiologia , Espectrometria de Massas em Tandem , Regulação Bacteriana da Expressão Gênica , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genéticaRESUMO
Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).
Assuntos
Proteínas de Bactérias , Stenotrophomonas maltophilia , Sistemas de Secreção Tipo IV , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/enzimologia , Stenotrophomonas maltophilia/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Conformação ProteicaRESUMO
Confronting the environmental threat posed by textile dyes, this study highlights bioremediation as a pivotal solution to mitigate the impacts of Crystal Violet, a widely-utilized triphenylmethane dye known for its mutagenic and mitotic toxicity. We isolated and identified several bacterial strains capable of degrading Crystal Violet under various environmental conditions. Newly identified strains, including Mycolicibacterium nivoides, Chryseobacterium sp., Agrobacterium rhizogenes, Pseudomonas crudilactis, and Pseudomonas koreensis demonstrated significant decolorization activity of Crystal Violet, complementing the already known capabilities of Stenotrophomonas maltophilia. Initial experiments using crude extracts confirmed their degradation potential, followed by detailed studies that investigated the impact of different pH levels and temperatures on some strains' degradation efficiency. Depending on the bacteria, the degree of activity change according to pH and temperature was different. At 37 °C, Chryseobacterium sp. and Stenotrophomonas maltophilia exhibited higher degradation activity compared to 25 °C, while Pseudomonas crudilactis and Mycolicibacterium nivoides did not exhibit a statistically significant difference between the two temperatures. Mycolicibacterium nivoides performed optimally at pH 8, while Pseudomonas crudilactis showed high activity at pH 5. Stenotrophomonas maltophilia's activity remained consistent across the pH range. These findings not only underscore the effectiveness of these bacteria as agents for Crystal Violet degradation but also pave the way for their application in large-scale bioremediation processes for the treatment of textile effluents, marking them as vital to environmental sustainability efforts.
Assuntos
Biodegradação Ambiental , Violeta Genciana , Violeta Genciana/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Pseudomonas/metabolismo , Pseudomonas/genética , Stenotrophomonas maltophilia/metabolismo , Corantes/metabolismo , Bactérias/metabolismo , Bactérias/genéticaRESUMO
Exploring the contribution of common microorganisms to spoilage is of great significance in inhibiting spoilage in lamb. This work investigated the extent of protein degradation and profile changes of free amino acids (FAAs), free fatty acids (FFAs) and volatile organic compounds (VOCs) in lamb caused by single- and co-culture of the common aerobic spoilage bacteria, P. paralactis, Ac. MN21 and S. maltophilia. Meanwhile, some key VOCs produced by the three bacteria during lamb spoilage were also screened by orthogonal partial least square discriminant analysis and difference value in VOCs content between inoculated groups and sterile group. Lamb inoculated with P. paralactis had the higher total viable counts, pH, total volatile base nitrogen and TCA-soluble peptides than those with the other two bacteria. Some FAAs and FFAs could be uniquely degraded by P. paralactis but not Ac. MN21 and S. maltophilia, such as Arg, Glu, C15:0, C18:0 and C18:1n9t. Co-culture of the three bacteria significantly promoted the overall spoilage, including bacterial growth, proteolysis and lipolysis. Key VOCs produced by P. paralactis were 2, 3-octanedione, those by Ac. MN21 were 1-octanol, octanal, hexanoic acid, 1-pentanol and hexanoic acid methyl ester, and that by S. maltophilia were hexanoic acid. The production of extensive key-VOCs was significantly and negatively correlated with C20:0, C23:0 and C18:ln9t degradation. This study can provide a basis for inhibiting common spoilage bacteria and promoting high-quality processing of fresh lamb.
Assuntos
Acinetobacter , Técnicas de Cocultura , Microbiologia de Alimentos , Pseudomonas , Carne Vermelha , Stenotrophomonas maltophilia , Compostos Orgânicos Voláteis , Animais , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo , Pseudomonas/metabolismo , Pseudomonas/crescimento & desenvolvimento , Acinetobacter/crescimento & desenvolvimento , Acinetobacter/metabolismo , Stenotrophomonas maltophilia/crescimento & desenvolvimento , Stenotrophomonas maltophilia/metabolismo , Carne Vermelha/microbiologia , Carne Vermelha/análise , Ovinos , Armazenamento de Alimentos , Temperatura Baixa , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos não Esterificados/análise , Aminoácidos/metabolismo , Aminoácidos/análise , Carneiro Doméstico/microbiologia , ProteóliseRESUMO
Immobilization technology is a promising way to improve effectiveness and stability of microbial remediation for polycyclic aromatic hydrocarbons (PAHs), in which carrier material is one of key factors restricting removal efficiency. In this study, fulvic acid-wheat straw biochar (FA/WS) composites were applied for immobilization of an efficient PAHs degrading bacterium Stenotrophomonas maltophilia (SPM). FA/WS&SPM showed superior degradation capacity than free bacteria and biochar-immobilized bacteria, with the removal efficiency of pyrene (20 mg L-1) reaching 90.5 % (7 days). Transcriptome analysis revealed that FA in the carrier materials can promote transportation and degradation of pyrene, and cell growth, as well as inhibit cell apoptosis. Enzyme activity and degradation products detection showed that SPM utilized both phthalic acid and salicylic acid metabolic pathways to degrade pyrene. Practicality of FA/WS&SPM for different kinds of PAHs remediation had been verified in contaminated soil, demonstrating a great potential in the field of PAHs polluted sites remediation.
Assuntos
Benzopiranos , Biodegradação Ambiental , Células Imobilizadas , Pirenos , Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/metabolismo , Pirenos/metabolismo , Benzopiranos/metabolismo , Células Imobilizadas/metabolismo , Carvão Vegetal/química , Carvão Vegetal/farmacologiaRESUMO
Introduction: The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of Stenotrophomonas maltophilia is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake. Methods: Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of S. maltophilia. Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction. Results: Smlt3896-hemU-exbB2-exbD2-tonB2 and tonB1-exbB1-exbD1a-exbD1b operons were selected as candidates for hemin acquisition. Compared with the parental strain, hemU and tonB1 mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the Smlt3896 and tonB2 mutants was comparable to that of the parental strain. HemA expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated hemA expression. Like hemA, hemU was repressed by Fur in iron-replete conditions; however, hemU was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike hemA and hemU, the TonB1-exbB1-exbD1a-exbD1b operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression. Conclusion: HemA, HemU, and TonB1 contribute to hemin acquisition in S. maltophilia. Fur represses the expression of hemA and hemU in iron-replete conditions. HemA expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. HemU expression is regulated by low iron and hemin levels in a hemP-dependent manner.
Assuntos
Hemina , Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ferro/metabolismoRESUMO
Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.
Assuntos
Anti-Infecciosos , Infecções por Bactérias Gram-Negativas , Stenotrophomonas maltophilia , Humanos , Stenotrophomonas maltophilia/metabolismo , Bactérias Gram-Negativas , Biofilmes , Membrana Celular , Anti-Infecciosos/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologiaRESUMO
BACKGROUND: SmeYZ is a constitutively expressed efflux pump in Stenotrophomonas maltophilia. Previous studies demonstrated that: (i) smeYZ inactivation causes compromised swimming, oxidative stress tolerance and aminoglycoside resistance; and (ii) the ΔsmeYZ-mediated pleiotropic defects, except aminoglycoside susceptibility, result from up-regulation of entSCEBB'FA and sbiAB operons, and decreased intracellular iron level. OBJECTIVES: To elucidate the modulatory role of SmeQ, a novel cytoplasmic protein, in ΔsmeYZ-mediated pleiotropic defects. METHODS: The presence of operons was verified using RT-PCR. The role of SmeQ in ΔsmeYZ-mediated pleiotropic defects was assessed using in-frame deletion mutants and functional assays. A bacterial adenylate cyclase two-hybrid assay was used to investigate the protein-protein interactions. Gene expression was quantified using quantitative RT-PCR (RT-qPCR). RESULTS: SmeYZ and the downstream smeQ formed an operon. SmeQ inactivation in the WT KJ decreased aminoglycoside resistance but did not affect swimming and tolerance to oxidative stress or iron depletion. However, smeQ inactivation in the smeYZ mutant rescued the ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. In the WT KJ, SmeQ positively modulated SmeYZ pump function by transcriptionally up-regulating the smeYZQ operon. Nevertheless, in the smeYZ mutant, SmeQ exerted its modulatory role by up-regulating entSCEBB'FA and sbiAB operons, decreasing intracellular iron levels, and causing ΔsmeYZ-mediated pleiotropic defects, except for aminoglycoside susceptibility. CONCLUSIONS: SmeQ is the first small protein identified to be involved in efflux pump function in S. maltophilia. It exerts modulatory effect by transcriptionally altering the expression of target genes, which are the smeYZQ operon in the WT KJ, and smeYZQ, entSCEBB'FA and sbiAB operons in smeYZ mutants.
Assuntos
Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Aminoglicosídeos , Ferro/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Stenotrophomonas maltophilia J2, a highly efficient pyridine-degrading bacterium, was isolated from the aerobic tank of a pesticide-contaminated wastewater treatment plant. The strain J2 demonstrated an impressive pyridine degradation rate of 98.34% ± 0.49% within 72 h, at a pyridine concentration of 1100 mg·L-1, a temperature of 30 °C, a pH of 8.0, and a NaCl concentration of 0.5%. Notably, two new pyridine metabolic intermediates, 1,3-dihydroxyacetone and butyric acid, were discovered, indicating that J2 may degrade pyridine through two distinct metabolic pathways. Furthermore, the immobilized strain J2 was obtained by immobilizing J2 with biochar derived from the stem of Solidago canadensis L. In the pyridine-contaminated wastewater bioremediation experiment, the immobilized strain J2 was able to remove 2000 mg·L-1 pyridine with a 98.66% ± 0.47% degradation rate in 24 h, which was significantly higher than that of the control group (3.17% ± 1.24%), and remained above 90% in subsequent cycles until the 27th cycle. High-throughput sequencing analysis indicated that the J2 +B group had an elevated relative abundance of bacteria and functional genes that could be associated with the degradation of pyridine. The results offer a foundation for the effective use of immobilized strain in the treatment of recalcitrant pyridine-contaminated wastewater.
Assuntos
Stenotrophomonas maltophilia , Águas Residuárias , Stenotrophomonas maltophilia/metabolismo , Biodegradação Ambiental , Bactérias/metabolismo , Piridinas/metabolismoRESUMO
The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.
Assuntos
Oxitetraciclina , Stenotrophomonas maltophilia , Humanos , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Cristalografia por Raios X , Antibacterianos/farmacologia , Antibacterianos/química , Tetraciclina/farmacologia , Tetraciclina/metabolismo , Oxitetraciclina/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299-356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299-356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299-356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299-356. KJΔOmpA299-356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299-356. Next, the involvement of the three σ factors in the ΔompA299-356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299-356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299-356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal ß-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to ß-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.
Assuntos
Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Regulon , Peptidoglicano/metabolismo , Fator sigma/metabolismo , Estresse Oxidativo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.
Assuntos
Stenotrophomonas maltophilia , Humanos , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Lipopolissacarídeos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Expressão GênicaRESUMO
BACKGROUND: Stenotrophomonas maltophilia is ubiquitous in the environment and is an important MDR opportunistic pathogen. Oxidative stress is an inevitable challenge to an aerobic bacterium. Accordingly, S. maltophilia has many capabilities to face variable oxidative stress. Some of the oxidative stress alleviation systems cross-protect bacteria from antibiotics. In our recent RNA-sequencing transcriptome analysis, we documented the increased expression of a three-gene cluster, yceA-cybB-yceB, in the presence of hydrogen peroxide (H2O2). The YceI-like, cytochrome b561 and YceI-like proteins encoded by yceA, cybB and yceB are located in the cytoplasm, inner membrane and periplasm, respectively. OBJECTIVES: To characterize the role of the yceA-cybB-yceB operon of S. maltophilia in oxidative stress tolerance, swimming motility and antibiotic susceptibility. METHODS: The presence of the yceA-cybB-yceB operon was verified by RT-PCR. The functions of this operon were revealed by in-frame deletion mutant construction and complementation assay. Expression of the yceA-cybB-yceB operon was assessed by quantitative RT-PCR. RESULTS: The yceA, cybB and yceB genes form an operon. Loss of function of the yceA-cybB-yceB operon compromised menadione tolerance, enhanced swimming motility and increased susceptibility to fluoroquinolone and ß-lactam antibiotics. The expression of the yceA-cybB-yceB operon was up-regulated by oxidative stress, such as H2O2 and superoxide, and not impacted by antibiotics, such as fluoroquinolone and ß-lactams. CONCLUSIONS: The evidence strongly supports the view that the physiological function of the yceA-cybB-yceB operon is to alleviate oxidative stress. The operon provides an additional example that oxidative stress alleviation systems can cross-protect S. maltophilia from antibiotics.
Assuntos
Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Natação , Peróxido de Hidrogênio/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Estresse Oxidativo , Fluoroquinolonas/metabolismo , ÓperonRESUMO
A number of multidrug-resistant bacterial pathogens code for S1-P1 nucleases with a poorly understood role. We have characterized a recombinant form of S1-P1 nuclease from Stenotrophomonas maltophilia, an opportunistic pathogen. S. maltophilia nuclease 1 (SmNuc1) acts predominantly as an RNase and is active in a wide range of temperatures and pH. It retains a notable level of activity towards RNA and ssDNA at pH 5 and 9 and about 10% of activity towards RNA at 10 °C. SmNuc1 with very high catalytic rates outperforms S1 nuclease from Aspergillus oryzae and other similar nucleases on all types of substrates. SmNuc1 degrades second messenger c-di-GMP, which has potential implications for its role in the pathogenicity of S. maltophilia.
Assuntos
Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , GMP Cíclico/metabolismo , Endonucleases/metabolismo , RNA/metabolismoRESUMO
Stenotrophomonas maltophilia is an emerging opportunistic respiratory pathogen in people with cystic fibrosis (CF). S. maltophilia is frequently observed in polymicrobial infections, and we have previously shown that Pseudomonas aeruginosa promotes colonization and persistence of S. maltophilia in mouse respiratory infections. In this study, we used host and bacterial RNA sequencing to further understand the molecular underpinnings of this interaction. To evaluate S. maltophilia transcript profiles, we used a recently described method for selective capture of bacterial mRNA transcripts with strain-specific RNA probes. We found that factors associated with the type IV pilus, including the histidine kinase subunit of a chemotactic two-component signaling system (chpA), had increased transcript levels during dual-species infection. Using immortalized CF respiratory epithelial cells, we found that infection with P. aeruginosa increases adherence of S. maltophilia, at least in part due to disruption of epithelial tight junctions. In contrast, an isogenic S. maltophilia chpA mutant strain lacked cooperative adherence to CF epithelia and decreased bacterial burden in vivo in dual-species infections with P. aeruginosa. Similarly, P. aeruginosa lacking elastase (lasB) failed to promote S. maltophilia adherence or bacterial colonization and persistence in vivo. Based on these results, we propose that disruption of lung tissue integrity by P. aeruginosa facilitates adherence of S. maltophilia to the lung epithelia, likely in a type IV pilus-dependent manner. These data lend insight into S. maltophilia colonization and persistence in people in later stages of CF disease and may have implications for interactions with other bacterial opportunists. IMPORTANCE Despite advances in treatment options for people with CF, complications of bacterial infections remain the greatest driver of morbidity and mortality in this patient population. These infections often involve more than one bacterial pathogen, and our understanding of how interspecies interactions impact disease progression is lacking. Previous work in our lab found that two CF pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, can work together in the lung to cause more severe infection. In the present study, we found that infection with P. aeruginosa promotes persistence of S. maltophilia by interfering with epithelial barrier integrity. Depolarization of the epithelial cell layer by P. aeruginosa-secreted elastase increased S. maltophilia adherence, likely in a type IV pilus-dependent manner. Ultimately, this work sheds light on the molecular mechanisms governing an important multispecies interaction seen in pulmonary diseases such as CF.
Assuntos
Fibrose Cística , Infecções por Bactérias Gram-Negativas , Stenotrophomonas maltophilia , Humanos , Animais , Camundongos , Pseudomonas aeruginosa/genética , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Células Epiteliais/microbiologia , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Mucosa Respiratória , Infecções por Bactérias Gram-Negativas/microbiologiaRESUMO
Iron is an essential micronutrient for various bacterial cellular processes. Fur is a global transcriptional regulator participating in iron homeostasis. Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has emerged as an opportunistic pathogen. To elucidate the novel regulatory mechanism behind iron homeostasis in S. maltophilia, wild-type KJ and KJΔFur, a fur mutant, were subjected to transcriptome assay. A five-gene cluster, sbiBA-sbiTRS, was significantly upregulated in KJΔFur. SbiAB is an ATP type efflux pump, SbiT is an inner membrane protein, and SbiSR is a two-component regulatory system (TCS). The sbiTRS operon organization was verified by reverse transcription-PCR (RT-PCR). Localization prediction and bacterial two-hybrid studies revealed that SbiT resided in the inner membrane and had an intramembrane interaction with SbiS. In iron-replete conditions, SbiT interacted with SbiS and maintained SbiSR TCS in a resting state. In response to iron depletion stress, SbiT no longer interacted with SbiS, leading to SbiSR TCS activation. The iron source utilization assay demonstrated the contribution of SbiSR TCS to stenobactin-mediated ferric iron utilization but notto the utilization of hemin and ferric citrate. Furthermore, SmeDEF and SbiAB pumps, known stenobactin secretion outlets, were members of the SbiSR regulon. Collectively, in an iron-depleted condition, SbiSR activation is regulated by Fur at the transcriptional level and by SbiT at the posttranslational level. Activated SbiSR contributes to stenobactin-mediated ferric iron utilization by upregulating the smeDEF and sbiAB operons. SbiSR is the first TCS found to be involved in iron homeostasis in S. maltophilia. IMPORTANCE Therapeutic options for Stenotrophomonas maltophilia infections are limited because S. maltophilia is intrinsically resistant to several antibiotics. Iron is an essential element for viability, but iron overload is a lethal threat to bacteria. Therefore, disruption of iron homeostasis can be an alternative strategy to cope with S. maltophilia infection. The intricate regulatory networks involved in iron hemostasis have been reported in various pathogens; however, little is known about S. maltophilia. Herein, a novel sbiTRS operon, a member of Fur regulon, was characterized. SbiT, an inner membrane protein, negatively modulated the SbiSR two-component regulatory system by intramembrane protein-protein interaction with SbiS. In response to iron-depleted stress, SbiSR was activated via the regulation of Fur and SbiT. Activated SbiSR upregulated smeDEF and sbiAB, which contributed to stenobactin-mediated ferric iron utilization. A novel fur-sbiT-sbiSR-smeDEF/sbiAB regulatory circuit in S. maltophilia was revealed.
Assuntos
Ferro , Stenotrophomonas maltophilia , Ferro/metabolismo , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , ÓperonRESUMO
OmpA, the most abundant porin in Stenotrophomonas maltophilia KJ, exists as a two-domain structure with an N-terminal domain of ß-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJΔOmpA299-356, an ompA mutant of S. maltophilia KJ with a truncated OmpA devoid of 299 to 356 amino acids (aa), was able to stably embed in the outer membrane. KJΔOmpA299-356 was more susceptible to ß-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the ΔompA299-356-mediated increase in ß-lactam susceptibility (abbreviated as "ΔOmpA299-356 phenotype"). KJΔOmpA299-356 displayed a lower ceftazidime (CAZ)-induced ß-lactamase activity than KJ. Furthermore, KJ2, a L1/L2 ß-lactamases-null mutant, and KJ2ΔOmpA299-356, a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable ß-lactam susceptibility. Both lines of evidence indicate that decreased ß-lactamase activity contributes to the ΔOmpA299-356 phenotype. We analyzed the transcriptome results of KJ and KJΔOmpA299-356, focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJΔOmpA299-356. Deletion of the nagA gene from the chromosome of KJΔOmpA299-356 restored ß-lactam susceptibility and CAZ-induced ß-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJΔOmpA299-356 contributes to the ΔOmpA299-356 phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJΔOmpA299-356. The deletion mutant construction, ß-lactam susceptibility, and ß-lactamase activity analysis demonstrated that σP, but not σE, was involved in the ΔOmpA299-356 phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the σP regulon. The involvement of the σP-NagA-L1/L2 regulatory circuit in the ΔOmpA299-356 phenotype was manifested. IMPORTANCE Porins of Gram-negative bacteria generally act as channels that allow the entry or extrusion of molecules. Moreover, the structural role of porins in stabilizing the outer membrane by interacting with peptidoglycan (PG) and the outer membrane has been proposed. The linkage between porin deficiency and antibiotic resistance increase has been reported widely, with a rationale for blocking antibiotic influx. In this study, a link between porin defects and ß-lactam susceptibility increase was demonstrated. The underlying mechanism revealed that a novel σP-NagA-L1/L2 regulatory circuit is triggered due to the loss of the OmpA-PG interaction. This study extends the understanding on the porin defect and antibiotic susceptibility. Porin defects may cause opposite impacts on antibiotic susceptibility, which is dependent on the involvement of the defect. Blocking the porin channel role can increase antibiotic resistance; in contrast, the loss of porin structure role may increase antibiotic susceptibility.
Assuntos
Stenotrophomonas maltophilia , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Ceftazidima/farmacologia , Porinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Antibiotic resistance is threatening the medical industry in treating microbial infections. Many organisms are acquiring antibiotic resistance because of the continuous use of the same drug. Gram-negative organisms are developing multi-drug resistance properties (MDR) due to chromosomal level changes that occurred as a part of evolution or some intrinsic factors already present in the organism. Stenotrophomonas maltophilia falls under the category of multidrug-resistant organism. WHO has also urged to evaluate the scenario and develop new strategies for making this organism susceptible to otherwise resistant antibiotics. Using novel compounds as drugs can ameliorate the issue to some extent. The ß-lactamase enzyme in the bacteria is responsible for inhibiting several drugs currently being used for treatment. This enzyme can be targeted to find an inhibitor that can inhibit the enzyme activity and make the organism susceptible to ß-lactam antibiotics. Plants produce several secondary metabolites for their survival in adverse environments. Several phytoconstituents have antimicrobial properties and have been used in traditional medicine for a long time. The computational technologies can be exploited to find the best compound from many compounds. Virtual screening, molecular docking, and dynamic simulation methods are followed to get the best inhibitor for L1 ß-lactamase. IMPPAT database is screened, and the top hit compounds are studied for ADMET properties. Finally, four compounds are selected to set for molecular dynamics simulation. After all the computational calculations, withanolide R is found to have a better binding and forms a stable complex with the protein. This compound can act as a potent natural inhibitor for L1 ß-lactamase.
Assuntos
Stenotrophomonas maltophilia , Vitanolídeos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Stenotrophomonas maltophilia/metabolismo , beta-Lactamases/química , beta-Lactamas/metabolismoRESUMO
Bacteria are candidates for the biotransformation of environmental arsenic (As), while As metabolism in bacteria is not yet fully understood. In this study, we sequenced the genome of an As-resistant bacterium strain Stenotrophomonas maltophilia SCSIOOM isolated from the fish gut. After arsenate (As(V)) exposure, S. maltophilia transformed As(V) to organoarsenicals, along with the significant change of the expression of 40 genes, including the upregulation of arsH, arsRBC and betIBA. The heterogeneous expression of arsH and arsRBC increased As resistance of E. coli AW3110 by increasing As efflux and transformation. E. coli AW3110 (pET-betIBA) could transform inorganic As into dimethylarsinate (DMA) and nontoxic arsenobetaine (AsB), which suggested that AsB could be synthesized through the synthetic pathway of its analog-glycine betaine. In addition, the existence of arsRBC, betIBA and arsH reduced the reactive oxygen species (ROS) induced by As exposure. In total, these results demonstrated that S. maltophilia adopted an As metabolism strategy by reducing As accumulation and synthesizing less toxic As species. We first reported the production and potential synthetic pathway of AsB in bacteria, which improved our knowledge of As toxicology in microorganisms.
Assuntos
Arsênio , Stenotrophomonas maltophilia , Animais , Arseniatos/metabolismo , Arseniatos/toxicidade , Arsênio/metabolismo , Arsênio/toxicidade , Arsenicais , Betaína/metabolismo , Ácido Cacodílico/metabolismo , Escherichia coli/metabolismo , Peixes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Stenotrophomonas maltophilia/metabolismoRESUMO
Synthetic conjugate vaccines are an important area of research for the prevention and occurrence of diseases caused by Gram-negative bacteria. For the development of such vaccines, access to the pure and homogeneous oligosaccharide fragments of the bacterial cell surface polysaccharides are necessary. Stenotrophomonas maltophilia is a typical opportunistic Gram-negative bacteria that causes severe pulmonary and other infections; often in hospitalized patients. With the emergence of multidrug resistant strains and increased virulence, new therapeutic strategies are needed to combat the threat. Herein, we report the syntheses of the trisaccharide repeating unit of S. maltophilia O6 antigen through stepwise and one-pot assemblies of the trisaccharide. The target trisaccharide was appended with a 2-aminoethyl linker that could provide the opportunity for conjugation to carrier proteins for the synthesis of vaccine candidates.