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1.
Emerg Infect Dis ; 30(10): 2056-2069, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39320153

RESUMO

In the United States in 2021, an outbreak of 4 cases of Burkholderia pseudomallei, the etiologic agent of melioidosis and a Tier One Select Agent (potential for deliberate misuse and subsequent harm), resulted in 2 deaths. The causative strain, B. pseudomallei ATS2021, was unintentionally imported into the United States in an aromatherapy spray manufactured in India. We established that ATS2021 represents a virulent strain of B. pseudomallei capable of robust formation of biofilm at physiologic temperatures that may contribute to virulence. By using mouse melioidosis models, we determined median lethal dose estimates and analyzed the bacteriologic and histopathologic characteristics of the organism, particularly the potential neurologic pathogenesis that is probably associated with the bimABm allele identified in B. pseudomallei strain ATS2021. Our data, combined with previous case reports and the identification of endemic B. pseudomallei strains in Mississippi, support the concept that melioidosis is emerging in the United States.


Assuntos
Burkholderia pseudomallei , Melioidose , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Melioidose/microbiologia , Melioidose/epidemiologia , Animais , Camundongos , Virulência , Estados Unidos/epidemiologia , Humanos , Feminino , Modelos Animais de Doenças , Biofilmes , Doenças Transmissíveis Importadas/microbiologia , Doenças Transmissíveis Importadas/epidemiologia
2.
Infect Immun ; 90(8): e0015922, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35862734

RESUMO

Burkholderia pseudomallei, the causative agent of melioidosis, is classified by the CDC as a tier 1 select agent, and work involving it must be performed in a biosafety level 3 (BSL-3) laboratory. Three BSL-2 surrogate strains derived from B. pseudomallei 1026b, a virulent clinical isolate, have been removed from the CDC select agent list. These strains, Bp82, B0011, and JW270, are highly attenuated in rodent models of melioidosis and cannot be utilized to identify virulence determinants because of their high 50% lethal dose (LD50). We previously demonstrated that the Madagascar hissing cockroach (MHC) is a tractable surrogate host to study the innate immune response against Burkholderia. In this study, we found that JW270 maintains its virulence in MHCs. This surprising result indicates that it may be possible to identify potential virulence genes in JW270 by using MHCs at BSL-2. We tested this hypothesis by constructing JW270 mutations in genes that are required (hcp1) or dispensable (hcp2) for B. pseudomallei virulence in rodents. JW270 Δhcp1 was avirulent in MHCs and JW270 Δhcp2 was virulent, suggesting that MHCs can be used at BSL-2 for the discovery of important virulence factors. JW270 ΔBPSS2185, a strain harboring a mutation in a type IV pilin locus (TFP8) required for full virulence in BALB/c mice, was also found to be attenuated in MHCs. Finally, we demonstrate that the hmqA-G locus, which encodes the production of a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines, is important for JW270 virulence in MHCs and may represent a novel virulence determinant.


Assuntos
Burkholderia pseudomallei , Baratas , Melioidose , Animais , Baratas/metabolismo , Contenção de Riscos Biológicos , Modelos Animais de Doenças , Madagáscar , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
Microbiology (Reading) ; 168(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35293855

RESUMO

Type IV pili are involved in adhesion, twitching motility, aggregation, biofilm formation and virulence in a variety of Gram-negative bacteria. Burkholderia pseudomallei, the causative agent of melioidosis and a Tier 1 biological select agent, is a Gram-negative bacterium with eight type IV pili-associated loci (TFP1 to TFP8). Most have not been fully characterized. In this study, we investigated BPSS2185, an uncharacterized TFP8 gene that encodes a type IVB pilus protein subunit. Using genetic deletion and complementation analysis in B. pseudomallei JW270, we demonstrate that BPSS2185 plays an important role in twitching motility and adhesion to A549 human alveolar epithelial cells. Compared to JW270, the JW270 ΔBPSS2185 mutant failed to display twitching motility and did not adhere to the epithelial cells. These phenotypes were partially reversed by the complementation of BPSS2185 in the mutant strain. The study also shows that BPSS2185 is expressed only during the onset of mature biofilm formation and at the dispersal of a biofilm, suggesting that the motility characteristic is required to form a biofilm. Our study is the first to suggest that the BPSS2185 gene in TFP8 contributes to twitching motility, adhesion and biofilm formation, indicating that the gene may contribute to B. pseudomallei virulence.


Assuntos
Burkholderia pseudomallei , Proteínas de Fímbrias , Biofilmes , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Células Epiteliais/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo
4.
J Biol Chem ; 294(36): 13248-13268, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31350337

RESUMO

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a ß-Gal-(1,3)-α-GalNAc-(1,3)-ß-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan-specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Proteínas de Bactérias/genética , Cromatografia Líquida , Biologia Computacional , Glicoproteínas/genética , Glicosilação , Humanos , Espectrometria de Massas , Mutação , Polissacarídeos/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie
5.
Infect Immun ; 86(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29109172

RESUMO

Burkholderia pseudomallei, the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging, and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell surface polysaccharides and cell-associated and cell-secreted proteins. Based on those findings, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated, and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate, while immunization with Hcp1 and TssM produced high-titer IgG and robust gamma interferon-secreting T cell responses against the proteins. Extending upon these studies, we found that when mice were vaccinated with a combination of CPS-CRM197 and Hcp1, 100% of the mice survived a lethal inhalational challenge with B. pseudomallei Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers, or spleens, indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights toward the development of a safe, affordable, and effective melioidosis vaccine.


Assuntos
Vacinas Bacterianas/imunologia , Melioidose/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Burkholderia pseudomallei , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/imunologia , Vacinas de Subunidades Antigênicas
6.
Microbiology (Reading) ; 164(9): 1196-1211, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30052173

RESUMO

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2-lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6-lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2-lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.


Assuntos
Antibacterianos/farmacologia , Burkholderia pseudomallei/efeitos dos fármacos , Burkholderia pseudomallei/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Família Multigênica , Fatores de Transcrição/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Burkholderia pseudomallei/genética , Perfilação da Expressão Gênica , Regulon , Fatores de Transcrição/genética
7.
Infect Immun ; 85(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27799332

RESUMO

Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia-infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia.


Assuntos
Antiácidos/farmacologia , Burkholderia mallei/efeitos dos fármacos , Burkholderia pseudomallei/efeitos dos fármacos , Cloroquina/farmacologia , Células Gigantes/efeitos dos fármacos , Sistemas de Secreção Tipo VI/efeitos dos fármacos , Virulência/efeitos dos fármacos , Animais , Proteínas de Bactérias/metabolismo , Burkholderia mallei/metabolismo , Burkholderia pseudomallei/metabolismo , Linhagem Celular , Mormo/tratamento farmacológico , Mormo/microbiologia , Concentração de Íons de Hidrogênio , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Camundongos , Sistemas de Secreção Tipo III/efeitos dos fármacos , Fatores de Virulência/metabolismo
8.
BMC Bioinformatics ; 17: 387, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27650316

RESUMO

BACKGROUND: Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized. RESULTS: We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users' search of detailed information for orthologous proteins related to secretion systems of the two pathogens. CONCLUSIONS: The updates of DBSecSys 2.0 provide unique capabilities to access comprehensive information about secretion systems of B. mallei and B. pseudomallei. They enable studies and comparisons of corresponding proteins of these two closely related pathogens and their host-interacting partners. The database is available at http://dbsecsys.bhsai.org .


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Burkholderia mallei/patogenicidade , Burkholderia pseudomallei/patogenicidade , Bases de Dados de Proteínas , Animais , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Burkholderia mallei/genética , Burkholderia mallei/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Humanos , Camundongos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
9.
PLoS Comput Biol ; 11(3): e1004088, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25738731

RESUMO

Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections and intracellular spread.


Assuntos
Burkholderia mallei/fisiologia , Burkholderia mallei/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Algoritmos , Animais , Proteínas de Bactérias/fisiologia , Análise por Conglomerados , Biologia Computacional , Adesões Focais , Mormo/microbiologia , Mormo/fisiopatologia , Humanos , Camundongos , Mapas de Interação de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Fatores de Virulência/metabolismo
10.
Annu Rev Microbiol ; 64: 495-517, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20528691

RESUMO

Burkholderia pseudomallei and Burkholderia mallei are closely related gram-negative bacteria that can cause serious diseases in humans and animals. This review summarizes the current and rapidly expanding knowledge on the specific virulence factors employed by these pathogens and their roles in the pathogenesis of melioidosis and glanders. In particular, the contributions of recently identified virulence factors are described in the context of the intracellular lifestyle of these pathogens. Throughout this review, unique and shared virulence features of B. pseudomallei and B. mallei are discussed.


Assuntos
Burkholderia mallei/patogenicidade , Burkholderia pseudomallei/patogenicidade , Mormo/microbiologia , Melioidose/microbiologia , Melioidose/veterinária , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Citoplasma/microbiologia , Mormo/patologia , Humanos , Melioidose/patologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
11.
Mol Cell Proteomics ; 12(11): 3036-51, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23800426

RESUMO

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes.


Assuntos
Burkholderia mallei/metabolismo , Burkholderia mallei/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia mallei/genética , Feminino , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Insercional , Mapeamento de Interação de Proteínas , Proteômica , Técnicas do Sistema de Duplo-Híbrido , Virulência/genética , Virulência/fisiologia , Fatores de Virulência/genética
12.
BMC Bioinformatics ; 15: 244, 2014 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-25030112

RESUMO

BACKGROUND: Bacterial pathogenicity represents a major public health concern worldwide. Secretion systems are a key component of bacterial pathogenicity, as they provide the means for bacterial proteins to penetrate host-cell membranes and insert themselves directly into the host cells' cytosol. Burkholderia mallei is a Gram-negative bacterium that uses multiple secretion systems during its host infection life cycle. To date, the identities of secretion system proteins for B. mallei are not well known, and their pathogenic mechanisms of action and host factors are largely uncharacterized. DESCRIPTION: We present the Database of Burkholderia malleiSecretion Systems (DBSecSys), a compilation of manually curated and computationally predicted bacterial secretion system proteins and their host factors. Currently, DBSecSys contains comprehensive experimentally and computationally derived information about B. mallei strain ATCC 23344. The database includes 143 B. mallei proteins associated with five secretion systems, their 1,635 human and murine interacting targets, and the corresponding 2,400 host-B. mallei interactions. The database also includes information about 10 pathogenic mechanisms of action for B. mallei secretion system proteins inferred from the available literature. Additionally, DBSecSys provides details about 42 virulence attenuation experiments for 27 B. mallei secretion system proteins. Users interact with DBSecSys through a Web interface that allows for data browsing, querying, visualizing, and downloading. CONCLUSIONS: DBSecSys provides a comprehensive, systematically organized resource of experimental and computational data associated with B. mallei secretion systems. It provides the unique ability to study secretion systems not only through characterization of their corresponding pathogen proteins, but also through characterization of their host-interacting partners.The database is available at https://applications.bhsai.org/dbsecsys.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Burkholderia mallei/fisiologia , Bases de Dados de Proteínas , Animais , Burkholderia mallei/metabolismo , Burkholderia mallei/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Fatores de Virulência/metabolismo
13.
Infect Immun ; 82(8): 3214-26, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24866793

RESUMO

Burkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei ΔgspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ΔgspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Burkholderia pseudomallei/química , Proteoma/análise , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Espectrometria de Massas em Tandem
14.
J Am Chem Soc ; 136(26): 9484-90, 2014 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-24884988

RESUMO

Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics.


Assuntos
Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidade , Metabolismo Secundário , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Animais , Burkholderia pseudomallei/química , Burkholderia pseudomallei/genética , Feminino , Genoma Bacteriano , Recombinação Homóloga , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Lisina/farmacologia , Melioidose/microbiologia , Camundongos Endogâmicos BALB C , Família Multigênica , Mutação , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Regiões Promotoras Genéticas , Fatores de Virulência/genética
15.
BMC Microbiol ; 14: 98, 2014 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-24750902

RESUMO

BACKGROUND: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. RESULTS: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. CONCLUSIONS: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines.


Assuntos
Burkholderia pseudomallei/fisiologia , Células Gigantes/citologia , Células Gigantes/microbiologia , Processamento de Imagem Assistida por Computador , Macrófagos/citologia , Macrófagos/microbiologia , Imagem Óptica , Animais , Automação Laboratorial , Linhagem Celular , Técnicas Citológicas , Camundongos , Fenótipo
16.
Appl Environ Microbiol ; 79(19): 5830-43, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23872555

RESUMO

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.


Assuntos
Burkholderia/fisiologia , Resposta SOS em Genética , Transcriptoma , Antibacterianos/farmacologia , Bacteriófagos/genética , Burkholderia/efeitos dos fármacos , Burkholderia/genética , Ciprofloxacina/farmacologia , Mitomicina/farmacologia , Mutagênicos , Biossíntese de Proteínas , Análise de Sequência de DNA , Siphoviridae/genética , Transcrição Gênica
17.
BMC Microbiol ; 12: 117, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22892068

RESUMO

BACKGROUND: Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. RESULTS: Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was <10 colony-forming units (cfu) for all three species. In comparison, the LD50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect's open circulatory system in vivo. CONCLUSIONS: The results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system.


Assuntos
Burkholderia/patogenicidade , Baratas/microbiologia , Modelos Animais , Animais , Interações Hospedeiro-Patógeno , Virulência , Fatores de Virulência/metabolismo
18.
BMC Microbiol ; 12: 289, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23217012

RESUMO

BACKGROUND: Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. RESULTS: We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. CONCLUSIONS: The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/fisiologia , Burkholderia mallei/virologia , Burkholderia pseudomallei/virologia , Especificidade de Hospedeiro , Bacteriófagos/isolamento & purificação , DNA Viral/química , DNA Viral/genética , Genoma Viral , Dados de Sequência Molecular , Receptores Virais/fisiologia , Análise de Sequência de DNA , Ligação Viral
19.
Antibiotics (Basel) ; 11(12)2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36551342

RESUMO

The microbial pathogens Burkholderia pseudomallei and Bacillus anthracis are unrelated bacteria, yet both are the etiologic agents of naturally occurring diseases in animals and humans and are classified as Tier 1 potential biothreat agents. B. pseudomallei is the gram-negative bacterial agent of melioidosis, a major cause of sepsis and mortality globally in endemic tropical and subtropical regions. B. anthracis is the gram-positive spore-forming bacterium that causes anthrax. Infections acquired by inhalation of these pathogens are challenging to detect early while the prognosis is best; and they possess innate multiple antibiotic resistance or are amenable to engineered resistance. Previous studies showed that the early generation, rarely used aminocoumarin novobiocin was very effective in vitro against a range of highly disparate biothreat agents. The objective of the current research was to begin to characterize the therapeutic efficacy of novobiocin in mouse models of anthrax and melioidosis. The antibiotic was highly efficacious against infections by both pathogens, especially B. pseudomallei. Our results supported the concept that specific older generation antimicrobials can be effective countermeasures against infection by bacterial biothreat agents. Finally, novobiocin was shown to be a potential candidate for inclusion in a combined pre-exposure vaccination and post-exposure treatment strategy designed to target bacterial pathogens refractory to a single medical countermeasure.

20.
Front Microbiol ; 13: 965572, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36060756

RESUMO

Burkholderia pseudomallei, the gram-negative bacterium that causes melioidosis, is notoriously difficult to treat with antibiotics. A significant effort has focused on identifying protective vaccine strategies to prevent melioidosis. However, when used as individual medical countermeasures both antibiotic treatments (therapeutics or post-exposure prophylaxes) and experimental vaccine strategies remain partially protective. Here we demonstrate that when used in combination, current vaccine strategies (recombinant protein subunits AhpC and/or Hcp1 plus capsular polysaccharide conjugated to CRM197 or the live attenuated vaccine strain B. pseudomallei 668 ΔilvI) and co-trimoxazole regimens can result in near uniform protection in a mouse model of melioidosis due to apparent synergy associated with distinct medical countermeasures. Our results demonstrated significant improvement when examining several suboptimal antibiotic regimens (e.g., 7-day antibiotic course started early after infection or 21-day antibiotic course with delayed initiation). Importantly, this combinatorial strategy worked similarly when either protein subunit or live attenuated vaccines were evaluated. Layered and integrated medical countermeasures will provide novel treatment options for melioidosis as well as diseases caused by other pathogens that are refractory to individual strategies, particularly in the case of engineered, emerging, or re-emerging bacterial biothreat agents.

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