Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
1.
Biochim Biophys Acta Biomembr ; 1864(10): 184000, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35798072

RESUMO

Secreted proteins contain an N-terminal signal peptide to guide them through the secretion pathway. Once the protein is translocated, the signal peptide is removed by a signal peptidase, such as signal peptidase I. The signal peptide has been extensively studied and reviewed; however, the mature region has not been the focus of review. Here we cover the experimental evidence that highlights the important role of the mature region amino acid residues in both the efficiency and the ability of secreted proteins to be successfully exported via secretion pathways and cleaved by signal peptidase I.


Assuntos
Escherichia coli , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Escherichia coli/genética , Escherichia coli/metabolismo , Sinais Direcionadores de Proteínas/genética
2.
PLoS Negl Trop Dis ; 14(1): e0008017, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978058

RESUMO

The infectious disease melioidosis is caused by the bacterium Burkholderia pseudomallei. Melioidosis is characterised by high mortality and morbidity and can involve the central nervous system (CNS). We have previously discovered that B. pseudomallei can infect the CNS via the olfactory and trigeminal nerves in mice. We have shown that the nerve path is dependent on mouse strain, with outbred mice showing resistance to olfactory nerve infection. Damage to the nasal epithelium by environmental factors is common, and we hypothesised that injury to the olfactory epithelium may increase the vulnerability of the olfactory nerve to microbial insult. We therefore investigated this, using outbred mice that were intranasally inoculated with B. pseudomallei, with or without methimazole-induced injury to the olfactory neuroepithelium. Methimazole-mediated injury resulted in increased B. pseudomallei invasion of the olfactory epithelium, and only in pre-injured animals were bacteria found in the olfactory nerve and bulb. In vitro assays demonstrated that B. pseudomallei readily infected glial cells isolated from the olfactory and trigeminal nerves (olfactory ensheathing cells and trigeminal Schwann cells, respectively). Bacteria were degraded by some cells but persisted in other cells, which led to the formation of multinucleated giant cells (MNGCs), with olfactory ensheathing cells less likely to form MNGCs than Schwann cells. Double Cap mutant bacteria, lacking the protein BimA, did not form MNGCs. These data suggest that injuries to the olfactory epithelium expose the primary olfactory nervous system to bacterial invasion, which can then result in CNS infection with potential pathogenic consequences for the glial cells.


Assuntos
Burkholderia pseudomallei , Melioidose/microbiologia , Bulbo Olfatório/microbiologia , Nervo Olfatório/microbiologia , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Animais , Antitireóideos/administração & dosagem , Antitireóideos/farmacologia , Genes Reporter , Células Gigantes , Humanos , Melioidose/patologia , Metimazol/administração & dosagem , Metimazol/farmacologia , Camundongos , Camundongos Transgênicos , Mucosa Respiratória/lesões , Mucosa Respiratória/microbiologia , Subunidade beta da Proteína Ligante de Cálcio S100/genética
3.
Infect Immun ; 84(9): 2681-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27382023

RESUMO

Infection with Burkholderia pseudomallei causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that B. pseudomallei can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice, B. pseudomallei caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers. B. pseudomallei rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 µm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of B. pseudomallei that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that B. pseudomallei can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity.


Assuntos
Tronco Encefálico/microbiologia , Burkholderia pseudomallei/patogenicidade , Cavidade Nasal/microbiologia , Medula Espinal/microbiologia , Nervo Trigêmeo/microbiologia , Administração Intranasal/métodos , Animais , Melioidose/microbiologia , Camundongos
4.
Infect Immun ; 84(7): 1941-1956, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27091931

RESUMO

Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.


Assuntos
Cápsulas Bacterianas/imunologia , Burkholderia pseudomallei/imunologia , Interações Hospedeiro-Patógeno/imunologia , Melioidose/imunologia , Melioidose/microbiologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Animais , Cápsulas Bacterianas/genética , Carga Bacteriana , Burkholderia pseudomallei/genética , Células Cultivadas , Biologia Computacional/métodos , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunidade Inata , Melioidose/genética , Melioidose/metabolismo , Camundongos , Mutação , Infiltração de Neutrófilos , Neurônios Receptores Olfatórios/imunologia , Neurônios Receptores Olfatórios/metabolismo , Neurônios Receptores Olfatórios/microbiologia , Infecções Respiratórias/genética , Infecções Respiratórias/metabolismo , Transdução de Sinais , Virulência , Fatores de Virulência
5.
Nat Chem Biol ; 11(12): 955-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26565989

RESUMO

We report the structural and functional characterization of a novel heparanase (BpHep) from the invasive pathogenic bacterium Burkholderia pseudomallei (Bp), showing ∼24% sequence identity with human heparanase (hHep). Site-directed mutagenesis studies confirmed the active site resi-dues essential for activity, and we found that BpHep has specificity for heparan sulfate. Finally, we describe the first heparanase X-ray crystal structure, which provides new insight into both substrate recognition and inhibitor design.


Assuntos
Burkholderia pseudomallei/enzimologia , Glucuronidase/química , Glucuronidase/metabolismo , Cristalografia por Raios X , Glucuronidase/isolamento & purificação , Humanos , Modelos Moleculares , Conformação Proteica
6.
Clin Microbiol Rev ; 27(4): 691-726, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25278572

RESUMO

The brain is well protected against microbial invasion by cellular barriers, such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). In addition, cells within the central nervous system (CNS) are capable of producing an immune response against invading pathogens. Nonetheless, a range of pathogenic microbes make their way to the CNS, and the resulting infections can cause significant morbidity and mortality. Bacteria, amoebae, fungi, and viruses are capable of CNS invasion, with the latter using axonal transport as a common route of infection. In this review, we compare the mechanisms by which bacterial pathogens reach the CNS and infect the brain. In particular, we focus on recent data regarding mechanisms of bacterial translocation from the nasal mucosa to the brain, which represents a little explored pathway of bacterial invasion but has been proposed as being particularly important in explaining how infection with Burkholderia pseudomallei can result in melioidosis encephalomyelitis.


Assuntos
Infecções do Sistema Nervoso Central/microbiologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/microbiologia , Infecções do Sistema Nervoso Central/imunologia , Infecções do Sistema Nervoso Central/transmissão , Humanos , Vigilância Imunológica , Cavidade Nasal/microbiologia , Nervo Olfatório/microbiologia , Nervo Trigêmeo/microbiologia
7.
mBio ; 5(2): e00025, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24736221

RESUMO

ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity.


Assuntos
Encéfalo/microbiologia , Burkholderia pseudomallei/fisiologia , Interações Hospedeiro-Patógeno , Melioidose/microbiologia , Nervo Olfatório/microbiologia , Nervo Trigêmeo/microbiologia , Animais , Encéfalo/patologia , Feminino , Imuno-Histoquímica , Melioidose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Cavidade Nasal/microbiologia , Nervo Olfatório/patologia , Fatores de Tempo , Nervo Trigêmeo/patologia
8.
Biochem Biophys Res Commun ; 436(3): 362-5, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23726917

RESUMO

Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.


Assuntos
Asparaginase/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Anaerobiose , Asparaginase/genética , Asparagina/metabolismo , Meios de Cultura/metabolismo , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Citoplasma/enzimologia , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fumaratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Isoenzimas/genética , Isoenzimas/fisiologia , Nitrogênio/metabolismo , Transcrição Gênica
9.
PLoS One ; 8(5): e63394, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23704903

RESUMO

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen.


Assuntos
Burkholderia pseudomallei/crescimento & desenvolvimento , Células Gigantes/patologia , Espaço Intracelular/microbiologia , Macrófagos/microbiologia , Macrófagos/patologia , Percepção de Quorum , Acil-Butirolactonas/metabolismo , Administração Intranasal , Animais , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Linhagem Celular , Deleção de Genes , Genes Bacterianos/genética , Humanos , Ligases/deficiência , Ligases/metabolismo , Melioidose/microbiologia , Melioidose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Virulência
10.
J Med Microbiol ; 61(Pt 5): 607-614, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22301613

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in areas of South-East Asia and northern Australia, and is classed as a category B select agent by the Centers for Disease Control and Prevention (CDC). Factors that determine whether host infection is achieved or if disease is chronic or acute are unknown but the type of host immune response that is mounted is important. B. pseudomallei can replicate within macrophages, causing them to multinucleate. In light of the common lineage of macrophages with dendritic cells (DCs), and the role played by DCs in orchestration of the immune response, we investigated the interactions of a variety of B. pseudomallei and B. thailandensis strains with DCs. This study demonstrates that, in the majority of cases, infection of human monocyte-derived dendritic cells is dramatically decreased or cleared by 12 h post-infection, showing a lack of ability to replicate and survive within DCs. Additionally we have shown that B. pseudomallei activates DCs, as measured by cytokine secretion, and live bacteria are not required for activation.


Assuntos
Burkholderia/imunologia , Burkholderia/patogenicidade , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Adolescente , Adulto , Burkholderia/isolamento & purificação , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Melioidose/imunologia , Melioidose/microbiologia , Viabilidade Microbiana , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/microbiologia , Adulto Jovem
11.
Biotechnol J ; 6(6): 660-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21567959

RESUMO

Proteins destined for export via the Sec-dependent pathway are synthesized with a short N-terminal signal peptide. A requirement for export is that the proteins are in a translocationally competent state. This is a loosely folded state that allows the protein to pass through the SecYEG apparatus and pass into the periplasm. In order to maintain pre-secretory proteins in an export-competent state, there are many factors that slow the folding of the pre-secretory protein in the cytoplasm. These include cytoplasmic chaperones, such as SecB, and the signal recognition particle, which bind the pre-secretory protein and direct it to the cytoplasmic membrane for export. Recently, evidence has been published that non-optimal codons in the signal sequence are important for a time-critical early event to allow the correct folding of pre-secretory proteins. This review details the recent developments in folding of the signal peptide and the pre-secretory protein.


Assuntos
Códon , Escherichia coli , Biossíntese de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Códon/genética , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Biológicos , Periplasma/genética , Periplasma/metabolismo , Biossíntese de Proteínas/fisiologia , Dobramento de Proteína , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/genética , Proteínas/genética , Proteínas/metabolismo , Canais de Translocação SEC , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo
12.
J Infect Dis ; 199(12): 1761-70, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19456230

RESUMO

BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis, is generally considered to be acquired via inhalation of dust or water droplets from the environment. In this study, we show that infection of the nasal mucosa is potentially an important portal of entry in melioidosis. METHODS: After intranasal inoculation of mice, infection was monitored by bioluminescence imaging and by immunohistological analysis of coronal sections. The bacterial loads in organ and tissue specimens were also monitored. RESULTS: Bioluminescence imaging showed colonization and replication in the nasal cavity, including the nasal-associated lymphoid tissue (NALT). Analysis of coronal sections and immunofluorescence microscopy further demonstrated the presence of infection in the respiratory epithelium and the olfactory epithelium (including associated nerve bundles), as well as in the NALT. Of significance, the olfactory epithelium and the brain were rapidly infected before bacteria were detected in blood, and a capsule-deficient mutant infected the brain without significantly infecting blood. CONCLUSIONS: These data suggest that the olfactory nerve is the route of entry into the brain and that this route of entry may be paralleled in cases of human neurologic melioidosis. This study focuses attention on the upper respiratory tract as a portal of entry, specifically focusing on NALT as a route for the development of systemic infection via the bloodstream and on the olfactory epithelium as a direct route to the brain.


Assuntos
Burkholderia pseudomallei , Tecido Linfoide/microbiologia , Melioidose/microbiologia , Mucosa Olfatória/microbiologia , Animais , Burkholderia pseudomallei/citologia , Divisão Celular , Feminino , Proteínas Luminescentes , Melioidose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Nariz/anatomia & histologia , Nariz/microbiologia , Bulbo Olfatório/microbiologia , Células Receptoras Sensoriais/microbiologia
13.
Trends Microbiol ; 17(4): 146-50, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19307122

RESUMO

The signal peptide of proteins exported via the general secretory pathway encodes structural features that enable the targeting and export of the protein to the periplasm. Recent studies have shown biased codon usage at the second amino acid position and a high usage of non-optimal codons within the signal peptide. Altering these biases in codon usage can have deleterious effects on protein folding and export. We propose that these codon-usage biases act in concert to optimize the export process through modulating ribosome spacing on the transcript. This highlights a new aspect of protein export and implies that codon usage in the signal peptide encodes signals that are important for protein targeting and export to the periplasm.


Assuntos
Códon , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Dobramento de Proteína , Transporte Proteico
14.
J Microbiol Methods ; 76(3): 320-3, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19150470

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic or emerging world-wide. Here we report unmarked allele-replacement mutagenesis using efficient sacB counter-selection. Despite being genotypically sacB(+), most commonly used B. pseudomallei strains are sucrose-resistant and efficient sacB counter-selection is demonstrated in both resistant and sensitive strains.


Assuntos
Burkholderia pseudomallei/genética , Mutagênese Sítio-Dirigida/métodos , Sacarose/metabolismo , Alelos , Burkholderia pseudomallei/metabolismo , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Deleção de Sequência
15.
FEMS Immunol Med Microbiol ; 54(1): 144-53, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18657105

RESUMO

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 h after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days. This delayed diagnosis is a major contributing factor to high mortality rates. Rapid diagnosis is vital for successful management of the disease. This study describes the purification and evaluation of three recombinant antigenic proteins, BPSL0972, BipD and OmpA from B. pseudomallei 08, for their potential in the serodiagnosis of melioidosis using an indirect enzyme-linked immunosorbent assay (ELISA) method. The recombinant proteins were evaluated using 74 serum samples from culture-confirmed melioidosis patients from Malaysia, Thailand and Australia. In addition, 62 nonmelioidosis controls consisting of serum samples from clinically suspected melioidosis patients (n=20) and from healthy blood donors from an endemic region (n=18) and a nonendemic region (n=24) were included. The indirect ELISAs using BipD and BPSL0972 as antigens demonstrated poor to moderate sensitivities (42% and 51%, respectively) but good specificity (both 100%). In contrast, the indirect ELISA using OmpA as an antigen achieved 95% sensitivity and 98% specificity. These results highlight the potential for OmpA to be used in the serodiagnosis of melioidosis in an endemic area.


Assuntos
Antígenos de Bactérias , Burkholderia pseudomallei/imunologia , Melioidose/diagnóstico , Proteínas Recombinantes , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Melioidose/imunologia , Melioidose/microbiologia , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos , Fatores de Tempo
16.
Appl Environ Microbiol ; 74(4): 1064-75, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18156318

RESUMO

Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P(BAD) promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P(S12) or synthetic bacterial P(EM7) promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked delta(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agent-compliant genetic manipulations of B. pseudomallei and other Burkholderia species.


Assuntos
Burkholderia pseudomallei/genética , Técnicas Genéticas , Plasmídeos/genética , Transformação Bacteriana/genética , Sequência de Bases , Bleomicina , Clonagem Molecular , Primers do DNA/genética , Farmacorresistência Bacteriana/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Replicon/genética , Transposases/genética
17.
Cell Microbiol ; 9(2): 514-31, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16987331

RESUMO

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a spectrum of potentially fatal diseases endemic in Northern Australia and South-East Asia. We demonstrate that B. pseudomallei rapidly modifies infected macrophage-like cells in a manner analagous to osteoclastogenesis. These alterations include multinucleation and the expression by infected cells of mRNA for factors required for osteoclastogenesis: the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 gamma (MIP-1gamma), 'regulated on activation normal T cell expressed and secreted' (RANTES) and the transcription factor 'nuclear factor of activated T-cells cytoplasmic 1' (NFATc1). An increase in expression of these factors was also observed after infection with Burkholderia thailandensis. Expression of genes for the osteoclast markers calcitonin receptor (CTR), cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) was also increased by B. pseudomallei-infected, but not by B. thailandensis-infected cells. The expression by B. pseudomallei-infected cells of these chemokine and osteoclast marker genes was remarkably similar to cells treated with RANKL, a stimulator of osteoclastogenesis. Analysis of dentine resorption by B. pseudomallei-induced osteoclast-like cells revealed that demineralization may occur but that authentic excavation does not take place under the tested conditions. Furthermore, we identified and characterized lfpA (for lactonase family protein A) in B. pseudomallei, which shares significant sequence similarity with the eukaryotic protein 'regucalcin', also known as 'senescence marker protein-30' (SMP-30). LfpA orthologues are widespread in prokaryotes and are well conserved, but are phylogenetically distinct from eukaryotic regucalcin orthologues. We demonstrate that lfpA mRNA expression is dramatically increased in association with macrophage-like cells. Mutation of lfpA significantly reduced expression of the tested host genes, relative to the response to wild-type B. pseudomallei. We also show that lfpA is required for optimal virulence in vivo.


Assuntos
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/imunologia , Células Gigantes/fisiologia , Melioidose/imunologia , Osteoclastos/metabolismo , Receptores da Calcitonina/metabolismo , Animais , Burkholderia pseudomallei/fisiologia , Calcitonina/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Cricetinae , Modelos Animais de Doenças , Células Gigantes/metabolismo , Melioidose/genética , Melioidose/microbiologia , Mesocricetus , Osteoclastos/citologia
18.
Infect Immun ; 74(9): 5374-81, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16926432

RESUMO

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease that is endemic to Northern Australia and Southeast Asia and is acquired from soil or water. Adherence of B. pseudomallei 08 to cultured cells increases dramatically following prior growth at 30 degrees C or less compared to that following prior growth at 37 degrees C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27 degrees C but not at 37 degrees C, which considerably enhances bacterial association with eukaryotic cells. Further, we demonstrate that the type IVA pilin-encoding gene, pilA, is essential for microcolony development by B. pseudomallei 08, and thus optimum association with eukaryotic cells, but is not required for direct adherence (bacterium-cell interactions). In contrast, although the B. pseudomallei genome sequence strain, K96243, also contains transcriptionally active pilA, microcolony formation rarely occurs following growth at either 27 degrees C or 37 degrees C and cell association occurs significantly less than with strain 08. Analysis of pilA transcription in 08 identified that pilA is dramatically upregulated under microcolony-forming conditions, viz., growth at low temperature, and association with eukaryotic cells; the pattern of transcription of pilA in K96243 differed from that in 08. Our study also suggests that biofilm formation by B. pseudomallei 08 and K96243 on polyvinylchloride is not mediated by pilA. Adherence and microcolony formation, and pilA transcription, vary between strains, consistent with known genomic variation in B. pseudomallei, and these phenotypes may be relevant to colonization from the environment.


Assuntos
Aderência Bacteriana , Burkholderia pseudomallei/crescimento & desenvolvimento , Burkholderia pseudomallei/patogenicidade , Proteínas de Fímbrias/fisiologia , Temperatura , Aderência Bacteriana/genética , Biofilmes/crescimento & desenvolvimento , Burkholderia pseudomallei/genética , Células Cultivadas , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica , Variação Genética , Humanos , Cloreto de Polivinila/metabolismo , Transcrição Gênica
19.
Infect Immun ; 73(2): 1260-4, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15664977

RESUMO

The Burkholderia pseudomallei K96243 genome contains multiple type IV pilin-associated loci, including one encoding a putative pilus structural protein (pilA). A pilA deletion mutant has reduced adherence to human epithelial cells and is less virulent in the nematode model of virulence and the murine model of melioidosis, suggesting a role for type IV pili in B. pseudomallei virulence.


Assuntos
Aderência Bacteriana/fisiologia , Burkholderia pseudomallei/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Animais , Aderência Bacteriana/genética , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Fímbrias Bacterianas/genética , Humanos , Camundongos , Fatores de Tempo
20.
FEMS Microbiol Lett ; 241(2): 243-8, 2004 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-15598539

RESUMO

The production of lipase and protease from psychrotrophic strains of Pseudomonas fluorescens may result in spoilage of dairy products. The lipase (lipA) and alkaline metalloprotease (aprX) genes of P. fluorescens B52 are regulated by temperature and are located at opposite ends of an operon which contains eight genes and spans 14 kb. In this report, we show that lipase activity in the supernatant of cultures of P. fluorescens strain B52 is also regulated by the homologue of the Escherichia coli EnvZ-OmpR two-component regulatory system. Differences in the regulation of lipase and protease may be related to the proximal and distal locations of aprX and lipA within the operon.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Complexos Multienzimáticos/metabolismo , Óperon , Pseudomonas fluorescens/enzimologia , Serina Endopeptidases/metabolismo , Transativadores/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Lipase , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/crescimento & desenvolvimento , Análise de Sequência de DNA , Serina Endopeptidases/genética , Transativadores/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA