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1.
J Biol Chem ; 292(5): 1705-1723, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-27974466

RESUMO

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.


Assuntos
Sistemas de Secreção Bacterianos/metabolismo , Shigella flexneri/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Mutação , Shigella flexneri/genética
2.
Mol Microbiol ; 95(1): 31-50, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25353930

RESUMO

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.


Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Sistemas de Secreção Bacterianos , Cisteína/metabolismo , Shigella flexneri/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Reagentes de Ligações Cruzadas/metabolismo , Imageamento Tridimensional , Microscopia Eletrônica , Modelos Moleculares , Simulação de Acoplamento Molecular , Multimerização Proteica , Estrutura Secundária de Proteína , Shigella flexneri/química , Shigella flexneri/genética
3.
J Struct Biol ; 192(3): 441-448, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26439285

RESUMO

T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a ∼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers.


Assuntos
Proteínas de Bactérias/genética , Dobramento de Proteína , Shigella flexneri/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
4.
Nat Genet ; 35(2): 139-47, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12973349

RESUMO

Schistosoma japonicum causes schistosomiasis in humans and livestock in the Asia-Pacific region. Knowledge of the genome of this parasite should improve understanding of schistosome-host interactions, biomedical aspects of schistosomiasis and invertebrate evolution. We assigned 43,707 expressed sequence tags (ESTs) derived from adult S. japonicum and their eggs to 13,131 gene clusters. Of these, 35% shared no similarity with known genes and 75% had not been reported previously in schistosomes. Notably, S. japonicum encoded mammalian-like receptors for insulin, progesterone, cytokines and neuropeptides, suggesting that host hormones, or endogenous parasite homologs, could orchestrate schistosome development and maturation and that schistosomes modulate anti-parasite immune responses through inhibitors, molecular mimicry and other evasion strategies.


Assuntos
DNA de Helmintos/genética , Evolução Molecular , Schistosoma japonicum/genética , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Genes de Helmintos , Interações Hospedeiro-Parasita , Humanos , Mamíferos , Dados de Sequência Molecular , Filogenia , Schistosoma japonicum/classificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
5.
Microbiology (Reading) ; 158(Pt 7): 1884-1896, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22575894

RESUMO

The type III secretion apparatus (T3SA), which is evolutionarily and structurally related to the bacterial flagellar hook basal body, is a key virulence factor used by many gram-negative bacteria to inject effector proteins into host cells. A hollow extracellular needle forms the injection conduit of the T3SA. Its length is tightly controlled to match specific structures at the bacterial and host-cell surfaces but how this occurs remains incompletely understood. The needle is topped by a tip complex, which senses the host cell and inserts as a translocation pore in the host membrane when secretion is activated. The interaction of two conserved proteins, inner-membrane Spa40 and secreted Spa32, respectively, in Shigella, is proposed to regulate needle length and to flick a type III secretion substrate specificity switch from needle components/Spa32 to translocator/effector substrates. We found that, as in T3SAs from other species, substitution N257A within the conserved cytoplasmic NPTH region in Spa40 prevented its autocleavage and substrate specificity switching. Yet, the spa40(N257A) mutant made only slightly longer needles with a few needle tip complexes, although it could not form translocation pores. On the other hand, Δspa32, which makes extremely long needles and also formed only few tip complexes, could still form some translocation pores, indicating that it could switch substrate specificity to some extent. Therefore, loss of needle length control and defects in secretion specificity switching are not tightly coupled in either a Δspa32 mutant or a spa40(N257A) mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Substâncias Macromoleculares/metabolismo , Shigella/metabolismo , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Especificidade por Substrato
6.
Infect Immun ; 78(12): 4999-5010, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20937761

RESUMO

Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacterial pathogens. Upon cell contact, they inject effector proteins directly into eukaryotic cells through a needle protruding from the bacterial surface. Host cell sensing occurs through a distal needle "tip complex," but how this occurs is not understood. The tip complex of quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. It binds needles via its extreme C-terminal coiled coil, thereby likely positioning a large domain containing its hydrophobic regions at the distal tips of needles. In this study, we used short deletion mutants within this domain to search for regions of IpaB involved in secretion regulation. This identified two regions, amino acids 227 to 236 and 297 to 306, the presence of which are required for maintenance of IpaB at the needle tip, secretion regulation, and normal pore formation but not invasion. We therefore propose that removal of either of these regions leads to an inability to block secretion prior to reception of the activation signal and/or a defect in host cell sensing.


Assuntos
Proteínas de Bactérias/fisiologia , Sistemas de Secreção Bacterianos/fisiologia , Disenteria Bacilar/microbiologia , Shigella flexneri/patogenicidade , Antígenos de Bactérias/genética , Antígenos de Bactérias/fisiologia , Aderência Bacteriana/fisiologia , Sistemas de Secreção Bacterianos/genética , Membrana Eritrocítica/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Células HeLa , Humanos , Microscopia de Fluorescência , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Deleção de Sequência/genética , Shigella flexneri/genética
7.
Biomed Environ Sci ; 21(2): 103-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18548848

RESUMO

OBJECTIVE: Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1. METHODS: By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis. RESULTS: Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS. CONCLUSION: Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".


Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Pseudomonas aeruginosa/metabolismo , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Sequência de Bases , Western Blotting , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Cinética , Chaperonas Moleculares/genética , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
PLoS One ; 11(5): e0155141, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27171191

RESUMO

Type III secretion systems (T3SSs) are central virulence devices for many Gram-negative bacterial pathogens of humans, animals & plants. Upon physical contact with eukaryotic host cells, they translocate virulence-mediating proteins, known as effectors, into them during infection. T3SSs are gated from the outside by host-cell contact and from the inside via two cytoplasmic negative regulators, MxiC and IpaD in Shigella flexneri, which together control the effector secretion hierarchy. Their absence leads to premature and increased secretion of effectors. Here, we investigated where and how these regulators act. We demonstrate that the T3SS inner membrane export apparatus protein MxiA plays a role in substrate selection. Indeed, using a genetic screen, we identified two amino acids located on the surface of MxiA's cytoplasmic region (MxiAC) which, when mutated, upregulate late effector expression and, in the case of MxiAI674V, also secretion. The cytoplasmic region of MxiA, but not MxiAN373D and MxiAI674V, interacts directly with the C-terminus of MxiC in a two-hybrid assay. Efficient T3S requires a cytoplasmic ATPase and the proton motive force (PMF), which is composed of the ΔΨ and the ΔpH. MxiA family proteins and their regulators are implicated in utilization of the PMF for protein export. However, our MxiA point mutants show similar PMF utilisation to wild-type, requiring primarily the ΔΨ. On the other hand, lack of MxiC or IpaD, renders the faster T3S seen increasingly dependent on the ΔpH. Therefore, MxiA, MxiC and IpaD act together to regulate substrate selection and secretion mode in the T3SS of Shigella flexneri.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Shigella flexneri/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Força Próton-Motriz , Especificidade por Substrato , Técnicas do Sistema de Duplo-Híbrido
9.
FEMS Microbiol Lett ; 239(1): 87-93, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15451105

RESUMO

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.


Assuntos
Aspergillus fumigatus/enzimologia , Lisofosfolipase , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Lisofosfolipase/química , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Dados de Sequência Molecular , Infecções Oportunistas/microbiologia , Fosfatidilcolinas/metabolismo , Análise de Sequência de DNA , Regulação para Cima
10.
Artigo em Zh | MEDLINE | ID: mdl-12884613

RESUMO

OBJECTIVE: To sub-clone and express the gene encoding Schistosoma japonicum calcium-binding protein (SjE16) and study its immunological response. METHODS: The specific primers were designed according to the expressed sequence tags (ESTs) sequence, which was used for amplification of the encoding sequence from the cDNA clone containing SjE16. The gene was subcloned into pGEX4T-1 plasmid and expressed. The rSjE16 was tested for its immunological response by ELISA. RESULTS: The gene encoding Schistosoma japonicum SjE16 was cloned and expressed successfully. The immunogenicity and diagnostic potential of rSjE16 were investigated. It was demonstrated by immunoassay in rabbits that the specificity and sensitivity of the test were 94.1% (16/17) and 88.2% (15/17), respectively, and the level of antibody titer of the untreated group reached a peak at 9-11 wk post infection and maintained high at least for 21 wk post infection, while the antibody level in the treated group rapidly decreased to pre-infection level in 11 wk after treatment. In human, the specificity of the test was 98.3% (57/58); the sensitivity of acute and chronic patient serum assay was 85.5% (53/62) and 70.2% (40/57), respectively. CONCLUSION: The recombinant protein of SjE16 (rSjE16) was acquired. It can be recognized by the sera from schistosomiasis patients, and the level of antibodies decreased quickly after treatment in experimental rabbits, which implicates the potential value for the evaluation of chemotherapy and detection of active infection.


Assuntos
Proteínas de Ligação ao Cálcio/imunologia , Proteínas Recombinantes de Fusão/imunologia , Schistosoma japonicum/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Ensaio de Imunoadsorção Enzimática , Etiquetas de Sequências Expressas , Testes Imunológicos , Proteínas Recombinantes de Fusão/biossíntese , Schistosoma japonicum/genética , Esquistossomose Japônica/diagnóstico , Sensibilidade e Especificidade
11.
Microbiology (Reading) ; 154(Pt 8): 2195-2208, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18667553

RESUMO

The Pseudomonas aeruginosa type III secretion system (T3SS) is known to be a very important virulence factor in acute human infections, but it is less important in maintaining chronic infections in which T3SS genes are downregulated. In vitro, the activation of T3SS expression involves a positive activating loop that acts on the transcriptional regulator ExsA. We have observed that in vivo T3SS expression is cell density-dependent in a manner that does not need known quorum-sensing (QS) signals. In addition, stationary-phase culture supernatants added to exponential-phase growing strains can inhibit T3SS expression. The analysis of transposon insertion mutants showed that the production of such T3SS-inhibiting signals might depend on tryptophan synthase and hence tryptophan, which is the precursor of signalling molecules such as indole-3-acetic acid (IAA), kynurenine and Pseudomonas quinolone signal (PQS). Commercially available tryptophan-derived molecules were tested for their role in the regulation of T3SS expression. At millimolar concentrations, IAA, 1-naphthalacetic acid (NAA) and 3-hydroxykynurenine inhibited T3SS expression. Inactivation of the tryptophan dioxygenase-encoding kynA gene resulted in a decrease in the T3SS-inhibiting activity of supernatants. These observations suggest that tryptophan catabolites are involved in the downregulation of T3SS expression in the transition from a low- to a high-cell-density state.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Triptofano/metabolismo , Proteínas de Bactérias/genética , Ácidos Indolacéticos/metabolismo , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Mutação , Transporte Proteico , Pseudomonas aeruginosa/genética , Triptofano/análogos & derivados , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
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