RESUMEN
Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Endorribonucleasas/metabolismo , ARN Bacteriano/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Endorribonucleasas/genética , Humanos , ARN Bacteriano/genética , Sistemas de Secreción Tipo III/genética , Yersinia pseudotuberculosis/genéticaRESUMEN
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd ) of 3.8 µm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.
Asunto(s)
Mapas de Interacción de Proteínas , Sistemas de Secreción Tipo III/metabolismo , Infecciones por Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Especificidad por Sustrato , Sistemas de Secreción Tipo III/química , Yersinia pseudotuberculosis/química , Infecciones por Yersinia pseudotuberculosis/microbiologíaRESUMEN
All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca(2+)-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas Bacterianas/química , Glutatión Transferasa/metabolismo , Proteínas de la Membrana/químicaRESUMEN
The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Membrana Celular/química , Lípidos de la Membrana/química , Desplegamiento Proteico , Yersinia/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos , Lípidos de la Membrana/metabolismo , Micelas , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Electricidad EstáticaRESUMEN
Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , ARN Bacteriano/genética , Transactivadores/genética , Yersiniosis/genética , Yersinia/genética , Yersinia/patogenicidad , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Femenino , Técnicas de Inactivación de Genes , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Temperatura , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cisâ yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopDA(263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Virulencia , Yersiniosis/microbiología , Yersiniosis/patología , Yersinia pseudotuberculosis/genéticaRESUMEN
Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Western Blotting , Calcio/metabolismo , Membrana Celular/ultraestructura , Citosol/microbiología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Microscopía Inmunoelectrónica , Mutación , Neutrófilos/metabolismo , Neutrófilos/microbiología , Plásmidos/genética , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/fisiologíaRESUMEN
Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.
Asunto(s)
Proteínas Bacterianas/genética , Chlamydia trachomatis/efectos de los fármacos , Chlamydia trachomatis/genética , Hidrazinas/farmacología , Mutación , Protoporfirinógeno-Oxidasa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Chlamydia trachomatis/enzimología , Chlamydia trachomatis/patogenicidad , Farmacorresistencia Bacteriana , Células HeLa , Hemo/metabolismo , Humanos , Hierro/metabolismo , Hierro/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Protoporfirinógeno-Oxidasa/química , Protoporfirinógeno-Oxidasa/metabolismoRESUMEN
Bacteria that have sustained long-standing close associations with eukaryotic hosts have evolved specific adaptations to survive and replicate in this environment. Perhaps one of the most remarkable of those adaptations is the type III secretion system (T3SS)--a bacterial organelle that has specifically evolved to deliver bacterial proteins into eukaryotic cells. Although originally identified in a handful of pathogenic bacteria, T3SSs are encoded by a large number of bacterial species that are symbiotic or pathogenic for humans, other animals including insects or nematodes, and plants. The study of these systems is leading to unique insights into not only organelle assembly and protein secretion but also mechanisms of symbiosis and pathogenesis.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Chaperonas Moleculares/fisiología , Imitación Molecular , Complejos Multiproteicos/fisiología , Señales de Clasificación de Proteína/fisiología , Salmonella typhimurium/fisiologíaRESUMEN
Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.
Asunto(s)
Presentación de Antígeno/inmunología , Autofagia/inmunología , Endosomas/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/inmunología , Animales , Presentación de Antígeno/fisiología , Autofagia/fisiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Sistemas de Secreción Bacterianos/inmunología , Sistemas de Secreción Bacterianos/fisiología , Western Blotting , Línea Celular , Endosomas/fisiología , Técnica del Anticuerpo Fluorescente , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos/inmunología , Macrófagos/fisiología , Ratones , Yersiniosis/inmunología , Yersinia pseudotuberculosis/fisiología , Infecciones por Yersinia pseudotuberculosis/fisiopatologíaRESUMEN
YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca(2+) conditions) and calcium-depleted medium (-Ca(2+) conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca(2+)-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca(2+) conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under -Ca(2+) conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Yersinia pseudotuberculosis/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Proteínas Bacterianas/genética , Western Blotting , Calcio/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Células HeLa , Humanos , Modelos Biológicos , Mutación , Yersinia pseudotuberculosis/efectos de los fármacos , Yersinia pseudotuberculosis/genéticaRESUMEN
Recent work has shown that a domain of YopE of Yersinia pseudotuberculosis ranging from amino acids 54 to 75 (R. Krall, Y. Zhang, and J. T. Barbieri, J. Biol. Chem. 279:2747-2753, 2004) is required for proper localization of YopE after ectopic expression in eukaryotic cells. This domain, called the membrane localization domain (MLD), has not been extensively studied in Yersinia. Therefore, an in cis MLD deletion mutant of YopE was created in Y. pseudotuberculosis. The mutant was found to secrete and translocate YopE at wild-type levels. However, the mutant was defective in the autoregulation of YopE expression after the infection of HeLa cells. Although the mutant translocated YopE at wild-type levels, it showed a delayed HeLa cell cytotoxicity. This delay was not caused by a change in GTPase activating protein (GAP) activity, since the mutant showed wild-type YopE GAP activity toward Rac1 and RhoA. The MLD mutant displayed a changed intracellular localization pattern of YopE in HeLa cells after infection, and the YopEDeltaMLD protein was found to be dispersed within the whole cell, including the nucleus. In contrast, wild-type YopE was found to localize to the perinuclear region of the cell and was not found in the nucleus. In addition, the yopEDeltaMLD mutant was avirulent. Our results suggest that YopE must target proteins other than RhoA and Rac1 and that the MLD is required for the proper targeting and hence virulence of YopE during infection. Our results raise the question whether YopE is a regulatory protein or a "true" virulence effector protein.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Datos de Secuencia Molecular , Transporte de Proteínas/fisiología , Yersinia pseudotuberculosis/genética , Infecciones por Yersinia pseudotuberculosis/genéticaRESUMEN
The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte de Proteínas/fisiología , Yersinia pseudotuberculosis/metabolismo , ADP Ribosa Transferasas/metabolismo , Proteínas de la Membrana Bacteriana Externa/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Retroalimentación Fisiológica , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Humanos , L-Lactato Deshidrogenasa/metabolismo , Mutación , Virulencia , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad , Infecciones por Yersinia pseudotuberculosis/fisiopatologíaRESUMEN
BACKGROUND: Streptococcus pneumoniae is a genetically diverse major human pathogen, yet a common colonizer of the nasopharynx. Here we analyzed the influence of defects affecting in vitro growth rate, on the ability of S. pneumoniae to colonize and to cause invasive disease in vivo. RESULTS: Of eleven different clinical isolates one serotype 14 carrier isolate showed a significantly longer generation time as compared to other isolates, and was severely attenuated in mice. To directly investigate the impact of growth rate on virulence, a panel of mutants in five non-essential housekeeping genes was constructed in the virulent TIGR4 background by insertion-deletion mutagenesis. Three of these mutants (ychF, hemK and yebC) were, to different degrees, growth defective, and showed a reduced invasiveness in an intranasal murine challenge model that correlated to their in vitro growth rate, but remained capable of colonizing the upper airways. The growth defect, as well as virulence defect of the hemK insertion-deletion mutant, was mediated by polarity effects on the downstream yrdC gene, encoding a probable chaperone in ribosome assembly. CONCLUSION: We conclude that large fitness defects are needed to completely prevent pneumococci from causing invasive disease after intranasal challenge. However, even severe growth defects still allow pneumococci to persistently colonize the upper airways.
Asunto(s)
Proteínas Bacterianas/genética , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/genética , Animales , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Humanos , Mutación INDEL , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Operón , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/patogenicidad , Factores de Tiempo , VirulenciaRESUMEN
Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/efectos de los fármacos , Yersinia pseudotuberculosis/efectos de los fármacos , Animales , Antibacterianos/química , Proteínas Bacterianas/genética , Proliferación Celular/efectos de los fármacos , Chlamydia trachomatis/citología , Chlamydia trachomatis/efectos de los fármacos , Chlamydophila pneumoniae/citología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos , Células HeLa , Humanos , Ratones , Transcripción Genética/efectos de los fármacos , YersiniosisRESUMEN
Type III secretion systems are utilised by numerous Gram-negative bacteria to efficiently interact with a host. Appropriate expression of type III genes is achieved through the integration of several regulatory pathways that ultimately co-ordinate the activity of a central transcriptional activator usually belonging to the AraC family. The complex regulatory cascades allow this virulence strategy to be utilised by different bacteria even if they occupy diverse niches that define a unique set of environmental cues. Simulating the appropriate combination of signals in vitro to allow a meaningful interpretation of the type III assembly and secretion regulatory cascade remains a common goal for researchers. Pieces of the puzzle slowly emerge to provide insightful views into the complex regulatory networks that allow bacteria to assemble and utilise type III secretion to efficiently colonise a host.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Bacterias Gramnegativas/metabolismo , Factores de Transcripción , Animales , Factor de Transcripción de AraC , Proteínas Bacterianas/genética , Bacterias Gramnegativas/patogenicidad , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Plantas/microbiología , Proteínas Represoras/genética , VirulenciaRESUMEN
Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y. pseudotuberculosis in which this pathogen up-regulates the plasmid copy number during infection. We found that an increased dose of plasmid-encoded genes is indispensable for virulence and substantially elevates the expression and function of the T3SS. Remarkably, we observed direct, tight coupling between plasmid replication and T3SS function. This regulatory pathway provides a framework for further exploration of the environmental sensing mechanisms of pathogenic bacteria.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Plásmidos/genética , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/genética , Infecciones por Yersinia pseudotuberculosis/microbiología , Yersinia pseudotuberculosis/patogenicidad , Animales , Dosificación de Gen , Humanos , Ratones , Virulencia , Yersinia pseudotuberculosis/genéticaRESUMEN
Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.