scholarly journals Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Katelyn R. Soules ◽  
Scott D. LaBrie ◽  
Benjamin H. May ◽  
P. Scott Hefty
Keyword(s):  
Transcriptional Activation ◽  
Type Iii Secretion ◽  
Atp Hydrolysis ◽  
Developmental Cycle ◽  
Regulatory Domain ◽  
Atpase Domain ◽  
Type Iii ◽  
Sigma 54 ◽  
Expression Of Genes ◽  
Membrane Reorganization

ABSTRACT Chlamydia bacteria are obligate intracellular organisms with a phylum-defining biphasic developmental cycle that is intrinsically linked to its ability to cause disease. The progression of the chlamydial developmental cycle is regulated by the temporal expression of genes predominantly controlled by RNA polymerase sigma (σ) factors. Sigma 54 (σ54) is one of three sigma factors encoded by Chlamydia for which the role and regulon are unknown. CtcC is part of a two-component signal transduction system that is requisite for σ54 transcriptional activation. CtcC activation of σ54 requires phosphorylation, which relieves inhibition by the CtcC regulatory domain and enables ATP hydrolysis by the ATPase domain. Prior studies with CtcC homologs in other organisms have shown that expression of the ATPase domain alone can activate σ54 transcription. Biochemical analysis of CtcC ATPase domain supported the idea of ATP hydrolysis occurring in the absence of the regulatory domain, as well as the presence of an active-site residue essential for ATPase activity (E242). Using recently developed genetic approaches in Chlamydia to induce expression of the CtcC ATPase domain, a transcriptional profile was determined that is expected to reflect the σ54 regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ54 promoter elements. Reporter gene analyses using these putative σ54 promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ54 controls expression of genes that are critical for conversion of Chlamydia from replicative reticulate bodies into infectious elementary bodies. IMPORTANCE The factors that control the growth and infectious processes for Chlamydia are still poorly understood. This study used recently developed genetic tools to determine the regulon for one of the key transcription factors encoded by Chlamydia, sigma 54. Surrogate and computational analyses provide additional support for the hypothesis that sigma 54 plays a key role in controlling the expression of many components critical to converting and enabling the infectious capability of Chlamydia. These components include those that remodel the membrane for the extracellular environment and incorporation of an arsenal of type III secretion effectors in preparation for infecting new cells.

scholarly journals Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Chunfu Yang ◽  
Tregei Starr ◽  
Lihua Song ◽  
John H. Carlson ◽  
Gail L. Sturdevant ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Actin Polymerization ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Cytoskeletal Proteins ◽  
Host Cells ◽  
Genetic Mechanism ◽  
Developmental Cycle ◽  
Obligate Intracellular ◽  
Type Iii

ABSTRACTChlamydia trachomatisis an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknownpgp4-regulated chromosomal T3S effector gene.IMPORTANCEChlamydia's obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

scholarly journals Activation of the Pseudomonas aeruginosa Type III Secretion System Requires an Intact Pyruvate Dehydrogenase aceAB Operon

2002 ◽  
Vol 70 (7) ◽  
pp. 3973-3977 ◽  
Author(s):  
Denis Dacheux ◽  
Olivier Epaulard ◽  
Arjan de Groot ◽  
Benoit Guery ◽  
Rozen Leberre ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Activation ◽  
Pyruvate Dehydrogenase ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Polymorphonuclear Neutrophils ◽  
Type Iii ◽  
New Genes ◽  
Cystic Fibrosis Isolate

ABSTRACT Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in trans of ExsA restored TTSS function and cytotoxicity.

scholarly journals Expression of ExsA in trans Confers Type III Secretion System-Dependent Cytotoxicity on Noncytotoxic Pseudomonas aeruginosa Cystic Fibrosis Isolates

2001 ◽  
Vol 69 (1) ◽  
pp. 538-542 ◽  
Author(s):  
Denis Dacheux ◽  
Ina Attree ◽  
Bertrand Toussaint
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Transcriptional Activation ◽  
Type Iii Secretion ◽  
Ex Vivo ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
In Trans

ABSTRACT Twelve Pseudomonas aeruginosa cystic fibrosis isolates that are not able to exert a type III secretion system (TTSS)-dependent cytotoxicity towards phagocytes have been further studied. The strains, although possessing TTSS genes and exsA, which encodes a positive regulator of the TTSS regulon, showed no transcriptional activation of the exsCBA regulatory operon. The expression of exsA in trans restored the in vitro secretion of TTSS proteins and ex vivo cytotoxicity.

scholarly journals Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions

2011 ◽  
Vol 79 (8) ◽  
pp. 3036-3045 ◽  
Author(s):  
B. Chellas-Géry ◽  
K. Wolf ◽  
J. Tisoncik ◽  
T. Hackstadt ◽  
K. A. Fields
Keyword(s):  
Type Iii Secretion ◽  
De Novo ◽  
Ectopic Expression ◽  
Effector Proteins ◽  
Developmental Cycle ◽  
Inclusion Membrane ◽  
Content Type ◽  
Type Iii ◽  
Intracellular Parasites ◽  
The Many

ABSTRACTChlamydiaspp. are among the many pathogenic Gram-negative bacteria that employ a type III secretion system (T3SS) to overcome host defenses and exploit available resources. Significant progress has been made in elucidating contributions of T3S to the pathogenesis of these medically important, obligate intracellular parasites, yet important questions remain. Chief among these is how secreted effector proteins traverse eukaryotic membranes to gain access to the host cytosol. Due to a complex developmental cycle, it is possible that chlamydiae utilize a different complement of proteins to accomplish translocation at different stages of development. We investigated this possibility by extending the characterization ofC. trachomatisCopB and CopB2. CopB is detected early during infection but is depleted and not detected again until about 20 h postinfection. In contrast, CopB2 was detectible throughout development. CopB is associated with the inclusion membrane. Biochemical and ectopic expression analyses were consistent with peripheral association of CopB2 with inclusion membranes. This interaction correlated with development and required both chlamydialde novoprotein synthesis and T3SS activity. Collectively, our data indicate that it is unlikely that CopB serves as the sole chlamydial translocation pore and that CopB2 is capable of association with the inclusion membrane.

scholarly journals Characterization of the Yersinia enterocolitica Type III Secretion ATPase YscN and Its Regulator, YscL

2006 ◽  
Vol 188 (10) ◽  
pp. 3525-3534 ◽  
Author(s):  
Bill Blaylock ◽  
Kelly E. Riordan ◽  
Dominique M. Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Atp Hydrolysis ◽  
Biochemical Properties ◽  
Host Cells ◽  
Gram Negative Bacteria ◽  
Type Iii ◽  
Bacterial Proteins

ABSTRACT Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

scholarly journals InvF Is Required for Expression of Genes Encoding Proteins Secreted by the SPI1 Type III Secretion Apparatus inSalmonella typhimurium

1999 ◽  
Vol 181 (16) ◽  
pp. 4949-4954 ◽  
Author(s):  
K. Heran Darwin ◽  
Virginia L. Miller
Keyword(s):  
Type Iii Secretion ◽  
Secreted Proteins ◽  
Intergenic Sequence ◽  
Type Iii ◽  
Expression Of Genes ◽  
Island Type ◽  
Cultured Epithelial Cells ◽  
Putative Operon ◽  
Genes Encoding ◽  
Culture Supernatants

ABSTRACT The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of theinv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion ininvF was constructed and tested for the expression of Φ(sigD-lacZYA),sipC::Tn5lacZY, and a plasmid-encoded Φ(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion ofSalmonella typhimurium into cultured eucaryotic cells.sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of thesigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.

scholarly journals Regulation of Pseudomonas aeruginosa virulence by the ExsACDE Signaling Cascade

2014 ◽  
Vol 70 (a1) ◽  
pp. C1393-C1393
Author(s):  
Robert Bernhards ◽  
Xiao Yi ◽  
Manisha Shrestha ◽  
Florian Schubot
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Type Iii Secretion ◽  
Negative Regulator ◽  
Signaling Cascade ◽  
Regulation Of Transcription ◽  
Regulatory Domain ◽  
Type Iii ◽  
Dimerization Interface

Pseudomonas aeruginosa requires its type III secretion system (T3SS) to facilitate acute infections. T3SS-related gene expression is controlled by the AraC-type transcriptional activator ExsA. A signaling cascade involving ExsA and three additional proteins -ExsC, ExsD, and ExsE directly ties the up-regulation of transcription to the activation of the type III secretion apparatus. In order to characterize the molecular interactions underlying the signaling process the crystal structures of the unique T3SS chaperone ExsC in complex with its cognate effector ExsE, the structure of the negative regulator ExsD and that of the regulatory domain of ExsA have been determined. The ExsC-ExsE structure revealed two Arg-X-Val-X-Arg motifs in ExsE that form identical interactions along opposite sides of the ExsC dimer. The structure of ExsD not only provided insights into the interactions of how ExsD is sequestered by ExsC but also revealed surprising similarities between ExsD and DNA binding proteins. Based on these findings, a new model for the ExsC-ExsD complex is proposed to explain its distinctive 2:2 stoichiometry and why ExsC displays a weaker affinity for ExsD than for ExsE. Lastly, we have determined the structure of ExsA regulatory domain revealing the protein's dimerization interface. The position of the interface appears to postulate interesting conformational changes upon binding of ExsA to DNA. The crystal structures of ExsD and the ExsA domain also serve as road maps for determining the interface for the critical interactions between these two proteins at the bottom of this unique signaling cascade.

scholarly journals Interactions between CdsD, CdsQ, and CdsL, Three Putative Chlamydophila pneumoniae Type III Secretion Proteins

2008 ◽  
Vol 190 (8) ◽  
pp. 2972-2980 ◽  
Author(s):  
Dustin L. Johnson ◽  
Chris B. Stone ◽  
James B. Mahony
Keyword(s):  
Protein Interactions ◽  
Type Iii Secretion ◽  
Bacterial Pathogen ◽  
Integral Membrane Protein ◽  
Chlamydophila Pneumoniae ◽  
Polyclonal Antiserum ◽  
Immunofluorescent Staining ◽  
Developmental Cycle ◽  
Type Iii ◽  
Genes Encoding

ABSTRACT Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidine- and glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus.

scholarly journals Temporal Expression of Type III Secretion Genes of Chlamydia pneumoniae

2003 ◽  
Vol 71 (5) ◽  
pp. 2555-2562 ◽  
Author(s):  
Anatoly Slepenkin ◽  
Vladimir Motin ◽  
Luis M. de la Maza ◽  
Ellena M. Peterson
Keyword(s):  
Type Iii Secretion ◽  
Chlamydia Pneumoniae ◽  
Early Stage ◽  
Effector Proteins ◽  
Developmental Cycle ◽  
Temporal Expression ◽  
Type Iii ◽  
Ifn Γ ◽  
Obligate Intracellular Pathogen ◽  
Late Stages

ABSTRACT Chlamydia pneumoniae has been shown to possess at least 13 genes that are homologous with other known type III secretion (TTS) systems. Upon infection of HEp-2 cells with C. pneumoniae, the expression of these genes was followed by reverse transcriptase PCR throughout the developmental cycle of this obligate intracellular pathogen. In addition, expression was analyzed when C. pneumoniae was grown in the presence of human gamma interferon (IFN-γ). The groEL-1, ompA, and omcB genes were used as markers for the early, middle, and late stages of the developmental cycle, respectively, and the inhibition of expression of the fstK gene was used as a marker for the effect of IFN-γ on the maturation of C. pneumoniae. In the absence of IFN-γ, the TTS genes were expressed as follows: early stage (1.5 to 8 h), yscC, yscS, yscL, yscJ and lcrH-2; middle stage (by 12 to 18 h), lcrD, yscN, and yscR; and late stage (by 24 h), lcrE, sycE, lcrH-1, and yscT. Of the genes expressed early, the lcrH-2 gene was detected the earliest, at 1.5 h. Expression of the yscU gene was not detected at any of the time points examined. Under the influence of IFN-γ, the cluster of TTS genes that were normally not expressed until the middle to late stages of the developmental cycle, namely, lcrD, lcrE, and sycE, as well as lcrH-1, were down-regulated, and expression could not be detected up to 48 h. In contrast, the expression of the other TTS genes appeared to be unchanged in the presence of IFN-γ. The lcrH-1 and lcrH-2 genes differed from one another in both their temporal expression and response to IFN-γ. In other TTS systems, these genes code for proteins that function in regulation of effector protein synthesis as well as serve as chaperones for proteins that provide for the translocation of the effector proteins into the host cell. In summary, the expression pattern of the TTS genes of C. pneumoniae examined suggests that they are temporally regulated throughout the developmental cycle. Furthermore, paralleling the inhibition of the maturation of the reticulate body to the elementary body, TTS genes expressed in the later stages of the cycle appear to be down-regulated when the organism is grown in the presence of IFN-γ.

Sign in / Sign up

Export Citation Format

Share Document