Chain-Selective Isotopic Labeling of the Heterodimeric Type III Secretion Chaperone, Scc4:Scc1, Reveals the Total Structural Rearrangement of the Chlamydia trachomatis Bi-Functional Protein, Scc4

Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ66-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the 15N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4. 1H,15N-heteronuclear single quantum coherence spectra of the 15N-Scc4:Scc1 and Scc4:15N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4′s functional switching mechanism and Scc4:Scc1 association in CT.

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Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle

Molecular Microbiology ◽

10.1111/j.1365-2958.2006.05347.x ◽

2006 ◽

Vol 61 (6) ◽

pp. 1543-1555 ◽

Cited By ~ 111

Author(s):

K. Wolf ◽

H. J. Betts ◽

B. Chellas-Gery ◽

S. Hower ◽

C. N. Linton ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Small Molecule ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Small Molecule Inhibitor ◽

Secretion System ◽

Developmental Cycle ◽

Type Iii ◽

Molecule Inhibitor

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Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated

mBio ◽

10.1128/mbio.01648-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 23

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.

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Developing Cyclic Peptomers as Broad-Spectrum Gram negative Bacterial Type III Secretion System Inhibitors

10.1101/2020.08.03.235622 ◽

2020 ◽

Author(s):

Hanh N. Lam ◽

Tannia Lau ◽

Adam Lentz ◽

Jessica Sherry ◽

Alejandro Cabrera-Cortez ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Chlamydia Trachomatis ◽

Broad Spectrum ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Type Iii ◽

Bacterial Type

ABSTRACTAntibiotic resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from non-pathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs, but do not inhibit bacterial growth. Here we describe identification of an isomer, 4EpDN, that is two-fold more potent (IC50 4 μM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated Twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injestisome T3SS. 4EpDN reduced the number of T3SS basal bodies detected on the surface of Y. enterocolitica, as visualized using a fluorescent derivative of YscD, an inner membrane ring with low homology to flagellar protein FliG. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.IMPORTANCETraditional antibiotics target both pathogenic and commensal bacteria, resulting in a disruption of the microbiota, which in turn is tied to a number of acute and chronic diseases. The bacterial type III secretion system (T3SS) is an appendage used by many bacterial pathogens to establish infection, but is largely absent from commensal members of the microbiota. In this study, we identify a new derivative of the cyclic peptomer class of T3SS inhibitors. These compounds inhibit the T3SS of the nosocomial ESKAPE pathogen Pseudomonas aeruginosa and enteropathogenic Yersinia and Salmonella. The impact of cyclic peptomers is specific to the T3SS, as other bacterial secretory systems are unaffected. Importantly, cyclic peptomers completely block replication of Chlamydia trachomatis, the causative agent of genital, eye, and lung infections, in human cells, a process that requires the T3SS. Therefore, cyclic peptomers represent promising virulence blockers that can specifically disarm a broad spectrum of Gram-negative pathogens.

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Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous system

BMC Microbiology ◽

10.1186/1471-2180-14-40 ◽

2014 ◽

Vol 14 (1) ◽

pp. 40 ◽

Cited By ~ 32

Author(s):

Maria da Cunha ◽

Catarina Milho ◽

Filipe Almeida ◽

Sara V Pais ◽

Vítor Borges ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Yersinia Enterocolitica ◽

Type Iii Secretion ◽

Type Iii ◽

Heterologous System

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Correction: The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

PLoS Pathogens ◽

10.1371/journal.ppat.1004094 ◽

2014 ◽

Vol 10 (3) ◽

pp. e1004094

Author(s):

Keyword(s):

Chlamydia Trachomatis ◽

Type Iii Secretion ◽

Innate Immune ◽

Immune Signaling ◽

Phosphorylated Protein ◽

Type Iii ◽

Innate Immune Signaling ◽

Tyrosine Phosphorylated Protein ◽

Secretion Chaperone

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Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP

Molecular Microbiology ◽

10.1111/j.1365-2958.2011.07802.x ◽

2011 ◽

Vol 82 (1) ◽

pp. 131-144 ◽

Cited By ~ 23

Author(s):

Amanda J. Brinkworth ◽

Denise S. Malcolm ◽

António T. Pedrosa ◽

Katarzyna Roguska ◽

Sevanna Shahbazian ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Type Iii Secretion ◽

Type Iii ◽

Secretion Chaperone

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

Infection and Immunity ◽

10.1128/iai.00159-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3036-3045 ◽

Cited By ~ 14

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.

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