Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding

ABSTRACT We have previously shown that individuals infected with Chlamydia trachomatis can develop a robust antibody response to a Chlamydia type III secretion effector protein called Tarp and that immunization with Tarp induces protection against challenge infection in mice. The current study aimed to map the immunodominant regions of the Tarp protein by expressing 11 fragments of Tarp as glutathione S-transferase (GST) fusion proteins and detecting the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms. A major immunodominant region was strongly recognized by all antibodies. This region covers amino acids 152 to 302, consisting of three repeats (amino acids 152 to 201, 202 to 251, and 252 to 302). Each of the repeats contains multiple tyrosine residues that are phosphorylated by host cell kinases when Tarp is injected into host cells. Several other minor immunodominant regions were also identified, including those comprising amino acids 1 to 156, 310 to 431, and 582 to 682 (recognized by antisera from both humans and rabbits), that comprising amino acids 425 to 581 (recognized only by human antisera), and that comprising amino acids 683 to 847 (preferentially recognized by rabbit antisera). This immunodominance was also confirmed by the observations that six out of the nine monoclonal antibodies (MAbs) bound to the major immunodominant region and that the other three each bound to one of the minor fragments, comprising amino acids 1 to 119, 120 to 151, and 310 to 431. The antigenicity analyses have provided important information for further understanding the structure and function of Tarp.

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Deciphering molecular details of the RAC–ribosome interaction by EPR spectroscopy

Scientific Reports ◽

10.1038/s41598-021-87847-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sandra J. Fries ◽

Theresa S. Braun ◽

Christoph Globisch ◽

Christine Peter ◽

Malte Drescher ◽

...

Keyword(s):

Protein Folding ◽

Epr Spectroscopy ◽

De Novo ◽

Contact Point ◽

Ribosomal Subunit ◽

Paramagnetic Resonance ◽

40S Ribosomal Subunit ◽

Site Directed Spin Labeling ◽

Α Helix ◽

Chaperone Complex

AbstractThe eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC–ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

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Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages

Infection and Immunity ◽

10.1128/iai.00183-08 ◽

2008 ◽

Vol 76 (9) ◽

pp. 4299-4310 ◽

Cited By ~ 24

Author(s):

Yue Zhang ◽

James Murtha ◽

Margaret A. Roberts ◽

Richard M. Siegel ◽

James B. Bliska

Keyword(s):

Cell Death ◽

Type Iii Secretion ◽

Yersinia Pseudotuberculosis ◽

De Novo ◽

Host Cells ◽

Host Responses ◽

Intracellular Bacteria ◽

Wild Type ◽

Macrophage Response ◽

Type Iii

ABSTRACT Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV+ wild-type, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV+ Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV+ but not pYV-cured or ΔyopB Y. pseudotuberculosis within phagosomes: only a small fraction of the pYV+ bacteria internalized replicated by 24 h. In addition, ∼20% of macrophages infected with wild-type pYV+ Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV+ Y. pseudotuberculosis with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts the survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.

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Characterization, Phylogeny, and Genome Analyses of Nonpathogenic Xanthomonas campestris Strains Isolated from Brassica Seeds

Phytopathology ◽

10.1094/phyto-08-19-0319-r ◽

2020 ◽

Vol 110 (5) ◽

pp. 981-988 ◽

Cited By ~ 2

Author(s):

Yung-An Lee ◽

Pei-Yu Yang ◽

Shau-Chang Huang

Keyword(s):

Type Iii Secretion ◽

Xanthomonas Campestris ◽

Pathogenicity Island ◽

Whole Genome Sequence ◽

Effector Protein ◽

Type Iii Effector ◽

Type Iii ◽

Effector Genes ◽

Genome Analyses ◽

Xanthomonas Campestris Pv Campestris

Xanthomonads were detected by using the Xan-D(CCF) medium from the brassica seeds, and their pathogenicity was determined by plant inoculation tests. It was found that some seed lots were infested with Xanthomonas campestris pv. campestris, some with X. campestris pv. raphani, and some with nonpathogenic xanthomonads. The nonpathogenic xanthomonad strains were identified as X. campestris, and the multilocus sequence analysis showed that the nonpathogenic X. campestris strains were grouped together with pathogenic X. campestris, but not with nonpathogenic strains of X. arboricola. In addition, all isolated X. campestris pv. campestris and X. campestris pv. raphani strains were positive in the hrpF-PCR, but the nonpathogenic strains were negative. It was further found that nonpathogenic X. campestris strain nE1 does not contain the entire pathogenicity island (hrp gene cluster; type III secretion system) and all type III effector protein genes based on the whole genome sequence analyses. The nonpathogenic X. campestris strain nE1 could acquire the entire pathogenicity island from the endemic X. campestris pv. campestris and X. campestris pv. raphani strains by conjugation, but type III effector genes were not cotransferred. The studies showed that the nonpathogenic X. campestris strains indeed exist on the brassica seeds, but it could be differentiated by the PCR assays on the hrp and type III effector genes. Nevertheless, the nonpathogenic X. campestris strains cannot be ignored because they may be potential gene resources to increase genetic diversity in the endemic pathogenic X. campestris pv. campestris and X. campestris pv. raphani strains.

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Studying Conformational Changes of the Yersinia Type-III-Secretion Effector YopO in Solution by Integrative Structural Biology

Structure ◽

10.1016/j.str.2019.06.007 ◽

2019 ◽

Vol 27 (9) ◽

pp. 1416-1426.e3 ◽

Cited By ~ 5

Author(s):

Martin F. Peter ◽

Anne T. Tuukkanen ◽

Caspar A. Heubach ◽

Alexander Selsam ◽

Fraser G. Duthie ◽

...

Keyword(s):

Structural Biology ◽

Conformational Changes ◽

Type Iii Secretion ◽

Type Iii ◽

Integrative Structural Biology ◽

Type Iii Secretion Effector

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Global impact of Salmonella type III secretion effector SteA on host cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2014.05.056 ◽

2014 ◽

Vol 449 (4) ◽

pp. 419-424 ◽

Cited By ~ 10

Author(s):

Elena Cardenal-Muñoz ◽

Gabriel Gutiérrez ◽

Francisco Ramos-Morales

Keyword(s):

Type Iii Secretion ◽

Host Cells ◽

Global Impact ◽

Type Iii ◽

Type Iii Secretion Effector

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Construction of a reporter system to study Burkholderia mallei type III secretion and identification of the BopA effector protein function in intracellular survival

Transactions of the Royal Society of Tropical Medicine and Hygiene ◽

10.1016/s0035-9203(08)70029-4 ◽

2008 ◽

Vol 102 ◽

pp. S127-S133 ◽

Cited By ~ 11

Author(s):

Gregory C. Whitlock ◽

D. Mark Estes ◽

Glenn M. Young ◽

Briana Young ◽

Alfredo G. Torres

Keyword(s):

Type Iii Secretion ◽

Protein Function ◽

Intracellular Survival ◽

Effector Protein ◽

Burkholderia Mallei ◽

Reporter System ◽

Type Iii

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Characterization of the Xanthomonas axonopodis pv. glycines Hrp Pathogenicity Island

Journal of Bacteriology ◽

10.1128/jb.185.10.3155-3166.2003 ◽

2003 ◽

Vol 185 (10) ◽

pp. 3155-3166 ◽

Cited By ~ 94

Author(s):

Jung-Gun Kim ◽

Byoung Keun Park ◽

Chang-Hyuk Yoo ◽

Eunkyung Jeon ◽

Jonghee Oh ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Pathogenicity Island ◽

Inducible Promoter ◽

Core Region ◽

Effector Protein ◽

Trna Genes ◽

Secretion Systems ◽

Xanthomonas Axonopodis ◽

Type Iii

ABSTRACT We sequenced an approximately 29-kb region from Xanthomonas axonopodis pv. glycines that contained the Hrp type III secretion system, and we characterized the genes in this region by Tn3-gus mutagenesis and gene expression analyses. From the region, hrp (hypersensitive response and pathogenicity) and hrc (hrp and conserved) genes, which encode type III secretion systems, and hpa (hrp-associated) genes were identified. The characteristics of the region, such as the presence of many virulence genes, low G+C content, and bordering tRNA genes, satisfied the criteria for a pathogenicity island (PAI) in a bacterium. The PAI was composed of nine hrp, nine hrc, and eight hpa genes with seven plant-inducible promoter boxes. The hrp and hrc mutants failed to elicit hypersensitive responses in pepper plants but induced hypersensitive responses in all tomato plants tested. The Hrp PAI of X. axonopodis pv. glycines resembled the Hrp PAIs of other Xanthomonas species, and the Hrp PAI core region was highly conserved. However, in contrast to the PAI of Pseudomonas syringae, the regions upstream and downstream from the Hrp PAI core region showed variability in the xanthomonads. In addition, we demonstrate that HpaG, which is located in the Hrp PAI region of X. axonopodis pv. glycines, is a response elicitor. Purified HpaG elicited hypersensitive responses at a concentration of 1.0 μM in pepper, tobacco, and Arabidopsis thaliana ecotype Cvi-0 by acting as a type III secreted effector protein. However, HpaG failed to elicit hypersensitive responses in tomato, Chinese cabbage, and A. thaliana ecotypes Col-0 and Ler. This is the first report to show that the harpin-like effector protein of Xanthomonas species exhibits elicitor activity.

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