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

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.

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Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Journal of Bacteriology ◽

10.1128/jb.00893-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7549-7555 ◽

Cited By ~ 28

Author(s):

Dustin L. Johnson ◽

James B. Mahony

Keyword(s):

Amino Acid ◽

Type Iii Secretion ◽

Transmembrane Domain ◽

Integral Membrane Protein ◽

Kinase Domain ◽

Chlamydophila Pneumoniae ◽

Structural Protein ◽

E Coli ◽

Type Iii ◽

Layer Chromatography

ABSTRACT Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.

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Environmental Regulation and Virulence Attributes of the Ysa Type III Secretion System of Yersinia enterocolitica Biovar 1B

Infection and Immunity ◽

10.1128/iai.73.9.5961-5977.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5961-5977 ◽

Cited By ~ 47

Author(s):

Krista Venecia ◽

Glenn M. Young

Keyword(s):

Yersinia Enterocolitica ◽

Type Iii Secretion ◽

Chromosomal Locus ◽

Host Interactions ◽

Type Iii ◽

Genes Encoding ◽

Transposon Mutants ◽

Phase Temperature ◽

Mouse Model Of Infection ◽

Definitive Role

ABSTRACT Pathogenic biovars of Yersinia enterocolitica maintain the well-studied plasmid-encoded Ysc type III secretion (TTS) system, which has a definitive role in virulence. Y. enterocolitica biovar 1B additionally has a distinct chromosomal locus, the Yersinia secretion apparatus pathogenicity island (YSA PI) that encodes the Ysa TTS system. The signals to which the Ysa TTS system responds and its role in virulence remain obscure. This exploratory study was conducted to define environmental cues that promote the expression of Ysa TTS genes and to define how the Ysa TTS system influences bacterium-host interactions. Using a genetic approach, a collection of Y. enterocolitica Ysa TTS mutants was generated by mutagenesis with a transposon carrying promoterless lacZYA. This approach identified genes both within and outside of the YSA PI that contribute to Ysa TTS. Expression of these genes was regulated in response to growth phase, temperature, NaCl, and pH. Additional genetic analysis demonstrated that two regulatory genes encoding components of the YsrR-YsrS (ysrS) and RcsC-YojN-RcsB (rcsB) phosphorelay systems affect the expression of YSA PI genes and each other. The collection of Ysa TTS-defective transposon mutants, along with other strains carrying defined mutations that block Ysa and Ysc TTS, was examined for changes in virulence properties by using the BALB/c mouse model of infection. This analysis revealed that the Ysa TTS system impacts the ability of Y. enterocolitica to colonize gastrointestinal tissues. These results reveal facets of how Y. enterocolitica controls the function of the Ysa TTS system and uncovers a role for the Ysa TTS during the gastrointestinal phase of infection.

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Role of host cell polarity and leading edge properties in Pseudomonas type III secretion

Microbiology ◽

10.1099/mic.0.033241-0 ◽

2010 ◽

Vol 156 (2) ◽

pp. 356-373 ◽

Cited By ~ 13

Author(s):

Dacie R. Bridge ◽

Matthew J. Novotny ◽

Elizabeth R. Moore ◽

Joan C. Olson

Keyword(s):

Cell Migration ◽

Host Cell ◽

Type Iii Secretion ◽

Leading Edge ◽

Immunofluorescent Staining ◽

Cell Sensitivity ◽

Iq Motif ◽

Type Iii ◽

Cellular Changes ◽

Ht 29

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.

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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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Identification of Novel Type III Secretion Effectors in Xanthomonas oryzae pv. oryzae

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-1-0096 ◽

2009 ◽

Vol 22 (1) ◽

pp. 96-106 ◽

Cited By ~ 71

Author(s):

Ayako Furutani ◽

Minako Takaoka ◽

Harumi Sanada ◽

Yukari Noguchi ◽

Takashi Oku ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Regulatory Protein ◽

Effector Proteins ◽

Xanthomonas Oryzae ◽

Dependent Manner ◽

Plant Pathogenic Bacteria ◽

Type Iii ◽

Genes Encoding

Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB– and HpaP– mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

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Expression of the Bradyrhizobium japonicum Type III Secretion System in Legume Nodules and Analysis of the Associated tts box Promoter

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-8-1087 ◽

2008 ◽

Vol 21 (8) ◽

pp. 1087-1093 ◽

Cited By ~ 40

Author(s):

Susanne Zehner ◽

Grit Schober ◽

Mandy Wenzel ◽

Kathrin Lang ◽

Michael Göttfert

Keyword(s):

Type Iii Secretion ◽

Bradyrhizobium Japonicum ◽

Response Regulator ◽

Type Iii Secretion System ◽

Secretion System ◽

Symbiotic Efficiency ◽

Type Iii ◽

Genes Encoding ◽

Transcriptional Start Sites ◽

Type Three Secretion

In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.

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Salmonella pathogenicity island 1 (SPI-1) type III secretion of SopD involves N- and C-terminal signals and direct binding to the InvC ATPase

Microbiology ◽

10.1099/mic.0.038117-0 ◽

2010 ◽

Vol 156 (6) ◽

pp. 1805-1814 ◽

Cited By ~ 12

Author(s):

R. Boonyom ◽

M. H. Karavolos ◽

D. M. Bulmer ◽

C. M. A. Khan

Keyword(s):

Protein Interactions ◽

Type Iii Secretion ◽

Pathogenicity Island ◽

Effector Proteins ◽

Amino Acid Residues ◽

Type Iii ◽

Protein Protein Interaction ◽

Secretion Signal ◽

Serovar Typhimurium ◽

Bacterial Cytoplasm

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important pathogen and a causative agent of gastroenteritis. During infection, S. Typhimurium assembles molecular-needle complexes termed type III secretion (T3S) systems to translocate effector proteins from the bacterial cytoplasm directly into the host cell. The T3S signals that direct the secretion of effectors still remain enigmatic. SopD is a key T3S effector contributing to the systemic virulence of S. Typhimurium and the development of gastroenteritis. We have scrutinized the distribution of the SopD T3S signals using in silico analysis and a targeted deletion approach. We show that amino acid residues 6–10 act as the N-terminal secretion signal for Salmonella pathogenicity island 1 (SPI-1) T3S. Furthermore, we show that two putative C-terminal helical regions of SopD are essential for its secretion and also help prevent erroneous secretion through the flagellar T3S machinery. In addition, using protein–protein interaction assays, we have identified an association between SopD and the SPI-1 T3S system ATPase, InvC. These findings demonstrate that T3S of SopD involves multiple signals and protein interactions, providing important mechanistic insights into effector protein secretion.

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The Tripartite Type III Secreton of Shigella flexneri Inserts Ipab and Ipac into Host Membranes

The Journal of Cell Biology ◽

10.1083/jcb.147.3.683 ◽

1999 ◽

Vol 147 (3) ◽

pp. 683-693 ◽

Cited By ~ 363

Author(s):

Ariel Blocker ◽

Pierre Gounon ◽

Eric Larquet ◽

Kirsten Niebuhr ◽

Véronique Cabiaux ◽

...

Keyword(s):

Protein Secretion ◽

Type Iii Secretion ◽

Shigella Flexneri ◽

Coupled Processes ◽

Critical Threshold ◽

Secretion Systems ◽

Type Iii ◽

Rbc Membrane ◽

Genes Encoding ◽

Electron Microscopy Analysis

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-Å pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37°C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

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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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