scholarly journals The Chlamydial Plasmid-Encoded Protein pgp3 Is Secreted into the Cytosol of Chlamydia-Infected Cells

2008 ◽  
Vol 76 (8) ◽  
pp. 3415-3428 ◽  
Author(s):  
Zhongyu Li ◽  
Ding Chen ◽  
Youmin Zhong ◽  
Shiping Wang ◽  
Guangming Zhong
Keyword(s):  
Host Cell ◽  
Chlamydial Infection ◽  
Open Reading Frames ◽  
Cryptic Plasmid ◽  
Activity Factor ◽  
Infected Cells ◽  
Cell Cytosol ◽  
Endogenous Proteins ◽  
Chlamydial Genome ◽  
Host Cell Cytosol

ABSTRACT The chlamydial cryptic plasmid encodes eight putative open reading frames (ORFs), designated pORF1 to -8. Antibodies raised against these ORF proteins were used to localize the endogenous proteins during chlamydial infection. We found that the pORF5 protein (also known as pgp3) was detected mainly in the cytosol of Chlamydia-infected cells, while the remaining seven proteins were found inside the chlamydial inclusions only. The pgp3 distribution pattern in the host cell cytosol is similar to but not overlapping with that of chlamydial protease/proteasome-like activity factor (CPAF), a chlamydial genome-encoded protein known to be secreted from chlamydial inclusions into the host cell cytosol. The anti-pgp3 labeling was removed by preabsorption with pgp3 but not CPAF fusion proteins and vice versa, demonstrating that pgp3 is a unique secretion protein. This conclusion is further supported by the observation that pgp3 was highly enriched in cytosolic fractions and had a minimal presence in the inclusion-containing nuclear fractions prepared from Chlamydia-infected cells. The pgp3 protein was detected as early as 12 h after infection and was secreted by all chlamydial species that carry the cryptic plasmid, suggesting that there is a selection pressure for maintaining pgp3 secretion during chlamydial infection. Although expression of pgp3 in the host cell cytosol via a transgene did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection, purified pgp3 protein stimulated macrophages to release inflammatory cytokines, suggesting that pgp3 may contribute to chlamydial pathogenesis.

Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes

1999 ◽  
Vol 112 (5) ◽  
pp. 681-693
Author(s):  
U.E. Schaible ◽  
P.H. Schlesinger ◽  
T.H. Steinberg ◽  
W.F. Mangel ◽  
T. Kobayashi ◽  
...  
Keyword(s):  
Host Cell ◽  
Lucifer Yellow ◽  
Cysteine Proteinase ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Anion Transporter ◽  
Infected Cells ◽  
Lysobisphosphatidic Acid ◽  
Cell Cytosol ◽  
Host Cell Cytosol

The intracellular parasite Leishmania survives and proliferates in host macrophages. In this study we show that parasitophorous vacuoles of L. mexicana gain access to cytosolic material via two different routes. (1) Small anionic molecules such as Lucifer Yellow are rapidly transported into the vacuoles by an active transport mechanism that is sensitive to inhibitors of the host cell's organic anion transporter. (2) Larger molecules such as fluorescent dextrans introduced into the host cell cytosol are also delivered to parasitophorous vacuoles. This transport is slower and sensitive to modulators of autophagy. Infected macrophages were examined by two novel assays to visualize and quantify this process. Immunoelectron microscopy of cells loaded with digoxigenin-dextran revealed label in multivesicular endosomes, which appeared to fuse with parasitophorous vacuoles. The inner membranes of the multivesicular vesicles label strongly with antibodies against lysobisphosphatidic acid, suggesting that they represent a point of confluence between the endosomal and autophagosomal pathways. Although the rate of autophagous transfer was comparable in infected and uninfected cells, infected cells retained hydrolyzed cysteine proteinase substrate to a greater degree. These data suggest that L. mexicana-containing vacuoles have access to potential nutrients in the host cell cytosol via at least two independent mechanisms.

scholarly journals Intramolecular Dimerization Is Required for the Chlamydia-Secreted Protease CPAF To Degrade Host Transcriptional Factors

2004 ◽  
Vol 72 (7) ◽  
pp. 3869-3875 ◽  
Author(s):  
Feng Dong ◽  
Jyotika Sharma ◽  
Yanming Xiao ◽  
Youmin Zhong ◽  
Guangming Zhong
Keyword(s):  
Transcription Factors ◽  
Host Cell ◽  
Proteolytic Activity ◽  
Bacterial Species ◽  
Antigen Expression ◽  
Obligate Intracellular ◽  
Histocompatibility Complex ◽  
Activity Factor ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACT We previously identified a chlamydial protein designated CPAF (chlamydia protease/proteasome-like activity factor) that is secreted into host cell cytosol for degrading host transcription factors required for major histocompatibility complex antigen expression. Here we report that CPAF, synthesized as a 70-kDa proprotein, is processed into two fragments (designated CPAFn and CPAFc) to form intramolecular dimers that are much more stable than the naïve CPAF. Precipitation with antibodies that recognized CPAF dimers removed the proteolytic activity responsible for degrading host transcription factor RFX5 from chlamydia-infected host cell cytosol, while precipitation with antibodies that recognized free CPAF fragments alone did not remove this activity. Separation of CPAFn from CPAFc resulted in a loss of proteolytic activity. Furthermore, neither expressed full-length CPAF that was not processed nor coexpressed CPAFn and CPAFc fragments that failed to form dimers degraded RFX5. These observations demonstrate that intramolecular dimerization is required for CPAF to degrade host transcription factors, a strategy that is utilized by an obligate intracellular bacterial species to evade host defenses.

scholarly journals A Chlamydia trachomatis OmcB C-Terminal Fragment Is Released into the Host Cell Cytoplasm and Is Immunogenic in Humans

2011 ◽  
Vol 79 (6) ◽  
pp. 2193-2203 ◽  
Author(s):  
Manli Qi ◽  
Siqi Gong ◽  
Lei Lei ◽  
Quanzhong Liu ◽  
Guangming Zhong
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Content Type ◽  
Membrane Complex ◽  
Host Cell Cytoplasm ◽  
Complex Protein ◽  
Infected Cells ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACTTheChlamydia trachomatisouter membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals inC. trachomatis-infected cells permeabilized with saponin. Western blot analyses revealed that OmcB was partially processed into OmcBc and OmcBn fragments. The processed OmcBc was released into host cell cytosol, while the OmcBn and remaining full-length OmcB were retained within the chlamydial inclusions. The organism-associated OmcB epitopes became detectable only after theC. trachomatis-infected cells were permeabilized with strong detergents such as SDS. However, the harsh permeabilization conditions also led to the leakage of the already secreted OmcBc and chlamydia-secreted protease (CPAF) out of the host cells. The OmcBc processing and release occurred in all biovars ofC. trachomatis. Moreover, the released OmcBc but not the retained OmcBn was highly immunogenic inC. trachomatis-infected women, which is consistent with the concept that exposure of chlamydial proteins to host cell cytosol is accompanied by increased immunogenicity. These observations have provided important information for further exploring/optimizing OmcB as a target for the development of diagnosis methods and vaccines.

Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus

2001 ◽  
Vol 114 (15) ◽  
pp. 2747-2754
Author(s):  
David G. Swan ◽  
Rowena Stern ◽  
Sue McKellar ◽  
Kirsten Phillips ◽  
Chris A. L. Oura ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Cell ◽  
Cell Lines ◽  
Theileria Annulata ◽  
Open Reading Frames ◽  
Nuclear Extracts ◽  
Genes Encoding ◽  
Cell Gene Expression ◽  
Infected Cells ◽  
Host Nucleus

Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.

scholarly journals Chlamydia-Infected Cells Continue To Undergo Mitosis and Resist Induction of Apoptosis

2004 ◽  
Vol 72 (1) ◽  
pp. 451-460 ◽  
Author(s):  
Whitney Greene ◽  
Yangming Xiao ◽  
Yanqing Huang ◽  
Grant McClarty ◽  
Guangming Zhong
Keyword(s):  
Dna Synthesis ◽  
Host Cell ◽  
Chlamydial Infection ◽  
Host Cells ◽  
Infected Host ◽  
Apoptosis Induction ◽  
Infected Cells ◽  
Induction Of Apoptosis

ABSTRACT Both anti- and proapoptotic activities have been reported to occur during chlamydial infection. To reconcile the apparent controversy, we compared host cell apoptotic responses to infection with 17 different chlamydial serovars and strains. None of the serovars caused any biologically significant apoptosis in the infected host cells. Host cells in chlamydia-infected cultures can continue to undergo DNA synthesis and mitosis. Chlamydia-infected cells are resistant to apoptosis induction, although the extent of the antiapoptotic ability varied between serovars. These observations have demonstrated that an anti- but not proapoptotic activity is the prevailing event in chlamydia-infected cultures.

scholarly journals Persistent Chlamydia trachomatis Infection of HeLa Cells Mediates Apoptosis Resistance through a Chlamydia Protease-Like Activity Factor-Independent Mechanism and Induces High Mobility Group Box 1 Release

2011 ◽  
Vol 80 (1) ◽  
pp. 195-205 ◽  
Author(s):  
Jürgen Rödel ◽  
Christina Große ◽  
Hangxing Yu ◽  
Katharina Wolf ◽  
Gordon P. Otto ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Hela Cells ◽  
Persistent Infection ◽  
High Mobility ◽  
Apoptosis Resistance ◽  
Content Type ◽  
Activity Factor ◽  
Infected Cells ◽  
Ifn Γ

ABSTRACTIntracellular persistence ofChlamydia trachomatishas been implicated in the development of chronic infection that can result in pelvic inflammatory disease and tubal sterility. By inhibition of host cell apoptosis, chlamydiae have evolved a strategy to maintain the intracellular environment for replication and persistence. Both antiapoptotic host cell-derived factors and the chlamydial protease-like activity factor (CPAF) are involved inChlamydia-mediated apoptosis resistance. Here, we show that in HeLa cells infected with gamma interferon (IFN-γ)-induced persistentC. trachomatisserovar D, the expression of CPAF is downregulated, and proapoptotic protease substrates are not cleaved. Persistent infection protected HeLa cells from apoptosis when they were exposed to staurosporine. Small-interfering RNA-mediated inhibition of myeloid cell leukemia 1 (Mcl-1) protein upregulation sensitized persistently infected cells for apoptosis. The inhibitor of apoptosis protein 2 (IAP-2) seems not to be relevant in this context because IAP-2 protein was not induced in response to IFN-γ treatment. Although apoptosis was inhibited, persistent infection caused cell membrane disintegration, as measured by the increased release of cytokeratin 18 from HeLa cells. Moreover, persistently infected cells released significantly increased amounts of high mobility group box 1 (HMGB1) protein which represents a proinflammatory damage-associated pattern molecule. The data of this study suggest that cells infected with persistentC. trachomatisare protected from apoptosis independently of CPAF but may promote chronic inflammation through HMGB1 release.

scholarly journals Type II Secretion Substrates ofLegionella pneumophilaTranslocate Out of the Pathogen-Occupied Vacuole via a Semipermeable Membrane

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Hilary K. Truchan ◽  
Harry D. Christman ◽  
Richard C. White ◽  
Nakisha S. Rutledge ◽  
Nicholas P. Cianciotto
Keyword(s):  
Host Cell ◽  
Infected Host ◽  
Type Ii Secretion ◽  
Dependent Manner ◽  
Type Ii ◽  
Murine Macrophages ◽  
Intracellular Infection ◽  
Infected Host Cell ◽  
Cell Cytosol ◽  
Host Cell Cytosol

ABSTRACTLegionella pneumophilareplicates in macrophages in a host-derived phagosome, termed theLegionella-containing vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into the macrophage cytosol is well established, the location of type II secretion (T2S) substrates in the infected host cell is unknown. Here, we show that the T2S substrate ProA, a metalloprotease, translocates into the cytosol of human macrophages, where it associates with the LCV membrane (LCVM). Translocation is detected as early as 10 h postinoculation (p.i.), which is approximately the midpoint of the intracellular life cycle. However, it is detected as early as 6 h p.i. if ProA is hyperexpressed, indicating that translocation depends on the timing of ProA expression and that any other factors necessary for translocation are in place by that time point. Translocation occurs with allL. pneumophilastrains tested and in amoebae, natural hosts forL. pneumophila. It was absent in murine bone marrow-derived macrophages and murine macrophage cell lines. The ChiA chitinase also associated with the cytoplasmic face of the LCVM at 6 h p.i. and in a T2S-dependent manner. Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced by damage to host membranes, appeared within the LCV of infected human but not murine macrophages beginning at 6 h p.i. Thus, we hypothesize that ProA and ChiA are first secreted into the vacuolar lumen by the activity of the T2S and subsequently traffic into the macrophage cytosol via a novel mechanism that involves a semipermeable LCVM.IMPORTANCEInfection of macrophages and amoebae plays a central role in the pathogenesis ofL. pneumophila, the agent of Legionnaires’ disease. We have previously demonstrated that the T2S system ofL. pneumophilagreatly contributes to intracellular infection. However, the location of T2S substrates within the infected host cell is unknown. This report presents the first evidence of aL. pneumophilaT2S substrate in the host cell cytosol and, therefore, the first evidence of a non-T4S effector trafficking out of the LCV. We also provide the first indication that the LCV is not completely intact but is instead semipermeable and that this occurs in human but not murine macrophages. Given this permeability, we hypothesize that other T2S substrates and LCV lumenal contents can escape into the host cell cytosol. Thus, these substrates may represent a battery of previously unidentified effectors that can interact with host factors and contribute to intracellular infection byL. pneumophila.

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