Conservation of the biochemical properties of IncA from Chlamydia trachomatis and Chlamydia caviae: oligomerization of IncA mediates interaction between facing membranes

The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild-type strain requires direct interactions between IncA molecules at the inclusion and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.     

Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane

Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an approximately 120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable approximately 380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis.     

RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry

To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified approximately 226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRbeta may function as a receptor, as inhibition of PDGFRbeta by RNA interference or by PDGFRbeta neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRbeta or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRbeta and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite.     

Tom34: a cytosolic cochaperone of the Hsp90/Hsp70 protein complex involved in mitochondrial protein import

Most mitochondrial membrane proteins are synthesized in the cytosol and must be delivered to the organelle in an unfolded, import competent form. In mammalian cells, the cytosolic chaperones Hsp90 and Hsp70 are part of a large cytosolic complex that deliver the membrane protein to the mitochondrion by docking with the import receptor Tom70. These two abundant chaperones have other functions in the cell suggesting that the specificity for the targeting of mitochondrial proteins requires the addition of specific factors within the targeting complex. We identify Tom34 as a cochaperone of Hsp70/Hsp90 in mitochondrial protein import. We show that Tom34 is an integral component with Hsp70 and Hsp90 in the large complex. We also demonstrate the role of Tom34 in the mitochondrial import process, as the addition of an excess of Tom34 prevents efficient mitochondrial translocation of precursor proteins that have requirements for Hsp70/Hsp90. Tom34 exhibits an affinity for mitochondrial preproteins of the Tom70 translocation pathway as demonstrated by binding assays using in vitro translated proteins as baits. In addition, we examined the specificity and the size of different complex cytosolic machines. Separation of different radiolabeled cell-free translated proteins on Native-PAGE showed the presence of a high molecular weight complex which binds hydrophobic proteins. Importantly we show that the formation of the chaperone cytosolic complex that mediates the targeting of proteins to the mitochondria contains Tom34 and assembles in the presence of a fully translated substrate protein.     

The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane

The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology.     

Peptide immunization of guinea pigs against Chlamydia psittaci (GPIC agent) infection induces good vaginal secretion antibody response, in vitro neutralization and partial protection against live challenge

Immunization of female guinea pigs with a chimeric peptide consisting of variable domain IV (VDIV) and a region known as GP8 from the major outer membrane protein of Chlamydophila caviae, formerly Chlamydia psittaci guinea pig inclusion conjunctivitis strain, was performed to assess whether humoral immune responses could be elicited in the reproductive tracts of immunized animals. The C. caviae strain is able to cause a sexually transmitted infection in the guinea pig that closely parallels C. trachomatis infections in humans. The best anti-VDIV antibody response in vaginal secretions was achieved by intraperitoneal priming with subsequent intravaginal boosting (P < 0.001). Dot-blot analyses of vaginal secretions confirmed that these anti-VDIV antibodies, produced against a linear peptide, were able to recognize and bind to whole conformational C. caviae elementary bodies. Following live intravaginal challenge with C. caviae, a significant reduction in the intensity (P = 0.01) and an apparent reduction in the duration of the infection was evident between the guinea pigs immunized with VDIV-GP8 and non-immunized controls.     

Functional analysis of human metaxin in mitochondrial protein import in cultured cells and its relationship with the Tom complex

Metaxin is an outer membrane protein of mammalian mitochondria which is suggested to be involved in protein import into the organelle. RNA blot analysis showed that distribution of metaxin mRNA in human tissues differs from that of mRNA for the translocase component Tom20. Effect of overexpression of human metaxin on mitochondrial preprotein import and processing in COS-7 cells was studied. Overexpression of metaxin resulted in impaired mitochondrial import of natural and chimeric preproteins and in their accumulation. We previously reported that overexpression of Tom20 in cultured cells causes inhibition of import of mitochondrial preprotein. Coexpression of metaxin with Tom20 had no further effect on the preprotein import. Overexpression of the cytosolic domain of metaxin also caused inhibition of preprotein import, although less strongly than the full-length metaxin. In blue native PAGE, Tom40, Tom22, and a portion of Tom20 migrated as a complex of approximately 400 kDa, and the other portion of Tom20 migrated in smaller forms of approximately 100 and approximately 40 kDa. On the other hand, metaxin migrated at a position of approximately 50 kDa. These results confirm earlier in vitro results that metaxin participates in preprotein import into mammalian mitochondria, and indicates that it does not associate with the Tom complex.     

Chlamydia trachomatis infection alters the development of memory CD8+ T cells

The obligate intracellular bacterium Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide. Prior exposure to C. trachomatis has been shown to provide incomplete protection against subsequent infection. One possible explanation for the limited immunity afforded by prior C. trachomatis infection is poor activation of Chlamydia-specific memory CD8+ T cells. In this study, we examined the development of CD8+ memory T cell responses specific for the Chlamydia Ag CrpA. The percentage of CrpA63-71-specific T cells expressing an effector memory T cell phenotype (IL-7R+ CD62low) was dramatically diminished in mice immunized with C. trachomatis, compared with mice immunized with vaccinia virus expressing the CrpA protein. These alterations in memory T cell development were correlated with a significant reduction in the capacity of convalescent mice to mount an enhanced recall response to Chlamydia Ags, compared with the primary response. CrpA-specific memory T cells primed during VacCrpA infection also failed to respond to a challenge with Chlamydia. We therefore investigated whether C. trachomatis infection might have a global inhibitory effect on CD8+ T cell activation by coinfecting mice with C. trachomatis and Listeria monocytogenes and we found that the activation of Listeria-specific naive and memory CD8+ T cells was reduced in the presence of C. trachomatis. Together, these results suggest that Chlamydia is able to alter the development of CD8+ T cell responses during both primary and secondary infection, perhaps accounting for the incomplete protection provided by prior Chlamydia infection.     

The adaptor protein Tom1L1 is a negative regulator of Src mitogenic signaling induced by growth factors

The Src family of protein-tyrosine kinases (SFK) play important roles in mitogenesis and morphological changes induced by growth factors. The involved substrates are, however, ill defined. Using an antiphosphotyrosine antibody to screen tyrosine-phosphorylated cDNA expression library, we have identified Tom1L1, an adaptor protein of the Tom1 family and a novel substrate and activator of the SFK. Surprisingly, we found that Tom1L1 does not promote DNA synthesis induced by Src. Furthermore, we report that Tom1L1 negatively regulates SFK mitogenic signaling induced by platelet-derived growth factor (PDGF) through modulation of SFK-receptor association: (i) Tom1L1 inhibits DNA synthesis induced by PDGF; (ii) inhibition is overcome by c-myc expression or p53 inactivation, two regulators of SFK mitogenic function; (iii) Src or Fyn coexpression overrides Tom1L1 mitogenic activity; (iv) overexpression of the adaptor reduces Src association with the receptor; and (v) protein inactivation potentiates receptor complex formation, allowing increased SFK activation and DNA synthesis. However, Tom1L1 affects neither DNA synthesis induced by the constitutively active allele SrcY527F nor SFK-regulated actin assembly induced by PDGF. Finally, overexpressed Tom1 and Tom1L2 also associate with Src and affected mitogenic signaling in agreement with some redundancy among members of the Tom1 family. We concluded that Tom1L1 defines a novel mechanism for regulation of SFK mitogenic signaling induced by growth factors.     

Dissection of the mitochondrial import and assembly pathway for human Tom40

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.     

The immunoglobulin superfamily in Drosophila melanogaster and Caenorhabditis elegans and the evolution of complexity

Drosophila melanogaster is an arthropod with a much more complex anatomy and physiology than the nematode Caenorhabditis elegans. We investigated one of the protein superfamilies in the two organisms that plays a major role in development and function of cell-cell communication: the immunoglobulin superfamily (IgSF). Using hidden Markov models, we identified 142 IgSF proteins in Drosophila and 80 in C. elegans. Of these, 58 and 22, respectively, have been previously identified by experiments. On the basis of homology and the structural characterisation of the proteins, we can suggest probable types of function for most of the novel proteins. Though overall Drosophila has fewer genes than C. elegans, it has many more IgSF cell-surface and secreted proteins. Half the IgSF proteins in C. elegans and three quarters of those in Drosophila have evolved subsequent to the divergence of the two organisms. These results suggest that the expansion of this protein superfamily is one of the factors that have contributed to the formation of the more complex physiological features that are found in Drosophila.     

Cell death, BAX activation, and HMGB1 release during infection with Chlamydia

Infection by a number of Chlamydia species leads to resistance of the host cell to apoptosis, followed by induction of host-cell death. In a population of infected cells that displays protection against staurosporine-induced apoptosis among the adherent cells, we find that cells that had been recovered from the supernatant share characteristics of both apoptosis and necrosis, as assayed by the propidium iodide (PI)-annexin V double-labeling technique. Cell death was observed in both an epithelial cell line and primary fibroblasts, although the primary cells had a higher propensity to die through apoptosis than the immortalized cell line. Staurosporine-mediated activation of the pro-apoptotic BCL-2 family member, BAX, was inhibited in the epithelial cell line infected for 32 h with the lymphogranuloma venereum (LGV/L2) but not the murine pneumonitis (MoPn) strain of C. trachomatis, but inhibition of staurosporine-mediated BAX activation disappeared after 48 h of infection with the LGV/L2 strain. Conversely, infection with MoPn (C. muridarum) but not LGV/L2 led to BAX activation after 72 h, as previously reported for shorter (48 h) infection with the guinea pig inclusion conjunctivitis (GPIC) serovar of C. psittaci (C. caviae). These results suggest that the ability to inhibit staurosporine-mediated BAX activation or to activate BAX due to the infection itself may vary as a function of the chlamydial strain. Interestingly, both the epithelial cells and the fibroblasts also released high mobility group box 1 protein (HMGB1) during infection, although much less HMGB1 was released from fibroblasts, consistent with the higher level of apoptosis observed in the primary cells. HMGB1 is released preferentially by necrotic or permeabilized viable cells, but not apoptotic cells. In the extracellular space, HMGB1 promotes inflammation through interaction with specific cell-surface receptors. Higher levels of HMGB1 were also measured in the genital-tract secretions of mice infected vaginally with C. trachomatis, compared to uninfected controls. These results suggest that cells infected with Chlamydia release intracellular factors that may contribute to the inflammatory response observed in vivo.     

Single-cell RNA sequencing identifies novel cell types in Drosophila blood

Drosophila has been extensively used to model the human blood-immune system,as both systems share many developmental and immune response mechanisms.However,while many human blood cell types have been identified,only three were found in flies:plasmatocytes,crystal cells and lamellocytes.To better understand the complexity of fly blood system,we used single-cell RNA sequencing technology to generate co mprehensive gene expression profiles for Drosophila circulating blood cells.In addition to the known cell types,we identified two new Drosophila blood cell types:thanacytes and primocytes.Thanacytes,which express many stimulus response genes,are involved in distinct responses to different types of bacteria.Primocytes,which express cell fate commitment and signaling genes,appear to be involved in keeping stem cells in the circulating blood.Furthermore,our data revealed four novel plasmatocyte subtypes(Ppn+,CAH7~+,Lsp~+ and reservoir plasmatocytes),each with unique molecular identities and distinct predicted functions.We also identified cross-species markers from Drosophila hemocytes to human blood cells.Our analysis unveiled a more complex Drosophila blood system and broadened the scope of using Drosophila to model human blood system in development and disease.     

Immune-deficient Drosophila melanogaster: a model for the innate immune response to human fungal pathogens

We explored the host-pathogen interactions of the human opportunistic fungus Candida albicans using Drosophila melanogaster. We established that a Drosophila strain devoid of functional Toll receptor is highly susceptible to the human pathogen C. albicans. Using this sensitive strain, we have been able to show that a set of specific C. albicans mutants of different virulence in mammalian infection models are also impaired in virulence in Drosophila and remarkably display the same rank order of virulence. This immunodeficient insect model also revealed virulence properties undetected in an immunocompetent murine model of infection. The genetic systems available in both host and pathogen will enable the identification of host-specific components and C. albicans genes involved in the host-fungal interplay.     

Drosophila melanogaster S2 cells: a model system to study Chlamydia interaction with host cells

Chlamydia spp. are major causes of important human diseases, but dissecting the host-pathogen interactions has been hampered by the lack of bacterial genetics and the difficulty in carrying out forward genetic screens in mammalian hosts. RNA interference (RNAi)-based methodologies for gene inactivation can now be easily carried out in genetically tractable model hosts, such as Drosophila melanogaster, and offer a new approach to identifying host genes required for pathogenesis. We tested whether Chlamydia trachomatis infection of D. melanogaster S2 cells recapitulated critical aspects of mammalian cell infections. As in mammalian cells, C. trachomatis entry was greatly reduced by heparin and cytochalasin D. Inclusions were formed in S2 cells, acquired Golgi-derived sphingolipids, and avoided phagolysosomal fusion. Elementary body (EB) to reticulate body (RB) differentiation was observed, however, no RB to EB development or host cell killing was observed. RNAi-mediated inactivation of Rac, a Rho GTPase recently shown to be required for C. trachomatis entry in mammalian cells, inhibits C. trachomatis infection in S2 cells. We conclude that Drosophila S2 cells faithfully mimic early events in Chlamydia host cell interactions and provides a bona fide system to systematically dissect host functions important in the pathogenesis of obligate intracellular pathogens.     

Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus

A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.