RNA interference in mosquito: understanding immune responses, double-stranded RNA delivery systems and potential applications in vector control.

RNA interference (RNAi) refers to the process of post-transcriptional silencing of cellular mRNA by the application of double-stranded RNA (dsRNA). RNAi strategies have been widely employed to regulate gene expression in plants and animals including insects. With the availability of the full genome sequences of major vector mosquitoes, RNAi has been increasingly used to conduct genetic studies of human pathogens in mosquito vectors and to study the evolution of insecticide resistance in mosquitoes. This review summarizes the recent progress in our understanding of mosquito-pathogen interactions using RNAi and various methods of dsRNA delivery in mosquitoes at different stages. We also discuss potential applications of this technology to develop novel tools for vector control.

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway.

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.

Molecular characterization of guinea - pig (Cavia porcellus) CD8alpha and CD8beta cDNA.

CD8 is expressed on cytotoxic T cells (CTL) and functions as a coreceptor for recognition of major histocompatibility complex (MHC) class I peptide complexes by the T-cell receptor (TCR). The CD8 molecule consists of two subunits (alpha and beta) and exists either as a heterodimer (alphabeta) or a homodimer (alphaalpha). We report the cloning of full-length cDNAs of guinea pig CD8alpha and CD8beta. The deduced amino acid sequence of CD8alpha and CD8beta reveals characteristic structural features including a signal peptide, an immunoglobulin (Ig) variable-like region, hinge region, transmembrane, and cytoplasmic domains. In addition to the full-length cDNA, a splice variant of CD8beta cDNA was observed, suggesting splicing events as reported for human CD8beta. The sequence homology of guinea pig CD8 indicates greater homology to human, canine, and feline counterparts than to rodent CD8. As the guinea pig serves as an ideal non-primate animal model to several human infectious diseases, such as syphilis, tuberculosis, and chlamydial genital and ocular infection, the CD8 sequence information provides a necessary molecular tool for studying the cell-mediated immune response.

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.

Associations and interactions between bare lymphocyte syndrome factors.

The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes.

Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, an MHC class II immunodeficiency.

The bare lymphocyte syndrome (BLS) is characterized by the absence of MHC class II transcription and humoral- and cellular-mediated immune responses to foreign antigens. Three of the four BLS genetic complementation groups have defects in the activity of the MHC class II transcription factor RFX. We have purified the RFX complex and sequenced its three subunits. The sequence of the smallest subunit describes a novel gene, termed RFX-B. RFX-B complements the predominant BLS complementation group (group B) and was found to be mutant in cell lines from this BLS group. The protein has no known DNA-binding domain but does contain three ankyrin repeats that are likely to be important in protein-protein interactions.