Expression of HIV receptors, alternate receptors and co-receptors on tonsillar epithelium: implications for HIV binding and primary oral infection.

BACKGROUND: Primary HIV infection can develop from exposure to HIV in the oral cavity. In previous studies, we have documented rapid and extensive binding of HIV virions in seminal plasma to intact mucosal surfaces of the palatine tonsil and also found that virions readily penetrated beneath the tissue surfaces. As one approach to understand the molecular interactions that support HIV virion binding to human mucosal surfaces, we have examined the distribution of the primary HIV receptor CD4, the alternate HIV receptors heparan sulfate proteoglycan (HS) and galactosyl ceramide (GalCer) and the co-receptors CXCR4 and CCR5 in palatine tonsil. RESULTS: Only HS was widely expressed on the surface of stratified squamous epithelium. In contrast, HS, GalCer, CXCR4 and CCR5 were all expressed on the reticulated epithelium lining the tonsillar crypts. We have observed extensive variability, both across tissue sections from any tonsil and between tonsils, in the distribution of epithelial cells expressing either CXCR4 or CCR5 in the basal and suprabasal layers of stratified epithelium. The general expression patterns of CXCR4, CCR5 and HS were similar in palatine tonsil from children and adults (age range 3-20). We have also noted the presence of small clusters of lymphocytes, including CD4+ T cells within stratified epithelium and located precisely at the mucosal surfaces. CD4+ T cells in these locations would be immediately accessible to HIV virions. CONCLUSION: In total, the likelihood of oral HIV transmission will be determined by macro and micro tissue architecture, cell surface expression patterns of key molecules that may bind HIV and the specific properties of the infectious inoculum.

Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus.

Type IV secretion is used by pathogenic microorganisms to transfer effector macromolecules to eukaryotic target cells. The VirB/D4 apparatus of Agrobacterium tumefaciens transfers DNA and proteins to plant cells. We postulated that the cell pole is the site of assembly of the A. tumefaciens type IV apparatus. Using immunofluorescence microscopy, we now demonstrate that 10 of the VirB proteins localized primarily to one cell pole and a macromolecular VirB complex is assembled at the pole. Neither the assembly of the complex nor polar localization of a VirB protein requires ATP utilization by the VirB ATPases. The requirement of other VirB proteins for the polar localization of at least six VirB proteins indicates an essential role of protein-protein interaction in polar targeting. Four proteins (VirB3, VirB4, VirB8, and VirB11) could target themselves to a cell pole independent of a VirB protein. We provide evidence that VirB6-VirB10 are the structural components of the type IV apparatus. Using strains that express defined subsets of the virB genes, we demonstrate that VirB7-VirB10 are the minimum components sufficient for the assembly of a polar VirB complex. VirB6 associates with this complex to form the type IV secretion apparatus. VirB8 functions as the assembly factor and targets the apparatus to the cell pole.

The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole.

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells. To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy. In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes. Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6. We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization. Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm. Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein. The mutations abolished the DNA transfer function of the protein as well. The C-terminus of VirB6, in silico, can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole. We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole. To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments. Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus.

Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4.

Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants. VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore. To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes. In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence microscopy, cells probed with anti-VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location of VirD4 was not dependent on T-DNA processing, the formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large cytoplasmic region contains a nucleotide-binding domain. To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer. A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer. Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide-binding domains are required for polar localization of VirD4. The periplasmic domain mutant VirD4Delta36-61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.