Evolution of the CD4 family: teleost fish possess two divergent forms of CD4 in addition to lymphocyte activation gene-3

The T cell coreceptor CD4 is a transmembrane glycoprotein belonging to the Ig superfamily and is essential for cell-mediated immunity. Two different genes were identified in rainbow trout that resemble mammalian CD4. One (trout CD4) encodes four extracellular Ig domains reminiscent of mammalian CD4, whereas the other (CD4REL) codes for two Ig domains. Structural motifs within the amino acid sequences suggest that the two Ig domains of CD4REL duplicated to generate the four-domain molecule of CD4 and the related gene, lymphocyte activation gene-3. Here we present evidence that both of these molecules in trout are homologous to mammalian CD4 and that teleosts encode an additional CD4 family member, lymphocyte activation gene-3, which is a marker for activated T cells. The syntenic relationships of similar genes in other teleost and non-fish genomes provide evidence for the likely evolution of CD4-related molecules in vertebrates, with CD4REL likely representing the primordial form in fish. Expression of both CD4 genes is highest in the thymus and spleen, and mRNA expression of these genes is limited to surface IgM- lymphocytes. consistent with a role for T cell functionality. Finally, the intracellular regions of both CD4 and CD4REL possess the canonical CXC motif involved in the interaction of CD4 with p56LCK, implying that similar mechanisms for CD4+ T cell activation are present in all vertebrates. Our results therefore raise new questions about T cell development and functionality in lower vertebrates that cannot be answered by current mammalian models and, thus, is of fundamental importance for understanding the evolution of cell-mediated immunity in gnathosomes.     

Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis

The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K(m) value for Mn(2+) 170-fold. In contrast, Asp(75) is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V(max) decreased 1.4-4-fold. K(m) values for PEP and Mn(2+) increased 3-9- and 1.2-10-fold, respectively. K(m) values for GDP and bicarbonate changed little. 2) For PEP formation, V(max) increased 1.5-2.7-fold. K(m) values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.     

Feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine

Nipah virus (NiV) and Hendra virus (HeV) are paramyxoviruses capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. Case reports from outbreaks and previous challenge experiments have suggested that cats were highly susceptible to NiV infection, responding with a severe respiratory disease and systemic infection. Here we have assessed the cat as a model of experimental NiV infection and use it in the evaluation of a subunit vaccine comprised of soluble G glycoprotein (sG). Two groups of two adult cats each were inoculated subcutaneously with either 500 or 5,000 50% tissue culture infective dose(s) (TCID(50)) of NiV. Animals were monitored closely for disease onset, and extensive analysis was conducted on samples and tissues taken during infection and at necropsy to determine viral load and tissue tropism. All animals developed clinical disease 6 to 9 days postinfection, a finding consistent with previous observations. In a subsequent experiment, two cats were immunized with HeV sG and two were immunized with NiV sG. Homologous serum neutralizing titers were greater than 1:20,000, and heterologous titers were greater than 1:20,000 to 16-fold lower. Immunized animals and two additional naive controls were then challenged subcutaneously with 500 TCID(50) of NiV. Naive animals developed clinical disease 6 to 13 days postinfection, whereas none of the immunized animals showed any sign of disease. TaqMan PCR analysis of samples from naive animals revealed considerable levels of NiV genome in a wide range of tissues, whereas the genome was evident in only two immunized cats in only four samples and well below the limit of accurate detection. These results indicate that the cat provides a consistent model for acute NiV infection and associated pathogenesis and an effective subunit vaccine strategy appears achievable.     

Cutting edge: CD4 is the receptor for the tick saliva immunosuppressor, Salp15

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.     

A modular design for the clathrin- and actin-mediated endocytosis machinery

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.     

Orientation within the exosporium and structural stability of the collagen-like glycoprotein BclA of Bacillus anthracis

Bacillus anthracis spores, which cause anthrax, are enclosed by an exosporium consisting of a basal layer and an external hair-like nap. The filaments of the nap are composed of BclA, a glycoprotein containing distinct N-terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region. In this study, we used immunogold electron microscopy to show that the CTD of BclA forms the distal end of each filament of the hair-like nap, indicating that the NTD is attached to the basal layer. Ten randomly chosen anti-BclA monoclonal antibodies, raised against spores or exosporium, reacted with the CTD, consistent with its exterior location. We showed that recombinant BclA (rBclA), encoded by the B. anthracis Sterne strain and synthesized in Escherichia coli, forms a collagen-like triple helix as judged by collagenase sensitivity and circular dichroism spectroscopy. In contrast, native BclA in spores was resistant to collagenase digestion. Thermal denaturation studies showed that the collagen-like region of rBclA exhibited a melting temperature (T(m)) of 37 degrees C, like mammalian collagen. However, rBclA trimers exhibited T(m) values of 84 degrees C and 95 degrees C in buffer with and without sodium dodecyl sulfate, respectively. CTD trimers exhibited the same T(m) values, indicating that the high temperature and detergent resistances of rBclA were due to strong CTD interactions. We observed that CTD trimers are resistant to many proteases and readily form large crystalline sheets. Structural data indicate that the CTD is composed of multiple beta strands. Taken together, our results suggest that BclA and particularly its CTD form a rugged shield around the spore.     

Critical determinants of the interactions of capsule-expressing Neisseria meningitidis with host cells: the role of receptor density in increased cellular targeting via the outer membrane Opa proteins

Neisseria meningitidis capsule is an important virulence determinant required for survival in the blood but is reportedly involved in inhibiting cellular interactions mediated by meningococcal outer membrane adhesins. However, evidence from our previous studies suggested that target receptor density on host cells may determine whether or not capsulate bacteria can adhere via outer membrane proteins such as Opa. To confirm this and evaluate the impact of capsulation on bacterial interactions, we used Opa(+) and Opa(-) derivatives of capsulate and acapsulate meningococcal isolates and transfected cell lines expressing CEACAM1, a receptor targeted by Opa proteins. To assess the extent and rate of cell association, subpopulations of stably transfected Chinese hamster ovary cells with different receptor levels were derived. A quantitative correlation of CEACAM1 levels and Opa-dependent binding of both capsulate and acapsulate bacteria was demonstrated, which was accelerated at high receptor densities. However, it appears that invasion by Opa(+) capsulate bacteria only occurs when a threshold level of CEACAM density has been reached. Target cells expressing high levels of CEACAM1 (MFI c. 400) bound threefold more, but internalized 20-fold more Opa(+) capsulate bacteria than those with intermediate expression (MFI c. 100). No overall selection of acapsulate phenotype was observed in the internalized population. These observations confirm that capsule may not be an adequate barrier for cellular interactions and demonstrate the role of a host factor that may determine capsulate bacterial invasion potential. Upregulation of CEACAMs, which can occur in response to inflammatory cytokines, could lead to translocation of a small number of fully capsulate bacteria across mucosal epithelium into the bloodstream sufficient to cause a rapid onset of disseminated disease. Thus the data also suggest a novel rationale for the epidemiological observations that individuals with prior infectious/inflammatory conditions carry a high risk of invasive meningococcal disease.