A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxyterminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.Mycoplasmas are wall-less bacteria that represent the smallest organisms in nature capable of self-replication (28, 30). Many species are pathogens causing diseases in humans and animals, which are often chronic in nature and display major elements of immunopathology (36,43). Most of these pathogens adhere tenaciously to the epithelial linings of the respiratory or urogenital tract, rarely invading tissues. Adhesion of mycoplasmas to host cells is a prerequisite for successful colonization and ensuing pathogenesis (29,30). An important group of mycoplasmas, including the human pathogens Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma pirum, possess a flask cell shape with a protruding tip or bleb organelle (28-30). This organelle has been shown to mediate the intimate interaction of M. pneumoniae with the host's ciliated respiratory epithelium (a process known as cytadherence) (15,29). Extensive analysis of the cytadherence process in this species has demonstrated that the process is multifactorial, involving the coordinate action of primary adhesin molecules (P1 and P30) and several high-molecular-weight accessory membrane proteins that act in concert with cytoskeletal elements to facilitate the lateral movement and concentration of the adhesin molecules at the attachment organelle (4,9,15,16,29,32,38,39).Mycoplasma ga...

Removal of glutamate from the synaptic cleft by (Na ؉ ؉ K ؉ )-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30 -100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.Glutamate transporters in the brain ensure that the synaptic levels of the transmitter are kept below the neurotoxic levels (1-6). These transporters, located in the plasma membrane of nerve and glial cells, play an important role in limiting the duration of synaptic excitation (7-10). The uptake process is electrogenic (11-13), involving co-transport of three sodium ions, a proton, and a glutamate molecule and countertransport of a potassium ion (14 -16). In addition to the coupled flux, glutamate transporters mediate a thermodynamically uncoupled chloride flux activated by two of the molecules they transport, sodium and glutamate (17, 18).The five known eukaryotic glutamate transporters, GLT-1 (19), GLAST-1 (20), EAAC-1 (21), , and EAAT-5 (22), have an overall amino acid identity of ϳ50%. The homology is significantly higher in the carboxyl-terminal half of the transporters. Topology studies suggest that this region of the protein has an intriguing arrangement containing two oppositely oriented reentrant loops, the two transmembrane domains (TMs) 1 7 and 8, and an outward-facing hydrophobic region (Refs. 23-25, and see Fig. 1). Some of the features of the membrane topology remain under debate (26).Several amino acid residues critical for the function of glutamate transporters are located in the carboxyl-terminal half. Two adjacent amino acid residues of GLT-1, Tyr-403 and Glu-404, are located in TM7 (Fig. 1) and are conserved in all other glutamate transporters. Both were implicated in the binding of potassium ions (16,27) and appear to be close to one of the sodium binding sites (27). A conserved arginine residue, Arg...

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.

Glutamate transporters are essential for terminating synaptic transmission. Glutamate is translocated together with three sodium ions. In the neuronal glutamate transporter EAAC1, lithium can replace sodium. To address the question of whether the coupling ion interacts with the 'driven' substrate during co-transport, the kinetic parameters of transport of the three substrates, L-glutamate and D-and L-aspartate by EAAC-1 in sodium-and lithium-containing media were compared. The major effect of the substitution of sodium by lithium was on K m . In the presence of sodium, the values for K m and I max of these substrates were similar. In the presence of lithium, the K m for L-aspartate was increased around 13-fold. Remarkably, the corresponding increase for L-glutamate and D-aspartate was much larger, around 130-fold. In marked contrast, the K i values for a non-transportable substrate analogue were similar in the presence of either sodium or lithium. The preference for L-aspartate in the presence of lithium was also observed when electrogenic transport of radioactive substrates was monitored in EAAC1-containing proteoliposomes. Our results indicate that, subsequent to substrate binding, the co-transported solutes interact functionally in the binding pocket of the transporter.

No abstract