Evasion of Toll-like receptor 2 activation by staphylococcal superantigen-like protein 3.

Toll-like receptors (TLRs) are crucial for our host defense against microbial infections. TLR2 is especially important to fight bacterial infections, as it specifically recognizes bacterial lipoproteins of both Gram-positive and Gram-negative origin. Present on a variety of immune cells, TLR2 is critical for host protection against several bacterial infections, including those caused by Staphylococcus aureus. This major human pathogen causes increasing health care problems due to its increased resistance to antibiotics. S. aureus secretes a wide variety of proteins that inhibit innate immune responses. Recently, several staphylococcal superantigen-like proteins (SSLs) have been described to mediate immune evasive properties. Here, we describe that SSL3 specifically binds and inhibits TLR2 activation on human and murine neutrophils and monocytes. Through binding of the extracellular TLR2 domain, SSL3 inhibits IL-8 production by HEK cells expressing TLR1/2 and TLR2/6 dimers, stimulated with their specific ligands. The SSL3-TLR2 interaction is partially glycan dependent as binding of SSL3 to TLR2 is affected upon removal of sialic acid residues. Moreover, the SSL3(R308A) mutant lacking glycan-binding properties shows lower TLR2 inhibition. An SSL3 mutant, lacking the N-terminal 126 amino acids, still retains full TLR2 inhibiting activity. Of other SSLs tested, only SSL4, which shares the highest homology with SSL3, blocks TLR2 activation. SSL3 is the first-described bacterial protein that blocks TLR2 activation through direct extracellular interaction with the receptor. This unique function of SSL3 adds to the arsenal of immune evasive molecules that S. aureus can employ to subvert both innate and adaptive immunity.

Roles of NapF, NapG and NapH, subunits of the Escherichia coli periplasmic nitrate reductase, in ubiquinol oxidation.

The nap operon of Escherichia coli K-12, encoding a periplasmic nitrate reductase (Nap), encodes seven proteins. The catalytic complex in the periplasm, NapA-NapB, is assumed to receive electrons from the quinol pool via the membrane-bound cytochrome NapC. Like NapA, B and C, a fourth polypeptide, NapD, is also essential for Nap activity. However, none of the remaining three polypeptides, NapF, G and H, which are predicted to encode non-haem, iron-sulphur proteins, are essential for Nap activity, and their function is currently unknown. The relative rates of growth and electron transfer from physiological substrates to Nap have been investigated using strains defective in the two membrane-bound nitrate reductases, and also defective in either ubiquinone or menaquinone biosynthesis. The data reveal that Nap is coupled more effectively to menaquinol oxidation than to ubiquinol oxidation. Conversely, parallel experiments with a second set of mutants revealed that nitrate reductase A couples more effectively with ubiquinol than with menaquinol. Three further sets of strains were constructed with combinations of in frame deletions of ubiCA, menBC, napC, napF and napGH genes. NapF, NapG and NapH were shown to play no role in electron transfer from menaquinol to the NapAB complex but, in the Ubi+Men- background, deletion of napF, napGH or napFGH all resulted in total loss of nitrate-dependent growth. Electron transfer from ubiquinol to NapAB was totally dependent upon NapGH, but not on NapF. NapC was essential for electron transfer from both ubiquinol and menaquinol to NapAB. The results clearly established that NapG and H, but not NapF, are essential for electron transfer from ubiquinol to NapAB. The decreased yield of biomass resulting from loss of NapF in a Ubi+Men+ strain implicates NapF in an energy- conserving role coupled to the oxidation of ubiquinol. We propose that NapG and H form an energy- conserving quinol dehydrogenase functioning as either components of a proton pump or in a Q cycle, as electrons are transferred from ubiquinol to NapC.

Regulation of maltose transport in Saccharomyces cerevisiae.

Solute transport in Saccharomyces cerevisiae can be regulated through mechanisms such as trans-inhibition and/or catabolite inactivation by nitrogen or carbon sources. Studies in hybrid membranes of S. cerevisiae suggested that the maltose transport system Mal61p is fully reversible and capable of catalyzing both influx and efflux transport. This conclusion has now been confirmed by studies in a S. cerevisiae strain lacking the maltase enzyme. Whole cells of this strain, wherein the orientation of the maltose transporter is fully preserved, catalyze fully reversible maltose transport. Catabolite inactivation of the maltose transporter Mal61p was studied in the presence and absence of maltose metabolism and by the use of different glucose analogues. Catabolite inactivation of Mal61p could be triggered by maltose, provided the sugar was metabolized, and the rate of inactivation correlated with the rate of maltose influx. We also show that 2-deoxyglucose, unlike 6-deoxyglucose, can trigger catabolite inactivation of the maltose transporter. This suggests a role for early glycolytic intermediates in catabolite inactivation of the Mal61 protein. However, there was no correlation between intracellular glucose-6-phosphate or ATP levels and the rate of catabolite inactivation of Mal61p. On the basis of their identification in cell extracts, we speculate that (dideoxy)-trehalose and/or (deoxy)-trehalose-6-phosphate trigger catabolite inactivation of the maltose transporter.

Catabolite inactivation of wild-type and mutant maltose transport proteins in Saccharomyces cerevisiae.

The maltose transporter of Saccharomyces cerevisiae is subject to rapid, irreversible inactivation in the presence of glucose. Loss of transport function was paralleled by a decrease in amount of transporter protein and most likely involves endocytosis and degradation of the protein in the vacuole. This (catabolite) inactivation of Mal61p was triggered not only by glucose but also by 2-deoxy-D-glucose, which cannot be metabolized beyond 2-deoxy-D-glucose phosphate. The signal that targets membrane proteins specifically for catabolite inactivation is unknown. To investigate whether or not specific modification of Mal61p triggers the inactivation, putative protein kinase A and C phosphorylation sites were removed, and the transport activities and levels of the mutant proteins upon addition of glucose were followed in time. Three Mal61p mutants, i.e. S295A, T363A, and S487A, exhibited significantly reduced rates of inactivation in the presence of glucose. Likewise, in wild-type Mal61p the rate of inactivation and degradation of the protein paralleled each other in the case of T363A. On the contrary, for the S295A and S487A mutants the rates of protein degradation were slowed down more profoundly than was the loss of transport activity. These observations indicate that (i) some form of modification (e.g. phosphorylation) of the protein precedes breakdown, (ii) the modification inactivates Mal61p, and (iii) the inactivation of Mal61p is not necessarily followed by proteolytic degradation.

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis.

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.