Structural features of a zinc binding site in the superantigen strepococcal pyrogenic exotoxin A (SpeA1): implications for MHC class II recognition.

Streptococcal pyrogenic exotoxin A (SpeA) is produced by Streptococcus pyogenes, and has been associated with severe infections such as scarlet fever and Streptococcal Toxic Shock Syndrome (STSS). In this study, the crystal structure of SpeA1 (the product of speA allele 1) in the presence of 2.5 mM zinc was determined at 2.8 A resolution. The protein crystallizes in the orthorhombic space group P2(1)2(1)2, with four molecules in the crystallographic asymmetric unit. The final structure has a crystallographic R-factor of 21.4% for 7,031 protein atoms, 143 water molecules, and 4 zinc atoms (one zinc atom per molecule). Four protein ligands-Glu 33, Asp 77, His 106, and His 110-form a zinc binding site that is similar to the one observed in a related superantigen, staphylococcoal enterotoxin C2. Mutant toxin forms substituting Ala for each of the zinc binding residues were generated. The affinity of these mutants for zinc ion confirms the composition of this metal binding site. The implications of zinc binding to SpeA1 for MHC class II recognition are explored using a molecular modeling approach. The results indicate that, despite their common overall architecture, superantigens appear to have multiple ways of complex formation with MHC class II molecules.

Recombinant Staphylococcus aureus exfoliative toxins are not bacterial superantigens.

Staphylococcal scalded-skin syndrome is an exfoliative dermatitis characterized by the separation of the epidermis at the stratum granulosum. This disruption is mediated by one of two Staphylococcus aureus exotoxins, exfoliative toxins A and B (ETA and ETB). Both ETA and ETB have been reported to be bacterial superantigens. A controversy exists, however, as other data indicate that these exotoxins are not superantigens. Here we demonstrate that recombinant exfoliative toxins produced in Escherichia coli do not act as T-cell mitogens and thus are not bacterial superantigens. These data fit the clinical profile of the disease, which is not associated with the classic symptoms of a superantigen-mediated syndrome.

Structural basis for the recognition of superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules and T-cell receptors.

Streptococcal pyrogenic exotoxin A (SpeA) is a superantigen produced by Streptococcus pyogenes and is associated with severe infections characterized by rash, hypotension, multiorgan failure and a high mortality rate. In this study, an allelic form of this toxin, SpeA1, was crystallized with four molecules in the crystallographic asymmetric unit and its crystal structure was determined at 2.6 A resolution. The crystallographic R-factor was 19.4% (33 497 reflections) for 7031 protein atoms and 88 water molecules. The overall structure of SpeA1 is considerably similar to that of other prototype microbial superantigens, either of staphylococcal or streptococcal origin, but has greatest similarity to staphylococcal enterotoxin C (SEC). Based on structural and mutagenesis data, we have mapped several important residues on the toxin molecule, which are involved in the recognition of major histocompatibility complex (MHC) class II molecules and T-cell receptors. Also, the toxin appears to possess a potential zinc-binding site which may have implications in binding to particular MHC class II molecules. Finally, we propose models for SpeA1-MHC class II and SpeA1-T-cell receptor association and the relevance of this phenomenon to the superantigenic action of this toxin is considered.

Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes.

Synthesis of urease by Klebsiella species is known to be induced when the nitrogen source of the growth medium is limiting, suggesting that urease gene expression is controlled by the nitrogen regulatory (ntr) system. This study showed that K. pneumoniae with mutations in either ntrA or ntrC, two integral components of the ntr system, were phenotypically urease-negative. These mutants could be complemented back to a urease positive phenotype with recombinant plasmids encoding the corresponding ntr gene. A series of ure-lacZYA transcriptional fusions, in conjunction with primer extension analysis, identified a DNA region that encoded a nitrogen-regulated promoter. This promoter region controlled transcription of ureD, the first gene in the Klebsiella pneumoniae urease gene cluster, and ureA, a gene that resides immediately downstream of ureD. A high level of transcription from the ureD promoter required NAC, a recently characterized member of the nitrogen regulatory cascade. NAC is a Lys R-like transcriptional regulator that can act at sigma 70 promoters; expression from nac itself is dependent upon NTRA. Therefore, expression of K. pneumoniae urease was dependent upon the nitrogen regulatory cascade, and transcription of at least two urease genes was from a promoter that was positively regulated by NAC.

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.