Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

Identification of novel staphylococcal virulence genes by in vivo expression technology.

We have applied in vivo expression technology (IVET) to the study of staphylococcal virulence. Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model. Of these, only six were known previously; 11 others have homology to known non-staphylococcal genes. The known staphylococcal genes include agrA, part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses. We constructed 11 strains containing mutations in previously unknown ivi genes. Of these strains, seven were significantly attenuated in virulence compared with the wild-type parent. The mutagenized ivi genes may encode novel staphylococcal virulence factors.

Clinical and neuroradiographic manifestations of eastern equine encephalitis.

BACKGROUND: Eastern equine encephalitis occurs principally along the east and Gulf coasts of the United States. Recognition of the neuroradiographic manifestations of eastern equine encephalitis could hasten the diagnosis of the illness and speed the response to index cases. METHODS: We reviewed all cases of eastern equine encephalitis reported in the United States between 1988 and 1994. The records of 36 patients were studied, along with 57 computed tomographic (CT) scans and 23 magnetic resonance imaging (MRI) scans from 33 patients. RESULTS: The mortality rate was 36 percent, and 35 percent of the survivors were moderately or severely disabled. Neuroradiographic abnormalities were common and best visualized by MRI. Among the patients for whom MRI scans were available, the results were abnormal for all eight comatose patients as well as for all three noncomatose patients who subsequently became comatose. The CT results were abnormal in 21 of 32 patients with readable scans. The abnormal findings included focal lesions in the basal ganglia (found in 71 percent of patients on MRI, and in 56 percent on CT), thalami (found in 71 percent on MRI and in 25 percent on CT), and brain stem (found in 43 percent on MRI and in 9 percent on CT). Cortical lesions, meningeal enhancement, and periventricular white-matter changes were less common. The presence of large radiographic lesions did not predict a poor outcome, but either high cerebrospinal fluid white-cell counts or severe hyponatremia did. CONCLUSIONS: Eastern equine encephalitis produces focal radiographic signs. The characteristic early involvement of the basal ganglia and thalami distinguish this illness from herpes simplex encephalitis. MRI is a sensitive technique to identify the characteristic early radiographic manifestations of this viral encephalitis.

Sequence of the toxic shock syndrome toxin gene (tstH) borne by strains of Staphylococcus aureus isolated from patients with Kawasaki syndrome.

To explore whether a novel staphylococcal clone or structural variant of toxic shock syndrome toxin 1 is associated with Kawasaki syndrome, six toxigenic strains of Staphylococcus aureus from Kawasaki syndrome patients were studied. The strains were divisible into two groups based on phenotypic and genotypic characteristics and are therefore unequivocally not clonal. Portions of the tstH genes of each strain were sequenced. Three were sequenced in their entirety, while the remainder were sequenced from codon 66 to codon 137 of the mature protein only. Two of the former group differed slightly in the sequences of their signal peptides relative to the sequence published for the tstH signal peptide. Those differences did not affect toxin processing or secretion. The sequenced portions of the regions encoding mature toxic shock syndrome toxin 1 were identical in all six strains and corresponded exactly to the published sequence of tstH. No evidence was found for the existence of a structural variant of tstH uniquely associated with Kawasaki syndrome.

Pulmonary zygomycosis after allogeneic bone marrow transplantation.

The Zygomycetes are uncommon human pathogens. They cause illness in some patients who are immunosuppressed and can present as any of several syndromes, including rhinocerebral, pulmonary, gastrointestinal, or cutaneous disease, or as disseminated infection. Pulmonary zygomycosis can occur in any of several patterns and can mimic more common pneumonic processes. This mimicry, as well as the rarity of the disease, may delay diagnosis. We present a case of pulmonary zygomycosis that occurred in a patient who was a bone marrow transplant recipient. The case illustrates several of the common features of the disease in this patient group.

Mutations affecting the activity of toxic shock syndrome toxin-1.

Toxic shock syndrome toxin-1 (TSST-1), the potent staphylococcal exoprotein linked to most cases of the toxic shock syndrome, is a V beta-restricted T-cell mitogen (a so-called "superantigen"). TSST-ovine (TSST-O) is a natural variant of TSST-1, and is produced by certain ovine mastitis-associated strains of Staphylococcus aureus. Compared to TSST-1, TSST-O is only weakly mitogenic for leporine or murine splenocytes. It differs from TSST-1 at 7 amino acid residues over its 194 amino acid length. Terminus shuffling between the two proteins has suggested that their C-terminal differences (T69, Y80, E132, and I140 in TSST-1; 169, W80, K132, and T140 in TSST-O) are in part responsible for their discrepant mitogenic properties. In order to explore further the functional consequences of altering TSST-1 at residues 132 and 140, we engineered point mutants of TSST-1 at those positions. The mutant proteins were purified to homogeneity from culture supernants of a nontoxigenic strain of S. aureus using a combination of ultrafiltration, liquid-phase isoelectric focusing, and ion-exchange chromatography. The mutants retained global structural integrity as evidenced by circular dichroism spectroscopy, their preserved resistance to trypsin digestion, and their preserved binding to a neutralizing murine monoclonal antibody. The mutants were then tested for mitogenicity for human T-cells: The mutant I140T was approximately as active as wild-type TSST-1, while the mutant E132D was about 10-fold attenuated. On the other hand, the mutants E132A or E132K were each at least 1000-fold attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Eastern equine encephalitis presenting as focal neuroradiographic abnormalities: case report and review.

We describe a well-documented case of Eastern equine encephalitis (EEE) in a patient who presented with fever and altered mental status and who had focal cranial lesions that were evident on both computed tomography (CT) and magnetic resonance imaging. Only 15 CT scans performed for patients with EEE have been described previously; findings on most were normal or revealed diffuse cerebral edema. It is not widely appreciated that EEE can manifest as discrete lesions on CT. We review the radiographic experience with EEE and interpret it in light of certain clinical and pathological features of the disease.

Intracellular expression of toxic shock syndrome toxin 1 in Saccharomyces cerevisiae.

In order to search for an occult cytotoxic enzymatic activity of the toxic shock syndrome toxin 1 (TSST-1), we placed the gene encoding TSST-1 (tstH) under the control of an inducible promoter in the eukaryotic yeast Saccharomyces cerevisiae. Under similar circumstances, the known bacterial enzymatic cytotoxins Shiga-like toxin and diphtheria toxin are both highly lethal to the yeast host. Although full-length stable TSST-1 was demonstrated within the yeast cells and although it retained mitogenicity for human T cells, it had no apparent effect on the yeast cells' growth kinetics or on their gross morphology. Retrieval and sequencing of the toxin gene revealed the wild-type sequence throughout, thus demonstrating that the apparent lack of toxicity for the yeast cells was not due to a serendipitous attenuating mutation within the coding region of the toxin gene. Similar results obtained after a second transformation of the same strain and after transformation of an unrelated strain demonstrate that neither chance permissive host mutation nor intrinsic host resistance was likely to have obscured an existing cytotoxic property of TSST-1. We conclude that TSST-1 probably does not possess a discrete enzymatic property cytotoxic for eukaryotic cells.

The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain.

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-in-activating proteins (RIPs), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y114F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A1 form of Slt-IA (wild-type-delta). The same general relationships held: relative to wild type-delta, Y114F-delta was attenuated about 7-fold, and Y114S-delta about 300-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations affecting the activity of the Shiga-like toxin I A-chain.

Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catalytically depurinating adenosine 4324 in 28S rRNA. Although the primary structure of the enzymatic portion of the molecule (Slt-IA) is known to contain regions of significant homology to the ricin A chain (RTA), and although certain residues have been implicated in catalysis, the crystal structure of Slt-IA has not been solved nor has the geometry of its active site been well defined. In order to derive a more complete understanding of the nature of the Slt-IA active site, we placed the slt-IA gene under control of an inducible promoter in Saccharomyces cerevisiae. Induction of the cloned element was lethal to the host. This lethality was the basis for selection of an attenuated mutant of Slt-IA changed at tyrosine 77, a locus not previously linked to the active site. As well, it permitted evaluation of the toxicity of a number of mutant Slt-IA cassettes that we constructed in vitro. Putative active-site residues implicated in this fashion and in other studies were mapped to an energy-minimized computer model of Slt-IA that had been generated on the basis of the known crystal structure of RTA. A cleft was identified on one face of the protein in which all implicated residues clustered, irrespective of their distances from one another in the primary structure of the molecule. Many of the chemical features anticipated in the active site of an RNA N-glycosidase are indeed present on the amino acid side chains occupying the cleft.

Fatal disseminated mycobacterial infection following intravesical bacillus Calmette-Guerin.

We describe a fatal case of disseminated mycobacteriosis after intravesical bacillus Calmette-Guerin immunotherapy. We summarize the prior safety record of this therapeutic modality, discuss local and systemic pathophysiological mechanisms by which dissemination might have occurred, and review the reported clinical experience with antituberculous chemotherapy for significant bacillus Calmette-Guerin infection. Finally, we offer suggestions for prophylaxis of certain patients with a history of exposure to intravesical bacillus Calmette-Guerin.

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.