Jerry M. Buysse

Large-Scale Identification of Genes Required for Full Virulence of Staphylococcus aureus

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Mechanism of Action and Pharmacology of Patiromer, a Nonabsorbed Cross-Linked Polymer That Lowers Serum Potassium Concentration in Patients With Hyperkalemia

Hyperkalemia is a potentially life-threatening condition, and patients who have chronic kidney disease, who are diabetic, or who are taking renin–angiotensin–aldosterone system inhibitors are at increased risk. Therapeutic options for hyperkalemia tend to have limited effectiveness and can be associated with serious side effects. Colonic potassium secretion can increase to compensate when urinary potassium excretion decreases in patients with renal impairment, but this adaptation is insufficient and hyperkalemia still results. Patiromer is a novel, spherical, nonabsorbed polymer designed to bind and remove potassium, primarily in the colon, thereby decreasing serum potassium in patients with hyperkalemia. Patiromer has been found to decrease serum potassium in patients with hyperkalemia having chronic kidney disease who were on renin–angiotensin–aldosterone system inhibitors. Results of nonclinical studies and an early phase clinical study are reported here. Two studies with radiolabeled drug, one in rats and the other in dogs, confirmed that patiromer was not absorbed into the systemic circulation. Results of an in vitro study showed that patiromer was able to bind 8.5 to 8.8 mEq of potassium per gram of polymer at a pH similar to that found in the colon and had a much higher potassium-binding capacity compared with other resins, including polystyrene sulfonate. In a study in hyperkalemic rats, a decrease in serum potassium was observed via an increase in fecal potassium excretion. In a clinical study in healthy adult volunteers, a significant increase in fecal potassium excretion and a significant decrease in urinary potassium excretion were observed. Overall, patiromer is a high-capacity potassium binder, and the chemical and physical characteristics of patiromer may lead to good clinical efficacy, tolerability, and patient acceptance.

A targeted mutagenesis system for Actinobacillus pleuropneumoniae.

We describe methods for the mutagenesis of cloned Actinobacillus pleuropneumoniae (Ap) genes and for the construction of Ap mutants by allelic exchange. We used these methods to construct isogenic mutants of Ap which no longer synthesized a 48-kDa outer membrane protein (AopA). The native aopA locus was replaced with a mutated locus that had been inactivated by insertion of a gene (KmR) encoding kanamycin resistance from Tn903. The inactivated aopA locus was cloned into a conjugative, R6K-derived, lambda pir-dependent suicide vector and introduced into Ap using a filtermating technique. Southern and Western blot analyses indicated that the wild-type locus was replaced by the mutated locus through either single- or double-crossover events, and that AopA was no longer produced by either type of mutant. These methods were used successfully to construct AopA- mutants in Ap serotypes 1 and 5. These methods should be generally useful in constructing mutant loci which can be used to analyze the roles of various Ap genes in the pathogenesis of contagious pleuropneumonia in swine.

Molecular Basis for Fungal Selectivity of Novel Antimitotic Compounds

ABSTRACT Compounds that selectively disrupt fungal mitosis have proven to be effective in controlling agricultural pests, but no specific mitotic inhibitor is available for the treatment of systemic mycoses in mammalian hosts. In an effort to identify novel mitotic inhibitors, we used a cell-based screening strategy that exploited the hypersensitivity of a yeast α-tubulin mutant strain to growth inhibition by antimitotic agents. The compounds identified inhibited yeast nuclear division and included one structural class of compounds shown to be fungus specific. MC-305,904 and structural analogs inhibited fungal cell mitosis and inhibited the in vitro polymerization of fungal tubulin but did not block mammalian cell microtubule function or mammalian tubulin polymerization. Extensive analysis of yeast mutations that specifically alter sensitivity to MC-305,904 structural analogs suggested that compounds in the series bind to a site on fungal β-tubulin near amino acid 198. Features of the proposed binding site explain the observed fungal tubulin specificity of the series and are consistent with structure-activity relationships among a library of related compounds.

DNA Sequence Homology Among ipa Genes of Shigella spp. and Enteroinvasive Escherichia coli

Bacillary dysentery, caused by Shigellaand enteroinvasive Escherichia coli (EIEC), is endemic throughout the world, and it accounts for 10 to 20% of acute diarrheal disease worldwide (11). Clinical symptoms of the disease range from a mild diarrhea to a severe dysenteric syndrome characterized by fever, abdominal cramps, frequent defecation of blood and mucus, and, in extreme cases, hypotensive shock. In developing countries inadequate sanitation and malnutrition contribute to the prevalence of the disease, which can be caused by ingesting as few as 10 to 100 bacteria (10). Shigellosis is restricted to primate hosts and all serotypes of Shigella, as well as nine serotypes of EIEC, can cause the disease. Understanding the mechanism of dysentery pathogenesis should facilitate the construction of rapid diagnostic probes for Shigella and EIEC and will help to identify genes and gene products that may be essential in devising an effective dysentery vaccine.