Dynamic Pneumococcal Genetic Adaptations Support Bacterial Growth and Inflammation during Coinfection with Influenza.

Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to postinfluenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza virus-infected hosts. The majority of the 32 genes identified are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated mutants with single-gene deletions (SGD) of five of the genes identified, SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB), to investigate their effects on in vivo fitness, disease severity, and host immune responses. The growth of the SGD mutants was slightly attenuated in vitro and in vivo, but each still grew to high titers in the lungs of mock- and influenza virus-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed altered neutrophil distribution with reduced degeneration in the lungs of influenza virus-SGD mutant-coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.

Crystallization and preliminary X-ray analysis of Der f 2, a potent allergen derived from the house dust mite (Dermatophagoides farinae).

Although a number of allergens have been identified and isolated, the underlying molecular basis for the potent immune response is poorly understood. House dust mites (Dermatophagoides sp.) are ubiquitous contributors to atopy in developed countries. The rhinitis, dermatitis and asthma associated with allergic reactions to these arthropods are frequently caused by relatively small (125-129 amino acids) mite proteins of unknown biological function. Der f 2, a major allergen from the mite D. farinae, has been recombinantly expressed, characterized and crystallized. The crystals belong to the tetragonal space group I4(1)22, with unit-cell parameters a = b = 95.2, c = 103.3 A. An essentially complete (97.2%) data set has been collected to 2.4 A at a synchrotron source. Attempts to solve the crystal structure of Der f 2 by molecular replacement using the NMR coordinates for either Der f 2 or Der p 2 (the homologous protein from D. pteronyssinus) failed, but preliminary searches using the crystalline Der p 2 atomic coordinates appear to be promising.

Crystallization and preliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher's disease.

Acid beta-glucocerebrosidase (N-acylsphingosyl-1-O-beta-D-glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme (Genzyme Corporation, Cambridge, MA, USA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is used in the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. Here, the crystallization and the initial diffraction analysis of Cerezyme are reported. The crystals are C-centered orthorhombic, with unit-cell parameters a = 285.0, b = 110.2, c = 91.7 A. A 99.9% complete data set has been collected to 2.75 A with an R(sym) of 8.8%.