Pneumococcal serine-rich repeat protein (PsrP) is a putative adhesin encoded in the Streptococcus pneumoniae pathogenicity island psrP-secY2A2. Challenge of mice with serotype 4, strain TIGR4, and the isogenic mutants T4DeltapsrP and T4DeltapsrP-secY2A2 determined that PsrP was required for bacterial persistence in the lungs but not for colonization in the nasopharynx or replication in the bloodstream during sepsis. In vitro experiments corroborated this anatomical site-specific role; psrP mutants failed to bind to A549 and LA-4 lung cells, yet adhered normally to human nasopharyngeal epithelial cells and to cells from human and rodent capillary endothelial cell lines. We determined that the amino terminus of PsrP mediated adhesion. Microspheres coated with recombinant PsrP(SRR1-BR) (rPsrP(SRR1-BR)) adhered to A549 cells, and moreover, preincubation of cells with rPsrP(SRR1-BR) inhibited TIGR4 adhesion in vitro. Antibodies against rPsrP(SRR1-BR) also neutralized PsrP function; antiserum against rPsrP(SRR1-BR) blocked TIGR4 adhesion in vitro and, following passive immunization, it protected mice against challenge. We conclude that PsrP is an adhesin required for bacterial persistence in the lungs and that rPsrP(SRR1-BR) is a protective antigen.

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with ؊ ONOO and H 2 O 2 in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/ H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind copper. We find that single mutations at copper ligands, including H46R, H48Q, and a quadruple mutant H46R/H48Q/ H63G/H120G, also diminish the binding of radioactive copper. Further, using native polyacrylamide gel electrophoresis and a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define diseasecausing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry. Amyotrophic lateral sclerosis (ALS),3 which is characterized by progressive muscle weakness and motor neuron loss, presents as both sporadic and familial (FALS) illness. A subset of FALS cases is caused by missense mutations in the superoxide scavenging enzyme, Cu/Zn-superoxide dismutase 1 (SOD1) (1, 2). To date, over 100 different point mutations, and Ͼ5 early termination mutations have been linked to FALS (www. alsod.org) (for reviews see Refs. 2-4). Early studies of FALS-SOD1 enzymes demonstrated that some mutants retain high levels of activity and relatively long half lives (5). Moreover, mutant proteins that are inactive or short-lived do not exhibit evidence of dominant negative action with regard to the superoxide-scavenging activity of enzyme derived from the normal allele (6). In transgenic mice, the hyperexpression of the G93A and G37R variants of FALS-SOD1 increases superoxide scavenging activity, kills motor neurons, and causes paralysis (7,8). SOD1 knock-out mice do not develop ALS-like phenotypes but do show sensory and motor neuropathy (9). Together, these studies establish that SOD1 mutations cause ALS through a gained toxic property. Although expressed ubiquitously (8, 10), mutant SOD1 selectively damages motor neurons, by mechanisms yet to be fully understood.

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