Use of insertional inactivation to facilitate studies of biological properties of pneumococcal surface protein A (PspA).

Abstract

PspA is a cell surface protein of Streptococcus pneumoniae that is present on a number of clinical isolates as well as the nonencapsulated laboratory strain Rx1. In a previous report we have shown that mAbs directed against PspA can protect mice from at least some of the pneumococcal strains bearing this protein. In our present report we have produced insertional inactivation mutants that lack PspA and have used these mutants to demonstrate that PspA can play a role in pneumococcal virulence and that anti-PspA immunity can lead to protection against pneumococcal infection. PspA- mutants were obtained using derivatives of plasmid pVA891 carrying chromosomal fragments from Rx1. From one of the mutants, we cloned a 550 bp fragment of the pneumococcal gene into pVA891 and transferred this chimeric plasmid, designated pKSD300, into Escherichia coli. After transformation of pKSD300 into Rx1, PspA production is not detected. In colony hybridization experiments, the 550 bp fragment hybridizes specifically to pneumococcal isolates in a pattern consistent with the hypothesis that the fragment is a portion of the pspA structural gene that is different from the portions coding for the antigenic determinants detected by mAbs Xi64 or Xi126. When X-linked immunodeficient (xid) CBA/N mice were immunized with wild-type Rx1, they were resistant to challenge with type 3 strain WU2. However, when these mice were immunized with a PspA- mutant of Rx1, they failed to survive the subsequent challenge, indicating that immunity to PspA can contribute to the resistance to pneumococcal infection. Using pKSD300 we insertionally inactivated pspA in D39, a virulent strain of S. pneumoniae. When injected intravenously there was a 10-fold greater reduction of the mutant pneumococci in the blood, as compared to the wild-type D39.

Contribution of novel choline‐binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae

“…A pspA-deficient derivative of D39, LM91, was provided by Larry McDaniel, University of Alabama, Birmingham, AL, USA. This strain is a null mutant in pspA created by insertion duplication mutagenesis (McDaniel et al, 1987). S. pneumoniae was routinely plated on tryptic soy agar supplemented with sheep blood (3%, v/v).…”

The Virulence Function of Streptococcus pneumoniae Surface Protein A Involves Inhibition of Complement Activation and Impairment of Complement Receptor-Mediated Protection

“…Accordingly, in a murine model of bacteremia, PspA-deficient (PspA Ϫ ) pneumococci have significantly reduced virulence compared with pneumococci that express PspA (PspA ؉ ) (2,3). Earlier studies from our laboratory and others' have shown that PspA inhibits complement activation, thereby limiting opsonization of pathogens by complement protein 3 (C3) (4 -7).…”

“…Pneumococcal surface protein A (PspA) 3 is an important virulence factor for S. pneumoniae (1)(2)(3). Accordingly, in a murine model of bacteremia, PspA-deficient (PspA Ϫ ) pneumococci have significantly reduced virulence compared with pneumococci that express PspA (PspA ؉ ) (2,3).…”

Cationic pullulan nanogel as a safe and effective nasal vaccine delivery system for respiratory infectious diseases

“…Because it thus can induce cross-reactive immune responses among different strains, PspA is a promising candidate antigen for the development of a next-generation pneumococcal vaccine. 22 , 23 Nasal administration of a fusion protein comprising PspA and flagellin from Vibrio vulnificus induced antigen-specific IgG and IgA both in serum and at mucosal surfaces and provided efficient protective immunity in mice against lethal challenge with live S. pneumoniae . 24 In addition, co-administration of PspA antigen and IL-12 as a nasal adjuvant enhanced PspA-specific IgG and IgA responses, with increased protection from nasal carriage.…”

“…28 , 29 These results support PspA as a promising antigen candidate for an S. pneumoniae vaccine that is likely to be effective not only in adults but also in children and the elderly. 30 , 31 …”