Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae

Abstract

Insertion-duplication mutagenesis was used to construct a pneumolysin-negative derivative of Streptococcus pneumoniae. This was achieved by first transforming the nonencapsulated strain Rxl with a derivative of the vector pVA891 carrying a 690-base-pair DNA fragment from the middle of the pneumolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, pneumolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these produced pneumolysin. Southern blot analysis confirmed that the pneumolysin gene in these transformants had been interrupted by the plasmidderived sequences. The pneumolysin-negative mutants showed reduced virulence for mice compared with D39, as judged by survival time after intranasal challenge, intraperitoneal 50% lethal dose, and blood clearance studies. Pneumolysin production was reinstated in one of the mutants by transformation with the cloned pneumolysin gene, with the concomitant loss of erythromycin resistance; the virulence in mice of this isolate was indistinguishable from that of D39. These results confirm the involvement of pneumolysin in pneumococcal pathogenesis.

Programming stress‐induced altruistic death in engineered bacteria

“…By making this assumption, evolution of programmed death in microbes can be analyzed under the general framework of public-good cooperation (Ackermann et al, 2008). Figure 1A summarizes several natural examples that may fit in this framework: Streptococcus pneumoniae (Berry et al, 1989;Hirst et al, 2004) responds to its host environment and releases the virulence factor pneumolysin through cell lysis, which helps its host invasion. Salmonella typhimurim (Stecher et al, 2007;Ackermann et al, 2008) responds to competition in the host's microbiota by causing host inflammation through programmed death.…”

A role in cell-binding for the C-terminus of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae

“…Streptococcus pneumoniae is the primary agent of community-acquired pneumonia [1]. A proven virulence factor of the pneumococcus is pneumolysin [2][3][4]. Pneumolysin is a member of the thiol-activated family of toxins [5].…”

Streptococcal toxins: role in pathogenesis and disease

“…Contributes to proliferation in whole blood, and colonization of the nasopharynx and lungs (Berry et al, 1989b;Kadioglu et al, 2000;Reiss et al, 2011) Suilysin Streptococcus suis Cytotoxic to endothelial cells, epithelial cells, macrophages and neutrophils (Allen et al, 2001;Lun et al, 2003) β-Haemolysin/cytolysin…”

“…PLY activates p38 in vitro (Ratner et al, 2006) and the NLRP3 inflammasome in macrophages to stimulate the production of type I interferons following pneumococcal phagocytosis (Koppe et al, 2012). In murine infection models, PLY-deficient mutants have reduced proliferation in whole blood (Benton et al, 1995), diminished capacity to colonize the nasopharynx, induce less lung inflammation and neutrophil recruitment and are rapidly cleared from the lung, compared with WT (Berry et al, 1989b;Kadioglu et al, 2000). Genetically inactivated PLY toxoids are immunogenic and protective against lethal pneumococcal challenge in mouse vaccination models (Paton et al, 1991;Alexander et al, 1994;Kirkham et al, 2006).…”