R.A.C. Lock

ABSTRACT Exposure of freshwater trout (Salmo gairdneri) to waterborne Cd2+ results in accumulation of the metal in the branchial epithelial cells and its appearance in the blood. Cd2+ apparently enters the cells via Ca2+ channels in the apical membrane. Transfer of Cd2+ through the basolateral membrane is probably by diffusion. Inhibition by Cd2+ of transepithelial Ca2+ influx is time-and Cd2+-concentration-dependent. The inhibition of transepithelial Ca2+ influx is accompanied by blockage of apical Ca2+ channels. In line with the assumption that cytosolic Cd2+ inhibits Ca2+ uptake by inhibiting the basolateral Ca2+ pump, we hypothesize that the blockage of Ca2+ channels is an indirect effect of Cd2+ and results from a rise in cytosolic Ca2+ level caused by inhibition of the basolateral membrane Ca2+ pump.

Insertion-duplication mutagenesis was used to construct an autolysin-negative derivative of Streptococcus pneumoniae. This derivative was obtained by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 375-base-pair TaqI DNA fragment from the middle of the autolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, autolysin-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 transformants produced autolysin. Southern blot analysis confirmed that the autolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The autolysin-negative mutants showed markedly reduced virulence for mice compared with that of strain D39; intranasal and intraperitoneal 50% lethal doses were increased 10(2)- and 10(5)-fold, respectively. Autolysin production was reinstated in one of the mutants by back-transformation with the cloned autolysin gene, with the concomitant loss of erythromycin resistance; the virulence of this isolate for mice was indistinguishable from that of D39. The importance of autolysin in pathogenesis was confirmed by immunization-challenge studies. Mice immunized with purified autolysin survived significantly longer than did control mice after intranasal challenge with strain D39. This study provides direct evidence that the pneumococcal autolysin contributes to virulence and identifies it as a potential vaccine antigen.

Pneumolysin is the thiol-activated cytolysin produced by Streptococcus pneumoniae. Mice were immunized with a genetically engineered toxoid version of pneumolysin, which was derived from a serotype 2 pneumococcus. The toxoid carried the mutation Trp-433-->Phe. Alum was used as the adjuvant. Immunized mice had significantly increased levels of anti-pneumolysin antibodies, principally immunoglobulin G1. Mice were challenged intraperitoneally or intranasally with 12 strains covering capsular serotypes 1 to 6, 7F, 8, and 18C. Following challenge, the survival rate and/or the time of death of nonsurvivors (survival time) was significantly greater than that of sham-immunized mice for all nine serotypes. However, differences in the degree of protection were noted between different strains. The route of challenge also appeared to influence the degree of protection. Nevertheless, the significant, albeit in some cases partial, protection provided against all nine pneumococcal serotypes supports the conclusion that pneumolysin toxoids warrant consideration for inclusion in a human vaccine.

The effects of cadmium (Cd2+) on calcium (Ca2+) transport in the gills of rainbow trout (Salmo gairdneri) were studied. The gill epithelium of freshwater fish represents a model for a Ca2+-transporting tight epithelium. Unidirectional Ca2+ fluxes in the gills were estimated in an isolated saline-perfused head preparation. Ca2+ influx was not affected when up to 10 microM Cd were added to the ventilatory water at the start of flux determinations (in vitro exposure). However, after 16 h in vivo preexposure of the fish to 0.1 microM Cd in the water, a 79% inhibition of Ca2+ influx was observed. Ca2+ efflux was not affected when up to 10 microM Cd were added to the ventilatory water during the flux determination. Ca2+ efflux in fish preexposed to 0.1 microM Cd for 16 h was also not affected; a preexposure to 1 microM Cd, however, resulted in a 173% increase in Ca2+ efflux rates. Tracer retention in the gill tissue indicated that both Ca2+ and Cd2+ enter the gill epithelium via a lanthanum (La3+)-inhibitable pathway. It is concluded that Cd2+ readily enters the branchial epithelial cells, similarly as Ca2+ does via La3+-sensitive apical Ca2+ channels. The inhibitory action of Cd2+ on transepithelial Ca2+ influx seems to result from an inhibition of the basolateral Ca2+ transport, occurring after a critical intracellular Cd2+ concentration has been reached.

The role of the cytolytic toxin pneumolysin in the pathogenicity of Streptococcus pneumoniae was investigated. Pneumolysin was purified to homogeneity and used to immunize mice. When these mice were subsequently challenged via the nasal route with virulent S. pneumoniae, they survived significantly longer than control mice. The mean survival times were 5.52 and 2.48 days for immunized and control mice, respectively. This work provides direct evidence for the involvement of pneumolysin in pneumococcal pathogenicity.