Janet E. Alexander

Role of Manganese-Containing Superoxide Dismutase in Oxidative Stress and Virulence of Streptococcus pneumoniae

ABSTRACT Streptococcus pneumoniae was shown to contain two types of superoxide dismutase, MnSOD and FeSOD. Levels of MnSOD increased during growth in an aerobic environment. The sodA gene, encoding MnSOD, of virulent S. pneumoniae type 2 strain D39 was inactivated to give mutant D39HY1. Aerobically, D39HY1 had a lower growth rate than the wild type and exhibited susceptibility to the redox-active compound paraquat, but anaerobic growth of D39HY1 was identical to that of the wild type. Virulence studies showed that the median survival time of mice infected intranasally with D39HY1 was significantly longer than that of mice infected with the wild-type pneumococcus. In contrast to the wild type, D39HY1 did not multiply in lungs during the first 24 h but thereafter grew at the same rate as the wild type. Appearance in the bloodstream was also delayed, but growth in the blood was unimpaired by the sodA mutation. The pattern of inflammation in lungs infected with D39HY1 differed from that in wild-type-infected mice. After infection with D39HY1, neutrophils were densely packed around bronchioles, in contrast to the wild-type infection, where neutrophils were more diffusely localized.

Role of Genetic Resistance in Invasive Pneumococcal Infection: Identification and Study of Susceptibility and Resistance in Inbred Mouse Strains

ABSTRACT From a panel of nine inbred mice strains intranasally infected withStreptococcus pneumoniae type 2 strain, BALB/c mice were resistant and CBA/Ca and SJL mice were susceptible to infection. Further investigation revealed that BALB/c mice were able to prevent proliferation of pneumococci in the lungs and blood, whereas CBA/Ca mice showed no bacterial clearance. Rapidly increasing numbers of bacteria in the blood was a feature of CBA/Ca but not BALB/c mice. In the lungs, BALB/c mice recruited significantly more neutrophils than CBA/Ca mice at 12 and 24 h postinfection. Inflammatory lesions in BALB/c mice were visible much earlier than in CBA/Ca mice, and there was a greater cellular infiltration into the lung tissue of BALB/c mice at the earlier time points. Our data suggest that resistance or susceptibility to intranasal pneumococci may have an association with recruitment and/or function of neutrophils.

Molecular analysis of virulence factors of Streptococcus pneumoniae

glas 1981). The rate of disease within a population depends Streptococcus pneumoniae (the pneumococcus) remains a major on the frequency with which invasive serotypes are carried pathogen of man despite the advent of antibiotic therapy. (Riley and Douglas 1981), but in certain populations some The organism is the causative agent of several important serotypes are rarely carried but are frequently isolated from diseases including pneumonia, meningitis and otitis media. cases. These serotypes can be considered to be particularly In the developing countries it has been estimated that 5 virulent. Little information is currently available about the million children under the age of 5 years die each year from bacterial and host factors that allow progression from carrier pneumonia, with the pneumococcus being the most common state to disease. causative agent (Anon. 1985). It has been suggested (Austrian The pneumococcus is very sensitive to a range of anti1981) that more than 1 million cases of pneumococcal pneubiotics, including penicillin. However, although the intromonia occur in the United States each year and the case duction of antibiotics reduced the mortality due to fatality rate of these infections is 5–7%. In the case of menpneumococcal infection, mortality rates still remain high, ingitis caused by pneumococcus, the attack rate in the United especially in cases of pneumococcal meningitis. Fatality rates States in 1989 was 2 cases per 100 000 population and the of 5–22% reported for pneumococcal pneumonia have been mortality rate was 55% (Lee et al. 1991). The pneumococcus suggested to be due to irreparable damage caused during the is also a major cause of otitis media. It has been estimated early phase of the disease (Austrian 1984). Antibiotic therapy that more than 75% of children will suffer at least one attack does not reduce the mortality of infection during the first 3 of this disease by the age of 6 years and the pneumococcus is d of treatment. Even after therapy with antibiotics has been the cause of up to 67% of cases of bacterial otitis media in effective in eliminating bacteria, the sequelae to pneumothe USA (Austrian 1984). coccal infection can be serious. For example, more than 20% Although the pneumococcus is an important cause of of survivors of pneumococcal meningitis are left with perdisease, its normal habitat is the nasopharynx of man. It is manent deafness (Kennedy et al. 1991). Antibiotics may also highly likely that the entire human population is colonized not be effective in certain high risk populations (Austrian by the pneumococcus at one time or another and at any given 1981). The occurrence of penicillin-resistant strains of time the carriage rate can be as high as 60% of the population pneumococcus is increasing and strains with multiple drug (Austrian 1986). Colonization with the pneumococcus begins resistance patterns are also being isolated (Spika et al. 1991). shortly after birth and by 12 weeks of age carriage rates in Current vaccines against the pneumococcus, for use in

A major locus conferring susceptibility to infection by Streptococcus pneumoniae in mice.

We have studied the genetics of susceptibility to infection by Streptococcuspneumoniae in mice. Linkage analysis of the F2 generation from a cross between resistant BALB/cO1aHsd and susceptible CBA/CaO1aHsd strains allowed us to map a major locus controlling the development of bacteremia and survival after intranasal infection.

Development of an optimized system for electroporation of Listeria species.

Electroporation was used to facilitate transformation of Listeria species with plasmid DNA. Optimal conditions for transformation of L. monocytogenes were a field strength of 8·5 kV/cm, 200 Ohms resistance, 25 μF capacitor with a time constant of 5 ms. With these conditions, 3·9 × 106 transformants/pg DNA were obtained. Under the same conditions, L. innocua and L. iuanouii exhibited a frequency of transformation similar to that of L. monocytogenes but a somewhat lower level was obtained with L. seeligeri.

Electrotransformation of Listeria species

Electroporation offers an efficient system for the introduction of genetic material into listeriae. Conjugation and protoplast transformation of Listeria monocytogenes has been reported (Flamm et al. 1984, Vicente et al. 1987). However, transformation rates (∼ 103/ μg of DNA) produced by these methods are too low for use in many molecular techniques. To facilitate the entry of DNA by electroporation, sufficient intensity of current must be applied to produce the optimum number of permeablilized areas. The intensity of current is a function of the field strength and time constant used.