Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence

Using allelic exchange, we created S. pneumoniae mutants that were deficient in RD2, 5, 7, 8, 9, 12 and 13. Mutants deficient in RD8, 9 and 13 were attenuated in a mouse model of disease. RD8 is 40,358 nucleotides in length and encodes 37 genes. Using a panel of isogenic mutants, we determined that RD8b3 is the operon within RD8 that is responsible for virulence. Mice infected with mutants deficient in RD8, RD8b3, RD9 and RD13 had significantly less bacteria in the blood two days after intranasal challenge and improved survival over time versus mice infected with wild type. In all instances mutants colonized the nasopharynx at levels equivalent to wild type.Genes within RD1, 3, 4, 6, and 10 have previously been shown to contribute to virulence. This study demonstrates that genes within RD8, 9 and 13 also contribute to virulence. The ability of mutants deficient in RD2, 5, 7, 8, 9, 12, and 13 to colonize the nasopharynx indicates that genes within these RDs are not required for asymptomatic carriage. Nonetheless, the observation that mutants deficient in RD8b3, 9 and 13 are attenuated indicates that genes within these loci are necessary for spread of the bacteria beyond the nasopharynx to normally sterile sites.Streptococcus pneumoniae (the pneumococcus) is a leading cause of community-acquired pneumonia, sepsis, and meningitis. Primarily a commensal, invasive pneumococcal disease (IPD) is characterized by spread of the pneumococcus from the nasopharynx to normally sterile sites such as the lungs, blood, and central nervous system. At risk for IPD are young children, the elderly, and individuals who are immunocompromised or have underlying medical conditions such as sickle cell anemia. Worldwide, it is estimated that S. pneumoniae is responsible for 15 cases of IPD per 100,000 persons per year and over a million deaths annually [1,2]. Of note, the preponderance of invasive disease is the result of infection with a relatively few invasive clones [3], a finding that su