Brucella abortus 2308 derivatives with mini-Tn5 insertions in purE, purL, and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn5 insertions in pheA, trpB, and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.Brucella abortus is a facultative, gram-negative pathogen of humans and animals that inhabits macrophages (2). The capacity to withstand nutritional deprivation would be expected to be particularly important for the survival of B. abortus, since this organism does not escape from the phagosome into the nutrient-rich environment of the host cell cytoplasm (2). Indeed, previous studies suggest that the brucellae encounter a considerable degree of nutritional deprivation during their long-term residence in host macrophages. Brucella melitensis purE (5) and Brucella suis aroC (12) mutants, for example, are unable to maintain chronic infection in experimentally infected mice, and the attenuation of the B. melitensis purE mutant purE201 extends to the natural ruminant host (4) and nonhuman primates (M. Nikolich, personal communication). These observations are also consistent with those seen with auxotrophic mutants of Salmonella and Mycobacterium and indicate that the availability of certain nutrients is severely restricted in the phagosomal compartment of host macrophages (3,11,22).In an effort to gain a better understanding of the metabolic versatility required for sustained intracellular residence by the brucellae in host macrophages, transposon mutagenesis and an in vitro screen were employed to identify B. abortus 2308 derivatives with mini-Tn5 insertions in genes required for resistance to nutrient deprivation. Transposon mutagenesis of this strain was performed by conjugative transfer of the mini-Tn5 derivative Km1 by employing pUT as the delivery vector and Escherichia coli S17-1pir as the conjugal donor strain (6,14,28). Approximately 1,000 B. abortus mini-Tn5 mutants were patched with sterile toothpicks onto Schaedler agar supplemented with 5% defibrinated bovine blood (SBA), SBA supplemented with 45 g of kanamycin/ml (SBAk), and Gerhardt's minimal medium (13) supplemented with 1.5% agar (GMMA). Plates were incubated at 37°C with 5% CO 2 and examined for growth after 4, 7, and 10 days of incubation. Schaedler agar, the basal medium for SBA, is a complex culture medium containing enzymatic digests of casein, animal and plant tissues, yeast extract, glucose, cystine, and hemin (Difco manual, 10th ed., Difco Laboratories, Detroit, Mich.). In contrast, GMM, the base for GMMA, is a defined medium formulated during a study aimed at defining the minimal in vitro growth requirements of Brucella strains (1...

No abstract

To quantify complement depletion by pneumolysin during Streptococcus pneumoniae bacteremia, cirrhotic and control rats were infected intravenously with one of three isogenic mutant strains of S. pneumoniae expressing different forms of pneumolysin. Outcome measures included clearance of the organisms from the bloodstream, alterations in 50% serum hemolytic complement (CH 50 ) activity and complement C3 levels during infection, and serum opsonic capacity at 18 h postinfection. Cirrhotic rats had significantly lower CH 50 and C3 levels than control rats, both before and after infection. However, initial complement levels did not predict bacterial load after 18 h of infection. Changes in CH 50 and C3 levels over the 18-h period correlated with numbers of H؉C؉ but not H؉C؊ or PLY؊ organisms in the bloodstream at 18 h postinfection. The sera of cirrhotic rats infected with the H؉C؉ strain had significantly decreased levels of C3 and showed significantly lower opsonizing activity for S. pneumoniae than sera from H؉C؉-infected control rats. These studies suggest that under limiting concentrations of complement, the expression of pneumolysin by pneumococci has a significant, negative effect on serum complement levels and reduces serum opsonic activity.

We investigated the role of pneumolysin’s complement-activating activity during Streptococcus pneumoniae bacteremia in a hypocomplementemic, cirrhotic host. Isogenic mutant pneumococcal strains, in which pneumolysin was expressed from a plasmid, were used. These strains included H+C+, expressing wild-type pneumolysin with both cytolytic and complement-activating activity; PLY−, carrying the plasmid without the pneumolysin gene; and, H+C−, expressing pneumolysin with cytolytic activity only. In control rats, intravenous infection with 2.0 × 107 CFU of H+C+ per ml of blood resulted in a decrease in bacteremia of 3.5 log units by 18 h postinfection and 55% mortality. By contrast, cirrhotic rats infected similarly with the H+C+ strain demonstrated a 0.2-log-unit increase in bacteremia by 18 h postinfection and 100% mortality. Both control and cirrhotic rats cleared the PLY− strain more effectively from their bloodstreams by 18 h postinfection (6.2 and 5.6 log unit decreases, respectively). Infection with the PLY− strain also resulted in low mortality (0 and 14%, respectively) for control and cirrhotic rats. When infected with the H+C− strain (without complement-activating activity), both groups cleared the organism from their bloodstreams nearly as well as they did the PLY− strain. Furthermore, the mortality rate for control and cirrhotic rats was identical after infection with the H+C− strain. These studies suggest that pneumolysin production contributes to decreased pneumococcal clearance from the bloodstream and higher mortality in both control and cirrhotic rats. However, pneumolysin’s complement-activating activity may uniquely enhance pneumococcal virulence in the hypocomplementemic, cirrhotic host.