Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

Zinc uptake by Streptococcus pneumoniae depends on both AdcA and AdcAII and is essential for normal bacterial morphology and virulence.

Zinc is an essential trace metal for living cells. The ABC transporter AdcABC was previously shown to be required for zinc uptake by Streptococcus pneumoniae. As we have recently described AdcAII as another zinc-binding lipoprotein, we have investigated the role of both AdcA and AdcAII in S. pneumoniae zinc metabolism. Deletion of either adcA or adcAII but not phtD reduced S. pneumoniae zinc uptake, with dual mutation of both adcA and adcAII further decreasing zinc import. For the Δ(adcA/adcAII) mutant, growth and intracellular concentrations of zinc were both greatly reduced in low zinc concentration. When grown in zinc-deficient medium, the Δ(adcA/adcAII) mutant displayed morphological defects related to aberrant septation. Growth and morphology of the Δ(adcA/adcAII) mutant recovered after supplementation with zinc. Dual deletion of adcA and adcAII strongly impaired growth of the pneumococcus in bronchoalveolar lavage fluid and human serum, and prevented S. pneumoniae establishing infection in mouse models of nasopharyngeal colonization, pneumonia and sepsis without altering the capsule. Taken together, our results show that AdcA and AdcAII play an essential and redundant role in specifically importing zinc into the pneumococcus, and that both zinc transporters are required for proper cell division and for S. pneumoniae survival during infection.

Biochemical characterization of the histidine triad protein PhtD as a cell surface zinc-binding protein of pneumococcus.

Zinc homeostasis is critical for pathogen host colonization. Indeed, during invasion, Streptococcus pneumoniae has to finely regulate zinc transport to cope with a wide range of Zn(2+) concentrations within the various host niches. AdcAII was identified as a pneumococcal Zn(2+)-binding protein; its gene is present in an operon together with the phtD gene. PhtD belongs to the histidine triad protein family, but to date, its function has not been clarified. Using several complementary biochemical methods, we provide evidence that like AdcAII, PhtD is a metal-binding protein specific for zinc. When Zn(2+) binds (K(d) = 131 ± 10 nM), the protein displays substantial thermal stabilization. We also present the first direct evidence of a joint function of AdcAII and PhtD by demonstrating that their expression is corepressed by Zn(2+), that they interact directly in vitro, and that they are colocalized at the bacterial surface. These results suggest the common involvement of the AdcAII-PhtD system in pneumococcal zinc homeostasis.