Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide

Abstract

PsaA of Streptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+ transporter. Mutations in psaA cause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. A psaBC deletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather, psaA mutations indirectly affect this process through the disruption of Mn2+ transport. The deficiency in Mn2+ transport also causes hypersensitivity to oxidative stress from H2O2 and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+ and Mn2+ cations after a lag of about 15 h. Addition of Mn2+ alone or together with Fe2+ allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+ alone extended the 15-h lag phase to 25 h. Thus, while Fe2+ adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2 was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.

FIG. 1.

Construction of psa mutants. (A) The wild-type strain TIGR4 contains an intact psa operon. JEN15 (psaB) has an insertion-duplication mutation in psaB, created with plasmid pJJ046. JEN16 has the majority of psaB and psaC deleted, with a 30-residue fusion protein. (B) PCR products amplified with primers that span the psa operon. Lane 1, 1-kb DNA ladder (Invitrogen); lane 2, TIGR4; lane 3, JEN16.

FIG. 2.

Western blot assay for PsaA expression. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose blots, and probed with anti-PsaA antiserum. PsaA runs at 37 kDa. Lane 1, TIGR4; lane 2, JEN13 (psaA); lane 3, JEN16 (ΔpsaBC). The procedure used is described in Materials and Methods.

FIG. 3.

Immunofluorescence microscopy. Intact TIGR4 cells were viewed by phase-contrast (A) and confocal (B) microscopy. Protoplasts made from TIGR4 were viewed by phase-contrast (C) and confocal (D) microscopy. Samples were stained with rabbit anti-PsaA followed by anti-rabbit immunoglobulin conjugated to fluorescein isothiocyanate.

FIG. 4.

Adherence of TIGR4 (squares), psaA (triangles), and ΔpsaBC (circles) strains to D562 nasopharyngeal cells. The assay was performed with confluent D562 monolayers. Serial dilutions of TIGR4, ΔpsaBC, and psaA strains were added to triplicate wells and incubated for 2.5 h at 37°C under 5% CO₂. The wells were washed, and the cells were detached prior to dilution plating to determine the CFU bound per well.

FIG. 5.

Virulence of TIGR4 and ΔpsaBC strains in a mouse model for systemic infection. Mice were infected intravenously, and survival time was monitored. The horizontal line designates the median time until the mice became moribund.

FIG. 6.

Oxidative stress response. (A) TIGR4 (open bars) and ΔpsaBC (solid bars) were treated with various concentrations of H₂O₂ for 30 min, and percent survival was determined. (B) TIGR4 (squares) and ΔpsaBC (circles) were incubated with 50 mM paraquat (solid symbols) or without (open symbols), and the number of surviving CFU was determined at various time points. Both assays were performed in triplicate.

FIG. 7.

Growth of TIGR4 (squares) and ΔpsaBC (circles) strains: (A) growth curves of TIGR4 and ΔpsaBC in THY; (B) growth curves of TIGR4 and ΔpsaBC grown in CDM with 0.1 μM MnSO₄ (solid symbols) or 50 μM MnSO₄ (open symbols). Results are representative of multiple experiments.

FIG. 8.

Growth of TIGR4 in CDM. TIGR4 was grown in CDM alone (squares) or supplemented with 0.1 μM MnSO₄ (upward triangles), 50 μM MnSO₄ (downward triangles), 0.1 μM MnSO₄ and 50 μM FeSO₄ (diamonds), 50 μM MnSO₄ and 50 μM FeSO₄ (circles), or 50 μM FeSO₄ (open squares). Results are representative of multiple experiments.

FIG. 9.

Effect of Tiron on growth. TIGR4 was grown in CDM alone with no MnSO₄ (solid squares), or with 50 μM FeSO₄ (solid circles). Tiron (5 mM) was added to the CDM with no MnSO₄ (open squares) and the same medium with 50 μM FeSO₄ (open circles). The results are representative of at least three independent experiments.