Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis

Abstract

Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD(+) The oxidation of NADH to NAD(+) was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence.

FIG 1

Diminished inhibition of S. mutans in the nox mutant. Δnox, the nox mutant; Δnox_compl, the complemented strain of the nox mutant; ΔspxB, the spxB mutant; CAT, catalase spread on plate prior to inoculation at ∼880 U cm⁻².

FIG 2

NADH oxidase activity and assessment of H₂O₂ production by the Nox protein. (A) NADH oxidase activities of rNox and recombinant SSA_0375 (rSSA_0375) protein (a negative control). (B) Hydrogen peroxide production of rNox, Bacillus licheniformis NADH oxidase protein (Nox_Bli), and rSSA_0375 protein. ***, P < 0.001. Data were obtained at least in biological triplicate.

FIG 3

Decrease in NADH oxidase activity in the nox mutant. Cell lysates were examined for NADH oxidase activity. Δnox, the nox mutant; Δnox_compl, the complemented strain of the nox mutant. **, P < 0.01. Data were obtained at least in biological triplicate.

FIG 4

Extracellular (A) and intracellular (B) hydrogen peroxide production in the nox mutant. H₂O₂ production was measured in culture supernatants (A) or cell lysates (B) of cultures containing SK36 and mutant strains. Δnox, the nox mutant; Δnox_compl, the complemented strain of the nox mutant; ΔspxB, the spxB mutant. An asterisk indicates a significant difference at a P value of <0.05 compared to SK36. Data were obtained at least in biological triplicate.

FIG 5

Attenuation in competitive index in vivo but not in vitro in the nox mutant. Vegetation, bacteria obtained from rabbit heart vegetation postinoculation; BHI, bacteria cultured in BHI broth. Data were obtained at least in biological triplicate.

FIG 6

Decrease in blood killing (A) and growth in human serum (B) in the nox mutant. JFP36, an erythromycin-resistant derivative of SK36; Δnox, the nox mutant; Δnox_compl, the complemented strain of the nox mutant. An asterisk indicates significant difference at a P value of <0.05 compared to SK36 or JFP36. Data were obtained at least in biological triplicate.

FIG 7

Sensitivity to environmental stresses in the nox mutant. Reduction in bacterial survival upon exposure to exogenous H₂O₂ (A) and acid (B) in the nox mutant. Δnox, the nox mutant; Δnox_compl, the complemented strain of the nox mutant. An asterisk indicates a significant difference at a P value of <0.05 compared to SK36. **, P < 0.01. Data were obtained at least in biological triplicate.