Biosynthesis and degradation of H2O2 by vaginal lactobacilli

Abstract

Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H(2)O(2), we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H(2)O(2) concentrations. Generation of H(2)O(2) was found to be especially common and strong for L. jensenii strains, while it was variable among L. crispatus and L. gasseri strains. Biosynthesis of H(2)O(2) only occurred upon agitation of the cultures, but the H(2)O(2)-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H(2)O(2) production, while Cu(2+) inhibited the growth of Lactobacillus jensenii CECT 4306, which was chosen as a model strain. Cultures with Fe(3+), hemin, and hemoglobin did not accumulate H(2)O(2). Fe(3+) activated an extracellular peroxidase that destroyed the H(2)O(2) being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe(3+) ions being a necessary cofactor of the reaction.

FIG. 1.

Standard curves of the relation between the concentration of hydrogen peroxide and the intensity of color produced by its reaction with the chromophores tetramethylbenzidine (•), o-dianisidine (▪), and DA-64 (▿), catalyzed by horseradish peroxidase (the wavelengths used were 620, 405, and 727 nm, respectively). DA-64 was the most sensitive at low concentrations of hydrogen peroxide (inset). Error bars show standard deviations. O.D., optical density.

FIG. 2.

Production of hydrogen peroxide by exponential-phase cultures of L. jensenii CECT 4306 (8 h of incubation) subjected to aeration (▪) or maintained in static conditions (▴). The same culture was treated with chloramphenicol 30 min before the start of aeration (♦). Error bars show standard deviations.

FIG. 3.

Effect of the addition of cations (2 mM) and other cofactors on H₂O₂ generation by cultures of L. jensenii CECT 4306 grown at 37°C for 16 h and subjected afterwards to aeration. ♦, No additions; ▴, Mg³⁺; ▪, Ca³⁺; •, Zn³⁺; + (gray line), Mn³⁺; ○, Fe³⁺; □, 30 mM hemin; ▵, 1% hemoglobin. Error bars show standard deviations.

FIG. 4.

Effects of different concentrations of Fe³⁺ added to cultures of L. jensenii CECT 4306 on H₂O₂ generation upon aeration. ⋄, Control culture without added Fe³⁺; ○, 25 nM; •, 50 nM; ▵, 0.1 mM; ▴, 0.2 mM; ▪, 2 mM. Error bars show standard deviations.

FIG. 5.

Degradation of H₂O₂ by cultures of L. jensenii CECT 4306. Cultures were grown for 16 h in MRS medium prior to the addition of H₂O₂ (2.5 mM). Results shown are for replicate cultures that were supplemented with 2 mM Fe³⁺ (▪) or grown without supplements (×), the same unsupplemented cultures to which 2 mM Fe³⁺ and H₂O₂ were added with a 30-min interval (▴), and uninoculated MRS medium with (♦) and without (•) added Fe³⁺. Error bars show standard deviations.

FIG. 6.

Effect of inhibition of protein synthesis on H₂O₂ accumulation by aerated cultures of L. jensenii CECT 4306. Results shown are for replicate control cultures grown without Fe³⁺ (♦) and replicate cultures grown without Fe³⁺ and subjected successively to 100 μg/ml chloramphenicol, 2 mM Fe³⁺, and aeration at 30-min intervals (▪). Similar data for lack of H₂O₂ accumulation were obtained from cultures grown in the presence of Fe³⁺ and the same cultures treated with chloramphenicol. Error bars show standard deviations.

FIG. 7.

Effect of Fe³⁺ on the growth of L. jensenii CECT 4306 cultures in the presence of increasing H₂O₂ concentrations. The absorbance of the cultures after 6 h of incubation is represented. Light gray bars, cultures with added Fe³⁺; dark gray bars, cultures without added Fe³⁺. Error bars show standard deviations. O.D., optical density.