Mechanistic Insights Underlying Tolerance to Acetic Acid Stress in Vaginal Candida glabrata Clinical Isolates

Abstract

During colonization of the vaginal tract Candida glabrata cells are challenged with the presence of acetic acid at a low pH, specially when dysbiosis occurs. To avoid exclusion from this niche C. glabrata cells are expected to evolve efficient adaptive responses to cope with this stress; however, these responses remain largely uncharacterized, especially in vaginal strains. In this work a cohort of 18 vaginal strains and 2 laboratory strains (CBS138 and KUE100) were phenotyped for their tolerance against inhibitory concentrations of acetic acid at pH 4. Despite some heterogeneity has been observed among the vaginal strains tested, in general these strains were considerably more tolerant to acetic acid than the laboratory strains. To tackle the mechanistic insights behind this differential level of tolerance observed, a set of vaginal strains differently tolerant to acetic acid (VG281∼VG49 < VG99 < VG216) and the highly susceptible laboratory strain KUE100 were selected for further studies. When suddenly challenged with acetic acid the more tolerant vaginal strains exhibited a higher activity of the plasma membrane proton pump CgPma1 and a reduced internal accumulation of the acid, these being two essential features to maximize tolerance. Based on the higher level of resistance exhibited by the vaginal strains against the action of a β-1,3-glucanase, it is hypothesized that the reduced internal accumulation of acetic acid inside these strains may originate from them having a different cell wall structure resulting in a reduced porosity to undissociated acetic acid molecules. Both the vaginal and the two laboratory strains were found to consume acetic acid in the presence of glucose indicating that metabolization of the acid is used by C. glabrata species as a detoxification mechanism. The results gathered in this study advance the current knowledge on the mechanisms underlying the increased competitiveness of C. glabrata in the vaginal tract, a knowledge that can be used to guide more suitable strategies to treat infections caused by this pathogenic yeast.

Keywords: C. glabrata stress response; acetic acid tolerance; vaginal candidiasis.

FIGURE 1

Comparison of the susceptibility to acetic acid of laboratory, vaginal and intestinal Candida glabrata vaginal strains. Susceptibility to acetic acid of the two laboratory strains KUE100/CBS138 and of the vaginal isolates was compared by spot assays (A) and during cultivation in liquid growth medium (B). In the case of the spot assays, the strains were cultivated in agarized MM growth medium (at pH 4.5 or at pH 6.4) either or not supplemented with different concentrations of acetic acid. After 3 days of incubation at 30°C, pictures were taken and the cell density of each spot measured using the software ImageJ. The results shown here are representative of, at least, three independent experiments that gave essentially the same pattern of growth. For the comparison of growth in liquid medium, the strains were cultivated in 96-wells microplates for 24 h in MM growth medium (at pH 4) either or not supplemented with 60 or 80 mM acetic acid. After 24 h of growth the OD_600nm of all the cultures was measured. The results obtained are means of at least five different experiments.

FIGURE 2

Comparison of the growth curves of the tolerant C. glabrata vaginal isolates VG281, VG216, VG99, and VG49 and of the susceptible laboratory strains KUE100 and CBS138 in the presence of acetic acid. (A) Growth curve of the vaginal isolates and of the laboratory strains was compared in MM growth medium (at pH 4) (■ dark bars) or in this same growth medium supplemented with 60 mM ( gray bars) or 80 mM (△ white bars) of acetic acid. Growth of the different cultures was based on the increase of OD_600nm of the culture. The growth curves shown are representative of at least five independent experiments that resulted in a similar growth pattern. (B) Based on the growth curves shown in (A), the duration of the lag phase and the maximum growth rate of the different strains during cultivation in the presence or absence of acetic acid were calculated. The results shown are the average of the results obtained for the different replicas of the growth curves that were obtained for each strain in each condition. To assess the differences obtained for the strains statistical analysis of the data was performed using as a reference the VG281 strain. ^∗∗∗p-value below 0.001; ^∗∗p-value below 0.01; ^∗p-value below 0.05.

FIGURE 3

Comparison of the susceptibility of the laboratory and of the vaginal C. glabrata strains to heat stress and to H₂O₂. Susceptibility of the vaginal isolates and of the laboratory strains KUE100 and CBS138 to H₂O₂ and to heat stress compared by spot assays. To test susceptibility to H₂O₂ the strains were cultivated in MM growth medium (at pH 4.5) either or not supplemented with inhibitory concentrations of this chemical. After 3 days of incubation at 30°C pictures of the plates were taken and the cell density of each spot measured using the software ImageJ to reconstruct the heat map shown. Comparison of the susceptibility of the strains to heat stress was undertaken in the same conditions with the difference that in the latter case after inoculation the strains were incubated at 37 or 42°C.

FIGURE 4

Candida glabrata strains are able to consume acetic acid in the presence of glucose. The laboratory strain KUE100 and the vaginal strains VG281 and VG216 were cultivated in liquid MM medium (at pH 4.0) until mid-exponential phase and were then re-inoculated in fresh medium (at pH 4) either or not supplemented with 60 mM acetic acid. Growth was followed during approximately 40 h during which samples of culture supernatants were harvested and used for the quantification of acetic acid (□) and glucose (●) concentrations by HPLC, as detailed in Section “Materials and Methods.” The results shown are means of those obtained in three independent experiments that gave essentially the same results.

FIGURE 5

Acetic acid-tolerant vaginal isolates accumulate less acid intracelularly than susceptible strains Time-course representation of the accumulation ratio of [1-¹⁴C]-acetic acid in vaginal isolates VG281 (moderately tolerant to acetic acid), VG216 and VG99 (tolerant to acetic acid), in comparison with the susceptible laboratory strain KUE100 (●), during cultivation in MM (at pH 4.0) supplemented with 60 mM of cold acetic acid. The results obtained were representative of, at least, five independent experiments. ^∗∗p-value below 0.01; ^∗∗∗p-value below 0.001.

FIGURE 6

Comparison of C. glabrata laboratory strain KUE100 and of the differently acetic acid tolerant vaginal clinical isolates to lyticase (A), calcofluor and Congo Red (B). (A) To test susceptibility of the different strains to lyticase these were cultivated in unsupplemented liquid MM medium until mid-exponential phase at standardized OD_600nm of 0.8 ± 0.005 and then re-inoculated into 0.1 mM sodium phosphate buffer (pH 7.0) supplemented with 10 μg/ml lyticase from Arthrobacter luteus. The results obtained were representative of, at least, three independent experiments. Statistical analysis of the data was performed using the last time point and using the KUE100 strain as a reference ^∗p-value below 0.05; ^∗∗p-value below 0.01. (B) To test susceptibility of the strains to inhibitory concentrations of calcofluor white or Congo Red the strains were cultivated, at 30°C for 3 days, in agarised MM medium supplemented with the indicated concentrations of the two cell wall perturbing agents. The pictures shown are representative of the results obtained in three independent experiments that were performed.

FIGURE 7

External medium acidification promoted by CgPma1 H⁺-ATPase in laboratory strain KUE100 and in the differently acetic acid-tolerant vaginal clinical isolates. Cell suspensions of the different strains – KUE100 (□), VG216 (Δ), VG99 (♢), and VG281 (◌) – were cultivated in MM growth medium (at pH 4) until mid-exponential phase and then de-energized for 30 min, as detailed in Section “Materials and Methods.” After this time concentrated cell suspensions were energized with a pulse of glucose to assess the proton pumping capacity prompted by CgPma1 H⁺-ATPase activity, in the presence or absence of acetic acid. The results obtained are means of at least four independent experiments that gave, essentially, the same results.