In Vitro Incorporation of Helicobacter pylori into Candida albicans Caused by Acidic pH Stress

Abstract

Yeasts can adapt to a wide range of pH fluctuations (2 to 10), while Helicobacter pylori, a facultative intracellular bacterium, can adapt to a range from pH 6 to 8. This work analyzed if H. pylori J99 can protect itself from acidic pH by entering into Candida albicans ATCC 90028. Growth curves were determined for H. pylori and C. albicans at pH 3, 4, and 7. Both microorganisms were co-incubated at the same pH values, and the presence of intra-yeast bacteria was evaluated. Intra-yeast bacteria-like bodies were detected using wet mounting, and intra-yeast binding of anti-H. pylori antibodies was detected using immunofluorescence. The presence of the H. pylori rDNA 16S gene in total DNA from yeasts was demonstrated after PCR amplification. H. pylori showed larger death percentages at pH 3 and 4 than at pH 7. On the contrary, the viability of the yeast was not affected by any of the pHs evaluated. H. pylori entered into C. albicans at all the pH values assayed but to a greater extent at unfavorable pH values (pH 3 or 4, p = 0.014 and p = 0.001, respectively). In conclusion, it is possible to suggest that H. pylori can shelter itself within C. albicans under unfavorable pH conditions.

Keywords: Candida albicans; Helicobacter pylori; intracellular bacteria; pH; stress.

Figure 1

Growth curve of Helicobacter pylori J99 at pH 3 (■), pH 4 (●), and pH 7 (▲).

Figure 2

Growth curve of Candida albicans at pH 3 (■), pH 4 (●), and pH 7 (▲). In vitro entry of H. pylori J99 into C. albicans ATCC 90028.

Figure 3

Bacterium-like bodies (BLBs) within C. albicans ATCC 90028 vacuoles after 48 h of incubation at pH 3, pH 4, and pH 7, observed by optical microscopy using a 100× objective lens. (A) Negative control, C. albicans 90028 strain; (B) incubation at pH 3; (C) incubation at pH 4; and (D) incubation at pH 7.

Figure 4

Immunofluorescent assay using Fluorescein Isothiocyanate (FITC)-labeled anti-H. pylori IgG polyclonal antibodies. (A) C. albicans ATCC 90028 not co-cultured with the bacterium (negative control) observed by differential interference contrast (DIC); (B) absence of fluorescence in the negative control; (C) presence of fluorescence emitted by H. pylori J99 (white arrows) (positive control); and (D) fluorescence emitted by the intracellular presence of H. pylori J99 (white arrows) in yeasts.

Figure 5

Detection, in 2% agarose gel, of the 16S rDNA gene of H. pylori J99 in the total DNA extracted from C. albicans ATCC 90028 previously co-incubated with the bacterium for 48 h at pH 3, pH 4, and pH 7. (M) Molecular weight marker; (C⁺) positive control (H. pylori J99); (C⁻) negative control (C. albicans 90028 incubated in the absence of the bacterium); (B) blank (polymerase chain reaction (PCR) degree water); (1–3) amplicons from the total DNA of co-cultures incubated at pH 7, 4, and 3, respectively.