Nutrient Deficiency Promotes the Entry of Helicobacter pylori Cells into Candida Yeast Cells

Abstract

Helicobacter pylori, a Gram-negative bacterium, has as a natural niche the human gastric epithelium. This pathogen has been reported to enter into Candida yeast cells; however, factors triggering this endosymbiotic relationship remain unknown. The aim of this work was to evaluate in vitro if variations in nutrient concentration in the cultured medium trigger the internalization of H. pylori within Candida cells. We used H. pylori-Candida co-cultures in Brucella broth supplemented with 1%, 5% or 20% fetal bovine serum or in saline solution. Intra-yeast bacteria-like bodies (BLBs) were observed using optical microscopy, while intra-yeast BLBs were identified as H. pylori using FISH and PCR techniques. Intra-yeast H. pylori (BLBs) viability was confirmed using the LIVE/DEAD BacLight Bacterial Viability kit. Intra-yeast H. pylori was present in all combinations of bacteria-yeast strains co-cultured. However, the percentages of yeast cells harboring bacteria (Y-BLBs) varied according to nutrient concentrations and also were strain-dependent. In conclusion, reduced nutrients stresses H. pylori, promoting its entry into Candida cells. The starvation of both H. pylori and Candida strains reduced the percentages of Y-BLBs, suggesting that starving yeast cells may be less capable of harboring stressed H. pylori cells. Moreover, the endosymbiotic relationship between H. pylori and Candida is dependent on the strains co-cultured.

Keywords: Candida; Helicobacter pylori; endosymbiosis; fetal bovine serum; intracellular H. pylori; nutrient deficiency; starvation.

Figure 1

Typical culture and microbiological identification of H. pylori. (A) Culture of H. pylori in CA-5%FBS; (B) Gram-stained smear; (C) Urease test; (D) Catalase test; and (E) Oxidase test. CA-5%FBS: Columbia agar supplemented with 5% FBS.

Figure 2

Culture and purity verification of Candida ATCC 90030 strain as representative of the strains used in the present work. (A) Yeast culture on Sabouraud agar; (B) Gram-stain of yeast cells; (C) Wet mount of yeast cells obtained from pure cultures. Note the vacuoles of yeast cells (black arrows) lacking BLBs; (D) Urease test performed on yeast cultures. BLBs: bacteria-like bodies.

Figure 3

Growth curves of different H. pylori strains in the presence of BB supplemented with 1%, 5% or 20% FBS or SS. (A) H. pylori J99; (B) H. pylori G-27; (C) H. pylori SS1 and (D) H. pylori H707. BB: Brucella broth; FBS: fetal bovine serum; SS: saline solution.

Figure 4

Candida growth curves in the presence of BB supplemented with 1%, 5% or 20% FBS or its absence. (A) C. albicans ATCC 90028; (B) C. glabrata ATCC 90030; (C) C. albicans VT-3; (D) C. glabrata LEO-37. The highest growth level was achieved using 20% FBS, followed by the cultures with 1% and 5% FBS. In the latter, there was no significant difference identified between these two growth curves p > 0.05; however, a highly significant difference was found between the growth curves in SS when compared with the other treatments p < 0.0001.

Figure 5

(A) Wet mount of H. pylori H707–C. albicans VT-3 24 h co-culture in BB-1%FBS showing Y-BLBs (black arrows) and H. pylori cells attached to pseudohyphae (red arrows). (B) Wet mount of C. albicans VT-3 cells from a pure culture (negative control) showing the absence of Y-BLBs. Movement of BLBs within yeasts is shown in Video S1. BB-1%FBS: Brucella broth supplemented with 1% of fetal bovine serum.

Figure 6

Mean percentage of Y-BLBs when H. pylori and Candida spp. were co-cultured in BB supplemented with 1%, 5% or 20% FBS concentrations or in SS. Mean percentages of Y-BLBs remained above 50% in the co-cultures supplemented with 1% FBS. No significant differences were observed between the percentages of Y-BLBs obtained in the co-cultures evaluating the same strains of H. pylori in the presence of different FBS concentrations. Different letters indicate significant differences (p < 0.05).

Figure 7

Means of Y-BLBs were obtained for all bacterial strain and yeast strain combinations cultured in BB supplemented with 1%, 5% or 20% FBS or SS during 48 h. This figure shows the means of Y-BLBs obtained when co-culturing H. pylori J99 and C. glabrata ATCC 90030 strains. The higher means of Y-BLBs for this bacteria–yeast combination were observed at 24 h and 48 h. Culture medium supplemented with 1% or 5% FBS produced the higher means of yeast cells harboring bacteria.

Figure 8

Mean of Y-BLBs identified in the co-cultures incubated in BB-1%FBS for 48 h. There is no significant difference in Y-BLB means when comparing co-cultures carried out with H. pylori J99 or H707 strains with all yeast strains. The higher means of Y-BLBs were obtained in the co-cultures carried out with these two strains and the two C. glabrata strains. Results are expressed as mean ± SD. Means with different letters are significantly different (p < 0.05).

Figure 9

Images showing the results of FISH analysis using a fluorescent probe specific for H. pylori. Yeast cells obtained from a H. pylori G-27–C. glabrata ATCC 90030 strain co-cultured in BB-5%FBS. (A) Hybridization of the H. pylori specific probe within C. glabrata ATCC 90030 cells (white arrows); (B) C. glabrata ATCC 90030 from a pure culture (negative control) showing the absence of hybridization of the fluorescent probe; (C) Pure H. pylori G27 strain used as positive control (red fluorescence). Blue fluorescence corresponds to the binding of aniline blue to 1–3ℬ-glucans of yeast cells. BB-5%FBS: Brucella broth supplemented with 5% FBS.

Figure 10

Image of a 2% agarose gel showing the amplicons obtained after amplifying the H. pylori 16S rRNA gene from total DNA extracted from Candida cells co-cultured with the different strains of H. pylori cultured in BB supplemented with 1%, 5% or 20% FBS or in SS. M: molecular weight markers, B: blank (master mix, primers, PCR grade water), C−: negative control (DNA from pure C. glabrata ATCC 90030), C+: positive control (DNA from pure H. pylori J99). Lanes 1–4 correspond to the amplicons obtained from DNA extracted from yeasts co-cultured with H. pylori. (A) Co-cultures in BB-1%FBS; (B) co-cultures in BB-5%FBS; (C) co-cultures in BB-20%FBS; (D) co-cultures in SS.

Figure 11

A, B, C and D correspond to a same microscopic field photographed at 1 s intervals. Cell viability, indicated by green fluorescence, of H. pylori harbored within C. albicans VT-3 after co-culturing them during 48 h. White arrows indicate yeast cells harboring intravacuolar H. pylori. The change in the position of H. pylori, at 1 s intervals, within the yeast can be observed (white arrow). In addition, the figure also shows yeast cells lacking intracellular H. pylori (red arrows).