Temperatures Outside the Optimal Range for Helicobacter pylori Increase Its Harboring within Candida Yeast Cells

Abstract

Helicobacter pylori is capable of entering into yeast, but the factors driving this endosymbiosis remain unknown. This work aimed to determine if temperatures outside the optimal range for H. pylori increase its harboring within Candida. H. pylori strains were co-cultured with Candida strains in Brucella broth supplemented with 5% fetal bovine serum and incubated at 4, 25, 37 or 40 °C. After co-culturing, yeasts containing bacteria-like bodies (Y-BLBs) were observed by optical microscopy, and the bacterium were identified as H. pylori by FISH. The H. pylori 16S rRNA gene was amplified from the total DNA of Y-BLBs. The viability of intra-yeast H. pylori cells was confirmed using a viability assay. All H. pylori strains were capable of entering into all Candida strains assayed. The higher percentages of Y-BLBs are obtained at 40 °C with any of the Candida strains. H pylori also increased its harboring within yeast in co-cultures incubated at 25 °C when compared to those incubated at 37 °C. In conclusion, although H. pylori grew significantly at 40 °C, this temperature increased its harboring within Candida. The endosymbiosis between both microorganisms is strain-dependent and permits bacterial cells to remain viable under the stressing environmental conditions assayed.

Keywords: Candida; Helicobacter pylori; intracellular; stress; temperature.

Figure 1

Growth curves of H. pylori strains (A) J99, (B) SS-1, (C) G-27 and (D) H707 incubated at 4 °C, 25 °C, 37 °C or 40 °C in Brucella broth supplemented with 5% bovine serum. Results are expressed as mean ± SD. OD: optical density.

Figure 2

Growth curves of the four strains of Candida assayed: (A) C. albicans ATCC 90028 (B) C. glabrata ATCC 90030, (C) C. albicans of vaginal origin VT-3 and (D) C. glabrata of oral origin LEO-37 cultured at 4 °C, 25 °C, 37 °C or 40 °C in Brucella broth supplemented with 5% fetal bovine serum. Results are expressed as mean ± SD. OD: optical density.

Figure 3

Micrographs, obtained at 1 s intervals, of wet mounts from (A) pure culture of C. albicans VT-3 (yeast purity control), (B–D) H. pylori G-27 and C. albicans VT-3 co-cultures incubated at 37 °C, showing the change in position of bacteria-like bodies (BLBs) (red arrows). Additionally, it is possible to observe yeast cells lacking BLBs (blue arrows) and nuclei of yeast cells (black arrows). The movement of BLBs can be observed in Video S1.

Figure 4

Average of the mean percentages of yeasts cells harboring bacteria-like bodies (Y-BLBs) of the four Candida strains after co-incubation of H. pylori strains with Candida strains at 4 °C, 25 °C, 37 °C or 40 °C during 48 h. The highest percentage of BLBs was found in co-cultures incubated at 40 °C. It can be seen that the highest percentages were obtained with the co-cultures carried out with the H. pylori J99 strain in microaerobic conditions. Results are expressed as mean ± SD. Means with different letters are significantly different (p < 0.05).

Figure 5

Means of yeasts harboring bacteria-like bodies (Y-BLBs) obtained in H. pylori J99 and C. albicans ATCC 90028 co-cultures incubated for up to 48 h at 4 °C, 25 °C, 37 °C or 40 °C in Brucella broth supplemented with 5% fetal bovine serum. After calculating the Y-BLBs means for all combinations of H. pylori and Candida co-cultures, similar patterns were observed. Results are expressed as mean ± SD.

Figure 6

Means of yeasts harboring bacteria-like bodies (Y-BLBs) in H. pylori and Candida co-cultures incubated at 40 °C for 48 h. The higher means of Y-BLBs were observed when the H. pylori J99 strain was co-incubated with any of the four yeast strains assayed. Results are expressed as mean ± SD. Means with different letters are significantly different (p < 0.05).

Figure 7

FISH identification of intra-yeast H. pylori. (A) Pure culture of C. glabrata ATCC 90030, (B) H. pylori J99-C. glabrata ATCC 90030 co-culture after hybridization to detect H. pylori using the Hpy 5′-CACACCTGACTGACTATCCCG-3′ probe labeled with Cy3 (white arrows). Red arrow indicates a yeast cell lacking bacteria-like bodies. Green fluorescence corresponds to the ConA-FITC fluorochrome bind to chitin of the yeast cell wall.

Figure 8

Agarose gel (2%) showing amplicons obtained after PCR amplification of the H. pylori 16S rRNA gene from total DNA extracted from yeast cells after co-incubating bacterial and yeast cells for 24 h at (A) 4 °C, (B) 25 °C, (C) 37 °C or (D) 40 °C. M: molecular weight marker, B: blank (master mix, primers, PCR grade water), C-: negative control (pure C. albicans ATCC 90028 DNA), C+: positive control (pure H. pylori SS-1 DNA), lane 1: H. pylori J99-C. albicans ATCC 90028 co-culture, lane 2: H. pylori J99-C. glabrata ATCC 90030 co-culture, lane 3: H. pylori J99-C. albicans VT-3, lane 4: H. pylori J99-C. glabrata LEO-37.

Figure 9

Viability assay micrographs, obtained at 1 s intervals ((A) time 0 s, (B) time 1 s, (C) time 2 s, (D) time 3 s) a viable (green fluorescence) H. pylori J99 cell within the vacuole of C. glabrata LEO-37 strain yeast cell. Images depict the change in the position of the H. pylori cell within the vacuole of the yeast cell caused by the movement of the bacterium.