Localization of Staphylococcus inside the vacuole of Candida albicans by immunodetection and FISH.

In our previous study, two bacteria B1 and B2 were excised from two amphotericin B-treated Candida albicans Y1 and Y2, respectively. Bacteria were identified as B1: Staphylococcus hominis and B2: Staphylococcus haemolyticus according to their biochemical characteristics and detection and sequencing of Staphylococcus-specific genes. In this study the intracellular origin of staphylococci inside the vacuole of yeast was examined. Polyclonal antibodies against S. hominis and S. haemolyticus were raised in rabbit and used for detection of staphylococcal proteins in protein pool of yeasts by western blotting (WB). Fluorescein-isothiocyanate (FITC)-conjugated antibodies were used for bacterial localization inside yeast's vacuole by direct immunofluorescence (DIF). Fluorescent in situ hybridization (FISH) with Staphylococcaceae -specific probe was performed for validation of immunodetection results. WB results showed occurrence of several proteins in protein pool of yeasts that were similar to staphylococcal proteins such as those with molecular weight of 57.5 and 66 kDa. Fluorescent microscopy showed interactions of FITC-antibodies with intracellular staphylococci which appeared as green spots. Hybridization of staphylococcal- specific probe with bacteria inside yeasts' vacuole confirmed immunodetection results. Detection of staphylococcal proteins and genes inside Candida albicans yeast indicates existence of intracellular bacteria inside the vacuole of yeast. These results suggest C. albicans as the potential reservoir of medically important bacteria.

Localization of H.pylori within the vacuole of Candida yeast by direct immunofluorescence technique.

BACKGROUND: Reports indicate that H.pylori is able to invade the eukaryotic cells and establish inside their vacuoles. In this study, FITC-conjugated IgY-Hp was used to localize H.pylori inside the vacuole of Candida yeast. Presence of intracellular H.pylori inside the new generations of yeast cells was also examined by light microscopy and Live/Dead BacLight staining method. METHODS: A single colony of fresh yeast culture was cultivated in a 100-µl medium containing yeast extract and N-acetylglucoseamine supplemented with fetal bovine serum. After 12-hr incubation at 37℃, FITC-conjugated IgY-Hp was added. After 3 hours, 10 µL of yeast suspension was smeared on a glass slide, air-dried and examined by fluorescent microscopy. Wet mounts of yeast culture and Live/Dead BacLight stained preparations were examined by light and fluorescent microscopy, respectively. Photographs were taken from the fast-moving H.pylori inside the yeast vacuoles. RESULTS: Fluorescent microscopy showed that FITC-conjugated IgY-Hp could enter yeast cells and specifically react with H.pylori, localizing the bacterium inside the yeast vacuole. Photographs taken from wet mounts observed by  light and fluorescent microscopy showed fast-moving H.pylori cells in the vacuole of mother as well as daughter yeast cells. The intravacuolar H.pylori cells stained green, showing their viability. CONCLUSION: Intracellular life of prokaryotes inside eukaryotes has been described as an evolutionary phenomenon with a great impact on bacterial persistence despite environmental stresses. Results of this study demonstrated the specific interaction of FITC-conjugated IgY-Hp with H.pylori cells and the bacterial localization inside the Candida yeast vacuole. The intracellular bacteria were viable and existed in the vacuole of next generations of yeast cells. It appears that H.pylori is well-equipped to dwell within the vacuole of eukaryotic cells where it is protected from stressful conditions, including antibacterial therapy. Presence of H.pylori inside the vacuole of new generations of yeasts demonstrates the intimate relationship between the two microorganisms, resulting in bacterial inheritance as part of the vacuolar content of yeast cells.

Immunodetection of Helicobacter pylori-specific proteins in oral and gastric Candida yeasts.

BACKGROUND: Human gastric epithelium and immunocytes have been recognized as the sole specialized eukaryotic cells that host Helicobacter pylori (H. pylori). The aim of this study was to provide further evidence for our previous proposal regarding the occurrence of H.pylori inside the yeast vacuole, verifying the viability of the intravacuolar H.pylori by western blotting. METHODS: Light microscopy and polymerase chain reaction (PCR) were used for primary detection of nonculturable H.pylori in 11 Candida yeasts (six oral and five gastric). Boiling was used for extraction of proteins from yeasts and the control H.pylori. Western blot analysis was recruited to assess the occurrence of H.pylori-specific proteins in protein pool of yeasts, using IgY-Hp raised in hens and IgG1-Hp raised in mice. RESULTS: The fast-moving bacterium-like bodies (BLBs) were identified as H.pylori by amplification of H.pylori 16S rRNA, ureAB, vacA s1, and ahpC genes from the whole DNA of yeasts. Analysis of the sequenced products of 16S rRNA gene amplified from the yeast and H.pylori isolates of patient #2 showed 100 % homology with the corresponding sequences of the reference H. pylori strains in GenBank. According to published data, it was plausible to assign the H.pylori-specific proteins, detected by western blot analysis, as thiol peroxidase (21 kDa), peroxiredoxin (AhpC) (26 kDa), urease-A subunit (UreA) (32 kDa), vacuolating cytotoxin A (VacA) small subunit (36 kDa), and VacA large subunit (56 kDa). CONCLUSION: Results of this study show that inside yeast, H.pylori expresses proteins and is viable. These proteins appear to serve as powerful tools to help H.pylori to establish in the vacuole of yeast where it can reach nutrients and multiply. The intimate relationship between H.pylori and Candida yeast which began long time ago, could have led to the establishment of H.pylori inside the yeast vacuole before invading human cells.