Candida albicans Infection Decreases The Expression Of The Na+-K+-2Cl– Cotransporter 1 In T84 and Madin Darby Canine Kidney Cells

The commensal human fungal pathogen, Candida albicans, prior to infect the human body, must penetrate the intestinal mucosal barrier. To do so, it needs to bypass the different protective mechanisms such as fluid secretion. The basolateral Na+-K+-2Cl– cotransporter 1 (NKCC1) is a key protein regulating fluid secretion in the intestine. We hypothesize that C. albicans decreases fluid secretion prior to invasion by inducing NKCC1 internalization. In our experiments, we used Madin Darby canine kidney (MDCK) cells expressing a GFP-NKCC1 fluorescent tag and T84 cells, a human colonic cell line. Cells were infected with 100,000 C. albicans for varying lengths of time, fixed, stained and mounted for fluorescence microscopy. The number of internalized vesicles was evaluated using FIJI. Our results show that in MDCK cells, C. albicans only increased NKCC1 internalization at the 30-minute time point (P\u3c0.05), all subsequent time points were not significant. Similarly, infecting T84 cells with C. albicans significantly induced NKCC1 internalization at the 30-minute (P\u3c0.05), 1 hour (P\u3c0.05), and 90-minute (P\u3c0.05) time points. Past 90 minutes, we observed a sharp decline in the number of internalized vesicles that continued to decrease through 6 hours of exposure to C. albicans. Finally, in C. albicans-T84 infected cells, using an immunoblot approach, we found that total NKCC1 protein expression was decreased by ~20% (P\u3c0.05) compared to uninfected cells. Our results suggest that C. albicans induces internalization of NKCC1, and subsequent degradation of NKCC1, which would decrease fluid secretion and allow adhesion, and invasion of the epithelium

Effects of Environmental Factors on Candida albicans Morphology: A Focus on Estrogen and Microgravity

During body invasion, C.albicans change their morphology from yeast to filamentous, but the environmental factors responsible for the change in morphology are not well characterized. During Sepsis, high levels of estrogen (E2) are recorded (~0.1 nM), in addition during spaceflight it has been shown that C.albicans become virulent. In this study, we aimed at characterizing the effect of estrogen and microgravity as environmental factors inducing filamentous growth. In our experiments, we grow C.albicans in minimum liquid media and use brightfield microscopy to observe the morphology. Microgravity is simulated using a clinostat. In an experimental series, we tested the effect of FBS (positive control) and estrogen on filamentous growth. We found that in the control only 1 out of 10 slides showed filaments. In the presence of FBS, we found filamentous growth in 10 out of 10 slides. In the presence of estrogen, filaments were observed in 8 out of 10 slides. In addition to FBS, the combination of FBS + E2 we found filament growth in 10 out 10 of the slides. However, in the presence of microgravity, we observed filaments in 9 out of 10 slides, meanwhile, we only observed 4 out of 10 slides without microgravity. In addition to microgravity, the combination of E2 + Microgravity, filaments were observed in 3 out of 10 slides. Meanwhile, we only observed 4 out of 10 slides with just E2. In conclusion, estrogen does not inhibit filament growth stimulated by FBS, but it prevents filamentous growth in microgravity

The Behavior of Candida Albicans Filamentation in Response to Estrogen and Fetal Bovine Serum Exposure in Liquid Media

C. albicans is an opportunistic fungus which under not yet well defined cues shifts morphology from yeast to filamentous and becomes invasive. During sepsis, estrogen levels are elevated. Thus, we hypothesized that estrogen could act as a signal for C. albicans to become filamentous. In this study, we used 3 media: minimum, spider, and YEPD to test the effect of estrogen and fetal bovine serum (FBS, positive control) on C. albicans filamentous growth. Our experimental setup was: control, 0.1 nM estrogen, 10% FBS, and FBS with estrogen. Every day for 2 days, we took 5 random pictures using a brightfield microscope and scored one if filaments were visible or zero if filaments were not visible. Our results show in YEPD that FBS and FBS + estrogen had significantly more pictures with filaments compared to control at day 1 and 2 (P=0.001, ANOVA Dunnett post hoc). In minimum, FBS + E2 had significantly less pictures than control (

Candida albicans induces internalization of the Na + -K + -2Cl – cotransporter 1 expressed in Madin-Darby Canine Cells

Candida albicans (C. albicans) is one of the most common human fungal pathogens, causing life threatening systemic infections in critically ill and immunocompromised patients. In the gut, fluid secretion participates in the body defense by flushing microorganisms and by maintaining the surface mucus hydrated. It is still unknown whether C. albicans decreases the host defense mechanism prior to invading intestinal cells. Previous work in the lab has demonstrated that the basolateral Na+ -K+ -2Cl - cotransporter 1 (NKCC1) plays a key role in the regulation of fluid secretion, and internalization of NKCC1 represents a potent pathway to blunt fluid secretion. We hypothesize that C. albicans, before invading intestinal cells, decreases fluid secretion by causing NKCC1 internalization. To test our hypothesis, we utilized Madin-Darby Canine Kidney (MDCK) cells stably expressing eGFP-NKCC1. MDCK were cultured on coverslips in 6 well plate. On the day of the experiment, MDCK were exposed to 50,000 C. albicans, for 15, 30 min,1 and 2 h hours. MDCK were also exposed to 100 nM phorbol 12-myristate 13-acetate (PMA) for 15 min as a positive control for NKCC1 internalization. Coverslips were fixed in 1% paraformaldehyde for 30 min and mounted for fluorescence microscopy. Fluorescent images were acquired using an inverted IX83 Olympus microscope equipped with a CCD camera. Exposing MDCK cells to C. albicans for 15 and 30 min did not result in NKCC1 internalization as compared to 100 nM PMA. Conversely, after 1- and 2-hours exposure to C. albicans, NKCC1 internalization was observed in some cells. Our preliminary experiments suggest that C. albicans induces NKCC1 internalization and may represent one mechanism by which C. albicans lower the intestinal defense mechanism. Gaining a better understanding of how C. albicans invades the mucosal barrier will greatly help developing new strategies for fighting candidiasis

Effect of Candida albicans infection on the plasma membrane expression of the Na+-K+-2Cl– cotransporter 1 (NKCC1) in T84 and Madin Darby Canine Kidney cells (MDCK)

C. albicans is a commensal human fungal pathogens. To infect the human body, it must penetrate the intestinal mucosal barrier. Fluid secretion is one of the intestinal defense mechanisms and NKCC1 is a key protein regulating fluid secretion in the colon. We hypothesize that, C. albicans, before invasion decreases fluid secretion by causing NKCC1 internalization. In our experiments, we used MDCK cells expressing a GFP-NKCC1, and T84 a human colonic. Cells were infected with 100,000 C. albicans for different times, fixed, stained and mounted for fluorescence microscopy. Images were acquired using an Olympus IX83 microscope equipped with a DP80 CCD camera. The number of vesicles was evaluated using FIJI. Our preliminary results show that in MDCK cells, phorbol 12 myristate 13 acetate (PMA), a positive control, induced a significant increase of vesicles containing NKCC1 (P\u3c0.001), whereas C. albicans did not significantly increase NKCC1 internalization at all time points tested (ANOVA, Dunnett\u27s Multiple Comparison). Similarly, PMA induced a significant increase of NKCC1 internalization (P=0.007) in T84 Cells. Infecting T84 cells with C. albicans, significantly induced NKCC1 internalization only at 90 min (P=0.01), but not at other time points. Our results suggest that C. albicans causes internalization of NKCC1 which would decrease fluid secretion. The non-significance at some time points may be due to the low number of replicates at our early time points, whereas at later time points we suspect that NKCC1 is already degraded and cannot be detected

The Effects of Estrogen (E2) on the Filamentous Growth of Candida albicans on Assorted Solid Medias

Candida albicans is a commensal yeast that lives within the human body. When exposed to specific environmental stimuli (ex. pH, temperature) the fungi will undergo a morphological change from yeast to filamentous (hyphal). Currently, C. albicans is the most prevalent human fungal pathogen, causing both mucosal and systemic infections. This infection, called candidiasis, can endanger high-risk groups, such as immunocompromised patients. While studied extensively, the relationship between C. albicans hyphal growth and estrogen (E2) has yielded mixed results amongst researchers. In our research, we attempted to provide clarity to this relationship by using a variety of solid media and studying how each affects E2’s influence on hyphal development. We used three solid media; Minimum, Spider, and YEPD. In our experiments, we found that C. albicans undergoes three distinct morphologies and variable behavior between the three media types. In Minimum media, we found that E2 is blocking filamentous growth after day two compared to no E2. In Spider media, E2 is blocking filamentous growth up to day three compared to no E2. Finally, in YEPD, E2 inhibits filamentous growth only at day two compared to no E2. In our conditions, E2 has an inhibitory effect on filamentous growth. Furthermore, our observations suggest that the inhibition is media-dependent. Currently, we are testing fetal bovine serum (FBS), which is known to induce filamentation, as a positive control to compare to the effects of E2

Effect of estrogen on Candida albicans growth cultured on YEPD solid media

C. albicans is a commensal fungus which under certain environmental cues shifts morphology from spores to filamentous and becomes invasive within the human body. During sepsis, blood estrogen (E2) becomes elevated. E2 is also known to promote tissue growth, thus we hypothesize that E2 may influence C. albicans growth. In the present work, we investigated the effect of estrogen on C. albicans colony sizes grown on yeast extract peptone dextrose (YEPD). Using bright field microscopy, images of five colonies in each condition were captured on day one and pictures of the same colonies were captured on day two. The diameter of each colony was computed using ImageJ and the surface area was calculated using Excel. On day one, one-way ANOVA shows no difference (P=0.09) in colony size for the 4 conditions tested (control 0.19±0.09 mm2, 0.1 nM E2 0.20±0.08, fetal bovine serum (FBS) 0.23±0.11, FBS+ E2 0.24±0.12). On the second day, the size of each colony significantly increased compared to day one (P\u3c0.001, paired t-test) for each individual condition (i.e., control day 1 versus control day 2). Comparing colony size increase (surface area on day 2- surface area day1) a significant difference among the 4 conditions P\u3c0.001, one-way ANOVA. Dunnett post hoc test shows no significant difference between control 2.41±0.29 mm2 vs E2 2.46±0.35 (P=0.8) but a significant difference between control 2.41±0.29 versus FBS 2.69±0.27 (P\u3c0.001) and control 2.41±0.29 vs FBS+ E2 2.71±0.35 (P\u3c0.001). In conclusion, our results show that FBS significantly increased colony growth in YEPD, but E2 had no significant effect on colony growth. @font-face {font-family: Cambria Math ; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent: ; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family: Times New Roman ,serif; mso-fareast-font-family: Times New Roman ;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-family: Calibri ,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:Calibri; mso-fareast-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family: Times New Roman ; mso-bidi-theme-font:minor-bidi;}div.WordSection1 {page:WordSection1;