Difference between revisions of "Part:BBa K644001"

Revision as of 11:11, 26 October 2020

Candida albicans hyphal wall protein 1 (HWP1) gene

HWP1 is a Candida albicans cell surface protein gene. It is used in Candida albicans to mediate tight binding to oral epithelial cells. Hwp1 is covalently linked to cell wall glucan through a remnant of its GPI anchor. (Eukaryot Cell. 2006 October; 5(10): 1604–1610.) Hwp1 is known to cause biofilm formation in vivo in C. albicans.

We cloned HWP1 into the plasmid pCTCON2. We used homologous recombination and transformed it into EBY100 Yeast. We used pCTCON2 because it has Aga2, which helps express HWP1 on the surface of the yeast cell. pCTCON2 also has the pGal1 promoter so HWP1 is only expressed in the presence of Galactose. A TRP marker is used as a selectable marker to tell the difference between yeast cells with the pCTCON2 plasmid and yeast cells without the pCTCON2 plasmid. S. cerevisiae will express HWP1 in the presence of Galactose and will not when it isn't present.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 617
Illegal BamHI site found at 1
Illegal BamHI site found at 1053
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Using the pCTCON2 vector, we made a fusion protein of Hwp1 and Aga2, such that Hwp1 would be expressed and displayed on the surface of S. cerevisiae. pCTCON2 has a gal promoter such that the expression of Aga2-Hwp1 is controlled by the presence of galactose in the media. As shown below, only cells grown in the presence of galactose expressed Hwp1 and were able to adhere to one another in large clumps.

Figure 1: Control, non-induced Hwp1 expressing yeast. Dispersed, no major clustering.

Figure 2: Experiment, induced Hwp1 expressing yeast. 3-D cluster of yeast cells growing around and on top of other cells.

2020 XHD-Wuhan-China’s Contribution

We found three papers from literature related to the function of this part: BBa_K644001, and the results are as follows:

Introduction of paper1

In this paper，the author has studied Cigarette smoke condensate modulated HWP1, EAP1, and SAP2 expression

Results

Using quantitative RT-PCR, researchers demonstrated that CSC (Cigarette smoke condensate)-exposed C. albicans expressed high levels of HWP1 mRNA and that this gene expression increased with increasing concentrations of CSC. Based on the data showing that CSC increased C. albicans adhesion, growth, and biofilm formation, we sought to de- termine whether this took place through the regulation of certain genes. Figure 4 reveals that HWP1 gene expression significantly increased following exposure of C. albicans to CSC. The activation of this gene significantly (p<0.001) increased according to CSC concentration. As shown in Figure 4, a twofold increase in HWP1 gene expression was recorded with a concentration of 30% CSC, compared to that observed in the controls, and with 50% CSC, this also modulated by CSC.

T--XHD-Wuhan-China--literature figure 1.png

Reference

Semlali A , Killer K , Alanazi H , et al. Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1, HWP1 and SAP2 gene expression[J]. Bmc Microbiology, 2014, 14(1):1-9.

Introduction of paper2

In this paper，The author has studied HWP1, SAP6 and Rim101 gene expression pattern in C. albicans

Results

The effect of farnesol, CS and AL nanogels containing farnesol on the expression of HWP1, SAP6 and Rim101 genes was investigated using real-time PCR (Figure 7). The finding shows that expression of HWP1 and SAP6 genes in C. albicans treated with 300 mM concentration of farnesol, CS and AL nanogels containing farnesol decreased significantly in comparison with un-treated control group (p<.01). However, it was found the significant difference between the expression of SAP6 gene of C. albicans treated with CS nanogel and non-treated Candida was observed. The expression of Rim101 in Candida treated with CS nanogel significantly decreased as compared to the non-treated cells (p<.01), whereas, Rim101 C. albicans treated with farnesol and AL nanogel containing farnesol did not show any change in the gene expression level.

T--XHD-Wuhan-China--literature figure 2.jpeg

Reference

Fatemeh Nikoomanesh, et al.Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2019, VOL. 47, NO. 1, 64–72

Introduction of paper3

HCR is important for HWP1 expression under multiple growth conditions. HCR (HWP1 control region) is located 1410 bp upstream of the transcription start site of HWP1 gene. HCR is believed to be relevant to the regulation of the gene HWP1. The results showed that when HCR is knocked out, the expression of the gene HWP1 decrease under several growth condition.The reporter strain S is similar to strain -1902GFP except that the HCR region has been deleted.

Results

T--XHD-Wuhan-China--literature figure 3.png

Reference

Samin K , Bao N Q , Wolyniak M J , et al. Release of transcriptional repression through the HCR promoter region confers uniform expression of HWP1 on surfaces of Candida albicans germ tubes[J]. Plos One, 2018, 13(2):e0192260.

Introduction of paper4

In this paper，The author has studied Hwp1 requirement for biofilm integrity in vitro.

Results

We reported recently that an hwp1/hwp1 mutant produces biofilms in vitro with a slight reduction in biomass compared to the wild- type strain (11). To determine whether this defect is caused by the hwp1 mutation, we compared biofilm biomasses (means 􏰃 standard deviations of results from quadruplicate samples) from the wild-type (0.0089 􏰃 0.0023 g), mutant (0.0024 􏰃 0.0002 g), and reconstituted (0.0077 􏰃 0.0016 g) strains. The hwp1/hwp1 mutant produced a biofilm with threefold less bio- mass than the reconstituted strain (P 􏰂 0.006). The mutant biomass was also significantly reduced from that of the wild- type strain (P 􏰂 0.005). The reconstituted strain and wild-type control strain (CAI4-URA3) produced similar levels of biofilm biomasses. These results indicate that Hwp1 is required for normal biofilm formation. Biofilm visualization through CSLM confirmed the biofilm defect of the hwp1/hwp1 mutant (Fig. 1). The mutant produced a biofilm of 􏰆100 􏰊m in depth that contained few hyphae (Fig. 1A and C). Both hyphae and yeast cells were found in the medium surrounding the biofilm (Fig. 1E). Reconstitution with a wild-type HWP1 allele permitted production of a biofilm of 200 to 300 􏰊m in depth in which hyphae were readily apparent (Fig. 1 B and D). Therefore, the mutant defect in biofilm biomass is similar in magnitude to its defect in biofilm depth. The finding that cells are present in the biofilm supernatant suggests that Hwp1 may be required to retain cells within a biofilm.

T--XHD-Wuhan-China--literature figure 4.jpeg

FIG. 1. In vitro biofilm formation. Biofilms were grown under our standard conditions (13) in Spider medium and stained with concanavalin A for CSLM visualization. Artificially colored CSLM depth views, in which blue color represents cells closest to the silicone and red color represents cells farthest from the silicone, are shown in panels A and B, in which blue represents 0 􏰊m and red represents 300 􏰊m (panel A) or 500 􏰊m (panel B). CSLM side views are shown in lower panels C and D, in which the scale bars represent 50 􏰊m. Cells in the surrounding medium of the hwp1/hwp1 biofilm were visualized through phase-contrast microscopy at 􏰋400 magnification (panel E)

Reference

Clarissa J. Nobile, et al. Function of Candida albicans Adhesin Hwp1 in Biofilm Formation EUKARYOTIC CELL, Oct. 2006, p. 1604–1610

@@ Line 42: / Line 42: @@
 cells. </regulartext1> </div>
 </html>
-===<h1>2020 XHD-Wuhan-China’s Contribution</h1>===
-We found three papers from literature related to the function of this part: BBa_K644001, and the results are as follows:
-<h1> Introduction of paper1</h1>
-In this paper，the author has studied Cigarette smoke condensate modulated HWP1, EAP1, and SAP2 expression
-<h2>Results </h2>
-Using quantitative RT-PCR, researchers demonstrated that CSC (Cigarette smoke condensate)-exposed C. albicans expressed high levels of HWP1 mRNA and that this gene expression increased with increasing concentrations of CSC. Based on the data showing that CSC increased C. albicans adhesion, growth, and biofilm formation, we sought to de- termine whether this took place through the regulation of certain genes. Figure 4 reveals that HWP1 gene expression significantly increased following exposure of C. albicans to CSC. The activation of this gene significantly (p<0.001) increased according to CSC concentration. As shown in Figure 4, a twofold increase in HWP1 gene expression was recorded with a concentration of 30% CSC, compared to that observed in the controls, and with 50% CSC, this also modulated by CSC.
-<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--literature_figure_1.png |thumb|none|800px]]
-<h2>Reference</h2>
-Semlali A , Killer K , Alanazi H , et al. Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1, HWP1 and SAP2 gene expression[J]. Bmc Microbiology, 2014, 14(1):1-9.
-<h2> Introduction of paper2 </h2>
-In this paper，The author has studied HWP1, SAP6 and Rim101 gene expression pattern in C. albicans
-<h2>Results </h2>
-The effect of farnesol, CS and AL nanogels containing farnesol on the expression of HWP1, SAP6 and Rim101 genes was investigated using real-time PCR (Figure 7). The finding shows that expression of HWP1 and SAP6 genes in C. albicans treated with 300 mM concentration of farnesol, CS and AL nanogels containing farnesol decreased significantly in comparison with un-treated control group (p<.01). However, it was found the significant difference between the expression of SAP6 gene of C. albicans treated with CS nanogel and non-treated Candida was observed. The expression of Rim101 in Candida treated with CS nanogel significantly decreased as compared to the non-treated cells (p<.01), whereas, Rim101 C. albicans treated with farnesol and AL nanogel containing farnesol did not show any change in the gene expression level.
-<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--literature_figure_2.jpeg |thumb|none|800px]]
-<h2>Reference</h2>
-Fatemeh Nikoomanesh, et al.Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2019, VOL. 47, NO. 1, 64–72
-<h2> Introduction of paper3</h2>
-HCR is important for HWP1 expression under multiple growth conditions. HCR (HWP1 control region) is located 1410 bp upstream of the transcription start site of HWP1 gene. HCR is believed to be relevant to the regulation of the gene HWP1. The results showed that when HCR is knocked out, the expression of the gene HWP1 decrease under several growth condition.The reporter strain S is similar to strain -1902GFP except that the HCR region has been deleted.
-<h2>Results </h2>
-<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--literature_figure_3.png |thumb|none|800px]]
-<h2>Reference</h2>
-Samin K , Bao N Q , Wolyniak M J , et al. Release of transcriptional repression through the HCR promoter region confers uniform expression of HWP1 on surfaces of Candida albicans germ tubes[J]. Plos One, 2018, 13(2):e0192260.
-<h2> Introduction of paper4</h2>
-In this paper，The author has studied Hwp1 requirement for biofilm integrity in vitro.
-<h2>Results </h2>
-We reported recently that an hwp1/hwp1 mutant produces biofilms in vitro with a slight reduction in biomass compared to the wild- type strain (11). To determine whether this defect is caused by the hwp1 mutation, we compared biofilm biomasses (means 􏰃 standard deviations of results from quadruplicate samples) from the wild-type (0.0089 􏰃 0.0023 g), mutant (0.0024 􏰃 0.0002 g), and reconstituted (0.0077 􏰃 0.0016 g) strains. The hwp1/hwp1 mutant produced a biofilm with threefold less bio- mass than the reconstituted strain (P 􏰂 0.006). The mutant biomass was also significantly reduced from that of the wild- type strain (P 􏰂 0.005). The reconstituted strain and wild-type control strain (CAI4-URA3) produced similar levels of biofilm biomasses. These results indicate that Hwp1 is required for normal biofilm formation.
-Biofilm visualization through CSLM confirmed the biofilm defect of the hwp1/hwp1 mutant (Fig. 1). The mutant produced a biofilm of 􏰆100 􏰊m in depth that contained few hyphae (Fig. 1A and C). Both hyphae and yeast cells were found in the medium surrounding the biofilm (Fig. 1E). Reconstitution with a wild-type HWP1 allele permitted production of a biofilm of 200 to 300 􏰊m in depth in which hyphae were readily apparent (Fig. 1 B and D). Therefore, the mutant defect in biofilm biomass is similar in magnitude to its defect in biofilm depth. The finding that cells are present in the biofilm supernatant suggests that Hwp1 may be required to retain cells within a biofilm.
-<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--literature_figure_4.jpeg |thumb|none|800px]]
-FIG. 1. In vitro biofilm formation. Biofilms were grown under our standard conditions (13) in Spider medium and stained with concanavalin A for CSLM visualization. Artificially colored CSLM depth views, in which blue color represents cells closest to the silicone and red color represents cells farthest from the silicone, are shown in panels A and B, in which blue represents 0 􏰊m and red represents 300 􏰊m (panel A) or 500 􏰊m (panel B). CSLM side views are shown in lower panels C and D, in which the scale bars represent 50 􏰊m. Cells in the surrounding medium of the hwp1/hwp1 biofilm were visualized through phase-contrast microscopy at 􏰋400 magnification (panel E)
-<h2>Reference</h2>
-Clarissa J. Nobile, et al. Function of Candida albicans Adhesin Hwp1 in Biofilm Formation EUKARYOTIC CELL, Oct. 2006, p. 1604–1610
 ===<h1>2020 XHD-Wuhan-China’s Contribution</h1>===