Candida albicans' Loss of the MNN4 Gene Does Not Affect Virulence

The impact of Candida Albicans' pathogenesis on how well the organism can interact with the body's immune system. Neutrophils and activated macrophages are some of the components. The recognition of the protein ligands present on the organism is necessary for these immune agents to become activated. Both N-linked and O-linked mannan proteins, which have the potential to act as ligands, have been found to be present in the fungus's cell wall. The CaMNN4 gene, which is involved in mannosylphosphorylation, was disrupted, and mnn4 null mutants were produced. The negatively charged phosphate group necessary to connect with the positively charged Alcian Blue dye was absent from the mnn4 null mutants. Despite this, the mnn4 null mutant was still able to interact with macrophages causing an immune response. PCR was used to amplify the DNA of the mn4 null mutants to enable clearer results. The MNN4 gene proved to cause a disruption in the cell wall of the C. Albicans by inhibiting the transfer of the mannosyl phosphate which altered the binding of the beta-1,2 mannose oligosaccharides to the acid labile side chains of the N-mannan. Mannosylphosphorylation is therefore not required in the virulence of C. Albicans.









INTRODUCTION



Fungi can be divided into three organisms: molds, yeasts, and mushrooms. Molds form a unicellular organism with hyphae. A collection of molds forms yeast. Examples of yeast include the Saccharomyces cerevisiae and the Candida albicans. The organisms are responsible for both beneficial and harmful effects on human beings. They can divide by either splitting into two daughter cells or via budding. Beneficially the S. cerevisiae has been used in the fermentation process which leads to the formation of beer products. Ascomycetes have been used in the baking industry for fermentation which causes dough to rise through the production of bubbles. Also, yeast act as sources of vitamins, specifically the B vitamins thus supplementing diets that are nutritionally deficient. The most groundbreaking benefit would be the ability of yeasts to undergo genetic manipulation without losing their structures and activity thus enabling molecular studies to be conducted (Narins, 2003, p. 248). Organisms like the S. cerevisiae and the C. Albicans can be genetically modified into desirable mutants which then allow for studies in molecular characteristics and pathogenic processes. One of the studies that are being conducted is that of identifying various virulence factors in the organisms thus advancing medical care.

Genetic studies with yeast have led to numerous discoveries. While some of them show life cycles similar to plant cells, others replicate in a similar fashion as animal cells do. Animal studies mainly utilise S. cerevisiae while the plant studies mostly focus on S. pombe (Narins, 2003, p. 250). Working with yeast in laboratories is an easy task due to their rapid growth which allows for the study of numerous life cycles. It is estimated that the organisms take about 2 hours to grow (Narins, 2003, p. 250). In addition to this, there exist some yeast cells that can be maintained either in their haploid or diploid states. DNA sequences can also be introduced or removed from the yeast very easily allowing for the formation of mutant strains that are different from the wild type. The practical that was carried out utilised the above aspects.

C. Albicans, an opportunistic pathogen (Wilson, et al., 2000, p. 65) is part of the normal flora in the mucous membranes of the respiratory tract, the female genital tract, and the gastrointestinal tract (Levinson, 2012, p. 389). The pathogen is oval in shape and possesses a single bud appearing as pseudohyphae, true hyphae or as yeast in tissue (Levinson, 2012, p. 390). Since the Can. Albicans is present on the skin, activities that predispose one to infection include skin penetrating objects such as needles for intravenous administrations and catheters both new and indwelling. Some of the symptoms of the infection include oral thrush, vaginitis, loss of nails when fingers and nails are infected, and diaper rash in children may also occur (Levinson, 2012, pp. 390-391). A risk factor in women is the use of antibiotics. Broad spectrum antibiotics lead to the death of normal flora at the vaginal mucosa. The Lactobacillus found in this region are involved in maintaining a low pH as the normal flora. The death of these important organisms leads to a rise in pH leading to the growth of the pathogen thus causing vaginitis. Other complications brought about by the infection include Candida esophagitis of the stomach and the ileum, endocarditis of the right side, endophthalmitis, and subcutaneous nodules (Levinson, 2012, p. 391).

The pathogenesis of C. Albicans leads to a 50% mortality rate in immunocompromised neutropenic individuals (Hobson, et al., 2004, p. 39628). On the contrary, the innate immunity of healthy immunocompetent individuals can eliminate the pathogen through the action of the neutrophils and macrophages. The activation of the macrophages occurs when their surface receptors interact with surface ligands on the C. Albicans which later leads to a cascade of immunological defence responses by the body. The ligands on the pathogen that have been identified to mediate this interaction include mannan, beta-1-6-glucan ligand, and the beta-1-3-glucan ligand (Hobson, et al., 2004, p. 39628). At times, for survival purposes, the C. Albicans causes the macrophages to form glycosylase enzymes and other genes influencing the effectiveness of its pathogenicity. Studies have shown that the structure of the wall of the C. Albicans is made up of mannoproteins, glucan, and chitin which exist in varying positions, secretions and expressions (Chaffin, et al., 1998). Figure. 1 shows the major components of the cell wall. Apart from phosphorylation, other processes that are involved in modifying the proteins are ubiquitination and glycosylation. As a result, the organism can acquire different morphological forms thus is refers to as being pleomorphic (Chaffin, et al., 1998). The transcriptional factors involved in conferring this characteristic include the myc-like EFG1 factor, the TUP1, and the RBF1 transcription factor (Chaffin, et al., 1998). As a result, the pathogen can exist in different conditions, in the body and outside the body, thus enhancing its pathogenicity.





Figure 1: Structural components of the cell wall of C. Albicans.

The basic components of the cell wall include the mannoprotein, glucan and the chitin as seen in the diagram.



The glycoproteins that enrich the outer cell wall of C. Albicans are modified by branching N-linked glycosylations which contain a fraction of mannosyl phosphate attachment to the macrophage (Hobson, et al., 2004, p. 39628). The N-linked mannan is made up of a backbone and a side chain. Components of the backbone include an alpha-1-6 polymannose while an alpha-1,3-linked oligomannosides and an alpha-1-2-linked oligomannoside made up the side chains (Hobson, et al., 2004, p. 39628). On the other hand, the mannosylphosphate is made up beta-1,2-linked mannose residues which are joined, via phosphodiester linkages, to its side chains (Hobson, et al., 2004, p. 39628). Acid hydrolysis transforms the N-linked mannan into an acid-labile fraction which contains a phosphodiester. The fraction contains two mannose side chains: an alpha-1,2-side chain and beta-1,2-linked side chain. Apart from the above, phospholipomannan are also involved in the production of the residues of beta-1,2-linked mannose which are made up of two components which are joined by a monnosylphospahate: a lipid and an unbranched beta-1,2-oligomannoside chain (Hobson, et al., 2004, p. 39628). The mannosylphosphate together with the beta-1,2-oligomannosides perform several functions which include: 1) binding to complement proteins, 2) adhesion of organism to enterocytes, 3) macrophage recognition by binding to macrophages, 4) inhibition of nitric oxide production and 5) stimulation of the production of tumor necrosis factor-alpha (Hobson, et al., 2004, pp. 39628-39629). The surface proteins on macrophage that are homologous to gelatin-3-receptors are also bound by the oligomannosides. All these elements are useful in the pathogenicity and disease progression of the C. Albicans in the host.

Mannosylphosphorylation proves to be one of the requirements in the process of antigen recognition of C. Albicans by macrophages. In the S. cerevisiae, two genes are instrumental in the mannosylphosphorylation of the N-linked mannan and the O-linked mannan thus facilitating pathogenicity of the organism: the ScMNN4 and the ScMNN6 (Hobson, et al., 2004, p. 39629). Homologous of the Saccharomyces cerevisiae MNN4 were made from C. Albicans to try and investigate the function of the MNN4 gene in a null mutant C. Albicans. The function of incorporating the mannosylphosphate is the S. cerevisiae is done by the MNN4 gene, and the practical seeks to determine whether the same role occurs in the C. Albicans. To eliminate the MNN4 gene from the C. Albicans, the method of blasting was used which led to the generation of an Aberdeen Fungal Group (AFG). The AFG obtained was diploid with only one deletion. The remaining deletion was done to ensure the C. Albicans analogue was a mnn4 null mutant that lacks mannosylphosphate on its surface. It was concluded that although the MNN4 gene alters the structural integrity of the yeast, it did not alter the virulence of the viable mutant. Ultimately, it is evident that the neither the beta-1,2-oligomannoosides nor the mannosylphosphate are instrumental in the virulence or the macrophage activation of C. Albicans.



2.0. RESULTS



2.1. Transformation of C. Albicans



To obtain MNN4 null mutants of C. Albicans, the transformation. The initial step involved identifying a homologous sequence of ScMNN4 in C. Albicans. The ScMNN4 was used to blast the C. Albican genome database to search for CaMNN4. A homolog of ScMMN4 was found in C.Albicans. An alignment of the two sequences showed that the two, CaMNN4 and the ScMNN4 sequence were similar.

Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation

An AFG2 strain was obtained from the Ura blasting had only one gene deletion. Since it is homologous, the other gene needed to be deleted so as to ensure the mutant obtained does not have the MNN4 gene. The MNN4 gene is the one responsible for mannosylphosporylation of the C. Albicans cell wall. The C. Albicans strain here is the CAI4 and acts as the parental or wild-type organism. The transformation process took a lot of time since one had to pass the reagents through a heated stock. The recovery from his stage accounted for the increased amount of time that was taken to complete the transformation. The shaking of the mixtures was done for several days since the organism had to be in its logarithmic phase of growth for the reaction to take place.

The AFG2+ contents were inoculated onto media plates containing 5-FOA agar and growth of colonies was observed after five days. Three colonies grew on the AFG2+ while two colonies grew on the AFG2+ B inoculation. There were no colonies on the AFG2A- and AFG2B- inoculants. It was expected that the colonies containing the URA3 would cause growth in the media plates while the URA3- transformants would not.



2.2. Restriction enzyme digestion and ethanol digestion of plasmid DNA



To make the circular plasmids linear, the pDH2 plasmid of the AFG is digested using restriction enzymes SacI and SacII. The aim of this step was to remove the disruption cassette from the plasmid and to make it linear so that transformation enzymes could act on the open reading frame effectively.

The step was done after the AFG had been used to generate a pDH2. The plasmid that was obtained had around 7600b.p. After digestion, the hisG-URA3-hisG cassettes also known as the Ura-blaster cassettes were removed after a successful transformation to form the null mutant had occurred.



2.3. Alcian Blue screening for transformants



To determine the presence of null MNN4 mutants, Alcian blue test was carried out on the primary transformants which are the AFG2+. The test allowed for the confirmation of the success of the transformation process. For transformation to have occurred, the negative phosphate groups in the cell wall of the transformants would have to be absent. The positive copper in the Alcian was responsible for the colour changes. The result bellow was obtained:



Transformation Supernatant Pellet



AFG3 1 light blue light blue

AFG3 2 light blue light blue

AFG3 3 light blue dark blue

Control 1 light blue dark blue

Control 2 clear dark blue



URA - Control 1

CA18 - Control 2





AFG3 1 and AFG3 2 showed light blue pellets and light blue supernatants. This occurred because one copy of the MNN4 gene was still present in the transformants. Therefore, the negative phosphate reacted with the positive copper in the Alcian blue dye to give the colour changes seen. The CA18 showed a clear supernatant since it was the control experiment thus it contained the MNN4 gene. AFG3 3 showed light blue supernatant and dark blue pellets. The dark colour indicated that presence of the phosphate group since the MNN4 gene had not been removed. In all the results, the presence of the light blue colour in the supernatant indicated the presence of the URA3 gene.



2.4. Extraction of Genomic DNA from C. Albicans parental strains



DNA genomes from the parent C. Albicans were extracted to form AGF2 and CA18 strains. The aim was to show that the transformants obtained were from the parental strain. To achieve this, electrophoresis of the strains was done to match the strains to the parent. Two bands occurred after electrophoresis thus indicating the presence of the parent DNAs in them. Bands also occurred at the top of the CA18 and the AFG2. These bands corresponded to the standard marker band thus showing that the parental DNAs were present in the strains.

The integrity of the genomic DNAs from the transformants. To prove that the DNAs were of high quality, the electrophoresis results above were used. The formation of the bands corresponding to the standard band proved the integrity.

Using the plasmid map of pDH2, the number of the digested DNA that transformed the C. albicans were calculated as below:



Calculation of transformation efficiency

TE = Number of transformants / ug of DNA

Volume of Plasmid = 30ul

Concentration of plasmid was 1ug/ul 🡪30ug of DNA

Bp for Ura-blaster cassette: 7600-2955=4645 bps =61% of the plasmids

(61 x 30) / 100 = 18.3ug of DNA

We used 10ul out of 30ul for each transformation

18.3 / 3 = 6.1ug

TE = Number of colonies / 6.1ug DNA

(TE for E.Coli ~ x109 CFU/500ng pDNA



Two bands from the cut plasmids were observed at approximately 5000bp and 300bp as expected in the electrophoresis of the cut plasmids. The third band was approximately at 8000bp. The first two bands were for the cut plasmids which showed that they were strong since they contained base pairs which were above the average number. The electrophoresis results were as below:







Figure 2: Results of gel electrophoresis for the four genes.



2.5. Preparing genomic DNA from AFG4 1 and AFG4 1 cultures



The aim of this part was to prepare genomic DNA from the two cultures: AFG4 1 and AFG4 2. The sample was obtained from the growing colonies which had been inoculated onto the 5-FOA agar media. The strengths of the DNA that was obtained from the two strains was tested using gel electrophoresis. The result was as below:



AFG4/2 AFG4/1 AFG4/2 AFG4/1

Figure 3: Gel electrophoresis of AFG4/1 and AFG4/2 DNAs.



One band as expected was observed because the sample had parental DNA thus proving it was of good quality. Since strains were obtained from the same parent, the DNA from the transformants had good quality leading to only one band. If several bands had formed, contamination would have been confirmed.

After this, cultures of the AFG4/1 and the AFG4/2 were prepared. The cultures were grown on YPD tubes and used later for phenotypic analysis.



2.6. PCR analysis of transformant



The AFG4/1 and AFG4/2 transformants and the CAI8 and AFG2 strains PCR reaction. The reaction would check if there was any contamination in the samples, check if there was any MNN4 gene remaining while amplifying the DNAs. The CAI8 was the wild type; the AFG2 was heterozygous while the AFG4/1 and AFG4/2 were heterozygous. A water control experiment was included on the PCR tube to help give credible results. The PCR amplified the DNA occurred with high effectiveness.

The PCR amplification indicated that there were high concentrations of DNA were present in CAI8 since the strain was a homologous wild strain. The column with AFG2 indicated high concentrations of DNA since the strain was diploid heterozygous with both hisG and wildtype gene.In the last two column of AFG4/1 and AFG/2, a low concentration of DNA resulted because the wildtype was deleted and only hisG was present. These two strains were homologous.



.



Line: C 1 2 3 4

Line 1-CAI8

Line 2- AFG2

Line 3-AFG4/1

Line 4 –AFG4/2

Line C-ControlFigure 4: Electrophoresis results of PCR products: CAI8, AFG2, AFG4/1, AFG4/2 and Water.



2.7. Staining kidney sections for presence of C. Albicans





Figure 5: MC359: Kidney section from a mouse infected with wild-type C. Albicans cells.



The pink cell shows C. Albicans yeast and filamentous forms whereas the blue background indicates the kidney cell. Hyphal formation is a fundamental virulence factor in C. Albicans. Staining of the sections by haematoxylin and gross inspection of the kidneys shows that there are numerous visible foci covering the renal cortex of the mice infected with the wild-type C. Albicans cells. The cell infected by C.albicans shows up as pink cells in colour against the background that was coloured blue.

The virulence of the results showed that that the MNN4 mutants survived longer.In CA18, the hyphae was present and the colour of the cells was pink. In the mnn4 null mutants, the hyohae was still present with the presence of pink cells.C. Albicans cells are represented as pink ones (yeasts and filamentous forms) against a pale blue organ background. This proved that deletion of the MNN4 gene did not affect virulence. All three strains including the wildtype formed hyphae, with one null mutant forming fewer, suggesting that the MNN4 gene is not necessary in facilitating virulence.

Transmission electron micrograph (TEM) confirmed the presence of a thicker cell wall lacking a fibrillar mannoprotein layer when comparing 4A and 4B. CDH15, the mnn4 mutant strain, lacked phosphomannan, which was able to be distinguished from the wildtype, CAF2-1.

By comparing 4A and 4B, it has been found that thicker cell wall that lacking a fibrillar mannoprotein layer was present by using Transmission electron micrograph (TEM).









2.8 Quantitative Alcian blue binding assay

CAI8 AFG2 AFG4/1 AFG4/2



Figure 6: Graph of the Alcian blue assay results on the four PCR products



The assay showed that there was less little binding between the positive copper in the dye and the lowly negative AFG4/1 and AFG4/2 strains. The reason for this was because the two strains lacked the MNN4 gene that codes for the negative phosphates found in the structural proteins. The CAI8 was more negative since it contained the gene thus the phosphate. CAI8 ended up binding the most amount of due to the diploid homologous genes of MNN4 present. The AFG2 contained the MNN4 gene on one of its diploid strands thus showed some binding since it had some negative charge, but not as much as that of CAI8. The results confirm that AFG4.1 strain had the greatest Alcian Blue bound per cell (38μg/cell), in comparison to CAI4 that had 17μg/cell bound, and AFG2: 3.67μg/cell.

After this, the four strains were analysed using the PCR to assess which ones were homozygous, heterozygous, and transformants. The screening made use of the fact that mnn4 null mutants lack mannosylphosphate thus failing to bind the Alcian Blue dye.

By comparing 4A and 4B, it has been found that thicker cell wall that lacking a fibrillar mannoprotein layer was present by using Transmission electron micrograph (TEM).





References

Chaffin, W. L. et al., 1998. Cell Wall and Secreted Proteins of Candida Albicans: identification, Function, and Expression. Microbiology and Molecular Biology Reviews, 62(1), pp. 130-180.

Hobson, R. P. et al., 2004. Loss of Cell Wall Mannosylphosphate in Candida Albicans Does not Influence Macrophage Recognition. Journal of biological chemistry, 279(38), pp. 39628-39635.

Levinson, W., 2012. Review of Medical Microbiology and Immunology. 12th ed. New York: McGraw-Hill.

Narins, B., ed., 2003. World of Microbiology and Immunology. New York: Thomson Gale.

Wilson, R. B., Davis, D., Enloe, B. M. & Mitchell, A. P., 2000. A recyclable Candida albicans URA3 cassette for PCR production-directed gene disruptions. Yeast, 16(1), pp. 65-70.