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farah thesis for candida albicans pothogenesis
1. BY
Msc student: Farah Mohammed Abdullah
Prof. Dr. Laith AbdulHassan M.Jawad
A Study Of Potential Activity Of Cinnamomum
Verum Extracts Against Expression Of ERG3
And ERG11 Genes in Candida albicans During
The Oral Mucosal Infection
2.
3. Introduction
Fungi are eukaryotes that their cell structure and
cellular metabolism are similar to animal cells. Some
species of the fungus can cause serious infections which
are hard to both treat and diagnose. Candida albicans is a
potentially deadly fungal pathogen, particularly in immune-
compromised patients.
The therapeutic options for fungal infections are quite
limited compared to bacterial infections (Khodavandi,etal.,
2017).
Oral candidiasis is an opportunistic infection of the oral
cavity often caused by the overgrowth of Candida, a yeast-
like fungus commonly found in the gastrointestinal tract of
humans, as normal skin flora and in mucous
membranes, Candida albicans (C. albicans) accounts for
around 80% of infections and can colonise the cavity, either
alone or in combination with non-albican species,
including Candid aglabrata And Candida tropicalis
4. The typical colonization rate of C. albicans varies with age. In
neonates it is 45%; in healthy children 45–65%; in healthy
adults 30–45%; in denture wearers 50–65%; in patients living
in acute or long-term facilities,
such as nursing or residential homes, 65–88%; and in
immunocompromised patients, such as those with HIV and/or
undergoing chemotherapy for acute leukaemia, it is 95% and
90% respectively (Patil, et al 2015).
C. albicans and to a lesser extent
other Candida species are present in the oral cavity of
up to 75% of the population.6 In healthy individuals this
colonization generally remains benign. However, mildly
immunocompromised individuals can frequently suffer
from recalcitrant infections of the oral cavity.
5. The use of medicinal plants as a treatment option is an ancient practice
and continues in the modern world. Ancient Asians including Sri
Lankans, Indians, and Japanese people especially used these
phytochemicals as their therapeutic agents.
The use of medicinal plants as therapeutic alternatives has helped many
populations that do not have any access to novel treatments and drugs
of high cost and low availability. So, the use of plants for the treatment of
diseases/infections has been empirically employed worldwide.
These oral infections with Candida species are termed “oral candidiasis”
(OC). Such infections are predominantly caused by C. albicans and can
affect the oropharynx and/or the esophagus of persons with dysfunctions of
the adaptive immune system. Indeed, HIV is a major risk factor for
developing OC. Further risk factors for developing OC include the wearing
of dentures and extremes of age.
6. .
In the past few years, with scientific advances, more
research was conducted to introduce new compounds
with medicinal properties, allowing scientists to find
new, effective, alternative medicinal compounds with
low side effects (Wijesinghe et al,. 2020).
Increased fungal resistance to classical drugs, drug
toxicity, and the costs involved justify the search for
new approaches to developing antifungal drugs.
Among those new approaches, essential oils are one
of the most promising groups of natural compounds for
the prevention and treatment of fungal infection (Silva
et al., 2011).
7. Candida albicans, like any other pathogen, is influenced by the expression
of various genes that contribute to its virulence. Candida albicans
possesses a range of genes associated with pathogenicity, and
understanding their roles is crucial for comprehending the mechanisms by
which this fungus causes disease. Here are some key genes and their
relationship to Candida albicans pathogenicity:
Erg3 and Erg11 are two important genes involved in the ergosterol
biosynthesis pathway of Candida albicans. Ergosterol is a crucial
component of the fungal cell membrane, and its biosynthesis is a target for
antifungal drugs.
Erg3 (C-5 sterol desaturase): The Erg3 gene encodes the enzyme C-5
sterol desaturase, which is responsible for the introduction of a double bond
at the C-5 position of the sterol precursor molecule.
Erg11 (lanosterol 14α-demethylase): The Erg11 gene encodes the enzyme
lanosterol 14α-demethylase, which is a key enzyme in the ergosterol
biosynthesis pathway.
8. Both Erg3 and Erg11 are critical for the synthesis of ergosterol, which is
vital for maintaining the integrity and functionality of the fungal cell
membrane. Disruption of these genes or alterations in their expression can
impact the susceptibility of Candida albicans to antifungal agents and
contribute to the development of drug resistance. Understanding the
mechanisms underlying the function and regulation of Erg3 and Erg11 can
help in the development of new antifungal strategies and the identification
of potential drug targets.
9. Methodology
EXPERIMENTAL DESIGN
Collect 50 oral swab
samples (infected)
25 swab samples as
negative control
(healthy)
identification and
diagnosis by
different routs
devided five groups
and treated by five
concentration
(12.5,25,50,100,
200,400)mg/ml
determine MIC
isolation of RNA OF
CANDIDA
ALBICANS
cDNA extractio n of
RT-PCR ANALYSIS
FOR DETECTION
THE QUALNTITY
OF ERG11,ERG3
GENES
10. Media
1.Sabouraud's Dextrose Agar (SDA)
2. Cornmeal Agar (CMA)
3.Candida Agar
4. CHROMagar Candida
5. Yeast extract Agar (YEA)
6-Biochemical identification (ApiCandida)
7-Total RNA extraction using Easy-spin™ (DNA free) total RNA
extraction Kit
11. Sample Preparation
Firstly, purchased the cinnamon bark from markets then washed and
cut into small pieces. Cinnamon bark was dried using a drying cabinet at
40–42 C for 3–4 days. The dried plant material was ground into powder.
Preparation of Ethanolic Extract
As much as 300 g of cinnamon bark crude (the powder of cinnamon
bark) was weighed and placed into a flask. Ethanol solvent with a
concentration of 96% was added to the flask until the plant materials were
submerged by the solvent. The extraction process took 3 h. After that used
filter paper wattman 1 to filtration the solution. The ethanol extract was
combined and evaporated using oven at 45 C.
16. Experimental work
Preparation of plant extract
Figure (4-7): A:wash the plant powder with ethanol for 24 hours, B:Filter the soaked with gauze
and cotton to remove impurities, C:The liqued is poured into dishes, D:Dry at room temperature
for 3 days, E:Dry by oven at 40 degrees for 2-3 hours, F:weight the powder, G:Dissolve the
powder with DMSO and dilution
17. Figure (4-1) Inhibitory diameters of the( N)nystatine and
(A)amphotircine (B) against Candida yeast cells on the muller
hintinagar
18. Figure (4-2) Inhibitory diameters of the used the concentration group
alcoholic extract of cinnamon bark (3.1256.25,12.5 , 25 , 50 , 100 ,
200,400) against Candida yeast cells on the muller hintin agar
21. Figure (4-9): explain the gene expression of ERG3 and effect of
three different concentration of cinonmum extract
22. Multiple comparisons test to compare the gene expression of ERG3 gene with
and within the negative control and different concentration by graph prism
program version 8.
23. Figure (4-10): explain the gene expression of ERG11 gene and effect
of three different concentration of cinonmum extracts
24. multiple comparisons test to compared the gene expression of ERG11 gene with and with in the
negative control and different concentration by graph prism program version8ns: Not significant
25. Figure ( 4-11) showed ERG3 gene expression in (-) control (DMSO.) When
compared with different concentration of cinonmum . Statistics using
GRAPH PAD PRISM 6 software
26. Figure(4-12): showed gene expression of ERG11 gene whne
compared between negative control (DMSO) and three
concentration of extract . GRAPH PAD PRISM 6 software
27. Characteristic two bands of RNA were successfully obtained compared to a single
band of DNA (extracted individually for a comparison reason).
28. Gel electrophoresis for first strand cDNA, (Agarose 1%, 10min. at 100 voltage and then
lowered to 70 volts, 60min.) Visualized under U.V light after staining
29. Amplification plot of ERG3 &ERG11 as targets; and House Keeping
JaneMas a house-keeping gene by the Mx3005P Strata gene system
(in different concentrations 12.5,25,50 mg/ml)
30. Amplification plot of three genes: ERG3 &ERG11 and
ACT by the Mx3005P Stratagene system
31. Figure(4.17): linking the zone inhibition of different concentrations
(12.5, 25, and 50) mg/ml results with the molecular changes for
ERG3 gene that are shown
32. Figure (4.18): showed the relation between the zone inhibitions and fold
change of gene expression of ERG11 gene
33. Conclusions
According to the results of this study we
conclude:
1. The most prevalent type of Candida, according
to our findings, is the yeast (C.albicans) found in
the mouth and around the teeth.
2. The alcoholic extract of cinnamon bark is highly
effective and has a high inhibitory effect against
yeast
3. Through its inhibitory effect on the gene
encoding proteins its resistance to traditional
antifungals.
4. Which showed a decrease in the level of gene
expression in both ERG3, ERG11 genes.
34. Recommendations
1. Study of the chemical components involved in the
composition of cinonmum and isolation of the active
compound.
2. Testing the effect of the active substance in
cinonmum on genes related to virulence factors for
Candida yeast.
3. Researching the possibility of combining cinonmum
extract with another plant extract to be the efficacy is
double.
4. To test the effectiveness of cinonmum extract in
vivo.
5. Using cinonmum extract as a medicinal treatment
after combining it with mouthwash and toothpastes
for the treatment of various oral diseases using the
biotechnology technique.
