Abstract:

With the effects of pathogenic bacteria increasing in the world and lack of imuno-

defense aiding the cause, the lack of properly developed antibiotics to combat Candida

albacans is deficient. Having large side effects to the consumers the need for a

secondary alternative for those suffering from the adverse affects of conventional

antibiotics, K-500 a derivative strain toxin secreted by Wiliopsis mrakii grants the

beneficial qualities of an antibiotic without the invasive distinct qualities of antibiotics.

Causing there to be a break down of proteins and inhibition of G1 sequencing in the

cells development, K-500 brings inhibition at the level of Fluconazole the treatment for

systematic C. albancans infection. Using in terms a byproduct, K-500 grants the ability to

regain control of a hyperactive yeast infection test through vaginal flora depletion and

invasive C. albancans strains. Under the right conditions affective levels of K-500

presented with pH preference of 4.0 and a temperature of 30 degrees Celsius, marks a

great importance of which can be expanded into the body to aid against a systematic

infection with potential targeted patients suffering from HIV as conventional antibiotics

are largely invasive and could eliminate beneficial bacteria to host. With junction of

amino acid pre loaded cells, K-500 was able to disrupt sensitive yeast membrane

permeability, Purification of the killer factor from W. mrakii K-500 to homogeneity may

allow more complete characterization of the bioactive peptide(s) thus contributing to

the understanding and synthesis of a novel antifungal agent with the potential for

therapy of Candida infections.

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Index:

Title Page………………………………………………………………………………………………………………….i

Abstract……………………………………………………………………………………………………………………1

Index…………………………………………………………………………………………………………………………2

Introduction……………………………………………………………………………………………………………..3

Interactive Nature and Production………………………………………………………………………….3-4

Mechanism of action……………………………………………………………………………………………....4-6

Secondary affects of antibiotics……………………………………………………………………………….6-8

Presence of Candida albicans in body………………………………………………………………………..8

Mutation……………………………………………………………………………………………………………….8-10

Antibiotic of Choice for Candida albicans…………………………………………………………….10-11

Methods described: Isolation for toxin…………………………………………………………….....10-11

Wiliopsis mrakii: K-500 Toxin……………………………………………………………………………….11-12

Reactivity in Candida albacans: results of culturing...............................................12-14

Conclusion……………………………………………………………………………………………………………….15

Reference…………………………………………………………………………………………………………………16

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Introduction:

Over the course of time there have been numerous references to the existence

of pathogenic bacteria through either prokaryotic or eukaryotic descent causing great

dismay in human organisms. From ancient studies there of made by eastern medicine

to the modern pharmaceutical companies i.e. Pfizer and Roche to name a few. Medical

expansion has been fascinating humanity as a means of expanding our livelihood and

our longevity of life on this Earth with the variable of quality leading the forefront. With

the knowledge gained through extensive research and analytical approach, researchers

in the fields of science and health have been able to extrapolate valuable discoveries in

which to this date alter our very existence i.e. antibiotics, stem cell research, and

genomics.

Interactive Nature and Production:

Through these ground breaking discoveries longevity and life span of humanity

in specific has exponentially been prolonged with quality. Now fundamental

information, we are left with nothing else but to expand knowledge base. Fighting the

very essence of the problem with itself revolutionizing the dimensions for success in

combating disease. Antibiotics are sanctioned as history makers for their abilities to kill

once lethal bacterial illnesses. Providing organisms secondary assistance against

pathogenic bacteria, “antibiotics because of their metabolic capabilities, many fungi play

beneficial roles, including making bread rise, producing antibiotics, and decomposing

dead organisms.” (PP:Microbiology 7th ED, pg. 3) Examples of such popularized by

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modern medicine, “Penicillin are widely used to treat a variety of infections, including

skin infections, chest infections and urinary tract infections Cephalosporin can be used

to treat a wide range of infections but are also effective in treating more serious

infections such as septicemia and meningitis.” (USNLM, 2002). Degrading the structure

of these pathogenic organisms, antibiotics break down degenerative cells and allow for

immune systems to recover and interact invasively eliminating threat.

The use of Fungi and yeast are largely used in the spectrum of medicine to create

in other words antidotes from the source itself, and are not limited to only one type of

bacteria but have been transformed and developed into many more specific kinds

present above. “Many known antibiotics are produced by actinomycetes (particularly by

representatives of the genus Streptomyces), which are filamentous soil bacteria. The

antibiotics are produced via secondary metabolic pathways, and originate from a small

number of simple precursors, including amino acids, small fatty acids, sugars, and

nucleic acids.” (ASU, 2006) Largely enough the source of beneficial nature of this

treatment is extraordinarily helpful but the side effects at times may out way the

benefits.

Mechanism of action:

In observational investigations biochemists and microbiologist come together

and form the environments where testing of pathogenic organisms and antibiotics are

symbiotically analyzed for characteristics such as, “cell wall synthesis; for example,

penicillin inhibits transpeptidation. Vancomcyn inhibits transglycsylation and

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transpeptidation. Bacitracin inhibits the regeneration of the carrier required for moving

the building blocks of the cell wall across the membrane.” (Alcamo, Guilfoile pg. 25)

These mechanisms of action break down in a sense the bacteria tat have acquired

mutagenic properties that the body cannot break down and allows for it to be exposed

for the immune system to take action upon it through its phagocytes. Not only is it

limited to these mechanisms of cell wall development but also protein synthesis, “

streptomycin is an example of an antibiotic that targets the ribosome in which mRNA,

tRNA, and amino acids are targeted. Binding to the ribosomal protein, this interferes

with the movement of the ribosome along the mRNA. As a consequence protein

synthesis is less accurate.” (Alcamo, Guilfoile pg. 25-26). Pivotal to the development of

basic cell life, protein synthesis is key as without the building blocks, amino acids, the

existence of a cell begins to dwindle and is more prone to the outside world it inhabits

for in the case of humans our immunological system. Directly coming in contact with

cells these inhibitors can be manipulated and targeted by researchers for sole purposes

of which “Antibiotics are either bactericidal -- they are able to kill the bacteria directly,

or bacteriostatic -- they prevent bacteria from reproducing. Broad-spectrum antibiotics

are effective against a wide range of organisms, whereas narrow spectrum antibiotics

are only effective against specific microorganisms. Broad-spectrum antibiotics are able

to target a structure or processes shared by several different bacteria and are therefore

useful if the causative organism is not known. Narrow spectrum antibiotics, however,

are only able to act on a specific molecule or process in the metabolism of a particular

type of bacteria that is unique to that particular species. Narrow spectrum antibiotics

5

are therefore more appropriate if the causative bacterium is known. Antibiotics work by

interfering with a process or aspect of metabolism within the organism they are

attacking.”(Perry, Margaret, Practice Nurse, 09536612, November 18 2011, Vol. 41,

Issue 19) In due time although they may be selective their is always a means of which

they can cause an over whelming effect on the present host requiring the antibiotic.

Secondary affects of antibiotics:

Research has always taken steps to the drawing board building on the essence of

what is already present. Through the help of biochemists’ the ground that has been

gained is still used and placed through isomeric compounds. Most significantly with

relevance to yeast development treatment the presence of toxin proteinaceus secretion

is vital in the case leading development of Wiliiopsis mrakii as a means of fighting

infection through unconventional approach instead using toxic secretion. This yeast

strain has been witnessed as an expression highly suitable when in contact with Candida

albicans; the leading cause for vaginal, oral, and phalange yeast infections. Expressing a

great deal of inhibition under specific conditions of pH and temperature Wiliiopsis mrak

marks an advantageous series of research studies in the path to using high efficiency

pharmaceuticals to progress into more selective reactions rather than invasive as

general first line defense antibiotics as have been witnessed.

Although using generalized conventional means of treatment for disease has

been widely practiced, it is fallacious as there is variance in all organisms, both host and

pathogen regardless of relevance. The effects of antibiotics on the body could lead to

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more harm then benefit as they may be too invasive and be counter productive

depleting normal floral both malignant and benign as mentioned by the FDA, “risk for

severe liver injury, adrenal insufficiency, and adverse drug interactions, clinicians should

no longer prescribe ketoconazole (Nizoral, Janssen Pharmaceuticals) tablets as a first-

line therapy” (FDA, 2013). “With approximately more than ten per cent of patients

whom thought to be allergic to antibiotics, most notably to penicillin’s, exposure

resulted in reactions ranging from rashes to anaphylaxis.” (Perry, Margaret, Practice

Nurse, 09536612, November 18 2011, Vol. 41, Issue 19) With the population developing

these reaction either through genetic inheritance or through secondary exposure the

importance at hand is pivotal in the withholding of public health.

Observing the paradox at hand of such diversity now, selective study must be

made amongst the large generalization that is public health. With resistance of

medication and treatment growing, new methods to combat disease must arise as

stated by the World Health Organization, “The results show high levels of E. coli

resistance to third generation cephalosporin and fluoroquinolones–two important and

commonly used types of antibacterial medicine–in the Americas. Resistance to third

generation cephalosporin in K. pneumoniae is also high and widespread. In some

settings, as many as 90% of Staphylococcus aureus” (WHO, 2014)

The mentioned above are just a few of the many bacteria and antibiotic

treatments that are administered to individuals but because of incorrect diagnosis,

patient ireesponsibility, and environmental mutations new and more invasive antibiotics

have been synthesized. The FDA states that now apart from regular side effects, “risk for

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severe liver injury, adrenal insufficiency, and adverse drug interactions, clinicians should

no longer prescribe ketoconazole (Nizoral, Janssen Pharmaceuticals) tablets as a first-

line therapy” (FDA, 2013). With the lines of defense dwindling, substitutes need to be

produced as a means of compensation either through the bodies self defense

mechanism of adaptive mutation or synthetically produced antibiotics.

Presence of Candida albicans in body:

Commonalities in host organisms are subject to Candida albicans, through which

skin, mucus membranes, vagina, and stomach house this fungus. Expressing a dimorphic

phenotype Candida albicans can express oval shape yeast characteristics as well as

branching hypha present in most species of fungi. Living in symbiosis, Candida albicans

doesn’t become hyperactive until the depleting of natural bacterial flora is in occurrence

causing infection. This could happen through a numerous amount of process, commonly

in women that are pregnant the hormonal unbalance could lead to protect biofilm

within the vagina to deplete premising C. albacans to grow invasively. The use of

prolonged antibiotics in the body since the antibiotics used will kill only the bacteria and

not any fungus this allows for C. albicans to gain a foothold over the local mucosa that it

inhabits. (J.Graf, 2010)

Mutation:

Within the mutation realm of bacteria there is a present structure made

reference to a plasmid, “small, double stranded DNA molecules that can exist

8

independently of the chromosome. Both circular and linear plasmids have been

documented, but their information is not essential to the host”. (PP:Microbiology 7th ED,

pg. 74) Although this genetic material isn’t exactly required it serves a purpose, which is

to give in fact to a, “Resistance factors are other groups of important plasmids that

confer antibiotic resistance properties of which genes code for enzymes capable of

destroying or modifying antibiotics.” (PP:Microbiology 7th ED, pg. 74) Precedent to the

already congruent nature of mutation this factor isn’t only limited to the expression of

one bacteria but can spread to the rest of the cultured environment and allow them to

be resistant to the already present antibiotics. This is the pandemic of knowledge that is

occurring all across the world and is limiting more the affects of these pharmaceuticals

because of not only cell evolvement but because of human careless nature. The mere

fact of these substances being abused in a sense slows the health and degenerative

acceleration becomes exponentially increased.

With mutations being very present and part of our daily lives the extent of which

we use conventional treatment for individuals has become a large pending crisis. In

order to produce more efficient and patient friendly pharmaceuticals the development

of which they are produced and mechanized in the first place must be reconfigured to

attack and use other methods of degeneration. Effectiveness of antibiotics most recently

expressed the decline in the bacterial aid defense against Candida albicans. “Candidiasis

is a fungal infection caused by yeasts that belong to the genus Candida. There are over

20 species of Candida yeasts that can cause infection in humans, the most common of

which is Candida albicans. Candida yeasts normally live on the skin and mucous

9

membranes without causing infection; however, overgrowth of these organisms can

cause symptoms to develop. Symptoms of candidiasis vary depending on the area of the

body that is infected.” (CDC, 2014) Being part of the natural floral of the human body

once it begins to grow out of control either through variance in personal well being,

through hormonal changes, pregnancy, sexual hygiene, and excessive depletion of

vaginal flora could lead to a overdeveloped yeast infection.

Antibiotic of Choice for Candida albicans:

As with infections now we tend to have antibiotics that are established as

primary lines of defense once our bodies have failed to fend of the threat. In the case of

Candida albicans the antibiotic of choice is as established as Fluconazole as mentioned

by the Oxford Journal, Fluconazole (an orally active triazole agent) is well established as

a first-line management option for both localized and systemic C. albicans infections.”

(Goa, K. L. & Barradell, L. B. Drugs 50, 658–90). Possessing a notation of limited invasive

nature to the host this drug of choice has gotten use for more than 36 years since its

discovery as a means of also being incorporated through systematic infection,

“development of a broad-spectrum antifungal agent active by both the oral and the

intravenous routes for the treatment of superficial and systemic infections.”

(Cooper K, Richardson K, …1990). This medication was well suited for individuals that

were both through infancy to geriatric phases. The ability of it to be placed

systematically through an IV granted a much more precise quick treatment than

conventional oral antibiotics through mucus membranes. Despite the success of this

10

antifungal pharmaceutical, the nature again of variance prevailed and substitutes need

to be synthesized.

Methods described: Isolation for toxin

Having given an over view of mechanisms of antibiotic production, mechanisms

of action, and side effects, the methods of which Valerie J. Hodgson, David Button and

Graeme M. Walker of Great Britain were able to conduct their findings were through

isolation agar, inhibition measurements, freeze drying, agar diffusion bioassay,

microtitrate plate assay, membrane damage assay, centrifuging, and incubating to

observe efficacy in the laboratory to treat Candida albicans. A less invasive treatment

along the same lines of morphology and not a compound that would hinder or stunt the

growth of an individual, Wiliiopsis mrakii strain k-500 displays a 21 to 24 strain killer-

sensitive relationships observed respectively when placed in in contact with vaginal

swab containing C. albicans. With an effective rate of 34-66% the reduction in growth

was expressed in the isolated agar that had been incubated for 3 days at 37 degrees

Celsius to simulate the levy accordance comparison to human inner bodily environment.

The mechanisms of which the K-500 Killer toxin worked dealt with its characteristics that

made it selectively unique to the other 24 strains studied.

Wiliopsis mrakii: K-500 Toxin

The selectivity of K-500 displays one only to its own, with variables of

temperature, pH concentration, concentration levels, and agar favor in its discerning

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success. 10-100 microliters of toxic solute secretion per strain was acquired and was a

direct result of the “F-Factor growth-related response in a minimal medium through the

killer factor was produced as cells progressed from late-exponential to early-stationary

phase. Protein levels in the extracellular medium mirrored toxin production and an

agitated system led to the highest titres of killer factor.” (Hodgson, Button, Walker, 141,

pg. 2009, 2003-2012). Attempts to filter the toxin out using gel-filtration

chromatography rendered unstable products on temperature increase making it more

difficult to contain at room temperature for medical purposes, i.e. 4 degrees Celsius it

was stable and well suited but as it was introduced in cultured medium through

temperature increase the stability began to dwindle with 5% of toxicity of host cell at

1.8 kDa of molecular weight be the smallest previously recorded making it much more

safe than in comparison to the toxin produced by S. cervisiae which has a kDa molecular

weight of 20.

Reactivity in Candida albacans: results of culturing

Through the abilities to spear a defensive nature the mechanism through which

K-500 acts through is one that truly captivates. “Affecting the exerted yeast the killer

toxin notably leaks K+ and ATP and disruption of macromolecular synthesis and the

toxin is thought to arrest cell proliferation in the G1 phase of the cell cycle.” (Hodgson,

Button, Walker, 141, pg. 2009, 2003-2012) This extraordinary find was remarkable as it

inhibits the growth and replication cycle of somatic cells in organisms from the second

origin of which they are produced in mitosis. Inhibits (1,3)-Beta-glucan synthesis

through the toxic protein secreted, the wall defense of the yeast is left open and

12

susceptible to the phagocytes of the body already present as time passed expressed by

fig. 7. Not allowing for more development to occur from newly synthesized organisms

the infection can stagger at a much less invasive speed opposing a conventional

antibiotic as expressed by the low rate and gradual increase of the yeasts deterioration;

fig 4. (Hodgson, Button, Walker, 141, pg. 2009, 2003-2012) Killer toxin K-500 was found

to be more active against stationary phase cells than cells in an exponential phase of

growth. If the killer factor, in common with other killer yeasts, acted at the level of the

cell envelope it is possible that fewer receptors were available for interaction with toxin

molecules in immature exponential cells, or that receptors had less affinity for the toxin

during active growth. (Hodgson, Button, Walker, 141, pg. 2009, 2003-2012) The killer

toxin maintained cells in lag phase and prevented, or inhibited, their progression into an

exponential phase of growth and an increase in cell numbers. The killer factor may, like

the K. lactic toxin, cause a arrest early in the cell cycle thereby preventing cells from

enlarging and reaching a critical size necessary for continued growth and cell division.

(Hodgson, Button, Walker, 141, pg. 2009, 2003-2012) The differential response between

different strains of C. albicans may be a result of different numbers of receptors being

available on sensitive cell walls for toxin interaction. (Hodgson, Button, Walker, 141, pg.

2009, 2003-2012)

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Conclusion:

The effects of antibiotics decreasing the nature of which we have to administer

treatment to individuals have reached a crest of importance. With more individuals

expressing intolerance to generalized antibiotics, a substitute must be implemented to

suffice for the demand of illness. Wiliopsis mrakii a strain of yeast which was used to

produce a proteinaceus toxin, K-500, displayed a nature of inhibition and determent to

growth for Candida albacans a prevalent and frequent producer of yeast infections.

Largely this toxin, K-500, suppressed the growth and volume of Candida albacans to

such an extent that the membrane damage of Candida albacans was used for periods of

only 24hrs and at low concentrations such as 20microleters giving a 65% average based

on estimate of graphical data for toxic production, due to its qualities of interfering with

cell wall development (1,3)-Beta-glucan synthesis and ceasing of mitosis at G1. I really

would recommend for this drug to be pushed for clinical study more and human trials if

safely filtered because at such low quantities and such effectiveness, it could be used for

patients that have problematic immunological systems such as HIV, which suffer from

opportunistic ailments such as Candida albacans. Further research needs to be

administered as this strain toxin could help save lives and facilitate general health for

the public once again.

References:

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