Student Number: Seat Number:

RHODES UNIVERSITY

FACULTY OF PHARMACY

SUPPLEMENTARY/AEGROTAT EXAMINATION: JANUARY/FEBRUARY 2022

BIOCHEMISTRY, MICROBIOLOGY, AND IMMUNOLOGY - PC 221

PAPER 2

Examiner: Dr N. Mutingwende MARKS: 100

DURATION: 3 HOURS
Moderator: Professor R. Tandlich PASS: 50

INSTRUCTIONS

1) The use of an English concise dictionary is NOT allowed.

2) ANSWER ALL QUESTIONS.

3) Please start each new full question on a separate page.

4) Please do NOT use red ink anywhere on your script.

5) Remember to FILL in the question numbers on the FRONT COVER of your answer
book.

6) ALL answers, including drawings, must be written in ink. Work written in pencil
WILL NOT be marked.

7) Write your student and seat number on Each page of the answer book.

8) PLEASE HAND IN THE QUESTION PAPER.

PLEASE DO NOT TURN OVER THIS PAGE UNTIL TOLD TO DO

SO.

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QUESTION 1
Describe the cycle of a lysogenic virus. Provide a short and theoretical justification for your
answer.

 Attachment if the virus to the host

 Injection of viral DNA

 Viral DNA is integrated into host DNA

 Viral DNA is replicated with host DNA at cell division

In a lysogenic cycle, the phage genome also enters the cell through attachment and
penetration. Instead of killing the host, the phage genome integrates into the bacterial
chromosomes and become part of the host.

(8)

QUESTION 2

Environmental Health and Biotechnology Researchers (EHBR) at Rhodes University are

studying dormant ‘persister’ cells produced by Salmonella bacteria. Bacteria form these cells
when they are exposed to stresses such as antibiotics. By studying persister cells, EHB
researchers hope to understand the link between these dormant cells and antibiotic resistance,
as well as develop treatments that target persister cells directly. You are requested, in your
capacity as a PC221 student, to advise the EHBR group of researchers before they embark on
their research. Your discussion/advice leads should include but not limited to the following:
Mode of action of persister cells, difference between persisters and resistance, how persistent
infections lead to resistance, targeting persisters and persistent infection treatment. Begin
your introduction with the following phrase

Most antibiotics act only on…. Bacterial cell walls (e.g. penicillin). Bacterial cell walls have a
unique structure not found in eukaryotic cells. Persister cells are subpopulations of cells that
resist treatment and become antimicrobial tolerant by changing to a state of dormancy or
quiescence. In their dormancy, persister cells do not divide. The tolerance shown in persister
cells differs from antimicrobial resistance in that the tolerance is not inherited. The mechanism
of persister tolerance is distinct from the well understood mechanisms of antibiotic resistance.
Persisters during infections can contribute to antibiotic treatment failure. Put simply, persisters
may survive the antibiotics and cause relapse of the infection. Since persisters are such a small
fraction of a microbial population, they may be undetectable at the end of antibiotic treatment;
consequently, it may appear that the infection is cleared. However, when the antibiotic
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treatment is stopped, persisters can resume growing and re-establish the infection, leading to
relapse. The relapsed infection will likely require a second course of antibiotic treatment. In this
way, persisters can also contribute to the development of antibiotic resistance. It is well known
that the more antibiotics are used, the more resistance will develop.

(8)

QUESTION 3
Describe the structure of the SARS-CoV-2 virion and virocell. Provide a short and
theoretical justification for your answer.

virion, an entire virus particle, consisting of an outer protein shell called a capsid and
an inner core of nucleic acid (either ribonucleic or deoxyribonucleic acid—RNA or
DNA). The core confers infectivity, and the capsid provides specificity to the virus.
Many virions are spheroidal—icosahedral—the capsid having 20 triangular faces, with
regularly arranged units called capsomeres, two to five or more along each side; and the
nucleic acid is densely coiled within. Other virions have a capsid consisting of an
irregular number of surface spikes and the nucleic acid loosely coiled within. Virions of
most plant viruses are rod-shaped; the capsid is a naked cylinder (lacking a fatty
membrane) within which lies a straight or helical rod of nucleic acid.

The virocell concept posits that viruses are cellular organisms and highlights the
capacity of viruses to create new genes to manipulate their cellular environment and to
create novel biological mechanisms². The virocell (or ribovirocell) corresponds to the
‘living form’ of the virus, whereas virions are in fact the equivalent of seeds or spores
for multicellular organisms¹. (8)

QUESTION 4
What are the main structural differences between the virion and the ribovirocell? Provide a
short and theoretical justification for your answer.
The virion is the infectious particle of a virus that is composed of a nucleic acid genome
(either DNA or RNA) surrounded by a protein coat called a capsid. The capsid is made
up of protein subunits called capsomeres that can be arranged in different ways to form
different shapes³. The virion is the extracellular form of the virus that can infect cells³.

On the other hand, the ribovirocell is a cell that produces virions but can still divide by
binary fission¹. In the ribovirocell, two different organisms coexist in symbiosis in the
same cell. The virocells or ribovirocells are the living forms of the virus, which can be
considered to be a living organism.
(8)

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QUESTION 5
What do you mean by epitope? Discuss the differences between affinity and avidity of an
antigen antibody reaction.

The structures present over the antigen that stimulate the production of antibodies are
called antigenic determinants or epitopes.

The interaction of an antigen antibody is a reversible binding process that requires

several non-covalent interactions like hydrogen bonds, electrostatic forces and
hydrophobic interactions. Affinity and Avidity between the antigen antibodies also play a
major role in their interaction. The potency of the reaction between a specific antigenic
determinant and its single combining site on the antibody determines its affinity. The
overall potential of binding of an antigen with many antigenic determinants to its
multivalent antibody determines its avidity. Normally antigen-antibody binding site on
antibodies are more or less flat and hence spacious so that they can attach large
complexes or structures.
(8)

QUESTION 6
Human genes responsible for producing complex biological molecules such as hormones,
enzymes and cytokines can be inserted into bacterial cells. These cells are easily grown to
high cell densities in large volumes and the desired therapeutic materials produced on a large
scale. Using a human-derived gene of interest; bacterial DNA as a plasmid vector and
Escherichia coli as the host bacterium.
Outline and discuss, step by step, how you would make use of the host bacterium machinery
as a mechanism to produce the desired therapeutic materials from the gene of interest on a
large scale. Include all the necessary enzymes involved and materials.
(10)
To produce desired therapeutic materials on a large scale using the host bacterium
Escherichia coli and a human-derived gene of interest, the following steps can be
followed:

1. Gene Isolation: Isolate the gene of interest from human DNA. This can be done using
techniques such as PCR (polymerase chain reaction) or gene synthesis.

2. Plasmid Vector Construction: Obtain a plasmid vector, which is a small, circular DNA
molecule capable of self-replication in bacteria. The plasmid should contain elements
such as an origin of replication, antibiotic resistance gene, and a promoter sequence to
drive gene expression. The gene of interest can be inserted into the plasmid using
molecular cloning techniques, such as restriction enzyme digestion and ligation.

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3. Transformation: Introduce the recombinant plasmid into Escherichia coli cells
through a process called transformation. This involves exposing the bacteria to a high
concentration of calcium chloride and heat shock, which makes the cells temporarily
permeable to foreign DNA. Some cells take up the plasmid DNA, incorporating it into
their own genome.

4. Selection and Screening: To identify transformed bacterial cells, a selectable marker

such as an antibiotic resistance gene can be included in the plasmid. After
transformation, the bacterial culture is plated on agar containing the appropriate
antibiotic. Only the transformed cells that have taken up the plasmid will survive and
form colonies.

5. Scale-Up Culture: Select a single colony from the transformed cells and inoculate it
into a small culture medium. Allow the bacteria to grow and multiply in the culture
medium under controlled conditions, such as temperature and pH. This step ensures that
the bacterial population is homogeneous and contains the desired plasmid.

6. Induction of Gene Expression: To initiate the production of the therapeutic material,

induce the expression of the gene of interest. This can be achieved by adding specific
inducers, such as IPTG (isopropyl β-D-1-thiogalactopyranoside), to the bacterial culture
medium. The inducer molecule binds to the promoter sequence on the plasmid,
activating the gene for expression.

7. Cell Harvesting: Allow the bacterial culture to grow to high cell densities, usually
monitored by measuring the optical density. Once the desired cell density is reached, the
cells are harvested. This can be done by centrifugation, where the cells are spun down
and separated from the culture medium.

8. Cell Lysis: Break open the harvested bacterial cells to release the intracellular
contents, including the therapeutic material. Cell lysis can be achieved by physical
methods (such as sonication or high-pressure homogenization) or by using enzymes such
as lysozyme or detergents.

9. Purification: Isolate the desired therapeutic material from the mixture obtained after
cell lysis. The purification process depends on the nature of the molecule and may
involve techniques such as chromatography, filtration, precipitation, or affinity-based
methods. The goal is to obtain a highly pure form of the therapeutic material.

10. Characterization and Quality Control: Perform various analytical tests to confirm
the identity, purity, and activity of the purified therapeutic material. These tests may
include protein gel electrophoresis, mass spectrometry, bioactivity assays, and
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biochemical analysis.

11. Formulation and Storage: Once the therapeutic material is purified and
characterized, it can be formulated into the desired dosage form (e.g., liquid, solid, or
lyophilized). Suitable storage conditions should be determined to maintain the stability
and activity of the therapeutic material over an extended period.

By following these steps, the host bacterium machinery can be effectively utilized to
produce the desired therapeutic materials on a large scale, making use of the gene of
interest and the capabilities of Escherich

QUESTION 7
Which is more primitive, innate immunity or adaptive immunity? Justify.
(8)
Innate Immunity
The innate immunity is primitive because all animals possess a primitive system of defense
against the pathogens to which they are susceptible. The defense is called innate immunity, it
is part of the immune system we are born with, and it is also called natural immunity. It is the
1st line of defense against any pathogens and infection in the body. Adaptive immunity only
responds to a wide range of pathogens that passed through innate immunity. Innate has no
memory whereas the adaptive immunity does have the property of memory.

QUESTION 8

Briefly discuss the antigen processing by MHC class I or class II pathway.

(6)
 The antigen containing endosomes are fused with the lysosome to form endolysosome,
the acidic pH of the endolysosome helps in the degradation of proteins into smaller
peptides.
 MHC class II molecules are synthesized in the endoplasmic reticulum and transported
to the endosomes with the help of invariant chain, which binds to the peptide binding
cleft of a newly synthesized MHC class II molecule.
 Class II molecules with bound invariant chain (CLIP) are transported to endosomes
and are degraded by proteolysis to release the invariant chain.
 The remaining part of CLIP is removed by HLA-DM present in the endosomes in
order to create space for peptide.
 Once CLIP is removed, the peptides are loaded over the MHC class II molecule.
 The MHC class II molecule bound to peptides is delivered to the surface for their
recognition by CD4+ T lymphocytes (humoral immune response).

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QUESTION 9

Discuss how the body fights against a bacterial or viral infection. Think about all the probable
mechanisms possible.

(8)
 Extracellular bacterial infection:
- Innate immunity: phagocytes can be stimulated to go and digest the invading
microbe, if it succeeds, the pathogen is destroyed successfully.
- If microbe is Gram- they can induce an inflammatory response or
complementary system and lectin pathway will be activated by the mannose
expressing bacteria.
- If the microbe escapes phagocytosis: Dendritic cells will help capture and
present the microbe to T-cell lymphocytes.
- The T-cell lymphocytes will then differentiate into T-helper cells and T-
cytolytic cells.
- The T-helper cell will activate the B-cells to secrete antibodies.
- If the phagocyte engulfs the microbe but fails to kill it; the bacteria will
continue growing and secrete foreign antigens inside the cells, the Natural
Killer cells will detect this infected cell, secrete interferon gamma and destroy
the infected cell along with the microbe.
 Intracellular microbes:
- Cell-mediated immunity is activated: cytotoxic T-cells
- For an infected cell; nucleated cell will activate CD8+ T-lymphocyte
- APC infected cell: Will activate CD4+ T-lymphocytes.
 Viral Infections:
- Viruses can only replicate within the cells, so the immune response is
intracellular adaptive immune response.
- Cell-mediated immunity is activated and mediated by cytotoxic T-cells
- The cell will process the viral proteins and present them to the MHC I
molecule and the CD8+ T-cells will recognize the MHC I loaded with peptide
and will destroy infected cell.
- Upon viral re-exposure, the cell will have made memory cells that produce
antibodies that can recognize the virus and destroy it before infecting cell.

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 QUESTION 10

A patient is undergoing the treatment of bacterial infections with an antibiotic drug. For a
while, he was responding to treatment. However, after six months, the patient relapsed: the
infection worsens even when the antibiotic's dosage increases. Based on what you have
learned in class:

(a) Suggest what has led to this possible mechanism that allowed the pathogens to become
resistant to the drug. (8)
Persisters during infections can contribute to antibiotic treatment failure. Put simply,
Persisters may survive the antibiotics and cause relapse of the infection. Since Persisters
are such a small fraction of a microbial population, they may be undetectable at the end of
antibiotic treatment; consequently, it may appear that the infection is cleared. However,
when the antibiotic treatment is stopped, Persisters can resume growing and re-establish
the infection, leading to relapse. The relapsed infection will likely require a second course
of antibiotic treatment. In this way, Persisters can also contribute to the development of
antibiotic resistance. It is well known that the more antibiotics are used, the more
resistance will develop.

(b) What would you propose as a new drug that would target these pathogens? Support
your reasons. (6)

QUESTION 11

Apart from using micro-organisms as synthetic factories for medicines, recombinant DNA
(rDNA) technology is used to produce pharmaceutically useful compounds of biological
origin not produced naturally by microorganisms.

(a) What is a recombinant protein? (3)

Single chimeric DNA formed by combining two or more different fragments of DNA
from diverse organisms is generally called as recombinant DNA and the method applied
to create recombinant DNA is called recombinant DNA technology.
(b) Briefly discus the steps involved in the synthesis of a recombinant protein such as insulin
. (5)
Molecular cloning is a process for creating recombinant DNA and generally involves the
following steps:
(1) Selection of a cloning vector
(2) Selection of a host organism
(3) Preparation of a vector DNA
(4) Preparation of DNA to be cloned
(5) Creation of recombinant DNA vector (having foreign DNA)
(6) Introduction of recombinant vector into host organism
(7) Selection of clones having insert vector.
(8) Screening and multiplication of recombinant clones with desired DNA inserts.

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(c) A growth cycle of a bacteria cell involves the production of both the primary and
secondary metabolites. What is the difference between primary and secondary
metabolites? Give an example for each.
(6)
Primary metabolites are small chemical compounds that are directly involved in the
growth, development, and reproduction of living organisms. Examples of primary
metabolites include proteins, enzymes, carbohydrates, lipids, and nucleic acids.
Secondary metabolites are organic compounds that are not directly involved in the
normal growth, development or reproduction of the organism. They are species-specific
and thus are different in different organisms. Examples of secondary metabolites include
alkaloids, flavonoids, terpenoids, tannins, and phenolics.

TOTAL MARKS: 100

END OF EXAMINATION PAPER

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