INVESTIGATION OF THE PROTECTIVE ROLE OF COMPLEMENT SYSTEM

AGAINST CAMPYLOBACTER JEJUNI

STUDENT NAME: Sadia Akbar

STUDENT R. No. 43721
STUDY LEVEL: PHD
DEPARTMENT: Department of Microbiology

SUPERVISOR: Dr. Syed Kashif Haleem/Assistant Professor

DATE OF SUBMISSION: 25 06 2021

 CONTENTS
SUMMARY 2
1. INTRODUCTION 3
2. BACKGROUND AND GAP ANALYSIS/PROBLEM STATEMENT 5
2.1 Research Question/Hypothesis 6
3. OBJECTIVES 6
4. RESEARCH DESIGN AND METHODS 7
5. THE PROPOSED MODEL/FLOW CHART DIAGRAM 11
6. EXPECTED OUTCOMES 12
7. REFERENCES 13

1
SUMMARY
The human serum plays a critical role in the host innate defence against invading
microorganisms. An important element of serum-mediated immunity is a collection of more than
35 dissolved proteins, known as the complement system. These proteins usually circulate in
serum and are usually found in zymogen forms. Recognition molecules recognize the pathogens
and activate a cascade of reaction against the pathogen recognised. Activation of complement
results in the rapid clearance of infection by enhancing phagocytosis and generating
inflammatory responses as well as direct cell lysis via the formation of pores at the pathogen
surface. Campylobacter jejuni is the major cause of food born gastroenteritis known as
campylobacteriosis. The infection is usually mild in immunocompetent host and resolves within
several days, suggesting that the host innate mechanisms efficiently clear this pathogen.
Recently, it is gaining more attention because of associated postinfectious complications such as
irritable bowel syndrome, Guillain–Barré Syndrome and reactive arthritis as well as the
emergence of antibiotic-resistant Campylobacter spp. A better understanding of the protective
capacity of the complement system is of great interest to investigate alternative therapies to
treat infectious diseases.
The current knowledge about the role of complement proteins in providing protection against C.
jejuni is very limited. This study will examine the role of different complement proteins and the
contribution of different complement pathways in clearing C. jejuni from the body. The binding
of different recognition molecules will be investigated in order to identify the recognition
molecules most effective in recognising this pathogen and initiating the downstream
complement cascade. Further, the role of different complement activation pathways will be
investigated by tracing the downstream activation of the complement system till the Membrane
Attack Complex formation.
All the studies will be carried out in-vitro using Enzyme-Linked Immunosorbent Assay at the
Molecular Biology and Immunology Lab, Department of Microbiology, Hazara University
Mansehra. The binding of recognition molecules and complement activation will be detected by
using specific antibodies against these molecules.

2
1. INTRODUCTION
Campylobacter jejuni a gram-negative, motile, thermophilic comma-shaped bacilli, is by far the
most common Campylobacter spp. that is responsible for 90% of all foodborne campylobacter-
gastrointestinal illnesses, referred to as campylobacteriosis [1, 2]. C. jejuni asymptomatically
colonize the intestinal tracts of farm animals such as cattle, poultry, swine as well as domestic
pets including cats and dogs . Bacteria shed continuously in animal faeces and typically result in
contamination of food and water. Even though there are several sources of Campylobacter,
foodborne campylobacteriosis by C. jejuni mainly occurs through the ingestion of contaminated
dairy products, undercooked contaminated meat and meat products and/or handling of poultry
meat [3]. The disease is typically mild, characterized by abdominal cramps, bloody diarrhoea and
fever, and usually resolves in immunocompetent host within several days, suggesting that host
innate mechanisms contribute to efficiently control the pathogen. Often self-limited, C. jejuni
infections occasionally may lead to postinfectious complications including bacteraemia, reactive
arthritis, irritable bowel syndrome and Guillain-Barré Syndrome [4]. Moreover, many cases of
antibiotic-resistant Campylobacter spp have emerged recently which possess a serious threat to
human health [5, 6]. The current status of antibiotic resistance has been declared as a global
emergency by WHO and has warned that if the microbes continue to develop resistance at the
current rate, the majority of antibiotics will become ineffective within 20 years. Hence, exploring
the role of immunity in limiting microbial infections, to introduce other therapeutic strategies,
besides antibiotic therapy, is gaining more attention.
The human intestinal tract relies on both adaptive and innate immune systems to control
invasion across the mucosal barrier and to protect the internal milieu from harmful substances
and pathogens. One of the key components of the innate immune system, with direct bactericidal
activity, is the complement system. The importance of complement in defence against microbial
pathogens including diarrheal C. jejuni strains is well known. The complement system is a
complex innate immuno-surveillance system that provides protection against invading
microorganisms. It represents one of the key effector mechanisms of innate immunity that bridge
the innate and adaptive immune system [7]. The system is composed of more than 35 serum
proteins, normally present as inactive zymogens. This group of proteins is activated by a series of
the proteolytic cascade, that starts with the recognition of pathogen and leads to the production
of significant pro-inflammatory mediators (anaphylatoxins), labelling the pathogen for
phagocytosis (opsonization) with the help of various opsonins (e.g. C3b) and causing direct cell
lysis via the formation of membrane penetrating pores called Membrane Attack Complex (MAC)
[8]. Moreover, for an optimum response to the invading microorganisms, innate pattern
recognition receptors, such as Toll-like receptors (TLRs) are required. The complement system is
activated by three major pathways; the classical pathway, the lectin pathway and the alternative
pathway. The complement C1q initiates the classical pathway by binding to the antigen-antibody
complex. The lectin pathway is activated when the recognition molecules collectin 11 (CL11),
ficolins, and/or Mannan- binding lectin (MBL) adsorb to the carbohydrates moieties on pathogen

3
surface [8, 9]. The alternative pathway is initiated without any recognition molecule after the
spontaneous deposition of C3 protein on the surface of the pathogen. After activation, all three
pathways converge at the formation of C3 convertase, leading to a cascade of reaction, resulting
in the creation of a membrane attack complex [10].
Host innate defences undoubtedly play a role in clearing the infections caused by C. jejuni [11-
13]. However, it can be burdened by certain postinfectious complications such as irritable bowel
syndrome, Guillain–Barré Syndrome and reactive arthritis [4, 14, 15]. Recently, many cases of
antibiotic-resistant Campylobacter spp have been isolated from human infections [5, 6]. For
these reasons, understanding of the role of the Immune System in limiting Campylobacter
infections is gaining more attention [13]. Due to the emerging antibiotic resistance, many
scientists are diverting their attention towards immune therapy to seek alternative strategies to
overcome the antibiotic resistance issue. The complement system is one of the effective
strategies that has gained attention during recent years [16-19].

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2. BACKGROUND AND GAP ANALYSIS/PROBLEM STATEMENT
Currently, there is a considerable gap in our knowledge regarding complement-mediated
immunity against Campylobacter infections. There is a need to fully understand the role of
complement activation pathways and identify complement proteins that are useful in providing
protection against C. jejuni. So far, the roles of different complement pathways covering all
complement proteins have been identified against major human pathogens [20-24]. However,
Campylobacter species have remained neglected in respect to their interaction/response against
the complement system. Very few studies reported preliminary findings on the role of the
complement system as a whole during the late 20th Century and suggested that the complement
system may have a role in the clearance of the C. jejuni using human serum [25, 26]. Surprisingly,
none of the studies investigated or reported an in-depth picture on the role of the complement
system against this pathogen thereafter. During the last few decades, our understanding of the
complement system has improved significantly with the discovery of new pathways, recognition
molecules and regulators. With the latest developments in the complement system, their role
against different pathogens is regularly updated with the passage of time. Despite the new
findings, the knowledge of different complement system pathways and proteins remains limited
against C. jejuni. There is a need to investigate the in-depth role of the complement system
against this pathogen. This study will be focused on in-vitro activation of complement proteins,
involving the binding of different recognition molecules (C1q, MBL, Ficolin-L, Ficolin-H, Ficolin-M
and CL-11) at the surface of C. jejuni in order to determine the most effective recognition
molecules in activation of complement on the surface of this bacterium. Furthermore, the
complement cascade reaction will be traced down till the creation of membrane attack complex
(MAC) to highlight the importance of these recognition molecules in initiating complement attack
against this pathogen. Enzyme-Linked Immunosorbent Assay will be used for all these in-vitro
studies. To trace the binding of recognition molecules and complement activation assays (C3, C4,
C5 and MAC deposition assays), specific antibodies will be used against these molecules.
Elucidation of these aspects is critical for understanding the protective capacity of the
complement system and may provide new insights to the design of effective intervention
measures to treat C. jejuni infection paving the way for developing alternate therapeutic
approaches against antibiotic-resistant pathogens.

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2.1 Research Question
The complement activation patterns have been reported to vary against different
microorganism. For instance, complement protein having a protective role against one pathogen
are completely ineffective against others. Investigating these differences in complement
activation helps in understanding the host-microbe interaction, identifying susceptible groups
and designing immunotherapeutic strategies by boosting the effect of useful complement
proteins or regulating the effect of overreacting components causing self-damage.
The research question of the current project would be that whether there are any complement
protein which are more effective in activating complement system against C. jejuni.

2.2 Hypothesis
As evident from the patterns of complement activation of other pathogens, we hypothesise that
“some complement proteins may have a more effective role in the clearance of C. jejuni as
compared to others”.
It is important to identify these proteins and propose them for potential immunotherapeutic
candidates in future.

3. OBJECTIVES
i. To estimate the occurrence of C. jejuni in samples collected from local hospitals and
poultry farms.
ii. To investigate the binding of recognition molecules of the classical and the lectin pathway
of complement activation to C. jejuni.
iii. To further investigate the downstream activation cascade to determine the role of
pathways more effective against C. jejuni.
iv. To compare the complement recognition and activation patterns of the chicken and
human isolates with the control ATCC strain of C. jejuni.

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4. RESEARCH DESIGN AND METHODS
All the experiments will be performed in-vitro.

4.1 Bacteria
Different bacteria will be used in this study. Selected ATCC C. jejuni strain will be used as a control.
In addition, complement activation will also be assessed and compared on human and chicken
isolates of C. jejuni. Clinical isolates will be confirmed by different biochemical tests.
4.2 Stock Culture Preparation
Campylobacter blood-free selective agar base CCDA media will be used to culture C. jejuni from
the stock. For the stock culture preparation, a single colony of C. jejuni from Campylobacter
blood-free selective agar base (CCDA) media will be inoculated into a Nutrient broth, and
incubated overnight at 37oC. The selective supplement will be used in media to enhance the
growth of C. jejuni. After incubation, the bacterial growth culture will be mixed with 15% glycerol
and preserved at -80°C as 300 µl aliquots for future use.

4.3 Preparation of formalin-fixed C. jejuni

The C. jejuni strains used in this study will be fixed with 0.5% formalin. 10 µl of bacteria will be
taken from -80oC stock culture inoculated in liquid broth medium and incubated overnight at
37oC. The obtained culture will be centrifuged at 3000rpm for 10 min. Then, the supernatant will
be discarded and pellet will be washed with Phosphate Buffer Saline (PBS) three times and
suspended at room temperature for 1-3 hours in 0.5% formalin in PBS. The bacteria will be
centrifuged again after formalin fixation followed by washing twice with PBS, and resuspension
in the coating buffer (pH 9.6; 35mM NaHCO3, 15mM Na2CO3) by adjusting the OD at 0.6 as
described by Lynch et al. [27].

4.4 Human Serum collections

Blood will be collected from healthy individuals and immediately transferred on ice for 1-3 hours,
to prevent complement protein activation. The serum will then be separated from blood by
centrifuging at 7500 rpm for 7-10 min and will be stored at -80oC for further use.

4.5 Positive and negative controls

For the negative control, the heat-inactivated serum will be prepared by separating a small
portion of each serum sample and treating at 56oC for 30 min to denature the complement
proteins.
Specific positive control will be used for each recognition molecule, including Mannan (Sigma)
for MBL, N-acetyl BSA (Promega) for L-ficolin, FCN-1 specific mAb (Hycult) for M-ficolin, FCN-2
specific mAb GN4 (Hycult) for H-ficolin and zymosan (Sigma) for CL-11. For C3 and C4 activation
assays, mannan (Sigma) and/or zymosan will be used.

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4.6 Enzyme-Linked Immune Sorbent Assays (ELISAs)
Different ELISA techniques will be used throughout this project, including solid-phase binding
assays for the detection of the binding of complement system recognition molecules to C. jejuni,
and complement activation assays to detect the levels of downstream cascade products C3b,
C4b, C5b and Bb on the surface of C. jejuni.

4.6.1. Solid Phase binding assays

Solid-phase binding assays will be carried out according to the protocols as described by Ali et
al., [24]. To detect the binding of recognition molecules, on the surface of C. jejuni, ELISA
microtiter plates (Nunc Maxisorb) will be coated overnight at 4oC with 100 μl of formalin-fixed C.
jejuni (OD=0.6) along with 100 μl of each positive control suspended in the coating buffer (pH
9.6: 15mM Na2CO3, 35mM NaHCO3). The residual protein binding sites will then be saturated by
adding blocking buffer (250 μl of 1% Bovine serum albumin (BSA) (Sigma) in TBS (pH 7.4: 140mM
NaCl, 10mM Tris) to the plates and incubating for 1-3 hrs at room temperature. Plates will be
washed in three cycles with 250 μl of wash buffer (5mM CaCl2 and TBS with 0.05% Tween 20).
All the test sera will be serially diluted in blocking buffer, and 100 µl of test and control sera will
be incubated with the plates in duplicates at 37oC for 90 min, followed by three cycles of washing.
Mouse anti-human MBL, rabbit anti-human ficolin-H, rabbit anti-human ficolin-L, rabbit anti-
human ficolin-M, mouse anti-human CL-11, and rabbit anti-human C1q, diluted in washing
buffer, will be added to each well in concentration suggested by manufacturers, incubated at
room temperature for 90 min, then washed thrice with wash buffer. Then, the primary antibodies
will be detected by incubating the plates at room temperature for 90 min, with diluted alkaline
phosphatase (AP) conjugated goat α-mouse and goat α-rabbit antibodies (Sigma-Aldrich),
followed by washing. Finally, AP will be detected by adding 100μl of colorimetric substrate ƿ-
nitrophenyl phosphate (pNPP) (Sigma) and left for 10 min at room temperature. The optical
density (OD) will be measured at 405nm by BioRad microtitre plate reader.

4.6.2. Complement activation assays

Complement activation assay will be performed to measure C3, C4 and C5 cleavage on the
surface of C. jejuni using methods as described by Haleem et al., [23]. Nunc Maxisorb plates will
be coated with 100 μl/well of formalin-fixed C. jejuni (OD=0.6). Mannan or zymosan sugar (Sigma)
(10 μg/ml) suspended in coating buffer will be used as a positive control along with bacteria and
left overnight at 4oC. Then, the residual protein binding sites will be blocked with 1% BSA in TBS
buffer (blocking buffer). Plates will be washed three times, and serially diluted sera from healthy
individuals will be added to the plates [diluted in Barbital Buffer Saline (BBS) (145mM NaCl, 4mM
barbital, 1mM MgCl2, 2mM CaCl2, pH 7.4)] and incubated at 37oC for 90 min. After washing,
bound C3b, C4b, C5b, Fb and MAC will be detected by using rabbit anti-C3c, rabbit anti-C4c, rabbit
anti-C5b, rabbit anti-fB and rabbit anti-MAC in different experiments followed by incubation at
room temperature for 90 minutes and washing with wash buffer. Then, 100 μl of diluted AP-

8
conjugated goat anti-rabbit (Sigma) will be added to the plates followed by incubation for 90 min
at room temperature and washing. Then, the extent of C3b, C5b, C4c, Bb and MAC deposition
will be determined, each in a different experiment, by adding p-nitrophenyl phosphate (pNPP)
substrate (Sigma) in a volume of 100 μl, and left for 10 minutes at room temperature. Finally, the
OD of the reaction will be measured at 405nm using a microtiter plate reader.

4.7 Phagocytosis assay and bactericidal activity assay of serum complement

Phagocytosis and serum bactericidal assays will be performed by methods described by Ali et al.,
[24] to examine the effect of variation in complement activation among different isolates on
overall bacterial clearance by serum or neutrophils. Neutrophils will be isolated from healthy
human blood using Histopaque 1119 and 1077, as suggested by the manufacturer’s manual.
Isolated neutrophils will be washed with Hank’s balanced salt solution (Invitrogen) thrice and
resuspended at a concentration of 106 cells/ml. The in-vitro killing of C. jejuni will be measured
by a decrease in bacterial count. Bacteria will be opsonised by incubating them with 20% of
healthy human serum collected from individuals of different age groups. Neutrophils will be
mixed with pre-ionised and non-ionised C. jejuni with a 250 μl final volume. The mixture will be
incubated at 37oC in a shaking incubator. 20 μl of the mixture will be separated from the mixture
at different time points up to 2 hours, separated by an interval of 30 minutes, and plated onto
agar plates to determine the viable count of bacteria at different time points.
To investigate if serum complement alone can kill C. jejuni, and whether killing is in concurrence
with binding assays of recognition molecules and complement activation assays, bacteria will be
incubated with 20% sera from healthy individuals at 37oC in a shaking incubator. Samples will be
taken from the mixture at different time points up to two hours, separated by half an hour
interval. The decrease in the bacterial count will be determined by plating bacteria onto agar
plates.

9
4.7 Availability of facilities
This research will be performed at the Molecular Biology and Immunology laboratory at the
Department of Microbiology, Hazara University. This lab contains all the required consumables
and equipment i.e., -20 and -80 lab freezers, Refrigerator, Class II biosafety cabinet, normal
incubator, CO2 incubator, autoclave, ELISA microtiter plate reader, double beam
spectrophotometer etc. and other necessary chemicals.

4.8 Time frame

S.No Activity Duration

1 Sampling Already done

2 Experimental work 1 year

3 Data interpretation and thesis writing 6 months

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5. THE PROPOSED MODEL/FLOW CHART DIAGRAM

Preparation of Serum Preparation of formalin fixed C. jejuni

• Collection of blood from healthy • Overnight incubation of C. jejuni strain

individuals in liquid media
• Storage on ice for 1-3 hrs to avoid • Centrifugation at 3000 rpm for 10 min
complement activation • Washing pellet with BPS and
• Centrifugation at 7500rpm for 7-10 suspended in 0.5% formalin for 1-3 hrs
min • Centrifugation and resuspension of
• Storage of sera at -20 oC for future use pellet in coating buffer

Investigation of the
Complement’s role
against C. jejuni

Phagocytosis assay
Solid Phase Binding assay • Incubation of the bacteria with sera
(opsonization) and Neutrophils at
Use of ELISA technique to check the
37 oC in a shaking incubator
deposition of recognition molecules
(MBL, ficolin-H, ficolin-L, ficolin-M, C1q • Taking samples from the mixture at
and CL-11) on the surface of C. jejuni different time points and plating
onto agar plates, to determine the
decrease in the bacterial count
ELISA

Serum Bactericidal Assay (SBA)

Complement activation assay
• Incubation of bacteria with 20% sera
Use of ELISA technique for
at 37 oC in a shaking incubator
complement activation to determine
which of the complement pathway • Taking samples from the mixture at
contribute more in fighting C. jejuni different time points and plating
infection onto agar plates, to determine the
decrease in the bacterial count

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6. EXPECTED OUTCOMES
1. Detection of the complement pathways in general and complement proteins in particular
which are essential in clearing C. jejuni from body.
2. These complement proteins will be suggested for future research as potential candidates
for immunotherapy.
3. Designing such strategies for the future could offer alternate treatment approaches besides
conventional antibiotic therapy and help in controlling the trend of antibiotic overuse to
eventually overcome the increasingly high resistance of pathogens against antibiotics.

12
7. REFERENCES
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and prevention methods-new molecular targets for innovative antivirulence drugs?, Appl Microbiol
Biotechnol, 104 (2020) 10409-10436.
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(2015) 998-1012.
[3] Humphrey T., O'Brien S., Madsen M., Campylobacters as zoonotic pathogens: a food
production perspective, International journal of food microbiology, 117 (2007) 237-257.
[4] Blaser M.J., Campylobacter jejuni and related species, Principles and practice of infectious
disease, (2005) 2548-2557.
[5] Efimochkina N.R., Stetsenko V.V., Sheveleva S.A., Formation of the Resistance of
Campylobacter jejuni to Macrolide Antibiotics, Bulletin of experimental biology and medicine, 169
(2020) 351-356.
[6] Alaboudi A.R., Malkawi I.M., Osaili T.M., Abu-Basha E.A., Guitian J., Prevalence, antibiotic
resistance and genotypes of Campylobacter jejuni and Campylobacter coli isolated from chickens
in Irbid governorate, Jordan, International journal of food microbiology, 327 (2020) 108656.
[7] Hillion S., Arleevskaya M.I., Blanco P., Bordron A., Brooks W.H., Cesbron J.Y., Kaveri S.,
Vivier E., Renaudineau Y., The Innate Part of the Adaptive Immune System, Clinical reviews in
allergy & immunology, 58 (2020) 151-154.
[8] Abbas A.K., Lichtman A.H., Pillai S., Cellular and molecular immunology E-book, Elsevier
Health Sciences, 2021.
[9] Merle N., Church S., Fremeaux-Bacchi V., Roumenina L., Complement system Part I—
molecular mechanisms of activation and regulation, Front. Immunol. 6 (2015) 262, in, 2015.
[10] Merle N.S., Church S.E., Fremeaux-Bacchi V., Roumenina L.T., Complement System Part I
- Molecular Mechanisms of Activation and Regulation, Front Immunol, 6 (2015) 262.
[11] Iovine N.M., Pursnani S., Voldman A., Wasserman G., Blaser M.J., Weinrauch Y., Reactive
nitrogen species contribute to innate host defense against Campylobacter jejuni, Infection and
immunity, 76 (2008) 986-993.
[12] Zilbauer M., Dorrell N., Boughan P.K., Harris A., Wren B.W., Klein N.J., Bajaj-Elliott M.,
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defensins 2 and 3, Infection and immunity, 73 (2005) 7281-7289.
[13] Hameed A., Human Immunity Against Campylobacter Infection, Immune network, 19 (2019)
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[14] Butzler J.-P., Campylobacter, from obscurity to celebrity, Clinical microbiology and infection,
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[15] Skirrow M., Campylobacter enteritis: a" new" disease, Br Med J, 2 (1977) 9-11.
[16] Bordron A., Bagacean C., Tempescul A., Berthou C., Bettacchioli E., Hillion S., Renaudineau
Y., Complement System: a Neglected Pathway in Immunotherapy, Clinical reviews in allergy &
immunology, 58 (2020) 155-171.
[17] Schwaeble W.J., Lynch N.J., Clark J.E., Marber M., Samani N.J., Ali Y.M., Dudler T., Parent
B., Lhotta K., Wallis R., Farrar C.A., Sacks S., Lee H., Zhang M., Iwaki D., Takahashi M., Fujita
T., Tedford C.E., Stover C.M., Targeting of mannan-binding lectin-associated serine protease-2
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[18] Ali Y.M., Hayat A., Saeed B.M., Haleem K.S., Alshamrani S., Kenawy H.I., Ferreira V.P.,
Saggu G., Buchberger A., Lachmann P.J., Sim R.B., Goundis D., Andrew P.W., Lynch N.J.,
Schwaeble W.J., Low-dose recombinant properdin provides substantial protection against
Streptococcus pneumoniae and Neisseria meningitidis infection, Proceedings of the National
Academy of Sciences of the United States of America, 111 (2014) 5301-5306.
[19] Mastellos D.C., Ricklin D., Lambris J.D., Clinical promise of next-generation complement
therapeutics, Nature reviews. Drug discovery, 18 (2019) 707-729.
[20] Trouw L.A., Daha M.R., Role of complement in innate immunity and host defense, Immunol
Lett, 138 (2011) 35-37.
[21] Carroll M.V., Sim R.B., Complement in health and disease, Adv Drug Deliv Rev, 63 (2011)
965-975.
[22] Lambris J.D., Ricklin D., Geisbrecht B.V., Complement evasion by human pathogens, Nat
Rev Microbiol, 6 (2008) 132-142.
[23] Haleem K.S., Ali Y.M., Yesilkaya H., Kohler T., Hammerschmidt S., Andrew P.W., Schwaeble
W.J., Lynch N.J., The Pneumococcal Surface Proteins PspA and PspC Sequester Host C4-
Binding Protein To Inactivate Complement C4b on the Bacterial Surface, Infect Immun, 87 (2019).
[24] Ali Y.M., Lynch N.J., Haleem K.S., Fujita T., Endo Y., Hansen S., Holmskov U., Takahashi
K., Stahl G.L., Dudler T., Girija U.V., Wallis R., Kadioglu A., Stover C.M., Andrew P.W.,
Schwaeble W.J., The lectin pathway of complement activation is a critical component of the innate
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14
Appendix 1: Synopsis Approval by the Graduate Research Committee

The synopsis of MPhil/PhD of Mr./Ms/Mrs. __Sadia Akbar____ bearing Roll No. ___43721___ has
been evaluated in the form of presentation and in hard form in the GRC meeting held on _June
28, 2021_ and the committee:

● Approved the synopsis to be presented in the ASRB in its present form.

● Reject the Synopsis in its present form and recommend Revision and must be submitted on
or before____________________.

S. GRC Member Name Designation Department Signature

No.

1 Dr. Isf ahan Tauseef Convener Microbiology

2 Dr. Syed Kashif Haleem Assistant Prof essor Microbiology

3 Dr. Faisal Siddiqui Assistant Prof essor Microbiology

Chairperson/HOD Full Name: Dr. Isfahan Tauseef

Signature________________________ Official Stamp_______________________

15
Appendix 2: Supervisor/Co-Supervisor Consent Form for MPhil/PhD
[Instructions] Please fill this page, and you can modify it in case of supervision.

MPhil/PhD
Name of the Student: Sadia Akbar
Admitted in Session: 2017
Course Work cGPA: 4

For PhD Supervision

I MR./MS./Mrs. _Dr. Syed Kashif Haleem __ have the facilities and agree to supervise/co-
supervise the research work of the above mentioned student of PhD bearing roll/registration No.
__43721_ as per rule of the Hazara University/HEC policy guidelines adopted from time to time
in the allowed time.

Name (Full in Capital Letters): DR. SYED KASHIF HALEEM

Designation: ASSISTANT PROFESSOR
Department: MICROBIOLOGY
University: HAZARA UNIVERSITY
Signature: __________________

16