Peter Beaconsfield Prize Entry

The fight to survive:

A bacterium’s tale against the
immune system

Wearn Xin Yee

Sir William Dunn School of Pathology
Merton College
Started on Michaelmas 2018
Supervised by Professor Christoph Tang
Abstract
Neisseria gonorrhoeae has a long evolutionary association with humans. One reason for its success is its ability to
resist immune killing by evading the human complement system, the body’s first line of defense against
pathogens. Despite the discovery of the complement pathway in the 1900s, researchers continue to identify
novel regulators of complement activity. Here, I investigate how these human complement regulators can
inhibit bacterial evasion mechanisms and influence the outcome of infection with N. gonorrhoeae. My findings
provide insights into host:pathogen interactions, and indicate potential novel approaches for therapy which are
needed given the rise of antimicrobial resistance.
Introduction
Neisseria gonorrhoeae is a bacterium that exclusively infects humans. It has a long evolutionary association with
humans, with descriptions of gonococcal infection dating back to ancient times1. Currently, N. gonorrhoeae is a
leading cause of sexually transmitted infection (STI), with more than 87 million new cases in 2016 worldwide2.
The bacterium predominantly colonises the reproductive tract, but can also infect other mucosal sites, with
occasional spread in the bloodstream3. Colonisation is a critical first step in disease; specifically, to establish an
infection, N. gonorrhoeae must colonise and adhere to epithelial cells in the urogenital tract via the bacterium’s
surface proteins. In addition, the bacterium resists killing by innate immune cells such as neutrophils4.
Apart from neutrophils, the complement system is a critical component of immune defense against bacteria.
The complement system is active in human serum and consists of proteins synthesized by the liver. Activation
of the complement system can lead to bacterial opsonization, thereby promoting phagocytosis by neutrophils,
and/or lysis5. However, within the body, complement activation must be controlled to prevent damage to self-
tissues. Thus, the complement pathway can be downregulated by human complement factor H (CFH)6. CFH
consists of 20 short consensus repeat (SCR) domains ‘stringed’ together by linker sequences7. To evade the
complement system, N. gonorrhoeae binds CFH via the 18th - 20th SCR8,9. This interaction is mediated by PorB, a
porin on the surface of the bacterium which has eight surface exposed loops (Figure 1). In addition, the PorB-
CFH interaction is enhanced when the surface of the bacterium is modified. Specifically, the addition of a host-
derived sialic acid onto bacterial surface lipopolysaccharide (LPS) enhances N. gonorrhoeae binding to CFH
(Figure 1)10,11.
Interestingly, it has recently been discovered that the body produces proteins that antagonize CFH. These
include the complement factor H related proteins (CFHRs). Humans express five CFHRs (known as CFHR1
to 5) which are encoded downstream of the CFH gene, and are present in low levels in serum and on mucosal
surfaces11. CFHRs range from having four SCR domains (CFHR2) to nine unique SCR domains (CFHR5)12.
CFHRs have similar sequence and structure to CFH but are unable to downregulate the complement system13.
Therefore, they can compete with CFH for binding to the same sites and act as competitive antagonists of
CFH. For example, CFHR3 can inhibit CFH recruitment by N. meningitides, increasing the sensitivity of bacteria
to complement14.

Are there potential antagonists to CFH that binds to N. gonorrhoeae?

As N. gonorrhoeae is a close relative of N. meningitidis, my aim was to determine if N. gonorrhoeae binds to CFHR2-
5. As CFH binding to the gonococcus is enhanced when bacteria have sialic acid added to their LPS, I looked
at CFHR binding after growth with or without additional sialic acid. Of the four different CFHRs investigated,
no binding of CFHR2, CFHR3 and CFHR4 to N. gonorrhoeae was detected regardless of LPS sialylation. In
contrast, binding of both CFH and CFHR5 was observed only when LPS is sialylated (Figure 2). CFHR1 was
not investigated as no CFHR1-specific antibody is available.
Due to similarities in the domain structure of CFH and CFHR5 as well as the dependence on sialic acid for
their binding to N. gonorrhoeae, it was thought that CFHR5 might bind via PorB, which also binds CFH10,11. To
investigate whether CFHR5 binds to PorB, strains with mutations and deletions in the surface-exposed loops
of PorB were generated (Figure 3). These mutations and deletions were confirmed to have no effect on bacteria
viability or porin expression.

Using flow cytometry, I showed that strains with porB mutations affecting loops 3, 4, 5 and 7 (L3116-121 -> Ala,
L4Δ171-176, L5Δ203-224 and L7290-295 -> Ala ) exhibited a loss of both CFH and CFHR5 binding (Figure 4). In
contrast, mutations affecting loop 2 (L283-88 -> Ala) had no effect (Figure 4). This shows that several PorB
modifications have similar effects on CFHR5 and CFH binding. As such, CFHR5 and CFH bind to similar
sites on PorB and thus could compete with each other, with CFHR5 preventing CFH from binding to PorB.
Overall, I have shown that CFHR5 can potentially act as a competitive antagonist to CFH, preventing CFH
recruitment to N. gonorrhoeae. CFHRs may therefore influence the outcome of infection by reducing CFH
recruitment to bacteria on mucosal surfaces. This might be especially important for the survival of N. gonorrhoeae
in the urogenital tract. To confirm if this is indeed the case, it will be necessary to use biophysical methods to
determine the binding kinetics of CFH vs. CFHR5. Serum sensitivity assays could also provide information on
the physiological significance of CFHR5 binding.

Why is this project important?

In the human body, CFHRs prevent inappropriate, over-activation of the immune system12, while pathogens
such as N. gonorrhoeae have evolved to exploit such immunomodulatory effectors to their advantage10,11. My
work thus highlights how microbes have evolved to take advantage of the complex immune networks that are
required to defend against pathogens while avoiding self-inflicted damage.
Furthermore, with the increase in antimicrobial resistance in N. gonorrhoeae15, we require different approaches
to combat N. gonorrhoeae instead of relying on traditional antibiotics. My findings suggest that a possible
approach would be to manipulate the immune system; inhibiting the evasion mechanisms employed by bacteria
using host regulatory proteins would render them sensitive to immune killing. Therefore, understanding the
interaction between CFHRs, CFH and N. gonorrhoeae not only provides insights into host:pathogen interactions
but also highlights novel therapeutic avenues. With many antibiotics becoming redundant due to spreading
resistance, targeting the immune system would pave a way for the treatment of infectious diseases in the future.
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