Biology of Disease

Level 3 (BIOL3621)
BSc (Hon) Biological and Biomedical Sciences

Gary Sharples (gary.sharples@durham.ac.uk)

Objectives
you will be able to describe:
the incidence and symptoms of disease
caused by Chlamydia
how Chlamydia trachomatis replicates intracellularly
type III systems and effectors in Chlamydia
the epidemiology and symptoms of gonorrhoea
Neisseria gonorrhoeae attachment and invasion of
cells using type IV pili and Opa proteins
rearranging surface components by Neisseria
gonorrhoeae
Chlamydia: the hidden epidemic
insidious nature
few symptoms
especially in
women

infection and complications similar

to gonorrhoea
main cause of nongonococcal
urethritis (NGU)
caused by Chlamydia trachomatis
 epidemiology of chlamydia
women men

data from Public Health England Chlamydia data from 2012 onwards are not comparable to
data from previous years (includes all age groups now)

most commonly diagnosed bacterial sexually

transmitted infection in the UK 30% of STIs
women aged 15-24 years and men aged 20-
24 years have the highest rates
2-6% of the UK population estimated to be
infected with chlamydia
survey data suggests that people aged 16-24
years are most likely to report a new sex
partner or two or more sex partners of the
opposite sex in the past year rate per
100,000
people of this age group continue
to experience the highest rates of
chlamydia, genital herpes and genital warts
in 2015, of the heterosexuals aged 15 to
24 diagnosed with an STI, 62%
(62,191/100,165) had chlamydia
symptoms
incubation period of 1-3 weeks
males
50% asymptomatic
painful urination and watery discharge
inflammation of epididymis may cause sterility
females
70% asymptomatic
slight vaginal discharge, inflammation of
cervix and burning pain on urination due to
urethritis
longer term complications...
 pelvic inflammatory disease (PID)
infection spreads to Fallopian tubes (pelvic inflammatory
disease) causing adhesions that block the passageways
(salpingitis)
infection uterus
Fallopian tube

ovary

swelling
adhesions

caused by both Chlamydia and Gonorrhoea

What you dont know cant hurt you?
consequences of asymptomatic PID include
increased risk of:
1. infertility
scarring and blockage of Fallopian
tubes prevents egg traversing
from ovary to uterus
2. ectopic pregnancy
foetus develops in Fallopian
tube instead of the uterus
3. AIDS
damaged tissue allows access
for other sexually-transmitted infections
infection of infants during birth
chlamydial ophthalmia
conjuctivitis, usually self-
limiting but can be treated
with erythromycin
chlamydial pneumonia
potentially serious

trachoma
strains in Africa, Middle-East
and Asia cause corneal
scarring and blindness
spread by contact - not STI
 diagnosis and treatment
urine and swab tests available
based on antibodies detecting
chlamydial proteins
polymerase chain reaction also
used to detect DNA in urine
samples
azithromycin

treatable with antibiotics:

doxycycline - a tetracycline
azithromycin - a macrolide
Chlamydia trachomatis: the bacterium
tiny: only 0.25 m
E. coli is 2-3 m long
small genome: 895 genes
can only be grown in tissue
culture cells and no genetic
systems are currently available
Gram-negative type cell wall but
lacks peptidoglycan - can make it
but only used transiently in cell
division
cross-linked membrane proteins
 Chlamydia trachomatis: cell wall
proposed structure of elementary body membrane
divalent cation

MOMP
outer
membrane

large CRP P layer

small
CRP inner
membrane

superficially like other Gram-negative bacteria, however, the

outer membrane is stabilised by major outer membrane proteins
(MOMPs: OmpA, OmcA and OmcB) plus small and large
cysteine-rich proteins (CRPs); the P layer, composed of large
CRPs, usually contains no peptidoglycan
Chlamydia trachomatis
grows only in living human tissue inside
cells - strict intracellular parasite
two stage life cycle:
Reticulate Body - RB - is the replicative form
Elementary Body - EB - is the survival form

EB RB

RB RB

RB EB
intracellular
replication:
intracellular survival depends on blocking lysosome fusion, while
promoting fusion with nutrient-rich exocytic vesicles by
initial attachment
recruiting host factors using chlamydial Inc proteins mediated by OmpA
binding heparan
sulphate
HSPG: host heparan
sulfate proteoglycans Type III secretion
system (T3SS)
injects translocated
actin-recruiting
phospoprotein
(TarP), which
induces uptake
TarP is rapidly
phosphorylated
inside the cell at
tyrosine residues and
peptidoglycan
only synthesised
influences actin
at septum and is polymerisation
required for cell
division starvation or
Incs: inclusion membrane antibiotic treatment
proteins can lead to a latent,
MTOC: persister cell state
microtubule-
organizing takes 48-72 hours to
centre complete cycle
infection of the female genital tract with Chlamydia trachomatis

sIgA: secretory IgA

pIgA: polymeric IgA

the inflammatory reaction is characterized by an influx of macrophages and neutrophils and the
formation of immune inductive sites in the submucosa
these sites, which contain B cells, T cells, dendritic cells and macrophages, coordinate the
initiation of an acquired immune response, including the deployment of an sIgA response
 chlamydia: immune activation
endothelium or epithelium Chlamydia
infected cells release
proinflammatory signals
secretion of chemokines and growth factors
(IL-11, IL-8, IL-12, IL-6, GM-CSF)
including a variety of
interleukins and
B cell macrophage granulocyte-macrophage
inflammatory colony-stimulating factor
T cell
neutrophil (GM-CSF)
immune cell migration
and activation these chemicals attracts a
variety of immune cells
the chronic and intense
follicle necrosis cell damage
inflammatory response
leads to tissue damage,
fibrosis and scarring
scar formation
gonorrhoea
caused by Neisseria
gonorrhoeae
small, gram-negative,
diplococcus - gonococcus
oxidase positive
gonorrhoea derived from
Greek gonos (semen) and
rhoia (flow) referring to
release of pus
fragile organism
-disinfection, drying
 gonorrhoea in history
World War II, U.S. documented for more
government
poster than 1000 years
also known as the Clap
from clappoir the 16th
Century French word for
brothel
Captain Cook and his
crew introduced
gonorrhoea and others to
Hawaii in 1778
urethral washouts prior to
World War II
epidemiology of gonorrhoea in England
second most common
bacterial STI after
chlamydia

transmission amongst
heterosexuals showing a
more gradual increase after
a substantial increase in
diagnoses in the mid-1990s

high levels of gonorrhoea

transmission are of
particular concern, given the
emergence of gonococcal
resistance (including high-
level resistance) to
azithromycin
symptoms of gonorrhoea: females
incubation period of 2-6 days
50% of cases asymptomatic
invasion of epithelial surfaces
of cervix and urethra
discharge may be present

abdominal pain and burning sensation on passing urine

fallopian tubes may be infected and blocked (salpingitis)
causing pelvic inflammatory disease (PID)
leads to infertility and risk of ectopic pregnancies
symptoms of gonorrhoea: males
infection primarily in
urethra
symptoms more obvious in
males with pain or a
burning sensation when
urinating
thin watery discharge
initially, followed by thick,
white pus
infection may spread to the
testicles causing
epididymitis (less
 treatment
penicillin
originally treated with
cephalosporin
nucleus
penicillin but a resistant
strain arose in 1976
now treated using
azithromycin
dual therapy with
ceftriaxone and
azithromycin
infection does not
provide immunity
ceftriaxone
(a third generation
various vaccines
cephalosporin)
currently undergoing trials
Neisseria gonorrhoeae antimicrobial resistance

the numbers on the bars reflect the number of years the antibiotic
was in use before resistance was reported
% of isolates resistant to
ceftriaxone by MIC
England, 2007-2014
% of isolates resistant to
azithromycin by MIC and
gender/sexual orientation
England, 2014
MIC: Minimum Inhibitory Concentration
17April2016
a large proportion of
gonococci that are in
circulation worldwide are
only a few resistance
markers away from
developing into
extensively drug resistant
(XDR) strains
XDR strains are resistant
to two or more of the
antibiotic classes that are
generally recommended
for the treatment of
gonorrhoea or three or
more of the classes that
are less frequently used
for treatment
virulence mechanisms
1. attachment pili
2. Opa afimbrial
adhesins
3. intracellular
survival
4. changing surface
coat proteins
interaction with host epithelia

type IV pili and Opa proteins

(Opa proteins are similar to
the Opa and Opc afimbrial
adhesins in meningococcus)
mediate interaction
cells are taken up inside the
epithelial cells via cellular
1 m protrusions
 pili mediate
initial attachment
followed by tight
adherence by
the Opa proteins
binding
stimulates
invasion and
transcytosis
infection often
PMN,
leads to
polymorphonuclear inflammation
ECM, extracellular leucocyte
matrix
and PMN influx
N. gonorrhoeae
engulfed by
PMN are
secreted in
PMN-rich
exudate
 getting attached: type IV pili
type IV pili proteins pilus extends and
tip makes contact
produced by pil with host cell
genes and pilus retracts
processed by a type allowing bacterial
movement and
II secretion system intimate contact
with the target cell
can be assembled
and disassembled
binds human
epithelial cell surface
receptor CR3 and
CD46
 processing and recombination
transformation DNA uptake sequences (DUS)
are necessary for efficient uptake

binding and uptake

Pil adhesin
proteins
major pilin

secretin
(channel)
donation

peptidase

pilus
retraction type IV pilus
incoming dsDNA is converted to ssDNA by various
nucleases - RecA mediates recombination into the
gonococcal chromosome
10 bp DNA uptake sequence (DUS) 5-GCCGTCTGAA-3 is integrated
present in ~1900 copies in the genome DNA makes new variants
tight attachment: Opa proteins
Opa: Opaque
colonies (Opa+) on
agar plates
caused by increased
bacterial aggregation
may lose their
opacity (Opa)

Gc has 12 different opa genes

different Opa proteins can recognise different host
receptors and varying numbers of them can be
expressed - or even none at all
Opa interaction with host cell receptors
Neisseria
OpaHS
OpaHS
heparansulphate
Fibronectin proteoglycans
D Vitronectin
R
G (HSPG)
HSPGs
and
PC-PLC
cell
Integrins (fibronectin
Integrins
ASM
PKC
membrane + vitronection)
OpaCEA
carcinoembryonic
Tight adherence, receptor clustering, antigen cell adhesion
cytoskeletal rearrangement, internalisation
molecule (CEACAM)
together with type IV pili, assists in epithelial cell uptake
CEACAM1 CEACAM1 is the main receptor
carcino- targeted by Opa proteins
embryonic
antigen- CEACAM1 belongs to the
related cell immunoglobulin (Ig) superfamily and
adhesion contains an N-terminal Ig variable-
molecule 1 like domain that is targeted by Opa
the Opa-CEACAM1 interaction
results in cellular invasion

Opa type IV pili

CEACAM1

note that N. gonorrhoeae lacks a capsule

 the changing face
of the gonococcus
multiple mechanisms for constant change

naturally transformable
will actively take up DNA from other strains of N. gonorrhoeae

1. recombination: gene swapping

2. replication slippage: switches and variation
variation of cell surface components
pili Opa

all surface structures can be altered or switched on or off

thought to be capable of generating >1 million different
antigenic variants
 Neisseria pilin variation
Neisseria species
chromosome have an expressed
pilE pilin gene (pilE)
and multiple silent
Sma/Cla repeat pilin gene copies
(pilS) that are not
pilS pilS pilS pilS expressed

rearrangements between pilE and pilS by genetic

recombination within the homologous segments
leads to antigenic variability in pilus structures
this helps Neisseria gonorrhoeae and Neisseria
meningitidis maintain chronic infection
 exchange steps

hybrid pilE/pilS

recipient pilE

variant pilE

N. gonorrhoeae has 18-19 pilS gene copies

these are located between 1 and 900 kb from pilE
pilin antigenic variation: the pilE gene

glycosylation
site
 aberrant recombination: L-pilin
unequal exchange
between pilS and pilE
P+ results in multiple copies
of pilS in pilE
gene is longer than
normal
expresses L-pilin which
PL gets stuck in periplasm
no pilus formed

pilus production by phase variation i.e. an on/off switch

 aberrant recombination: S-pilin
signal sequence for
export of pilin normally
P+ removed before pilus
assembly
with S-pilin, processing
occurs at +40 leading to
secreted soluble pilin
a few normal pili
PS produced but S-pilin acts
as molecular decoy by
mopping up antibodies
 phase
variation
expansion or contraction of
homopolymeric or tetranucleotide repeats
affects transcription or translation
main mechanism is RecA-independent
slipped-strand mispairing during DNA
replication or repair
gonococcus has around 30-40 phase
variable genes
 phase variation in pilC adhesin
on/off switch caused by slipped-strand mispairing

5' pilC gene 3'

...TCCCATACCGGCGGGGGGGGGGGGGCGATGGCGCAAACC...
...SerHisThrGlyGlyGlyGlyGlyAlaMetAlaGlnThr...

variable number of G-residues

10 or 13 Gs: gene is ON
11 or 12 Gs: gene is OFF
 regulation of translation
N. gonorrhoeae OpaC phase variation
ON 1 2 3 4 5 6 7 8 9

translation
DNA replication

OFF 1 2 3 4 5 6 7 8

no translation

3, 6, 9 or 12 CTTCT repeats allow translation of opaC

2, 4, 7 or 8 repeats results in a frameshift
modification of lipopolysaccharide
sialylated LPS imparts capsule-like
properties making the bacteria more resistant
to antibody and complement-mediated killing
and more able to avoid phagocytosis
sialylated LPS also mimic host-cell surface
structures, which facilitates avoidance of the
host antibody response

modification of LPS by sialic acid can also mask Opa

antigens
however, Opa proteins now hidden and cannot now
adhere to cells so LPS is switched on and off

balance: protection in blood vs ability to invade epithelia

References
Bacterial pathogenesis: a molecular approach (2010)
3rd Edition. Wilson, B.L. et al. ASM press ISBN: 063203775X
(616.9201 BAC)
electronic version is available on the library catalogue:
http://lib.myilibrary.com.ezphost.dur.ac.uk/ProductDetail.aspx?id=303429
some helpful general information on chlamydia and gonorrhoea

Chlamydia
AbdelRahman, Y.M. and Belland, R.J. (2005) The chlamydial
developmental cycle. FEMS Microbiology Reviews 29: 949-959
Miyairi, I. and Byrne, G.I. (2006) Chlamydia and programmed cell
death. Current Opinion in Microbiology 9: 102-108
Elwell, C., Mirrashidi, K., Engel, J. (2016) Chlamydia cell biology
and pathogenesis. Nature Reviews in Microbiology 14: 385-400
References (continued)
Gonorrhoea
Virji, M. (2009) Pathogenic Neisseriae: surface modulation,
pathogenesis infection control. Nature Reviews in Microbiology
7: 274-286
Vink, C., Rudenko, G., Seifert, H.S. (2012) Microbial antigenic
variation mediated by homologous DNA recombination. FEMS
Microbiology Reviews 36: 917-948
Criss, A.K., Seifert, H.S. (2012) A bacterial siren song: intimate
interactions between Neisseria and neutrophils. Nature Reviews
in Microbiology 10: 178-190
Goire N, et al. (2014) Molecular approaches to enhance
surveillance of gonococcal antimicrobial resistance. Nature
Reviews in Microbiology 12: 223-229