2024-2025
VU Amsterdam
AM_470657
Contents
Lecture 1 What is a pathogen? Symbiosis vs pathogenesis ..............................2
Lecture 2 Which pathogen strain to use? Genetic variation of microbial
pathogens concept ............................................................................................................7
Lecture 3 Which infectious disease models do you use? ................................ 14
Lecture 4 how to identify virulence factors ........................................................... 24
Lecture 5 Virulence factors: immune system interplay red queen
hypothesis ........................................................................................................................... 33
Lecture 6 Virulence factors: adaptations in metabolism ................................. 40
Lecture 7 Virulence factors – regulation of gene expression ........................ 46
Lecture 8 Intracellular trafficking & survival ......................................................... 56
Lecture 9 Molecular detection of novel viruses .................................................. 64
Lecture 10 Pathogens in the gut microflora ......................................................... 69
1
,Lecture 1 What is a pathogen? Symbiosis vs pathogenesis
What is a pathogen?
A pathogen is a (micro)biological agent that causes disease or illness to its host.
Pathogens
- Viruses
- Bacteria
- Protozoa
- Yeasts/Fungi
- Worms
Normal microflora → real pathogens
The normal microflora in the body
Gut microflora
More than 500 species, mainly:
- Firmicutes
- Bacteroidetes
Gnotobiotic animals (germ-free):
- Need 30% more calories
- Less vascularisation and poor development of villi
- Underdeveloped mucosal immune response
Gut anaerobes degrade and ferment indigestible plant
material → release of small simplified carbohydrates
Gut microflora induce immune response, including
production of antimicrobial peptides
Diseases by normal microflora
Some examples of pathogens among the normal
microflora are E. coli, staphylococcus aureus (acne),
Gardnerella vaginalis. They can be divided into two categories.
Normal microflora at the wrong place
1. Damage to the epithelium
a. B. fragilis
b. Damage to colon epithelium (spontaneous, after surgery). Results in the
infiltration of gut microflora in peritoneum. So not pathogenic when gut is
intact.
c. Co-infection E.coli and B. fragilis → synergistic. Abscess formation, high
morbidity and mortality
2. New sites for normal microflora
a. E. coli (urinary tract infections)
b. Can result in bladder infections (cystitis) and kidney infections (pyelonephritis).
Mainly in women, because of close proximity urethra and anus.
2
, 3. Foreign bodies (surgical implants)
a. Staphylococcus epidermis. A harmless skin bacterium, however it causes a lot
of surgical implant diseases. This bacterium produces surface proteins and
exopolymers that facilitate adhesion and biofilm formation on human skin and
implanted devices. This biofilm on top is extremely hard to get rid of.
b. Skin colonization in the hair follicle primes the cutaneous immune response to
tolerate future commensal
colonization via activation
of regulatory T cells. S.
epidermis excludes S.
aureus (acne) from skin by
blocking autoinduction and
synergizing with
antimicrobial peptides
4. Wrong host
a. E. coli O157 (strain). An outbreak occurred in western US, fall of 1996. 70
persons infected, of which 1 toddler died. Pathogenic E. coli strains are
categorized based on surface structures that are present in abundance and
elicit antibody responses. The outbreak was traced back to unpasteurized
apple juice that used apples picked up from the ground where deer have
dropped over E. coli strains (via feces)
i. O antigen: part of lipopolysaccharide layer
ii. K antigen: capsule
iii. H antigen: flagellin
b. E. coli O157 is a natural inhabitant of the cattle microbiome and causes no
obvious disease in ruminants. Levels of
E. coli depend on seasonal variation
and food supply. For humans this E. coli
strain is highly virulent: an inoculation
of fewer than 100 CFU is sufficient to
cause infection (compared to over a
million CFU for other E. coli strains)
c. Different mammals have similar
composition of gut flora, however, with
strain-specific bacteria!
Abnormalities in host defense
1. Genetic defect
a. SCID (severe combined immune deficiency) boy in the bubble
b. ‘a child (and young adult) with a life-threatening infectious disease is not just
unlucky. A fault in one gene can leave our immune system unarmed against
that one infection.’
c. BCG vaccination: most widely used Tuberculosis vaccine. However, one woman
had all these weird symptoms like osteomyelitis but this was due to a lack of
IFNγ response. She developed mendelian susceptibility to mycobacterial
disease (MSMD)
3
, i. IFNγ circuit is necessary for an effective immune response to intra-
macrophagic pathogens by inducing apoptosis
d. Herpes simplex encephalitis (HSE) → HSE is a rare complication of HSV-1
(koortslip) infection. It is the most common type of sporadic viral encephalitis.
High mortality/morbidity rate: before the use of acyclovir: 70% mortality and
3% of survivors with normal brain function.
i. Problem is a mutation in the TLR3 pathway that regulates the IFN-1
anti-viral response. This cytokine should stimulate an infected cell to
go into apoptosis
1. Mutations associated
with susceptibility to
HSE are shown in blue.
2. Mutations associated
with susceptibility to
HSE and
(myco)bacteria are
shown in green.
2. Suppression of immune response
a. Transplantation, malnutrition (measles, malaria)
3. Other infections
a. HIV, influenza
4. Antibiotics
a. Healthy gut flora
b. Killing of antibiotic-sensitive species
c. Massive outgrowth of endemic species or colonization by
antibiotic-resistant new species. If they grow to high amounts, they
can become serious
Why are (some) bacteria of the normal microflora good pathogens?
Pathogen → symbiont → commensal
Mutualism
- They’re always present (opportunistic)
- Factors important for colonization also used for virulence!
o Adhesion (pili)
o Evade killing immune response (capsule)
- Adapted to metabolism in the host (pathogenic evolution)
- Some virulence factors are needed to withstand other organisms, such as grazing
protozoa (legionella pneumophila)
Exogenous infections
Colonization of mucosal surfaces
Cross anatomical barriers
Tissue invasion
Breach host defenses
Dissemination
Adapted human pathogens
4
,Are persistent infecting organisms real pathogens?
Mycobacterium tuberculosis
Epstein Barr virus
- DNA virus (Herpesviridae)
- 90% of adults have EBV antibodies
- Infected for life (reside in immortalized B cells)
- Causative agent of mononucleosis (ziekte van Pfeiffer)
- EBV-associated malignancies ((non) Hodgkin’s lymphoma)
Infection process of EBV: host cell type switching
EBV (just like HSV) is switching cell types during the infection process (host cell tropism)
- HSV: epithelial cell – nerve cell – epithelial cell
- EBV: epithelial cell – B cells – epithelial cell
o It makes the B cells live for very long
Is EBV a harmonious pathogen?
Herpesvirus latency confers symbiotic protection from bacterial infection
Mice that had the herpesvirus simultaneously with listeria, all died from infection. However,
mice that had the herpesvirus 4 weeks before listeria, all survived. This showed that the
immune system was changed after herpes infection and that it also plays a symbiotic role. A
chronic infection with herpes → saves you from listeria
5
,Protection mechanism: trained immunity
Mechanism independent of cross-reactivity.
Related to enhanced IFNγ production, through
persistent epigenetic changes of macrophages
and progenitor cells.
Tuberculosis: a chronic wasting disease
Infection by inhalation of infectious bacteria
- 5% develop active disease
- 5-10% of chronically infected people develop disease (1-50 years)
pulmonary infection, but also extrapulmonary dissemination (liver, kidneys,
brain)
Tuberculosis
Bacteria phagocytosed by alveolar macrophages → localized proinflammatory
response that leads to a granuloma, or tubercle → granulomas: ‘containment’
phase: no overt signs of disease but also no eradication → caseation of the center
of the granuloma → viable, infectious bacilli into the airways
Granuloma formation is good for the host:
Confinement of the bacteria
Granuloma formation is good for the bacteria:
Unable for the T-cells to reach the center of the granuloma. Caseation of granulomas (and
not systemic spread) is essential for transmission to a new host
Problems with M. tuberculosis virulence
- No clear factor (toxin) involved in disease symptoms
- Does tuberculosis cause a disease?
o More than 90% of the infected people never develop it.
➔ It is possible for M. tuberculosis to isolate variants with increased virulence!
Experiments performed with mycobacterium marinum
This is a tuberculosis kind (ESX-5) that is seen in fish and is closely related to the human
strain. On humans, it causes a rash on the skin. It grows relatively
rapid (8-10 days). It grows optimally at 28-32 degrees.
This ESX-5 is attenuated in macrophage cell death. Some ESX-5
substrates are anti-virulence proteins. A mutant of the strain shows
early granuloma formation and high CFU.
Live TB vaccine (BCG) protects against other pathogens
1. Children vaccinated with BCG have also reduced mortality
for other childhood diseases such as malaria
2. Mice vaccinated with BCG are more protected against a
lethal candida infection (70% protection)
6
,Lecture 2 Which pathogen strain to use? Genetic variation
of microbial pathogens concept
The question of this lecture is what strain do you use for your
study?
→It is about a Cystic Fibrosis (CF) infection. This has a cystic
fibrosis transmembrane conductance regulator that pumps out
chloride ions. Therefore the salt concentrations are too high.
A CF infection can be caused by pseudomonas aeruginosa
(PA01).
You can go for the sequenced strain? Or not
Yes:
Genetics
Proteomics
Exchange data/materials with other labs
- Isogenic mutants → you want to use it from the background. One mutation only and
then you know that the infection is due to one mutation
o They are genetically modified strains that differ from a parent strain by a
specific, intentional genetic change while maintaining an otherwise identical
genetic background.
No:
Attenuation by in vitro growth; are you still looking at
the pathogen?
- Alginate production makes it very slimy in in
vitro growth. These mutations you get
overnight.
- P. aeruginosa (PA01), isolated from burn wound
in 1955 by Bruce Holloway: passaged for
decades, spread from laboratory to laboratory
over the world. Will this result in point mutation / deletions / inversions?
o It is from a burn wound, so because it is not from a CF patient, the strain can
be totally different!
P. aeruginosa PA01 genome diversity
3 PA01 strains were sequenced:
- Duplication of a mobile 12-kb prophage region
- 18 deletions of 3 to 1,006 bp in size
- 39 single-nucleotide substitutions, 17 of which affect protein sequences
- Large genome inversion
By sending the strains around, you can get mutations
M. marinum M genome diversity (mycobacterium)
This bacterium Is a ‘tuberculosis model’ for zebrafish
7
, - This strain turned out to be not virulent in zebrafish, because it did not secrete the
crucial virulence factor EsxA. → so, they obtained another M. marinum M strain and
determined genome sequences: 12 point mutations, including a frameshift mutation
(+1) in gene of secretion system (eccCb1) responsible for EsxA secretion. So because
of this mutation, this specific strain cannot be used anymore in studies.
Remember that pathogens can change and therefore you can’t just use them easily.
Subculturing selects against different virulence factors
It selects against energetically costly structures: capsule, flagella,
protein secretion. It also selects against bacterial clumping and
biofilm formation
Attenuation by continuous culturing
Method to isolate attenuated strains for vaccination
→ mycobacterium bovis BCG
M. bovis was grown for 11 years (230 passages) in culture
medium, already attenuated after 15 passages. However, a couple of deletions in the genome
of which one is crucial that makes the bacterium avirulent (in the RD1
gene)
Genome plasticity; is it the pathogen you want to study?
First, what is a bacterial species?
- The bacterial line of descent is clonal
- Each daughter cell is (in principle) identical to the mother cell
- Chromosomal mutations are continued in the sub lineage, but
cannot be mixed with other lineages of the same species
It is a species when there is a
- 70% DNA-DNA hybridization
o Hybridization → a powerful technique that helps identify
species’ relatedness by measuring the degree of genetic
similarity between organisms.
- 98% 16S rRNA identitity (gold standard)
➔ However, if we apply the same rules to mammals, humans would be the same
species as chimpanzees, gorillas, orangutans and gibbons
➔ Also important to remember is that if they are a bacterial species, they can still
have very important metabolic/pathogenic differences!! (M. tuberculosis vs M.
bovis are >99% identical)
So the bacterial line of descent is clonal, but is diversified by horizontal gene
transfer (HGT). also called lateral gene transfer → never mixing of genomes,
but exchanging parts of genomes.
- Replacing genes
- Introducing new genes
8
, Causes of HGT:
1. Transformation: DNA is released by bacterium,
then the naked DNA is taken up by another
bacterium. So not an old bacterium
a. Extremely efficient process
b. Not in all bacteria
2. Conjugation: when one bacterium has an extra
chromosomal piece of DNA (plasmid), through a
contact point this plasmid is exchanged and
integrated into a new host
a. Plasmid dependent
b. Extremely efficient process
c. Transfer of chromosomal DNA possible
3. Transduction: bacteriophage infects one bacterium, takes up host’s DNA and transfers
it to another bacterium, that consist of chromosomal DNA.
a. Extremely efficient for transfer of phage DNA
b. Not very efficient for transfer of
chromosomal DNA
Recombination required after
transformation/transduction
- Dependent on RecA protein
- Dependent of DNA homology
o High homology, increased
recombination efficiency
The amount and impact of HGT is species dependent.
M. tuberculosis has no HGT. Every daughter cell is directly
from the parent (except for when they have a point
mutation). On the right, a number of deletions are seen.
Based on the presence of these deletions you can make a
map of the history of Tb. They first thought Tb came from
close contact with cows, but after mapping the history,
this was not the case.
Omnipresent HGT: helicobacter pylori & Neisseria
meningitidis ! so HGT that can be at all places all the time!
- If we look at N. meningitidis, the pillus can bind
DNA and can retract it. The DNA enters the cytosol and
there it becomes ssDNA and then it can be incorporated
into the genome.
How to determine HGT:
- GC content (take into account amino acid composition!)
9
