Molecular basis of infection Flashcards
why will number of deaths increase significantly by 2050 due to antimicrobial resistance?
won’t be able to carry out surgery and other treatment due to risk of infection
People don’t make antimicrobials as not profitable in a few years they aren’t viable anymore
anti virulence factors good as?
reduce selective pressure for resistance
Superficial pathogens?
Fungal infections on the surface
Mechanisms of adhesion?
Specific mechanisms:
Ligand receptor interactions
Involve cell surface molecules: Adhesins - typically lectin or protein-protein based interactions
Non specific mechanisms:
Electrostatic force
Aggregation
Cell surface hydrophobicity
Primary pathogens?
Inside the body and cause disease
Opportunistic pathogens?
Take advantage of the host being in a weakened immune system situation for example
Biggest killer
Features about candida?
Commensal organism
Superficial and systemic infections
Features about cryptococcosis?
Causative organisms:
Cryptococcus neoformans
Cryptococcus gatti
Basidiomycetes yeasts
Both capsulate yeast
Found in soil and avian habitats
Route of infection:
Inhalation (desiccated yeast or spores)
Sexual cycle produces small spores which can be inhaled
Cause pulmonary cryptococcosis and meningitis
What can candida adhere to?
Epithelial / mucosal layers Endothelial cells Inert medial devices Self association (biofilms) Other microbes in the population
Candida cell wall?
Outer wall - Manno proteins
Linker - B1,6- Glucan (linker)
Inner wall - B1,3-glucan
Cell membrane - Chitin skeleton
Covalently attached proteins:
GPI remnant anchored (GPI-CWP)
Pir proteins
Non covalently attached proteins:
Secreted proteins in transit
Surface associated cystolic proteins
Common GPI-CWP structure?
N side to C side
Signal seq, effector domain, low complexity Set/Thr rich (O-glycosylated) tandem repeats, then GPI anchor
Features of GPI anchor?
Anchors protein to plasma membrane
Anchor subsequently cleaved and remnant becomes covalently attached to B1,6-glucan
Role of GPI-CWPs?
Adhesion
Cell wall biogenesis and remodelling
Structural elements
Enzymatic activity - mainly degradative
Candida ALS family?
8 members ALS1-7, ALS9
First identified from cDNA library
Agglutinin-like-sequence
Cross hybridisation and genome sequencing identified other members
Share common structure
Signal seq - Conserved domain - tandem repeat domain - variable domain - GPI anchor
Consequences of TR number variation?
Presentation on cell surface
Might not show outside the cell wall matrix
Amyloid formation as well: TR region of ALS proteins can form amyloids Fibrous protein structures Form B-strand rich aggregates May strengthen cell adhesion
Long allele play a greater role in adhesion
Role of ALS family in adhesion?
Studied through heterologous expression in S. cerevisiae and generation of null mutants in C. albicans
Als3 role?
Role in adherence to endothelial and epithelial cells
Als1 role?
Subtle role in adherence to endothelial cells
Als2 and 4 mutants display?
Defect in endothelial but not epithelial adhesion
Also display compensatory expression, knock out one gene the other gets expressed more
Features of ALS5, 6 and 7?
Perhaps have an anti adhesive role
Null mutants showed an increase in adhesion
However this could be due to compensation, exposure of other adhesins, required for dispersion
Als9 played a role in?
Endothelial adhesion and displays allelic variability
It’s mutant has defect in endothelial adhesion and not epithelial cells or laminin
Adhesive role conferred N terminal domain of ALS9-2 allele
Which ALS is required for early stages of infection?
ALS1, maybe a bit of ALS3
It’s only subtle due to functional redundancy (other ones take control) and compensatory expression (up regulation of other ALS genes in mutant backgrounds
How does Als N-terminal peptide binding domain plays a key role in adhesion
Peptide binding domain
Binds 6 amino acids at the C-terminus of peptides
Provides broad peptide recognition
Point mutants have phenotype comparable to null mutants
Other functions of ALS3?
Biofilm formation
Adhesion to other microbes
Invasion of other cells
Iron acquisition
HWP1 does what?
Hyphal specific adhesin
originally isolated as a phyphal specific protein
Encodes a GPI anchored CWP
N terminal domain has a 10Aa imperfect repeat, rich in proline and glutamine
Has 2 copies of a 42 Aa repeat present in a number of C.albicans CWps
Plays a role in adhesion, biofilm formation and mating
Hwp1 is a substrate of TGase and is required for stabilised adhesion to epithelial cells
Tgase is involved in cross linking epithelial proteins
Other adhesins in C . albicans?
Hwp2 / Rbt1:
Hyphal specific expression
Related to Hwp1, share 42 Aa motif
Hwp2 implicated in adhesion, mutants have biofilm and mating defect hence have virulence defects
Eap1:
Expressed in both yeast and hyphae
Mutant has epithelial and polystyrene adhesion defect
Plays early role in adhesion leading to biofilm formation
iff/Hyr1 family:
12 membered gene family, enriched in pathogens
share common conserved N-terminal domain
Limited study to date, some suggestion of a role in adhesion
Ywp1:
Yeast wall protein
Anti adhesin, mutant are hyper adherent
Summary on C.albicans adhering?
C.albicans adherers to multiple surfaces
Cell wall proteins play a key role in adhesion
Wide range of adhesins expressed by C.albicans
Adhesins are generally encoded by gene families:
Families display allelic variation both within and between strains and species
Functional redundancy and compensatory expression of adhesins is common
Where does crypto coccus adhere?
Environmental reservoirs Lung epithelial layers Brain endothelial cells Inert medical devices Self association (biofilms)
Cryptococcus capsule?
GXM - 90% of capsule - Mannose backbone substituted with glucuronic acid and xylose
GXMGal - 10% capsule - Galactose backbone with side chains of galactose and mannose, side chains substituted with glucuronic acid and xylose
What regulates crypto coccus capsule thickness?
Environmental cues
Capsule role in virulence?
Mutants highly attenuated in virulence
Capsule inhibits phagocytosis
Provides stress protection (dehydration and free radicals)
Impacts on immune system
What plays a role in the adherence to lung epithelial cells?
GXM
Antibodies to GXM can block this adherence
What plays a role in adherence of acapsulate cells to lung epithelia
MP84
What is Hyaluronic acid?
Present on crypto coccus cell surface
Forms fibrous structures extending from the cell wall
Causes adhesion to brain endothelial cells
Treatment with hyaluronidase reduces this adherence
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What’s phospholipase?
Other adhesin for cyrptococcus
Secreted enzyme
Required for spread from the lung
Interaction between hyaluronic acid and CD44 can?
play a role in adhesion and invasion across the blood brain barrier
Mortality rate from fungal infection?
20-90%
400,000 blindness per year
50% of asthma related deaths
Fungal exam question will be on crytococcus or candida or general
ok
Problem with targeting adhesins in crypto coccus?
If you target one another will come and replace it, the same in candida
Overview of fungal invasion?
Pathogenic fungi interact with a variety of host cells during the disease process
Invasion allows the pathogen to traverse cellular barriers
Invade normally non-phagocytic host cells
Cellular invasion conceals the pathogen from professional phagocytic cells
Invaded host cell may provide a source of nutrients
In disseminated disease the pathogen must invade both epithelial and endothelial cells
Methods of fungal infection?
Induced endocytosis - makes the cell uptake it
Transcellular invasion - go through the cell
Paracellular invasion - passes between the cells
Trojan horse - becomes engulfed by the cell and transported to a new sterile tissue
Epithelial invasion by C.albicans?
Induced endocytosis or active penetration
Active penetration can be transcellular or paracellular
Dependant on epithelial lineage, both mechanisms on oral epithelia and just active penetration on intestinal epithelia
Both methods generally involve C.albicans hype
Is C.albicans polymorphic?
Yes
Yeast
Pseudohyphae
Hyphae
Chlamydospore
Opaque
How does c.albicans invade via induced endocytosis?
Fungal invasin interacts with component on host cell surface
Interaction triggers pseudopod formation and fungal engulfment ‘zipper like’ mechanisms
Induced endocytosis seen with multiple cell lines
It’s hyphal specific - uptake is triggered by hyphal associated factors
Describe engulfment for candida?
Its a passive process does not directly result in host damage
Live / dead cells are endocytosed at a similar rate
Process is inhibited by cytochalasin D (CD) confirming endocytosis
Damage is an active process following endocytosis
Als3 acts as an?
Invasin
Als3 – hyphal specific member of ALS family, with role in adhesion to epithelial and endothelial cells
Null mutant in als3Δ displays a severe defect in endocytosis (epithelial and endothelial)
Further work showed:
Latex beads coated with recombinant Als3 are endocytosed
Als3 heterologously expressed in S. cerevisiae promotes endothelial cell invasion
hence Als3 can act alone to cause endocytosis
Als3 allows causes damage once in the cell
What is Ssa1?
Invasin
Ssa1 is a surface protein of the Hsp70 family
Loss of Ssa1 results in reduced endocytosis
Acts in same pathway as Als3
Mutant ssa1Δ displays attenuated virulence in both a OPC & disseminated mouse model
E-caherin acts as a receptor for?
Induced endocytosis
It’s normally involved in host cell-cell adhesion and tight junctions
Other receptors involved in induced endocytosis?
EGF and HER2 receptors
Act alongside E-cadherin, and bind Als3
Inhibition of receptor activity results in reduced disease in a OPC model
ZO-1 tight junction protein
ZO-1 co-localises to invasion sites
Small GTPases (Cdc42, Rac1 & RhoA) recruited to invasion site and required for actin reorganisation and uptake
What are M cells?
Uptake of antigens from lumen of GI tract
Cocultures of enterocytes and M cells show increased adhesion and invasion by C. albicans
Invasion is through induced endocytosis
So it may use M cells to cross the intestinal barrier
Candida invasion by active penetration?
Active penetration
Transcellular - directly into epithelial cells
Paracellular - between cell junctions
Requires fungal viability – active process
Potentially involves:
Physical force from directional growth and tugor pressure
Secreted hydrolytic enzymes degrading host tissue, esp. secreted proteinases
Secretion of Candidalysin toxin
What is Secreted aspartyl proteinases (SAPs)?
10 membered gene family
Proteinase inhibitors can display a protective role in infection models
May play a role in virulence through:
Nutrient acquisition
Facilitating adhesion and invasion
Immune evasion
Role in invasion:
Paracellular – degrading tight junction proteins
Transcellular – activating C. albicans cell surface proteins
SAPs may act in transcellular invasion by altering surface properties of C. albicans?
Pepstatin A was seen to block internalisation of C. albicans following pre-incubation
Suggests SAPs role is to proteolytically activate Candida cell wall proteins required for invasion
SAPs play a role in paracellular invasion by?
Degrading E-cadherin
Protease inhibitors block the degradation of E-cadherin
Rim101
Transcription factor involved in the pH response
rim101Δ mutant displays reduced E-cadherin degradation
Rim101 controls Sap4, 5 & 6 expression
Candidalysin causes?
Epithelial cell damage
ECE1
Encodes hyphal specific secreted protein
Proteolytically processed in the Golgi to give 8 peptides
An ece1D mutant adheres and invades host tissue but fails to cause epithelial cell damage
Cryptococcus invasion?
Induced endocytosis
Paracellular invasion
Trojan horse
Induced endocytosis of cryptococcus?
Invasin:
Hyaluronic acid – present as fibrous structures in the capsule
CPS1 encodes a hyaluronic acid synthase, and mutants demonstrate reduced invasion plus attenuated virulence
Receptor:
CD44 – cell surface glycoprotein binds hyaluronic acid
Interaction of CD44 & hyaluronic acid triggers pseudopod formation & invasion
Cryptococcus paracellular invasion?
The enzyme Urease which helps convert urea into ammonia + carbonic acid acts in paracellular invasion
Recognised virulence factor in a number of bacteria and fungi
In Cryptococcus is required for invasion across the Brain blood barrier
Mutants in URE1 display a defect in invasion into the brain
Action of urease may result in local pH rise & degradation of tight junction proteins (e.g. ZO-1)
Mutants in Ure1 (plus accessory proteins Ure7 & Nic1) display virulence defects and reduced brain burden
Trojan horse mechanism of invasion for cryptococcus?
Trojan horse mechanism requires: Initial phagocytosis Survival of Cryptococcus during migration Expulsion from the phagocyte It supports other form of invasion
Polymorphism on virulence?
Switching forms can impact on: Adhesion Host invasion Regulation of virulence traits Biofilm formation Immuni-modulation and evasion mating
C.albicans morphologies in vivo?
A mixture of yeast, pseudohyphae and hyphae are seen in tissue sections
Opaque cells are seen on skin
Chlamydospores have not been seen in clinical samples
Environmental signals triggering hyphal morphogenesis?
Hyphal growth is promoted by:
Temperature (>35 ºC), serum, neutral pH (>6.5), high pCO2, low pO2, N- or C- starvation, matrix embedded growth
Yeast growth is promoted by: Acidic pH (<6.5), temperature (<35 ºC), NH4+ ions, farnesol, homoserine lactone
A combination of signals is often required
e.g. Serum + 37 ºC provides robust signal for hyphal development
Signalling pathway for hyphal development?
cAMP-PKA
MAPK
Efg1 plays a central role in?
Filamentation
Hence : Efg1 is an activator of hyphal development
His hyphal form needed to cause disease?
yes
Tupq and Nrg1 are?
Negative regulators of filamentation
When activated keep pathogen in the yeast form
Yeast cells can reach target organs but fail to cause?
Disease
Tissue burdens unaffected by blocking morphogenesis – yeast can reach target organs
Yeast in target organs retain pathogenic potential
Role of hyphal morphogenesis in virulence?
Ability to switch forms required for virulence
Yeast can persist in target organs
Hyphal development required to cause tissue damage
Hyphal formation is linked with the co-expression of potential virulence factors
Two potential views:
Hyphal formation per se is required for virulence
or
Hyphal-specific genes are required for virulence
Hgc1 is required for?
hyphal morphogenesis
Hgc1 - hypha‐specific G1 cyclin‐related protein
Demonstrates hyphal-specific expression
Required for maintenance of hyphal growth
Doesn’t affect the expression of hyphal specific genes
Mutant displays attenuated virulence and fails to form hyphae in infected tissue
Role of hyphae in virulence?
Direct:
Active penetration & tissue damage
Thigmotropism
Escape following host cell engulfment
Indirect: Through co regulated genes
Adhesion & invasion (induced endocytosis) Altered host immune response Hydrolytic enzyme production Biofilm formation Secretion of Candidalysin toxin
Definition of a titan cell?
In cryptoccocus
Size > 10µm Single large vacuole Highly polypolid (>2C) Thickened cell wall Altered capsule
Titan cells expel ‘yeast-like’ daughters
Daughters produced more rapidly than by yeast phase cells and bud from the same site
Daughters can be haploid, diploid or aneuploid
Titanisation is both positively and negatively regulated through?
Positive regulation through cAMP/PKA pathway
Mutants are highly attenuated & rapidly cleared
Negative regulation through Usv101
Prolonged pulmonary phase of infection
Both also regulate capsule & melanin biosynthesis
Titan cells promote?
Survival in the lung and provide an active source of infection
Resist phagocytosis
Display resistance to host stresses (oxidative & nitrosative)
Rapidly produce daughter cells
Induce non-protective immune response (Th2)
Aneuploidy in daughters may be linked with azole resistance
C. albicans is exposed to multiple host niches such as?
Commensal:
Part of gastrointestinal microbiome
High nutrient levels
Controlled through competition with other microbes
As a pathogen it infects multiple sites:
Superficial infections: Mucosa & Skin
Disseminated: multiple target organs
Disseminated infection
Epithelial invasion = access to bloodstream = invasion of various tissues
Survival following phagocytosis
Cryptococcus is also is exposed to multiple niches?
Environment
Commonly found in soil & guano of birds
Interacts with amoebae as a natural predator
Infection generally associated with the lungs and/or brain
Dissemination:
Epithelial invasion = access to bloodstream = invasion into brain
Growth as an intracellular pathogen
Metabolic adaptation is essential for virulence how?
Adaptation required for pathogen to flourish in host niches
Glycolysis, gluconeogenesis and starvation responses (glyoxylate cycle) all contribute to disease
Colonisation will probably alter the niche
Main nutrient sources: host-derived glucose, lipids, proteins & amino acids
Central carbon metabolism is important in establishing an?
Infection
What is important for the development of a systemic infection?
Glycolysis
What is activated to resist phagocytosis?
Glyoxylate cycle and gluconeogenesis
PCK1 gene is a main control point for the regulation of gluconeogenesis. The cytosolic enzyme encoded by this gene, along with GTP, catalyzes the formation of phosphoenolpyruvate from oxaloacetate, with the release of carbon dioxide and GDP
ICL1 catalyzes the formation of succinate and glyoxylate from isocitrate, a key step of the glyoxylate cycle, which operates as an anaplerotic route for replenishing the tricarboxylic acid cycle. Required for growth on ethanol or acetate, but dispensable when fermentable carbon sources are available.
Metabolic flexibility following phagocytosis?
Alternative Carbon source utilisation:
Glyoxylate cycle, b-oxidation, gluconeogenesis activated
Nitrogen utilisation:
Up-regulation of amino acid biosynthesis
Significant induction of arginine biosynthesis
Activation of uptake systems:
Oligopeptide transporters, permeases for Carbon compounds and amino acids
Recycling:
Up-regulation of vacuolar protease genes
Trace metal acquisition:
Iron, copper and zinc uptake systems activated
Do Carbon sources affect C.albicans virulence?
yes
Due to altered cell wall architecture and stress resistance
Alternative Carbon sources are required in vivo
Lactate is a potential alternative Carbon source:
Produced by host tissues
Produced by the microbiome in GI / vaginal tract
Present in common surgical fluids
Candida albicans uses glucose and lactate concurrently
Lactate grown cells display altered cell walls
Growth on lactate does what to the cell wall?
Increased adhesion and cell wall hydrophobicity
Resistance to osmotic and cell wall stress, and antifungals
Dampened cytokine response
Reduced uptake by macrophages and increased macrophage killing
Increased virulence in systemic and vaginal models
What do fungal secreted hydrolyses do?
Roles in virulence: Nutrient acquisition Facilitating adhesion & invasion Immune evasion Biofilm formation Cell wall maintenance
Secreted hydrolyses in C.albicans?
Commonly encoded by gene families, including:
SAPs – Secreted Aspartyl Proteinases
LIPs – Lipases
PLBs – Phospholipases
Secreted hydrolyses in C. neoformans?
Proteases (aspartyl, metallo-, carboxy- & serine)
Urease
Phospholipase
Describe Secreted aspartyl proteinases (SAPs)?
10 membered gene family in C. albicans:
Sap1-8 secreted
Sap9 & 10 cell wall proteins
Display differential expression in vitro / ex vivo and in vivo
Proteinase inhibitors can display a protective role in infection models
May play a role in virulence through:
Nutrient acquisition
Facilitating adhesion and invasion
Immune evasion
But overall role still unclear
Differential expression of SAP family?
SAP1, 3 – Opaque cell specific SAP2 – Major proteinase with protein as the N source SAP4, 5, 6 – Hyphal specfic SAP8 – Temperature regulated SAP9, 10 – Constitutive
What is pepstatin A?
Inhibitor of aspartyl proteinases:
provides protection in an intranasal systemic model
led to faster clearance in a rat vaginal model
Suggests SAPs play an important role in virulence
Pepstatin A was seen to block internalisation of C. albicans following pre-incubation
Suggests SAPs role is to proteolytically activate Candida cell wall proteins required for invasion
Use of HIV proteinase does what?
Provide protection against candidosis:
Use of HIV proteinase inhibitors in AIDS patients reduced prevalence of oral candidosis
HIV proteinase inhibitors
Inhibit Sap activity
Induce rapid clearance of fungal burdens in a rat vaginal model
Reduce adhesion to epithelial cells
SAPs play a role in paracellular invasion by degrading?
E-cadherin - The E-cadherins also known as the epithelial cadherins on the surface of one cell can bind with those of the same kind on another to form bridges
Protease inhibitors block the degradation of E-cadherin
The role of what in systemic infection is controversial?
SAP1-6
Early mutant studies:
Δsap1-3 mutants attenuated in mucosal model
Δsap4-6 mutants attenuated in systemic model
Issues identified with standard methods used to generate null mutants (expression of URA3 selection marker)
Re-assessment of controlled strains indicates Sap1-6 are largely dispensable in a systemic model of infection
Cryptococcus secreted metalloprotease (Mpr1) is required for?
CNS invasion
Mpr1 – identified in screen of protease mutants defective in in vitro model of Blood Brain Barrier invasion
MPR1 mutants:
Defective in adhesion and invasion of BBB
Attenuated virulence with reduced burdens in the brain
