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
`
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
Cryptococcus aspartyl protease (May1) required for?
growth at low pH and virulence
May1 – encodes the major aspartyl protease
Required for growth at low pH
Mutant displays attenuated virulence & fails to proliferate in macrophages
Iron uptake for fungi when they have infection?
Iron is essential for all life-forms, and can exist as Fe2+ or Fe3+
Iron is tightly isolated in the host
Free iron concentration in serum ~10-18 M
Under aerobic conditions iron can be extremely toxic Fenton reaction: Fe2+ + H2O2 + H+ Fe3+ + HO˙ + H2O
Iron sources in host (~40 mg / kg)
Haemoglobin in blood (66% of iron) Transferrin / lactoferrin (0.1% of iron) sequesters extracellular iron Ferritin (30% of iron) Maintains intracellular iron homeostasis
Candida and Cryptococcus strategies for iron exploitation within the host
3 main routes of acquisition:
Siderophore uptake
Reductive pathway uptake
Haem and haemoglobin utilisation
Siderophore uptake to acquire iron?
Siderophore – low MW iron chelators involved in scavenging iron
C. albicans & C. neoformans lack siderophore synthesis pathways
Both can utilise siderophores produced by other organisms via a siderophore transporter (Sit1)
Reductive pathway for iron uptake?
Utilises iron free in the environment and from transferrin and ferritin
3 Stage process:
Fe3+ to Fe2+ via ferric reductase:
Solubilising iron
Fe2+ to Fe3+ via multi-copper oxidase:
Prevents toxicity due to free radicals
Fe3+ through the membrane via iron permeate:
Transport Fe3+ into cell, essential in low iron and required for virulence
Use of ferritin as iron source?
Ferritin only binds to hyphal cells
Als3 required for ferritin binding, and use of ferritin as an iron source
Utilisation of hame by C.albicans?
C. albicans causes erythrocyte lysis
Resulting in release of haemoglobin / haem proteins
Rbt5 family 5 members (Rbt5, Rbt51, Csa2, Csa1, Pga7), associated with cell wall or secreted Extract haem from haemoglobin and internalise it by endocytosis Δpga7 – attenuated in systemic model
Hmx1 – Haem oxygenase
Degrades haem releasing Fe2+
Δhmx1 – attenuated in systemic model
Utilisation of hame by C.neoformans?
Secretes Cig1 - hemophore to sequester haem
Highly expressed following iron starvation
Secreted mannoprotein that binds haem
Contributes to virulence alongside reductive iron uptake
Features of biofilms?
Community of surface attached (sessile) microbes
Can develop on living or inert surfaces
Enclosed in extracellular matrix, forming a complex 3D architecture
Can be polymicrobial
80% microbes generally live as biofilms
65-80% human infections associated with biofilms
Candida biofilms?
Associated with either:
Biotic surfaces: oral or vaginal mucosa, target organs
Abiotic surfaces: dentures, catheters
Biofilm formation associated with
High level resistance to antifungals (azoles & polyenes)
Resistance to host immune factors (e.g. ROS)
Cryptococcus biofilms?
Associated with either:
Environmental reservoirs – soil/pigeon excreta
Abiotic surfaces – medical devices (shunts. catheters)
Biotic surfaces – Cryptococcomas in brain tissue
Biofilm formation associated with
Resistance to environmental stresses (temp, pH, UV, predation)
High level resistance to antifungals (azoles & polyenes)
Resistance to host immune factors (e.g. ROS)
Similar structure in vitro and in vivo but morphological forms more interspersed in vivo
There is a upper hyphal layer and a basal yeast layer, seeks normally encased in extracellular matrix
Antifungal resistance in candida biofilms?
MIC 30-2000x higher for biofilms
Affects: Azoles, Amphotericin B, 5-Flucytosine
Lipid formulations of AmB & echinocandins are active against biofilms
Molecular basis of resistance Activation of efflux pumps – early stage Altered sterol composition Persister cell formation ECM - β1,3-glucan sequesters azoles
What is required for biofilm formation?
Morphogenesis
Biofilm formation requires hyphal formation, plus expression of hyphal specific genes
Stages of biofilm formation?
Attachment – adherence of yeast cells to substrate:
Adherence to host or abiotic surfaces
Typically involves cell wall proteins
E.g Eap1, Als1, Als3, Hwp1
Eap1 – cell wall protein
Expressed in both yeast & hyphal cells
Plays a role in adhesion to polystyrene and epithelial cell layers
Transcriptome changes seen within 30 min of attachment
Initiation – formation of micro-colony (adhesion & proliferation):
Filamentation required for biofilm formation
efg1/cph1 double mutant displays clear biofilm defect
hgc1 mutant also displays a biofilm defect
BCR1 - Bcr1 controls hyphal adhesins required for biofilm integrity and causes hyphal growth
Transcription factor
Mutant identified from genome wide screen for biofilm defects
Maturation – hyphal development & ECM production:
Extracellular matrix components
Carbohydrate
Mainly glucose, but also Hexosamine & Uronic acid
Proteins
Phosphorus
eDNA
Production dependent on environmental cues
Plays a role in the structure of the biofilm
Protective role through blocking host immune factors and antifungals
Zap1 controls β1,3-glucan levels in the ECM
Mature biofilms do not stimulate ROS production by neutrophils & are resistant to killing
β1,3-glucan in ECM protects C. albicans from neutrophil attacks
ECM also blocks the release of Neutrophil extracellular traps (NETs)
Biofilm ECM sequesters azoles
Dispersal - release of non-adherent yeast cells
Dispersed cells
Predominantly yeast
Display elevated adhesion, biofilm formation and virulence
Dispersed cells adapted to form biofilms and establish new sites of infection
Nrg1, negative regulator of the yeast to hyphal switch, may play a role in dispersal
How if biofilm formation controlled by a transcriptional network?
6 transcription factors identified with biofilm defects
Interconnected network – positive feedback between regulators
Common set of genes, plus genes unique to each regulator
Network may allow fine tuning and response to different environmental conditions
Structure of crypto coccus biofilms?
Yeast cells encased in exopolymeric matrix - similar components with matrix containing capsule
Enhanced resistance to head cold and UV light
Antibodies can block biofilm formation
Addition of sub lethal EDTA/EGTA (Ca2+/Mg2+ chelators) blocks biofilm formation & GXM release
Mg2+ is also involved in regulation of GXM synthesis
Divalent cations therefore play both structural and regulatory roles in EPM production
Attachment – adherence to substrate (1,2)
Initiation - micro-colony formation (3)
Maturation – EPM production (4)
Dispersal - release yeast cells (5)
Biofilm derived cells display?
Thicker capsule and release more GXM
Biofilms display resistance to oxidative stress agents and specific antimicrobial peptides
Capsule of biofilm-derived cells is thicker
Biofilm-derived cells also release more GXM
Phenotypes associated with elevated expression of capsule-related genes
Biofilm-derived cells are less actively phagocytosed and killed by macrophages
3 types of innate response?
Physical barriers:
Skin
Mucosa
Cellular:
Monocytes
Macrophages
Humoral: Complement Antimicrobial Peptides Cytokines Chemokines
2 types of adaptive response?
Cellular:
Lymphocytes
T cells B cells
Dendritic cells
Humoral:
Antibodies
Cytokines
PAMPS?
Pathogen associated molecular pattern
Conserved motif on pathogen
Not present on host tissue
What are PRRs?
Pattern recognition receptor Recognise & responds to PAMP Activates immune response Induces release of cytokines Phagocytosis, Inflammation Variety of receptors PAMPs Toll-like receptors Mannose receptor Dectin-1
How is the cell wall important for host-fungal interactions?
protection adhesion wall permeability antigenicity immunomodulation phagocytosis
Candida albicans cell wall components?
50-60% b-glucan (b1,3- & b1,6- polymers of glucose) - Recognised by the receptor Dectin 1 which is a c type lectin and mediates phagocytosis and cytokine response
0.5-3% Chitin (b1,4- polymer of N-acetylglucosomine)
Essential skeletal components of cell wall
Outer surface enriched in cell wall proteins
Genome encodes 80-120 cell wall proteins
Approx. 15 present at any time
Has mannan on wall which is a PAMP - hence when removed the immune response is lowered
C.albicans cell wall epitopes are involved in?
Inducing cytokine release, so are PAMPS essentially its where the antibody binds
What can a capsule do for Cryptococcus?
Hides cell wall so capsular components not recognised by PRRs
Composed of:
Polymer 1: Long fibers of glucuronoxylomannan GXM
repeating 6 motifs (M1-M6/7) of 3xMannose backbone, decorated with Xylose and Glucuronic acid
Polymer 2: Fibers of galactoxylomann GalXM
Divalent cations (Mg2+ and Ca2+) bridge polymers and assist in capsule assembly and stability
What can opsonins do to cryptococcus capsules?
Opsonins can recognize capsule:
complement can deposit in capsule (and mediate phagocytosis)
However T and B cells have poor/decreased responses to sugar epitopes antibodies do exist, but in lower levels, less variety of antibodies mediate phagocytosis and facilitate complement deposition
Implication for vaccines: conjugating GXM with a carrier protein and using adjuvants
CAPSULE EFFICIENTLY AVOIDS SOME OF INNATE AND ADAPTIVE IMMUNE RECOGNITION
Fungal strategies toEvade and defend from immune detection?
Capsule production (C. neoformans)
Yeast-hyphal switch (C. albicans)
Masking of β-glucan (C. albicans)
Produce more α-glucan (H. capsulatum)
Melanin production (many fungi)- quenches Reactive oxygen species
Capsule in Cn absorbs Reactive oxygen species
Degradative attack- proteases
Abundant detoxifying enzymes- multiple SODs (superoxide dismutase), catalase, etc, in each genome
Metabolic flexibility: capable of using many different nutrients
Environmental lifestyle (outdoors) has made fungi very sturdy
C.albicans evasion?
Yeast – hyphal transition
Cell wall changes
Different PAMPs
Differential recognition of yeast and hyphae
Change in balance of pro- or anti-inflammatory response
Direct modification
Inhibition and degradation of immune system molecules
Dectin-1 mediates recognition of yeast but not hyphal cells – mannan masking, how does this work?
Dectin 1 binds to specific sites on yeast cells where b-glucan is exposed
b-glucan is masked by mannans
on hyphal cells, resulting in altered cytokine response
What does Caspofungin treatment result in?
Cell wall perturbation exposing b-glucan and chitin in vitro and in vivo
b-glucan is also exposed in vivo following damage from host factors
Recognition of C. albicans strains is Dectin-1 independent by what percentage?
50%
Alteration of chitin content in vivo impacts on Dectin-1 dependency
Dectin-1 independent strains display cell wall alterations in vivo
Elevated levels of chitin synthase, correlating with a higher chitin content and altered wall architecture
Elevated chitin levels reduce dependency on Dectin-1 for resistance and inflammatory response
How does chitin detection dampen the inflammatory response?
Purified chitin:
Does not elicit a cytokine response
Blocks recognition of C. albicans cells specifically
Chitin-PRR is not fully elucidated yet ( or not existent)
C.albicans cell surface variability?
Potential source of variability
Cell wall proteins
80-120 cell wall proteins encoded by Candida genomes
Approx. 15 present at any time
Some evidence for involvement in immune response
Cell wall carbohydrates
Alteration of chitin : glucan : mannan composition
Mannan
Encoded by multiple gene families
Variation in mannan structure may influence host response
Evasion of the complement system?
Pra1, Gpm1, Gpd2 & Hgt1
Present on C. albicans cell surface
Bind components of the complement system & potentially block activation
Pra1 – pH regulated antigen 1
Cell surface, secreted & host surface associated protein
Secreted form – binds C3 and prevents activation, and blocks C3b deposition inhibiting macrophage uptake
Blocking of complement system?
Secreted aspartyl proteinases
10 membered gene family
Sap1-3 degrade complement C3b, C4b and C5
Blocks C3b deposition on cell surface by degrading it
Prevents generation of C5a which acts as a inflammatory mediator and chemoattractant
App1 protein in C. neoformans
Inhibits phagocytosis of yeast
Recombinant protein: inhibits phagocytosis of erythrocytes, when opsonized with iC3b+Ab but not Ab alone
Inhibits phagocytosis of yeast:
binds to CD11b subunit of CR3 (CD11b/CD18)
binds to CR2 (CD21)
What does Candidalysin in C.albicans do?
Causes holes in host cells and subsequent cell death: only produced by hyphae, but independent of hyphae formation
even at sublytic concentrations it triggers inflammation
Essential for oral candidiasis
Dual role in systemic infections
What do metaloproteases in C.neoformans do?
Mpr1 required for traversing endothelial cell layers
Mpr1 when expressed in S. cerevisiae (brewer’s yeast) was capable of traversing endothelial barriers
In Candida albicans Regulation is required to respond to changes in the host environment?
Changes in environment C-, N-, P-, Fe, Cu, Zn-source Secondary metabolites pH temperature Host stress e.g. ROS, NO Biofilms, mating Antifungals
Signal / stimulus to receptor to transduction to responses in a biological process through DNA to RNA to protein to modified protein
Stress associated with immune response?
Phagocytosis - Change in pH and weak acid
Low amino acid availability
Lack of glucose
Lack of iron and other metals
Respiratory burst ˗ Reactive oxygen species
Reactive nitrogen species
Cytokine burst - Inflammatory response
Elevated temperature
Pathogen response overall will be – immune evasion or stress response
Recap what are the Fungal strategies to cope with stress Evading immune detection?
Capsule production (C. neoformans) Melanin production (C. neoformans, H. capsulatum, A. fumigatus) Yeast-hyphal switch (C. albicans) Produce more α-glucan (H. capsulatum)
Fungal strategies to cope with stress - expression of stress response pathways?
Oxidative stress response Nitrosative stress response Heat shock response Amino acid starvation response Carbon–source specific stress Osmotic stress response pH response
What are stress responses?
Stress responses are “fitness attributes”
Adaptation to host microenviroment
Required for physiological fitness
Fitness is essential for virulence
Importance of stress response in virulence
Stress responses are activated in vivo
Mutants display attenuated virulence
Stress may be sequential or combinatorial in vivo
What are the MAPK pathways in C.albicans?
Mkc1 and Cek1 pathways:
Respond to cell wall damage
Both pathways required for virulence
Describe oxidative stress in C.albicans?
Oxidative stress key to interaction of C. albicans with phagocytes
C. albicans – relatively resistant to oxidative stress
Hog1 & Cap1 pathways key to signalling oxidative stress
Signalling defects result in:
Sensitivity to oxidative stress
Increased killing by phagocytes
Reduced virulence
What is Hog1?
Hog1 is required for response to oxidative stress
Hog1 is phosphorylated following stress
Hog1 required for growth following stress
Hog1 enters nucleus following stress
hog1 mutant readily killed by macrophages, and displays virulence defect in mouse model
What is Cap1?
Cap1 – AP-1 like transcription factor
Cysteine residues in C- terminus oxidised following stress
Activated Cap1 enters nucleus
Gene response activated through YRE elements in promoters
Cap1 mutants are sensitive to oxidative stress and are more sensitive to phagocytic killing
Virulence defect seen in mini-host models but not following murine intravenous challenge
What is the antioxidant defence?
Antioxidants are compounds that inhibit oxidation. Oxidation is a chemical reaction that can produce free radicals, thereby leading to chain reactions that may damage the cells of organisms.
O2• is eliminated by:
Superoxide dismutases
Cytosolic, mitochondrial and secreted forms
H2O2 is eliminated by
catalases, glutathione reductases and glutathione peroxidases, and thioredoxins
All induced following oxidative stress & phagocytosis
Secreted superoxide dismutases are required for survival following macrophage engulfment
Response to nitrosative stress?
Reactive nitrogen species produced by phagocytes
Response involves small set of genes
Oxidative stress (catalase, glutathione)
Nitric oxide dioxygenase (Yhb1)
Yhb1
Detoxifies NO•
Mutant is sensitive to NO• and more susceptible to neutrophil killing
Mutant displays minor defect in systemic model of infection
Unknown secreted factors also inhibit NO• production
Response to thermal stress?
Hsf1 Phosphorylated in response to heat shock Activates expression of chaperones Refolding or degradation of damaged proteins Regulated through interaction with Hsp90
Role of thermal stress response in a warm-blooded host?
Mutants blocking Hsf1 activation demonstrate attenuated virulence
Modelling stress response suggests activation can result from slow thermal transitions (e.g. development of fever)
Stress in vivo may be sequential or combinatorial?
Stress in vivo may be sequential or combinatorial
Coupled with use of alternative carbon sources
Sequential stress can result in:
Adaptation & cross protection
Combinational stresses can act synergistically
Cationic plus oxidative stress are synergistic – increased killing
Combination results in “stress pathway interference”
What is cyclosporin/CsA?
Product of secondary metabolism of soil fungus (Tolypocladium inflatum)
inhibits nematodes, insects and other fungi
first isolated in 1970 antifungal agent
1971 discovered to have immunosuppressant effects
revolutionized transplants!
Calcineurin in mammals?
Serine-threonine-specific Ca2+–calmodulin-activated protein phosphatase
Calcineurin inhibition inhibits T cell function
Widely used as an immunosuppressant target (Cyclosporin/CsA, tacrolimus/FK506)
Inhibits fungal growth at high temperature and prevents antifungal resistance
Widely conserved between fungi (and mammals)
Secreted and surface proteins allow bacteria to?
Colonise host tissues
Grow within host tissues
Avoid host defence mechanisms
Cause damage to the host
What is type 11 secretion in bacteria?
T2S is a two-step pathway in which proteins are
translcated to the periplasm via the normal Sec or Tat export machinery and are then secreted to the external environment by the T2S
The Tat/Sec translocon
Signal peptide dependent secretion across cytoplasmic membrane
Tat system involved in transfer of folded co-factor containing proteins
What is the Tat/Sec translocon?
Signal peptide
dependent secretion across cytoplasmic membrane
Sec system translocates unfolded exoproteins
Both Sec and Tat secreted proteins are cleaved by signal peptidase 1 (lsp)
How are the proteins pushed out?
Proteins pushed out by the assembly of pilin subunits to form a piston like structure
Unsure how channel is gated, though GspC, N or B are candidates
Microbial effectors secreted by Type 11 secretion?
Cholera toxin
•Phospholipase A L. pneumophilia (Sec)
•Phospholipase C L. pneumophilia (tat)
What’s bacterial type V secretion?
Proteins secreted by the Type V system are also called autotransporters as they mediates their own secretion through the OM
Have three domains; a sec dependent amino terminus, a cargo region and a b region
Microbial effectors secreted by type V secretion?
H. pylori VacA, the vacuolating Cytotoxin
Features of the type VII secretion?
Chaperone-usher pathway (CU)
VII
secretion
The Type I and Type P pili of UPEC
Describe type 1 secretion?
The cytoplasmic membrane is an ABC transporter (can be a RND or MFS protein)
A is a broad family of proteins called membrane fusion proteins (MFP)
O is indicative of members of the outer membrane family (OMF) of proteins
Typical Type I system
involved in the secretion of E. coli hemolysin
TolC can interact with Membrane Fusion proteins (MFP) that bind to either ABC/RND or MFS transporters
Typical Type I system
involved in the secretion of E. coli hemolysin
TolC can interact with Membrane Fusion proteins (MFP) that bind to either ABC/RND or MFS transporters
Describe type IV secretion in bacteria/
T4SSs are multisubunit cell envelope-spanning structures comprising a secretion channel and often a pilus or other surface filament or protein.
Their substrates can be Sec/TAT dependent in some cases.
numerous modes of action a bit like VacA (a type V auto transporter)
What are pathogenicity islands?
First used to describe two large unstable regions on the chromosome of uropathogenic Escherichia coli (UPEC)
Properties
•carry one or more virulence-associated genes •range from 10 kb to more than 100 kb
•frequently flanked on one side by a tRNA gene and often by direct repeat (DR) sequences
•Often, PAIs have a G + C content and a codon usage that differs from that of the rest of the core genome
Type III, IV and VI are complex systems often encoded by pathogenicity islands
Describe bacterial type 111 secretion?
The T3Smachinery, also known as the
injectisome or needle complex, is composed of a basal body anchored in the bacterial membranes and an external needle. The delivery of the effectors into the host cell cytoplasm requires an additional set of proteins, called translocators, which are themselves exported by the injectisome.
Key point T3S injects proteins directly into host cells!!
Types of gram positive bacteria secretion systems?
Sec/TAT dependent secretion
WXG secretion
LPXTG motifs - sortase
Lipoproteins
Accessory Sec systems?
SecA2-only substrates with (as shown here) or without an obvious signal peptide are recognized by SecA2 and are translocated most likely through the same SecYEG pore used by the housekeeping Sec1 export pathway
The serine-rich substrates of the SecA2/SecY2 export pathway have an AST domain adjacent to the signal peptide. A complex of Asp1, Asp2, and Asp3 might be involved in keeping the nascent substrate competent for its glycosylation (yellow dots) by various glycosylation factors (GFs) and possibly also in targeting of the substrate to the SecA2 protein
The WXG 100 (Type VII secretion system)?
’It was recently suggested that ESAT-6 secretion should be referred to as a type VII secretion system. The numerical classification is derived from Gram-negative bacteria, where polypeptides are transported across double membrane envelopes using mechanisms that are either independent of or expand the canonical Sec Pathway. Mycobacterial cell walls also encompass a double membrane envelope, including the plasma membrane and the mycolic acid layer with long aliphatic lipids. Thus, the term type VII secretion appeared to fit with the previously discovered type I–VI pathways. Nevertheless, as already noted by Pallen, genes encoding for putative WXG100 proteins are also found in the genomes of Gram-positive bacteria lacking double membrane envelopes. For example, two small WXG100 proteins, EsxA and EsxB, are secreted by S. aureus in a manner depending on genes that are clustered with esxA and esxB. This gene cluster has been named for its function: ESAT-6 secretion system (Ess)’’
Describe the sorties system?
Sortase substrates (i.e. CWA proteins (cell wall anchoring)) of Gram-positive bacteria, such as LPxTG proteins of Staphylococcus, Streptococcus, Enterococcus, Listeria and other Firmicutes, constitute an amazing repertoire of colonizing factors, toxins, proteases and enzymes that enable these opportunistic pathogens to mount successful infections
Gram positive secretion systems recap?
Sec/TAT dependent and WXG secretion leads to an extracellular fate
Sortase is a way of anchoring proteins at the cell surface
Several similarities between protein secretion in both Gram +ve and –ve bacteria
Describe bacterial type III secretion?
The T3SS machinery, also known as the
injectisome or needle complex, is composed of a basal body anchored in the bacterial membranes and an external needle. The delivery of the effectors into the host cell cytoplasm requires an additional set of proteins, called translocators, which are themselves exported by the injectisome.
Key point T3SS injects proteins directly into host cells
What is EPEC disease?§
Classically associated with diarrhoea in young children
Significant cause of infant diarrhoea in developing countries High mortality rates of between 10% and 40%
Works by: Effacement of absorptive microvilli Induction of actin-rich pedestals Inhibition of water/nutrient transporter function Weak inflammatory response Disruption of tight junctions
4 stages of EPEC disease?
Initial adherence to epithelial cells
Signal transduction
Effacement, pedestal formation, intimate attachment
Disruption of tight junction intergrity
Describe the EPEC locus of enterocyte effacement (LEE) 35kb?
Esc - E. coli secretion
Esp - E. coli secreted proteins
Sep - Secretion of E. coli proteins
Ces - chaperones
Main aim of effectors in type III secretion in EPEC disease?
Inhibit phagocytosis – pedestal formation
Modulation of the immune response
Prevent cell death
Describe Inhibit phagocytosis – pedestal formation?
Translocated Intimin receptor - TIR
Receptor for Intimin – intimate attachment stage
Stmiulates clustering of TIR
Key in actin rearrangement
Inhibition of NF-kB transcription
Phosphorylation of C-terminal tyrosines leads to N-WASP recruitment and actin polymerisation
EspH is also involved in actin polymerization but mechanism is independent of but also synergetic with signaling cascades activated by Tir.
EspB inhibits phagocytosis by binding to myosins that interact with actin filaments in mediating cellular processes
Type III effectors modulating the immune response?
Effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity.
Describe EspF disrupting tight junctions?
allows material from the lumen to penetrate the adlumenal compartment of the epithelium causing inflammation, resulting in diarrhoea
EspF is sorted to mitochondria, with the disruption of mitochondrial membrane potential, the release of cytochrome c into the cytoplasm, all characteristic features of the mitochondrial death pathway
Features of type 6 secretion in bacteria?
T6SS gene clusters are recognisable on the basis that they all contain 13 ‘core’ essential and conserved genes which encode the proteins making up the basic secretion apparatus.
InVibrio cholerae, T6SS genes are co-regulated with genes involved in DNA uptake. Hence, T6SS-dependent killing of other bacteria is directed at neighboring cells, which release their DNA to be taken up by the killer, which can then integrate valuable genes and rapidly evolve, leading to antibiotic resistance or virulence
It has been shown that different combinations of Hcp, VgrG and PAAR proteins can assemble functional T6SSs, particularly specific VgrG–PAAR spike combinations. These different assemblies then lead to the delivery of alternative sets of effectors during individual firing events
Overall, different combi-nations of core Hcp, VgrG and PAAR components, together with cognate adaptor proteins, allow one T6SS to deliver multiple, structurally diverse effector proteins
What are the effectors of type six secretion system?
VgrG proteins can have an effector function.
‘Evolved VgrGs’ - C-terminal extension containing an effector, domain.
A range of predicted functions for VgrGs - actin cross-linking, ADP-ribosyltransferase, lipase, metalloprotease, peptidoglycan binding or hydrolysis.
Translocation of some
Vibrio cholerae type VI effectors requires bacterial endocytosis by the host cell.
These effectors can be classified into three categories:
Cell wall-degrading enzymes (murein hydro-lases)
membrane-targeting proteins (phospholipases and
pore-forming)
3. Nucleases
Co-expressed with immunity proteins
Types of toll like receptors?
TLR-1 and TLR-2.
Form a heterodimer at the cell surface which binds peptidoglycan
TLR-3.
Binds to the double-stranded RNA of viruses
TLR-4.
Activated by LPS
TLR-5.
Binds to the flagellin
TLR-6.
Forms a heterodimer with TLR-2 and responds to peptidoglycan and certain lipoproteins.
TLR-7 and TLR-8.
Form a heterodimer that binds single-stranded RNA
TLR-9.
Binds to unmethylated CpG DNA of bacteria
What type of receptor is TLR2?
Promiscuous
Against bacteria how does the immune response manifest itself?
Tb as an example - intracellular gram positive pathogen
IFN-y and TNFa key in activating macrophages and in presentation of TB peptides to T cells by MHC class 11 molecules T cells critical in clearing disease
IFN-y key in activating the presentation of TBpeptides to T cells by MHC molecules
ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic
reticulum where it sequesters β2M to inhibit cell surface
expression of MHC-I-β2M complexes, resulting in
downregulation of class I-mediated antigen presentation
What is ESAT-6?
A secreted
component of the
ESX-1 secretion
system (T7SS)
Steps of MHC class 11 molecules being presented
Uptake of extracellular proteins into vesicular compartments of antigen -presenting cells
Processing of microbial proteins into peptide fragments in lysosome/endosome
Biosynthesis and transport of MHC class 11 molecules to endoscopes
Fusion of vesicle carrying MHC class 11 molecules to endosomes with vesicles carrying processed peptides
Expression of MHC class 11 molecules with loaded peptide on cell surface
How TB inhibits this is on week 9 immunomodulation part 2
Interferences -
Inhibition of proteolysis within phagosome
Inhibition of gene expression for MHC class 11 including the normal up-regulation on activation with IFN-y
Disruption to assembly of MHC class 11 complex, or their transport to endosomal vesicles, prevention of co-localisation of MHC class 11 with processed antigen
Inhibition of peptide loading on to MHC class 11 molecules in endosome
Inhibition of endosomal transport of loaded peptide to cell membrane
So what do bacterial immunomodulators do?
Inhibit the immune response, for example this is what TB does to T cell activation
What are biofilms?
Biofilms are defined as matrix-enclosed bacterial populations adherent to
each other and/or to surfaces or interfaces. This definition embraces microbial aggregates and floccules and also adherent populations within the pore spaces of porous media
What can form a biofilm?
Several gram-positive (e.g. Bacillus subtilis) and gram-negative bacteria (e.g. Pseudomonas aeruginosa)
Fungi such as Candida, Fusarium and Malessezia species
Microalgae and diatoms
Archaea such as Sulfolobus acidocaldarius and Pyrodictium
Key properties of biofilms?
Microbial aggregates at interfaces
• Genetic response to surface adhesion
• Extracellular polymeric substances matrix
• Gradients resulting in heterogeneous microenvironments
• Retention of extracellular enzymes in a matrix
• Nutrient and water acquisition by sorption and retention
• Recycling of nutrients
• Enhanced tolerance to disinfectants, biocides and other stressors
• Enhanced intercellular communication
• Facilitated horizontal gene transfer
• Collective, coordinated behaviour
Flemming nature microbiology 2019
Sessile biofilms in the ocean features?
Sessile biofilms in the oceanic and continental subsurface perform biochemical reactions providing iron, hydrogen, methane and carbon dioxide
Mineral and sediment formation
Natural water purification and bioremediation
Intimately involved in global climate processes
Biofilm formation?
Biofilms arise from single founder cells
In the initial phase cells frequently change their directions of motion favouring lateral expansion
As the biofilm develops however, individual cells begin to engage in straight trajectories favouring vertical expansion
Features of the biofilm matrix?
The self-produced exopolymeric matrix comprises lipids, proteins, environmental DNA and exopolysaccharides
Many proteins exhibit amyloid-like properties. Amyloids are protein aggregates with fibrillar morphology and a β-sheet secondary structure
Hydrophobin forms a water-resistant ‘raincoat’ over the Bacillus subtilis
biofilm, and the cellulose produced by Escherichia coli increases the resistance of the community to desiccation
Autotransporter adhesins promote cell-to-cell adhesion and aggregation
The major protein constituents the biofilm matrix of several bacteria (including Proteobacteria, Bacteroidetes, Firmicutes, Halanaerobium and Thermodesulfobacteria) are curli fibres. Curli are amyloidous protein fibres assembled on the cell surface within the nutrient depleted zones of a biofilm and provide structural integrity.
Cellulose is made of linear glucose chains and it has a protective, architectural and regulatory function during biofilm formation and protection from predators. When overexpressed, cellulose inhibit biofilm formation
The morphology of the developing biofilm depends on the production of the flagella filament and on the ability of the flagella to rotate
The major biofilm exopolysaccharide in Vibrio cholera is VPS. It is mainly composed of glucose and galactose, it confers rugosity, infectivity and protection to the biofilm
There are three main proteins involved in the V. cholerae biofilm matrix structure and integrity namely Bap1, RbmA and RbmC located in outer membrane vesicles
The B. subtilis biofilm matrix predominantly comprises a large molecular weight soluble secreted polysaccharide, and extracellular proteins TasA, TapA and BslA
Molecular mechanisms governing biofilm formation?
Biofilm formation and dispersal are highly controlled processes regulated at the genetic level and by environmental signals.
The main molecular key players are:
- quorum sensing
- cyclic adenosine monophosphate
- small RNAs
newly adherent cells are loosely associated with a surface. Through successive surface interactions and detachments, cells become surface adapted due to a progressive increase in cyclic adenosine monophosphate levels and a gradual corresponding increase in type IV pili, a key sensor component for attachment
Attachment: an increased expression of the small noncoding regulatory RNAs rsmY and rsmZ results in enhanced initial attachment to abiotic surfaces. In fact, rsmZ and rsmY reduce the activity of effector protein RsmA.
RsmA is a negative post-transcriptional regulator of the biofilm matrix polysaccharide Psl. rsmZ and rsmY also downregulate another effector, RsmN, controlling the same functions as RsmA.
Therefore, high levels of rsmZ and rsmY induce hyperattachment of Pseudomonas aeruginosa biofilms by increasing the amount of Psl.
Development: subsequent biofilm development can be hampered by the suppression of rsmZ
Development: QS systems are required for biofilm development by controlling the synthesis of rhamnolipids which maintain the channels in mushroom-shaped structures, resulting in the proper distribution of nutrient and oxygen and removal of waste products
QS also plays a role in the release of a large amount of eDNA at the late stage of biofilm development, as a consequence of the autolysis of a bacterial subpopulation
Dispersal: The overproduction of rhamnolipids causes the biofilm detachment from the surface, leading to its dispersal
Example QS systems are the N-acylated homoserine lactone based LasIR
and RhlIR systems and the quinolone signal based system in Pseudomonas aeruginosa
Genotypic heterogeneity within biofilms?
Diversity protects communities from unstable environmental conditions (insurance effects)
At the genetic level some biofilm-derived variants exhibit an increased ability to disseminate, whereas others manifest accelerated biofilm formation. The presence of these functionally diverse bacteria increases the ability of biofilms to resist an environmental stress. In Pseudomonas aeruginosa these induced genetic changes are produced by a recA-dependent mechanism
Chemical heterogeneity within biofilms?
Biofilms contain bacterial cells that are in a wide range of physiological states due also to concentration gradients of metabolic substrates and products
Oxygen concentration decreases from the fluid to the biofilm into the biofilm depths
Solutes that are the size of oxygen diffuse in the matrix at a rate that is approximately 60% of the diffusion rate in pure water but it is also depleted by bacteria within the biofilm
Phenotypic heterogeneity within biofilms?
Bacterial growth and activity are slowed due to substrate limitation and accumulation of acidic waste products
Bacteria sense and adapt to environmental conditions such as anaerobiosis, starvation, oxidative stress, osmotic stress, antimicrobial challenges and pH stress
Stochastic gene expression provides a further mechanism to generate phenotypic diversity in a population (division of labour). For example in Bacillus subtilis the repressor sinR is expressed in almost all cells whereas the sinI anti-repressor is expressed in only a subset of cells inducing matrix production
Biofilm-based infections are extremely difficult to cure because of?
1 nutrient and antibiotic gradients
2 matrix
3 eDNA
4 stress responses
5 genetic variants
6 multidrug efflux pumps
7 intercellular interactions
8 persister cells
Antimicrobial treatment of biofilms?
Decreased antibiotic penetration through the biofilm matrix alone does not fully explain the increased resistance of biofilms to antimicrobials
2 The exopolysaccharides Psl and Pel play a role in resistance to colistin, polymyxin B, tobramycin and ciprofloxacin by physically sequesters antibiotics in the biofilm matrix via electrostatic interactions. Moreover, enzymes present in the biofilm matrix, namely secreted β-lactamases, can degrade antimicrobials, thereby preventing these agents from reaching their cellular targets
3 eDNA increases biofilm resistance to certain antimicrobial agents by chelating cations such as magnesium, leading to a decrease in the effective concentration of Mg2+ in the environment activating signalling pathways of antimicrobial resistance
4 - Hypoxia induces resistance by slowing down metabolic activity or upregulating efflux pumps
- Stringent response due to amino acid starvation confers resistance by stalling ribosomes that are one of the major antimicrobial targets
5-6 Genetic variants and efflux pumps:
- the membrane-bound NdvB glucosyltransferase confers resistance to tobramycin, gentamicin and ciprofloxacin by producing cyclic glucans that interact with antibiotics preventing them from breaching the membrane
- the transcriptional activator BrlR confers resistance to tobramycin, norfloxacin, trimethoprim, tetracycline, kanamycin and chloramphenicol by activating the efflux pumps MexAB-OprM and MexEF-OprN in Pseudomonas aeruginosa
7 Quorum sensing systems such as Pseudomonas aeruginosa LasR and RhlR confers resistance to antimicrobials by hindering biofilm formation
The use of combinatorial therapy is more preferable instead of antibiotic monotherapy
What is the difference between antimicrobial resistance, antimicrobial tolerance and persistence?
Genetic resistance to antibiotics is the ability of bacteria to replicate and pass on their immunity to their progeny and not just survive in the presence of a drug
The most common measure of the level of resistance is the minimum inhibitory concentration (MIC), which is the lowest concentration of the antibiotic required to prevent the replication of the bacteria.
A higher MIC corresponds with a higher level of resistance.
Resistance is inherited and may be acquired by horizontal gene transfer of resistance encoding genes or mutations that confer the resistance phenotype
Antibiotic tolerance is the general ability of a population to survive antibiotic treatments, for example, by having a lower killing rate, but without a change in the MIC
What characterizes their slower killing, even at high concentrations of the drug, is the time required to kill a large fraction of the population, for example, the MDK99, which is the minimum duration of treatment that kills 99% of the bacterial population
Antibiotic persistence discovered by Hobby and Bigger in 1942 is the ability of a subset of the population to survive exposure to antibiotics
- Not all bacteria in a clonal culture are killed at the same rate (biphasic killing)
- When persisters regrow without antibiotics, their progeny is susceptible
- The level of persistence weakly depends
on the concentration of the drug as long as it is far above the MIC. - Persisters survive different classes of antibiotics
Heteroresistance is the ability of a small subpopulation to transiently display a substantially (more than eightfold) higher MIC
The heritability of the increased MIC is long enough to create detectable colonies
Viable but non culturable bacteria are normally culturable cells that have
lost their ability to grow on media but remain viable
Clinical relevance of antibiotic tolerance?
Bacteria that are genetically susceptible, yet phenotypically tolerant to treatment, play a critical role in perpetuating chronic and recurrent infections
Furthermore, recent in vitro evidence supports the hypothesis that antibiotic tolerance can also cause treatment failure by facilitating the acquisition of antibiotic resistance
E. coli UTIs are frequently formed due to the translocation of gut strains to the urinary tract. This transition from the diverse nutritional environment of the gut to the nutrient-poor, nitrogen-rich, high-osmolarity environment of the urinary tract is associated with the activation of tolerance-conferring responses
S. aureus causing joint replacement infections forms biofilms that are antibiotic tolerant generating recurrent and persistent infections
Molecular mechanisms underlying antibiotic tolerance?
Stress response activation induces the alteration of multiple cellular processes, including lowering the proton-motive force, decreasing cell wall turnover, altering DNA replication and repair systems such as the SOS response, inducing biofilm formation, and establishing intracellular bacterial communities. The net effect is a physiological state that is unfavourable to the lethal actions of major classes of antibiotics
Toxin-antitoxin: an increased presence of free toxins from toxin-antitoxin modules results in slowed growth, and is linked to antibiotic tolerance
Quorum sensing down-regulates metabolism, reduce DNA replication, up-regulate efflux pump activity, and participate in biofilm formation conferring antibiotic tolerance to the infecting bacterium, for example in P. aeruginosa pulmonary infections
Antibiotic treatment against antibiotic tolerant bacteria?
A screen of select carbon sources identified that addition of pyruvate, mannitol, fructose, or glucose, resulted in potentiation of aminoglycoside lethality towards antibiotic-tolerant E. coli populations
Pentobra is a new aminoglycoside designed specifically to circumvent the antibiotic tolerance that is conferred by low energy cell states
Clinical relevance of bacterial persisters?
Bacterial persisters have a role in the relapse and recalcitrance of infections
It was found that Bacteria causing salmonellosis and tonsillitis or lung infections were never cleared from the host during treatment with antibioticsand did not change genetype, hence were persisters
Similar findings were obtained for fungal infections (i.e. Candida albicans) indicating that the persister phenomenon may also be an important factor in the recalcitrance of fungal infections
The uptake of bacteria (e.g. S. Typhimurium and
M. tuberculosis) by host cells has been linked to an increase in the relative number of persister cells
Molecular mechanisms underlying the formation of bacterial persisters?
Several cellular targets of persistence effectors have been implicated in persister formation:
Reduced cellular energy levels can be the result of inhibition of ATP production or leakage of ATP out of the cell.
Persistence can be elicited by halted DNA replication
Translation can be reduced in persisters either directly by disrupting mRNA, tRNA, rRNA or ribosomal proteins, or indirectly by impeding ribosome assembly.
Proteins involved in these processes may aggregate under persistence-inducing conditions.
Persisters can be characterized by reduced intracellular drug concentration due to inhibition of antibiotic uptake, antibiotic efflux, or to low levels of prodrug-activating enzymes.
Persisters can express active defense mechanisms against antibiotic-induced damage.
Antibiotic treatment against bacterial persisters?
Two main strategies: inhibition of the formation of persister cells or killing existing persister cells. The latter can be achieved through direct killing or sensitization of the persister cells. Stimulation of antibiotic targets or influx increase the sensitivity of persister cells to conventional antibiotics.
Inhibition of persister formation can be obtained through interference with quorum sensing (M64), the SOS-respons (lexA3 inh.), persister specific genes (cadaverin), stationary phase respiration (NO) and the stringent response (relacin)
Sensitization of persister cells to antibiotics can be achieved through stimulation of ROS production (Ag+, L-serine, clofazimine) or stimulation of the bacterial metabolism and thereby antibiotic targets (C10, spent medium, cis-2-decenoic acid).
Antibiotic uptake can be increased through modification of the pH gradient (L-arginine) and direct stimulation of aminoglycoside uptake (L-serine, hypoionic shock, metabolites).
Direct killing of persister cells can be obtained via stimulation of nonspecific
protease activity (ADEP4, lassomycin), DNA crosslinking (mitomycin C, cisplatin) or membrane depolarization (boromycin, HT61, NCK-10). The most common
strategy to directly kill persister cells is through extensive damage of the bacterial membrane.
Clinical relevance of viable but non culturable bacteria?
Many bacterial species have the ability to resuscitate from the viable but non culturable state back to the culturable state when the stress is removed posing risk for human health
If viable but non culturable cells are present, the total number of viable bacteria in a sample will be underestimated by the CFU count method. Even worse, if all bacteria in the sample are in viable but non culturable state, food industry, water or clinical samples may be regarded as germ-free due to non-detection
Molecular mechanisms underlying the formation of viable but non culturable bacteria?
Stringent response: the stringent response protein RelA is upregulated during the VBNC state in V. cholerae. Likewise, it has been shown that E. coli mutants unable to produce the alarmone ppGpp (synthesized by RelA) were less likely to enter the VBNC state
Intracellular proteases: a study on Legionella pneumophila reported that ClpP, a protease that degrades antitoxins of E. coli and S. aureus, accumulated in VBNC cells. Another study on Salmonella enterica reported that a ClpX mutant exhibited a reduced rate of VBNC cell formation indicating that proteolysis is an important mediator of the VBNC state
Toxin-antitoxin modules: induction of the RelE and ChpAK toxins of E. coli produced viable but non culturable cells. Ectopic expression of HigB-1 and HigB-2 toxins of V. cholerae caused a proportion of the population to lose culturability
Antibiotic treatment against viable but non culturable bacteria?
Because viable but non culturable cells have low metabolic activity, they effectively become resistant to antibiotics, and yet are able to resuscitate and reinitiate infections.
Viable but non culturable Haemophilus influenzae present in biofilms are able to initiate chronic ear infections.
Vancomycin was reported to be effective against viable but non culturable E. faecalis only when at 500 times the MIC.
Viable but non culturable H. pylori are antibiotic resistant, which likely accounts for the frequent reinfections suffered by persons who undergo remission despite antibiotic treatment.
Uropathogenic E. coli cells, typically not detected by standard methods, were not eliminated by antibiotic treatment.
The role of persister and viable but non culturable bacteria in biofilms
Biofilms promote the presence of viable but non culturable and persister cells. Indeed, central areas deep within biofilms tend to be hypoxic, resource-limited, and acidic due to deposits of metabolic waste.
These stresses are known for inducing both the viable but non culturable and the persister state.
Given that biofilm-related infections lead to significant morbidity and mortality, studying the role that viable but non culturable and the persister bacteria play in biofilm biology is of significant interest.
Different genetic susceptibility to infection?
Table of week 11 slide 7
The influence of CFTR polymorphisms on susceptibility to infectious diseases?
cftr mutations can positively and negatively influence the susceptibility of the human host to different infections Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae Mycobacterium tuberculosis
Cystic fibrosis – an introduction?
Most common life-threatening inherited disease in the UK
Autosomal recessive condition, caused by mutations in cftr
Cystic Fibrosis Transmembrane conductance Regulator (CFTR)
Chloride channel on apical surface of epithelial cells throughout body
Regulates the flow of ions, and consequently fluid
Wider impact of CFTR mutation on immune function?
Abnormal lung environment:
Depleted ASL causing impaired MCC
Increased mucus viscosity
Excess pro infamatori cytokines
Chronic infection:
Predominance of Pa
Release of virulence factors - biofilm formation
Pa exoproducts stimulate IL-8, NLRP3 inflammasome
Intrinsic CFTR defect: Impaired phagocyte function Th2/Th17 skewed T cell responses Abnormal pro-inflammatory signalling Reduced counter inflammatory molecules
The pseudomonas paradox in CF?
Mucociliary clearance and defects in macrophage/neutrophil function are non-specific immune defences that shouldn’t predispose to specific pathogens
The profile of respiratory pathogens in CF patients is very different from that observed in non-CF patients
Non-CF patients are dominated by Streptococcus pneumoniae
In contrast, approx. up to 80% of CF patients (by adulthood) are chronically-infected with Pseudomonas aeruginosa
Pseudomonas aeruginosa:
Gram-negative opportunistic pathogen
Commonly found in soil & water
Increasing global importance as a cause of healthcare-acquired infections
Urinary tract infections, respiratory infections, dermatitis, soft tissue infections, bacteraemia, bone & joint infections, GI infections and a variety of systemic infections (burns/AIDS/cancer)
Primary pathogen in CF
During infection of the CF lung, P. aeruginosa typically converts to a “mucoid phenotype”
Associated with overproduction of the polysaccharide, alginate
Influences biofilm architecture, antibiotic tolerance and interaction with immune cells
CFTR is a pattern-recognition receptor for P. aeruginosa
CFTR binds the LPS of P. aeruginosa
Mutants of P. aeruginosa with truncated LPS core are not internalised
Presence of exogenous LPS inhibits P. aeruginosa internalisation, as does a synthetic peptide corresponding to CFTR
The predisposition of CF patients to P. aeruginosa and the subsequent success of the organisms as a CF pathogen is due to a combination of host and bacterial factors
CFTR genotype influences the epithelial innate immune function within the airways, specifically in relation to the response to P. aeruginosa
CFTR deficiency specifically predisposes to P. aeruginosa infection
What’s Ivacaftor?
a CFTR potentiator
Increases the channel’s open probability and transport of chloride
Initially approved for patients with at least one G551D mutation
What’s lumacaftor?
Improves the processing of ΔF508 CFTR and its transport to cell surface
Approved as a combination therapy with Ivacaftor (Orkambi)
CFTR deficiency predisposes to infection through both specific and non-specific mechanisms
Specific: CFTR-Pseudomonas interaction
Non-specific: General immune defects associated with CFTR deficiency
Restoring CFTR function in CF patients helps overcome this susceptibility to infection
What’s the benefit of being a cftr heterozygote?
Carriers of CF have less functional CFTR on the surface of epithelial cells
The high carrier rate implies a selective advantage of being heterozygous (the “heterozygote advantage”)
Conclusions on host genotype and susceptibility to infection?
Notes on CTFR and susceptibility to other pathogens is part 3 of this lecture haven’t made notes
The ability to establish infection is complex, involving a multitude of host and microbial factors
As exemplified by CFTR, polymorphisms within individual host proteins can both positively and negatively influence susceptibility to infectious disease through a variety of specific and non-specific mechanisms
CFTR polymorphisms may offer partial protection against typhoid, cholera and tuberculosis
Of those, TB seems to have been the most likely selective pressure for cftr mutations in Caucasian populations
Features of polymicrobial interactions?
Particularly relevant in the context of biofilms, where multiple species frequently coexist
Cumulative evidence from patients and animal models suggest that multispecies infections can lead to delayed wound healing, increased inflammation and can influence virulence & antibiotic tolerance
Pseudomonas aeruginosa and Staphylococcus aureus
Both significant pathogens; chronic CF infections, HAIs, wound infections
Pa & Sa co-infection is commonly reported in CF and wound infections
Mechanisms underlying microbial interactions?
Microbial interactions can be facilitated by several distinct mechanisms, including:
Quorum sensing (QS)
Release of extracellular products, many of which are QS-regulated
Competition for nutrients
Shaping immune responses & host-pathogen interactions
What is quorum sensing?
A form of cell-to-cell communication
Characterized by the secretion & detection of autoinducers
Mechanism of secretion & detection varies according to the autoinducer molecule
Cells within a population produce and respond to autoinducers
Concentration of autoinducer is proportional to population density
Detection of autoinducer results in coordinated behaviours within the population (e.g. secretion of proteases or haemolysins)
AL-2 a universal language?
AI-2 signal molecule is produced by a wide range of bacterial species
LuxS produces the precursor molecule (DPD), which undergoes various spontaneous rearrangements to produce distinct AI-2 molecules
Over 1/3rd of bacterial genomes reportedly encode a LuxS homologue
Mediator of QS, or simply a metabolic side product?
AI-2 and virulence-associated traits?
AI-2 has been implicated in virulence traits…… Biofilm formation Motility &/or chemotaxis Virulence factor production Adherence
Can the Activity of P. aeruginosa’s own QS systems can also be enhanced by microbial interactions?
Yes
Lots of reasons on slide 15
Conclusions about polymicrobial interactions?
Interactions between microbial species can have a profound impact on phenotypes that shape infection outcomes, including virulence
Commensal organisms (normal flora) can shape behaviour of pathogens
Quorum sensing is a key mediator of polymicrobial interactions
P. aeruginosa exproducts do what?
Influences efficacy of antibiotics against S. aureus
S. aureus HG003 exposed to culture supernatants of P. aeruginosa (lab strains & clinical isolates), prior to addition of antibiotic; survival then assessed after 24 hr
HQNO promotes tolerance of S. aureus to tobramycin
?
Tobramycin uptake by S. aureus is dependent on a proton motive force (PMF)
Pa HQNO collapses the PMF of Sa by inhibiting electron transfer, thus abolishing tobramycin uptake
i.e. HQNO production tolerance of Sa to tobramycin
P. aeruginosa induces multidrug tolerance in S. aureus?
. aeruginosa inhibits S. aureus respiration, and depletes intracellular ATP
Effect is primarily attributable to HQNO, but also pyocyanin and HCN
Rhamnolipids can enhance killing by tobramycin?
Rhamnolipids of P. aeruginosa can increase cell permeability by interacting with cell membranes
Facilitate tobramycin entry into otherwise tolerant PMF-depleted cells
The LasA endopeptidase of P. aeruginosa enhances vancomycin killing of S. aureus during co infection
In summary, Radlinski et al. characterized 3 distinct P. aeruginosa-mediated pathways altering S. aureus antibiotic susceptibility
HQNO induces a low-energy multidrug-tolerant state
LasA overcomes this tolerance in cooperation with vancomycin
Rhamnolipids overcome this tolerance in cooperation with tobramycin
P. aeruginosa virulence is influenced by peptidoglycan shed by Gram-positive species?
ok
Polymicrobial interactions conclusions?
Interactions between microbial species can have a profound impact on virulence and antimicrobial tolerance
Two-way interactions exist between microbes – both pathogen-pathogen interactions, and pathogen-commensal interactions
Quorum sensing plays a critical role in such microbial interactions
Direct communication mediated by autoinducer molecules
Biological activity of QS-regulated exoproducts
The complexity of these interactions highlights the difficulty of extrapolating from in vitro studies of microbes grown in isolation
What signals do bacteria respond to?
ENVIRONMENTAL CUES INCLUDE: Temperature Acidic pH Ions – e.g. Mg2+, Fe3+ Population density (QS) Osmolarity Presence of antimicrobials Oxygen availability Nutrient availability Antibiotics Interaction with host cells Immunological components Human hormones (e.g. adrenaline)
What are two-component systems?
Phospho-relay systems that facilitate adaptive responses to environmental stimuli
Sensor kinases (SK) N-terminal ligand-binding domain linked to a C-terminal catalytic core (HK) HK comprises HisKA & HATPase domains HisKA contains a conserved histidine, which is phosphorylated using ATP by the HATPase domain. Catalytic core is usually bifunctional, having both kinase and phosphatase activities
Response Regulator (RR) N-terminal REC domain contains conserved aspartate, which receives the phosphate from the SK C-terminal output domain facilitates the response DNA-binding domain Enzymatic activity Protein-protein interactions
The role of multikinase networks?
MKNs detect and integrate different stimuli to enable complex decision making.
MKNs have been implicated in important ‘lifestyle switches’ including sporulation, virulence and biofilm formation
These major lifestyle switches cannot be made simply on the basis of the presence or absence of a single ligand
MKNs enable multiple signals to be evaluated and factored into decision making
Acute vs chronic lifestyles of P. aeruginosa?
Acute P. aeruginosa infections
e.g. nosocomial pneumonia
Typically invasive & cytotoxic; frequently result in tissue damage, systemic spread & sepsis
Associated with expression of many surface-exposed and secreted virulence factors, type IV pili, flagella and type III secretion system
Chronic P. aeruginosa infections
e.g chronic wound infections, cystic fibrosis (CF) lung infections
Minimally invasive & non-cytotoxic; rarely systemic spread
Unrelenting host inflammation and associated morbidity & mortality
Associated with biofilm formation and persistence
Exploring cross-regulation between TCSs?
Both copper and zinc are required for normal cellular function, but both show toxicity at high levels (copper especially)
Metal toxicity can be associated with:
Redox activity (particularly when combined with ROS & RNS)
Out-competing other metals from metalloproteins
Antagonism of the uptake of other key trace metal nutrients
Inhibition of enzymatic activities by binding non-specifically to proteins
Conclusions on two-component systems?
Two-component systems facilitate the adaptive response of bacteria to external stimuli, and are not as ‘stand-alone’ as previously thought
Multikinase networks
Cross-regulation
Such TCS complexity enables the integration of multiple signals to facilitate complex decision making, a process that underpins virulence-related adaptations
Factors driving microbial adaptation in vivo?
Immune evasion & thus persistence
Mucoid phenotype
Loss of PAMPs (e.g. flagellin & pilin) and thus reduced clearance
Selective pressure against immunogenic secreted products
Metabolic fitness in a competitive polymicrobial environment
Loss of quorum sensing activity
Dynamic nature of the host environment
Increasing lung damage, promoting hypoxic regions
Tissue destruction may increase availability of haem-bound iron
Therapeutic interventions
Antimicrobial selective pressures
Microbial adaptations conclusions?
Virulence factors are not required for all stages of infection
Regulatory networks can support complex decision making and control the reversible expression of virulence traits, including the switch between acute and chronic phenotypes
Genome sequencing and phenotypic screening reveals specific genes and pathways that are under negative selective pressure during chronic infection
This process of ‘pathoadaptation’ can profoundly shape infection outcomes and polymicrobial interactions
