Molecular basis of infection Flashcards

Question

HWP1 does what?

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

``` Environmental reservoirs Lung epithelial layers Brain endothelial cells Inert medical devices Self association (biofilms) ```

Answer 5

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

Answer 6

Environmental cues

Answer 7

Mutants highly attenuated in virulence Capsule inhibits phagocytosis Provides stress protection (dehydration and free radicals) Impacts on immune system

Answer 8

GXM Antibodies to GXM can block this adherence

Answer 9

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 `

Answer 10

Other adhesin for cyrptococcus Secreted enzyme Required for spread from the lung

Answer 11

play a role in adhesion and invasion across the blood brain barrier

Answer 12

20-90% 400,000 blindness per year 50% of asthma related deaths

Answer 13

If you target one another will come and replace it, the same in candida

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

Yes Yeast Pseudohyphae Hyphae Chlamydospore Opaque

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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

Answer 22

Induced endocytosis It's normally involved in host cell-cell adhesion and tight junctions

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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

Answer 29

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

Answer 30

Induced endocytosis Paracellular invasion Trojan horse

Answer 31

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

Answer 32

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

Answer 33

``` Trojan horse mechanism requires: Initial phagocytosis Survival of Cryptococcus during migration Expulsion from the phagocyte It supports other form of invasion ```

Answer 34

``` Switching forms can impact on: Adhesion Host invasion Regulation of virulence traits Biofilm formation Immuni-modulation and evasion mating ```

Answer 35

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

Answer 36

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

Answer 37

cAMP-PKA MAPK

Answer 38

Filamentation Hence : Efg1 is an activator of hyphal development

Answer 39

Negative regulators of filamentation When activated keep pathogen in the yeast form

Answer 40

Disease Tissue burdens unaffected by blocking morphogenesis – yeast can reach target organs Yeast in target organs retain pathogenic potential

Answer 41

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

Answer 42

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

Answer 43

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 ```

Answer 44

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

Answer 45

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

Answer 46

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

Answer 47

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

Answer 48

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

Answer 49

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

Answer 50

Glycolysis

Answer 51

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.

Answer 52

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

Answer 53

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

Answer 54

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

Answer 55

``` Roles in virulence: Nutrient acquisition Facilitating adhesion & invasion Immune evasion Biofilm formation Cell wall maintenance ```

Answer 56

Commonly encoded by gene families, including: SAPs – Secreted Aspartyl Proteinases LIPs – Lipases PLBs – Phospholipases

Answer 57

Proteases (aspartyl, metallo-, carboxy- & serine) Urease Phospholipase

Answer 58

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

Answer 59

``` 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 ```

Answer 60

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

Answer 61

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

Answer 62

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

Answer 63

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

Answer 64

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

Answer 65

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

Answer 66

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 ```

Answer 67

3 main routes of acquisition: Siderophore uptake Reductive pathway uptake Haem and haemoglobin utilisation

Answer 68

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)

Answer 69

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

Answer 70

Ferritin only binds to hyphal cells | Als3 required for ferritin binding, and use of ferritin as an iron source

Answer 71

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

Answer 72

Secretes Cig1 - hemophore to sequester haem Highly expressed following iron starvation Secreted mannoprotein that binds haem Contributes to virulence alongside reductive iron uptake

Answer 73

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

Answer 74

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)

Answer 75

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

Answer 76

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 ```

Answer 77

Morphogenesis Biofilm formation requires hyphal formation, plus expression of hyphal specific genes

Answer 78

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

Answer 79

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

Answer 80

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)

Answer 81

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

Answer 82

Physical barriers: Skin Mucosa Cellular: Monocytes Macrophages ``` Humoral: Complement Antimicrobial Peptides Cytokines Chemokines ```

Answer 83

Cellular: Lymphocytes T cells B cells Dendritic cells Humoral: Antibodies Cytokines

Answer 84

Pathogen associated molecular pattern Conserved motif on pathogen Not present on host tissue

Answer 85

``` 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 ```

Answer 86

``` protection adhesion wall permeability antigenicity immunomodulation phagocytosis ```

Answer 87

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

Answer 88

Inducing cytokine release, so are PAMPS essentially its where the antibody binds

Answer 89

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

Answer 90

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

Answer 91

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

Answer 92

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

Answer 93

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

Answer 94

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

Answer 95

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

Answer 96

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)

Answer 97

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

Answer 98

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

Answer 99

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)

Answer 100

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

Answer 101

Mpr1 required for traversing endothelial cell layers | Mpr1 when expressed in S. cerevisiae (brewer’s yeast) was capable of traversing endothelial barriers

Answer 102

``` 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

Answer 103

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

Answer 104

``` Capsule production (C. neoformans) Melanin production (C. neoformans, H. capsulatum, A. fumigatus) Yeast-hyphal switch (C. albicans) Produce more α-glucan (H. capsulatum) ```

Answer 105

``` Oxidative stress response Nitrosative stress response Heat shock response Amino acid starvation response Carbon–source specific stress Osmotic stress response pH response ```

Answer 106

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

Answer 107

Mkc1 and Cek1 pathways: Respond to cell wall damage Both pathways required for virulence

Answer 108

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

Answer 109

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

Answer 110

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

Answer 111

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

Answer 112

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

Answer 113

``` 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)

Answer 114

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”

Answer 115

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!

Answer 116

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)

Answer 117

Colonise host tissues Grow within host tissues Avoid host defence mechanisms Cause damage to the host

Answer 118

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

Answer 119

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)

Answer 120

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

Answer 121

Cholera toxin •Phospholipase A L. pneumophilia (Sec) •Phospholipase C L. pneumophilia (tat)

Answer 122

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

Answer 123

H. pylori VacA, the vacuolating Cytotoxin

Answer 124

Chaperone-usher pathway (CU) VII secretion The Type I and Type P pili of UPEC

Answer 125

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

Answer 126

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)

Answer 127

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

Answer 128

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!!

Answer 129

Sec/TAT dependent secretion WXG secretion LPXTG motifs - sortase Lipoproteins

Answer 130

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

Answer 131

’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)’’

Answer 132

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

Answer 133

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

Answer 134

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

Answer 135

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 ```

Answer 136

Initial adherence to epithelial cells Signal transduction Effacement, pedestal formation, intimate attachment Disruption of tight junction intergrity

Answer 137

Esc - E. coli secretion Esp - E. coli secreted proteins Sep - Secretion of E. coli proteins Ces - chaperones

Answer 138

Inhibit phagocytosis – pedestal formation Modulation of the immune response Prevent cell death

Answer 139

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

Answer 140

Effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity.

Answer 141

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

Answer 142

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. In Vibrio 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

Answer 143

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

Answer 144

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

Answer 145

Promiscuous

Answer 146

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

Answer 147

A secreted component of the ESX-1 secretion system (T7SS)

Answer 148

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

Answer 149

Inhibit the immune response, for example this is what TB does to T cell activation

Answer 150

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

Answer 151

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

Answer 152

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

Answer 153

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

Answer 154

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

Answer 155

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

Answer 156

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

Answer 157

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

Answer 158

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

Answer 159

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

Answer 160

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

Answer 161

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

Answer 162

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

Answer 163

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

Answer 164

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

Answer 165

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

Answer 166

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 antibiotics and 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

Answer 167

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.

Answer 168

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.

Answer 169

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

Answer 170

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

Answer 171

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.

Answer 172

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.

Answer 173

Table of week 11 slide 7

Answer 174

``` cftr mutations can positively and negatively influence the susceptibility of the human host to different infections Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae Mycobacterium tuberculosis ```

Answer 175

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

Answer 176

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 ```

Answer 177

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

Answer 178

a CFTR potentiator Increases the channel's open probability and transport of chloride Initially approved for patients with at least one G551D mutation

Answer 179

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

Answer 180

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”)

Answer 181

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

Answer 182

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

Answer 183

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

Answer 184

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)

Answer 185

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?

Answer 186

``` AI-2 has been implicated in virulence traits…… Biofilm formation Motility &/or chemotaxis Virulence factor production Adherence ```

Answer 187

Yes Lots of reasons on slide 15

Answer 188

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

Answer 189

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

Answer 190

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

Answer 191

. aeruginosa inhibits S. aureus respiration, and depletes intracellular ATP Effect is primarily attributable to HQNO, but also pyocyanin and HCN

Answer 192

Rhamnolipids of P. aeruginosa can increase cell permeability by interacting with cell membranes Facilitate tobramycin entry into otherwise tolerant PMF-depleted cells

Answer 193

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

Answer 194

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

Answer 195

``` 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) ```

Answer 196

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 ```

Answer 197

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

Answer 198

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

Answer 199

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

Answer 200

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

Answer 201

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

Answer 202

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

Molecular basis of infection Flashcards

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