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GAS virulence

Soheila AbachiFor more information you could download my thesis at the following link [https://dalspace.library.dal.ca/handle/10222/10559/browse?type=author&value=Abachi+Hokmabadi%2C+Soheila]

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Cross-section of the two types of bacterial cell wallsDepartment of crop and soil environmental sciences, Virginia Polytechnic Institute and State University

Structure of the Gram-positive cell wall Joubert, Quizlet LLC

Cell wall

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Colored trans- mission electron micrograph of GAS, cell diameter of one micronSport photo gallery website

Cell surface structure of GAS & secreted products involved in its virulence Todar's online textbook of bacteriology

Bacterial surface

4Surface proteome of GASwww.studyblue.com

Streptococcus colonization is facilitated byAdherenceSignalingNutritional adaptationHost modulation

Virulence

5Nobbs et al. 2009

Virulence

GAS virulence factors

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Antiphagocytic M protein M-protein-like

• M-related protein (Mrp)• Enn and others

Hyaluronic acid capsule C5a peptidase (a protein fragment released from cleavage complement component C5 by

protease C5-convertase into C5a and C5b fragments)Adherence to epithelial cells Lipoteichoic acid (oral epithelial cells) Fn binding proteins (oral epithelial, cutaneous Langerhans

cells) M protein (skin keratinocytes) Hyaluronic acid capsule (CD44-positive keratinocytes)

GAS virulence factors

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Internalisation M protein Protein F1Invasion Hyaluronic acid capsule M proteinSpread through tissues Hyaluronidase Streptokinase SpeB (cysteine protease) DNAses A-DSystemic toxicity Streptolysin O Streptolysin S Superantigenic exotoxin

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Fn binding proteins

Fn binding protein(s)

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100 kDa, anchored to cell wall at its LPxTG domain in C-terminal region of molecule

Environmentally regulated expression, expression of protein F1 enhanced in O2-rich environment & when GAS adhere to the cutaneous surface

various Fn-binding proteins have different N-terminal domains All contain a peptide that is repeated in tandem from three to

five times (R1, R2, etc) GAS has 5 Fn binding protein(s) protein F1 (Sfb1), protein F2,

PFBP, SOF, & Sfbx Utilize (i) peptide repeat domains to bind Fn (primarily to N-

terminal domain of Fn) & (ii) upper binding domain that reacts with collagen binding domain of Fn e.g. F1 & F2 [such interactions promote efficient entry of GAS into host cells]

Fn is large glycoprotein, 440 kDa, in human blood plasma & extracellular matrix

10 conformational change is a change in the shape of a macromolecule, often induced by environmental factors

Quantification of primary adherence of different strains of S. pyogenes (serotypes M6 and M49) and S. epidermidis (positive control) to uncoated polystyrene surfaces.

Cordula Lembke et al. Appl. Environ. Microbiol. 2006;72:2864-2875

Cordula Lembke et al. Appl. Environ. Microbiol. 2006;72:2864-2875

M1

M18

M6

M2

FIG.2. Quantification of characteristic primary adhesion profiles of different S. pyogenes serotype strains and S. epidermidis (positive control) to immobilized matrix proteins and polystyrene surfaces. (A) Serotype M1 GAS strain; (B) serotype M2 GAS strain; (C) serotype M6 GAS strain; (D) serotype M18 GAS strain; (E) S. epidermidis (positive control strain). Bacteria were grown in BHI under static conditions at 37°C in ambient air. Adhesion of the bacteria was quantified by safranin staining of potential biofilms and subsequently by measuring absorbance at 492 nm at the indicated time points. The mean values of six independent experiments and standard deviations are shown. Immobilized matrix proteins were as follows: Fn, fibronectin; Fo, fibrinogen; Co I, collagen type I; Co IV; collagen type IV; Lam, laminin; wPrt, without proteins (i.e., polystyrene surface).

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ATPase

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•ATP synthase regulates

intercellular & cytoplasmic pH

•Important molecular target for

drugs in the treatment of infectious

disease

ATP synthase schematic

University of Leeds, Faculty of Biological Sciences

ATPase, Acid tolerance, lack of TCA cycle

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Streptococci lack respiratory chains, relying on glycolysis or arginine fermentation for production of ATP (energy)

GAS lacks necessary enzymes for functional TCA cycle & oxidative-cytochromes for electron transport

Unable to generate a large proton potential Cell membrane physiology major role in acid base regulation Depend on permeability of cells to protons at various environmental

pH values Membrane ATPase important in cell permeability H+-ATPase (ATP synthase) hydrolyze ATP & form electrochemical

gradient of protons GAS extrude protons across plasma membrane establishing an

electrochemical potential providing driving force for various kinds of physiological work e.g. uptake of sugars, amino acids, other nutrients with aid of secondary porters (primary transport systems) & regulation of cytoplasmic pH & cytoplasmic concentration of potassium & other ions

Cytochromes are hemeproteins containing heme groups and are primarily responsible for the generation of ATP via electron transport.

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Biofilm

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S. pyogenes ATCC 19615 biofilm in vitro in antibiotic-free medium. (A) One-day culture (B) Four-day culture

Hirota et al., 1998

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Biofilm formation

Scanning electron microscopy of S. pyogenes biofilm development under continuous flow conditions in a flow chamber system.

Cordula Lembke et al. Appl. Environ. Microbiol. 2006;72:2864-2875

M18

M2

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Scanning electron microscopy of S. pyogenes biofilm development under continuous flow conditions in a flow chamber system. (A to C) Serotype M18 GAS strain; (D to F) serotype M2 GAS strain. Biofilms were formed for 72 h on coverslips coated with collagen type IV (A to C) or fibronectin (D to F) in a flow chamber system under continuous flow conditions for the medium. The development of the biofilm architecture is shown at magnifications of ×140 (D), ×350 (A), ×1,400 (B and E), and ×3,500 (C and F).

SEM of S. pyogenes biofilms.

Cordula Lembke et al. Appl. Environ. Microbiol. 2006;72:2864-2875

M49

M6

M18

SEM of S. pyogenes biofilms. (A and B) Serotype M49 GAS cells from a 72-h static culture on an uncoated plastic surface. Images reveal primary bacterial adherence without subsequent formation of typical biofilm structures, with magnifications of ×500 (A) and ×5,000 (B). (C and D) Serotype M6 GAS grown on plastic coverslips for 72 h in static culture, with magnifications of ×500 (C) and ×5,000 (D). (E and F) Biofilms of the serotype M18 GAS strain grown for 72 h in static culture on collagen type IV-coated coverslips, with magnifications of ×200 (E) and ×2,000 (F).

Biofilm

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3D biofilm structures consistof up to 46 bacterial layers

GAS mutants failed to form biofilm lacking transcription for regulators Mga & CovR

(CsrR) lacking M protein, hyaluronic acid capsule

Biofilm

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Biofilm

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Biofilm, Adhesion

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serotypes M1, M12, M28, M49 biofilm-negative strains serotypes M1, M4, M12, M49 form microcolonies when

grown in liquid medium. serotypes M1, M4, M12, M49 aggregation into

microcolonies mediated by conserved 19-amino-acid residue peptide present in M protein & protein H

serotypes M2, M18 preferentially adhere to human matrix protein-coated surfaces

serotype M6, M14 preferentially adhere to uncoated plastic or glass surfaces

Isolate-specific patterns within a certain serotype could be due to diverse regulatory mechanisms leading to differential expression of primary adhesins.

The adhesins & corresponding regulators enabling strains to directly interact with uncoated plastic are uncharacterized

Protein H: a surface protein with separate binding sites for IgG, albumin

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Rheumatic fever M serotpyes 5, 6, 18, 19, 24, …

Acute post streptococcal glomerulonephritis M serotpyes 12, 49, 55, 57, 60, 63, …

Pharyngitis causing M serotypes 1, 3, 5, 6, 12, 18, 19, 24,…

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Phytochemicals

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Anthocyanins Flavonols Flavan-3-ols cyanidin delphinidin malvidin pelargonidin peonidin petunidin

kaemferol myricetin quercetin syringetin

catechinproanthocyanidins

Hydrolysable tannins Phenolic acids Terpenes & Triterpene acidsellagitanninsgallotannins

benzoic acid caffeic acid chlorogenic acid ellagic acid ferulic acid gallic acid gentisic acid

α-pinene, β-pineneα-cadinolmyrcene sabinene betulinic acidoleanolic acidursolic acid

Characterization of phytochemicals

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Compound name Class Strain Conc. MOAUrsolic acid Oleanolic acid

Triterpene acid

S. mutans UA159

1024 µg/ml 100% AI

(-)-Epigallocatechin (-)-Epigallocatechin-3-O-gallate

Flavan-3-ol S. pyogenes DSM 2071

30 μg/ml 15-40% AI

Epicatechin-(4β→8, 2β→O→7)-epicatechin-(4β→8)-epicatehin

Flavan-3-ol S. mutans UA159

500 µg/ml 85% F-ATP AI

Morin Flavonol S. pyogenes MGAS6180

225 μM 50-60 % BR

AI; Adherence Inhibition, BR; Biofilm Biomass Reduction, F-ATPAI; F-ATPase Activity Inhibition

Phytochemicals with anti-infective effects against Streptococcus spp.

Zhou et al., 2013, Janecki et al., 2010, Duarte et al., 2006, Gregoire et al., 2007, Green et al., 2012, Prabu et al., 2006

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FEVER

HEADACHENAUSEA

VOMITING,

ABDOMINAL PAIN

SORE THROAT

Symptoms

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Resistance MOA

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Penicillinase, a group of -lactamase enzymes, inactivates lactam ring of penicillin molecule.

Erythromycin resistant Target modification, methylation of 23S rRNA

Mechanisms of antibiotic resistance in bacteriaTodar's online textbook of bacteriology

Leclercq et al. 2002

Antibiotic resistant

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Cross section of a biofilm with attachment to surface Antibiotic containing aqueous phase

Bacteria with activated stress responses Antagonized antibiotic action in zones with nutrient depletionBacteria differentiated into a protected phenotype

Antibiotic resistant Biofilms start forming:1)Cellular recognition of attachment sites on surface

2)Nutritional cues

3)Expose of planktonic to sub-MIC antimicrobial

Resistance

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Tolerance mechanism Shutting down targetResistance mechanism Prevent antibiotic from hitting target

Resistance

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Resistance mechanism Prevent antibiotic from binding to targetAllow cells to grow at an elevated level of

antibioticMain types of resistance are Target modification by mutationTarget modification by specialized

enzymatic changesTarget substitution, such as expressing an

alternative targetAntibiotic modificationAntibiotic effluxRestricted antibiotic permeation

Resistance

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Persister cells Are not antibiotic-resistant mutants Are slow dividing cells (dormants), little or no cell-wall synthesis,

translation or topoisomerase activity Temporarily give up propagation in favor of survival Able to survive a dose of antibiotic that kills regular cells Numbers in a growing population of bacteria rises at mid-log &

reaches max. Of 1% at stationary phase Are produced substantially in slow-growing biofilms Form when proteins toxic to cell, growth & essential functions are

overproduced/over expressed Resistance mechanisms prevent the antibiotic from

hitting/binding a target Tolerance mechanism shutting down the targets

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Quorum sensing

Quorum sensing

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Quorum-sensing consist of three components,

a small soluble signal peptide a two-component regulatory system

that has a membrane-bound histidine kinase sensor & an intracellular response

Quorum sensing

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Two-component regulatory system CsrRS (CovRS) regulates its own expression & virulence-associated genes;

has operonstreptokinasecysteine protease SpeBSLOMga activates its own transcription & several virulence

genes;M protein family (emm, mrp, arp, enn)C5a peptidaseserum opacity factor (sof; sbfII)Sic Collagen-like protein (sclA)

Quorum sensing

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Rgg (or RofB) regulates expression of; Genes encoding regulators Mga and CsrRSFasBCA (three-component system) regulates

expression of;Fibrinogen & fibronectin binding SLS encoding locus (sagA)SilA & SilB (two-component system) regulates

expression of;Proteins responsible for spreading of S.

pyogenes into deeper tissues during infection

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M protein

M protein

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Primary virulence factor Resist phagocytosis in blood & Attach to host cells Starts from surface of the bacteria a-helical, coiled-coiled protein (advantage: antigenic

variation, multiple functional domains) number & sequence of the A & B repeats vary depending

upon M type. C-repeats conserved among different M types. Adhesion to host cells mediated by either variable domain or conserved domain depending upon

the receptors expressed by host cells Close resemblance of its molecular design to certain

mammalian proteins formation of epitopes responsible for serological cross-reactions between microbial & mammalian proteins

M protein

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Expression of M protein enhanced at higher partial pressures of CO2 & GAS adheres to deeper tissues where more likely to encounter phagocytic cells

emm gene encodes M serotype specificity & M type-specific opsonic epitopes

Adhesion by the variable domain depend upon type of M protein expressed & on type of receptors expressed by the targeted tissue

Pharyngitis causing M serotypes (1, 3, 5, 6, 12, 18, 19, 24,…)

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Treatment

Treatment

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Treatment goals include Prevention of suppurative and nonsuppurative complications Reduction of clinical signs and symptoms Reduction of bacterial transmissionMinimization of antimicrobial adverse effects Antibiotic selection requires consideration of Patients’ allergies Bacteriologic and clinical efficacy Frequency of administrationDuration of therapyPotential side effectsCompliance Cost

Treatment

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Treatment failure

Treatment failure

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10-18% penicillin treatment failure Total treatment failure as high as 30% Non-compliance

Treatment failure

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Treatment failure defined as Detection of GABHS of same serotype, with or without

symptoms of pharyngitis, after recent completion of appropriate antibiotic therapy

Mechanisms for treatment failuresreinfection through various meanslack of compliance streptococcal tolerance to penicillin early initiation of antibiotics resulting in inadequate

immune response lack of protective microflora or its involuntary

eradication copathogenicity of beta-lactamase-producing flora

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Adhesion

Adhesion

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6 different mechanisms of adhesion for S. pyogenes Pharyngeal epithelial cells and HEp-2 cells M protein-

mediated adhesion Tissue culture cells infected with influenza virus

fibrinogen-mediated adhesion Bacterial attachment to different types of substrata with

fatty acid-binding domains LTA mediated adhesion Depend upon type of target substratum used in assay

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Capsule

Capsule

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GAS depend on HA capsule to evade phagocytosis & to interact with epithelial cells

HA capsule only plays a secondary role in infections caused by GAS strains pathogenic for humans

Digest tissue HA & facilitate spread of GAS4% of GAS and 2.7% of GCS positive HA12.5% of GAS, 72.1% of GBS, 84% of GCS

and 85.3% of GGS positive HY

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200 known M types divided into 4 major subfamilies based on sequence of peptidoglycan-spanning domain at the 3′ end of emm

Subfamilies A, B, C cause pharyngitisSubfamily D cause skin diseasesSubfamily E generalists, cause

symptomatic infection at either tissue, skin or throat

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Division

Cell wall, division

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Many virulence factors of G + anchored to PG by a sorting signal

Signal sequence of M protein, which contains an YSIRK-G/S motif (SP+YSIRK), is targeted to the division septum, while signal sequence of protein F lacking this motif (SP−YSIRK) is targeted to old pole

M protein is rapidly anchored at septum, simultaneously at the mother & daughter septa

By contrast SP−YSIRK SfbI protein accumulates gradually on peripheral PG resulting in a polar distribution impairment of septum assembly results in marked reduction in amount of M protein, but not of SfbI

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Hydrophobicity, LTA

Hydrophobicity

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Bacterial pathogens depend on hydrophobic interactions for successful colonization of a host

M+ GAS possess more negative surface charge & express extreme surface hydrophobic properties

Binding of fibrinogen & albumin decreased surface hydrophobicity of M+ GAS

GAS clinical isolates hydrophobic LTA release from cytoplasmic membrane, bind to surface proteins via

glycerol phosphate end, glycolipid end free to interact with various substrata utilizing the hydrophobic effect

Sub-MIC penicillin reduces hydrophobicity less adhesion GAS in Exponential phase much less hydrophobic than in stationary phase During exponential growth phase hyaluronate capsule cover LTA

hydrophobicity decrease GAS cells enter stationary phase, capsule no longer produced in high

quantities & HY degrade polymer hydrophobicity increase Major adhesin(s) of streptococci may be hydrophobins Stationary phase GAS superior to exponential GAS in adhesion to host cells

Hydrophobicity

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Sub-MIC antibiotics decrease cell surface hydrophobicity increase negative electric charge hinder interaction between GAS & pharyngeal Epi cells suppression of infection

Bacitracin & pristinamycin increased hydrophobicity no effect on adhesion to pharyngeal Epi cells

Induces excretion of LTA from GAS loss of ability to adhere

Fimbriae

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Type-specific M protein was removed from intact surface GAS showed increased susceptibility to phagocytosis, (b) lack of opsonic effect of homologous M antibody on the treated streptococci, and (c) loss of HCl- extractable M protein.

GAS lacking M protein adhered to human oral mucosal cells equally as well as untreated, fimbriated organisms which retained their M protein

Fatty acids ester linked with glycerol teichoic acid (fimbriae) rather than M protein of streptococci binds the organisms to epithelial cells

Fimbriae

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Structure-activity

relationship

Structure-activity; phenolic acids

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Greater antibacterial activity against gram-positive than negative (strain dependent activity) OM of gram-negative bacteria with hydrophobic surface structure excludes hydrophilic

molecules, so are inherently resistant to AM including phenolic acids Gram-positive enclosed in plasma membrane covered by thick peptidoglycan wall (No OM) Poorly absorbed in small intestine, some stimulate growth of gut microbes Structure-activity relationship Different alkyl chain length with hydroxyl groups important for AM actions, longer chain

better activity Presence of hydroxyl groups on phenol groups & oxidized status of phenol groups important

Disrupt fluidity of the cell membrane with increasing hydrophobic alkyl chains Enter molecular structure of membrane with polar hydroxyl group oriented into aqueous

phase by hydrogen bonding & nonpolar carbon chain aligned into lipid phase by dispersion forces, when hydrophilic force exceeds hydrophobic one, activity disappear

Number & position of substitutions in benzene ring & saturated side-chain length important Potency against Lactobacillus spp.

1.benzoic acids; 4-hydroxy- > 3-hydroxy- > non-substituted > 4-hydroxy-3-methoxy- > 3,4-dihydroxy-substituted acids2.phenylacetic acid: non-substituted > 3-hydroxy- > 4-hydroxy- > 3,4-dihydroxy-substituted acids3.phenylpropionic acids; non-substituted > 4-hydroxy- > 3-hydroxy > 3,4-dihydroxy-substituted acids

Structure-activity; Flavonoids

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Flavonoids; largest groups of secondary metabolites, constructed basically with an A & C ring of benzo-1-pyran-4-quinone & a B ring, commonly conjugated with sugars as glycosides

Main classes; (1) flavones (basic structures), e.g. luteolin, apigenin, diosmetin,

chrysoeriol, tangeretin, sinensetin, gardenin, vitexin and baicalein; (2) flavonols (having a hydroxyl group at the 3-position), e.g. kaempferol,

quercetin, galangin, datiscetin, morin, robinetin, isorhamnetin, tamarixetin, quercetagetin and myricetin;

(3) flavanones (2–3 bond saturated), e.g. hesperetin, taxifolin, eriodictyol and naringenin;

(4) flavan-3-ol, e.g. catechin and epicatechin; (5) isoflavone, e.g. genistein, daidzein and coumestrol; (6) anthocyanidins: cyanidin, delphinidin, pelargonidin and peonidin Gram-positive absorb more EGCG into peptidoglycan cell wall &

aggregate its presence, while Gram-negative do not aggregate & absorb less EGCG because of repulsive negative charge of lipopolysaccharides on surfaces of Gram-negative, binding of EGCG to peptidoglycan disrupts its function in osmotic protection, cell division, & cell wall biosynthesis

Structure-activity; Polyphenols

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Phenolic acids (ellagic & gallic acids) or flavonoids (flavan-3-ol, flavan-3-4-diol or flavan-4-ol) are esterified or polymerized into dimeric, oligomeric or polymeric compounds

Most abundant are tannins; hydrolysable tannins (HT) & condensed tannins (CT)

HT; complex molecules with a polyol as a central core such as glucose, glucitol, quinic acids, quercitol and shikimic acid, partially or totally esterified with a phenolic group, i.e. gallic acid (3,4,5-trihydroxy benzoic acid; gallotannins) or gallic acid dimmer hexahydroxydiphenic acid (ellagitannins)

CT (proanthocyanidins); polymers of flavan-3-ols (epi)catechin & (epi)gallocatechin units, linked by C4-C8 and C4-C6 interflavonoid linkages

prodelphinidin B-2 3′-O-gallate (a proanthocyanidin gallate); anti-HSV-2; inhibit attachment & penetration between cells & viruses through instability of viral glycoproteins

Gallotannins; strong affinity for iron & inactivation of membrane-bound proteins, morphological changes due to inhibition of cell division by binding of gallotannins to cell wall or inhibition of enzymes involved in cell separation

Phytochemicals Antibacterial MOA

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Phenols & phenolic acids; disruption of energy production due to enzyme inhibition by oxidized products, through reaction with sulfhydryl groups or through more nonspecific interactions with proteins

flavonoids (robinetin, myricetin and epigallocatechin gallate); inhibit the synthesis of nucleic acids of both Gram-negative & positive, B ring may play a role in intercalation or hydrogen bonding with stacking of nucleic acid bases which cause inhibitory action on DNA and RNA synthesis

Quercetin; binds to GyrB subunit of E. coli DNA gyrase and inhibits enzyme’s ATPase activity, cause an increase in permeability of inner bacterial membrane & a corruption of membrane potential

Epicatechin gallate & epigallocatechin gallate; inhibit antibiotic efflux pumps in MRSA, inhibit β-ketoacyl-ACP reductase (FabG) & trans-2-enoyl-ACP reductase (FabI) components in bacterial type II fatty-acid synthase system

Phytochemicals Antibacterial MOA

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Molecules that mimic AHL signals and affect quorum-sensing

Inhibit AHL-dependent gene expression, interfere with several AHL-regulated bacterial processes without any effect on bacterial growth or general protein synthesis capability

Inhibit interspecies coaggregationPrevent adhesion, inactivate mature single &

multi-species biofilms, decrease polysaccharide production

synergists/potentiators of antibiotics

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phytochemicals that have different antibacterial modes of action can potentiate activity of same antibiotic class. For instance, berberine (interact with the cytoplasmic membrane and with DNA) & epicatechin, epigallocatechin gallates (inhibit efflux activity and bacterial type II fatty acid synthesis) have distinct antibacterial mode of action, however, they potentiate antibacterial action of β-lactam antibiotics

piperine, reserpine, & triterpenoid saponins sensitize bacteria & potentiate action quinolones & polymyxins (chemotherapeutic strategy; sensitize bacteria with phytochemicals & modulate their susceptibility to antibiotics at reduced concentrations)

phytochemicals with membrane permeability effects, potentiate antibacterial activity of antibiotics that target intracellular sites (aminoglycosides, macrolides, quinolones, tetracyclines)

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Antibiotic MOA

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Antibiotic mode of action 1)Cell wall

synthesis inhibitor 2)Protein

synthesis inhibitor 3)Folate

synthesis inhibitor

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Erythromycin

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penicillin-binding proteins: catalyze cross-linking of bacterial cell wallsPBPs can be permanently inhibited by penicillin and other β-lactam antibiotics

Penicillin

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Permissions P. Dirckx, Center for Biofilm Engineering, Montana State University, Bozeman

Development of new antimicrobial agents

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Handling the infections less effective low survival rate long-term disability Immuno-compromised

Demand: discovery of natural compounds

with diverse chemical structures,

mechanisms of action

Threat

DANGERThreats

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Phytochemicals

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ATP synthase