The host microbe inter phase as a target for anti infective therapy
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The host-microbe inter-phase as a target for anti-infective therapy. Jordi Vila Department of Microbiology, Hospital Clinic, School of Medicine, University of Barcelona Barcelona, Spain. 5th ESCMID School of Clinical Microbiology and Infectious Diseases

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The host microbe inter phase as a target for anti infective therapy l.jpg
The host-microbe inter-phase as a target for anti-infective therapy

Jordi Vila

Department of Microbiology, Hospital Clinic,

School of Medicine, University of Barcelona

Barcelona, Spain

5th ESCMID School of Clinical Microbiology and Infectious

Diseases

Santander, Spain June 10-16, 2006


Slide2 l.jpg

COLONIZATION therapy

HOST DEFENSES AND

CYTOKINE RELEASE

INVASION

RELEASE OF

VIRULENCE FACTORS TISSUE INJURY

QUORUM SENSING

IL-6 TNF-a

IL-8 IL-1b


Colonization l.jpg

Most bacteria must first adhere to host cell surfaces to cause infection

Many pathogenic bacteria express specialized surface proteins called adhesins which mediate their binding to the cells.

COLONIZATION


Adhesins l.jpg

Long filamentous structures called cause infectionpili or fimbriae --. The adhesin may be:

Proteins at the tip

Pilin subunit itself

Afimbrial adhesins – tighter adherence

Fibrillar structures

Non-fibrillar adhesin, i.e: Protein F in S. pyogenes

ADHESINS

* In Gram-negative bacteria:

* In Gram-positive bacteria


Host cell receptors for adhesins l.jpg

Carbohydrate residues of glycoproteins or glycolipids cause infection(Protein-carbohydrate interaction)

- This binding is quite specific

Extracellular matrix proteins. (Protein-protein interaction)

- I.e. Fibronectin

In some cases the pathogen injects its own protein receptor into the host cell.

It is common for a pathogen to express and utilize more than one adhesin

HOST CELL RECEPTORS FOR ADHESINS


Fimbriae in upec l.jpg
FIMBRIAE in UPEC cause infection

  • Type 1 Fimbriae (FimH)- Glycoprotein with mannose chains (Uroplakin Ia)

  • P Fimbriae (PapG, GI, GII and GIII)- a-D-Galactopyranosyl-(1-4)-b-D-Galactopyranoside


Slide7 l.jpg

G cause infection

F

E

The pap operon

E

E

K

G

F

E

E

E

A

K

ME

EP

MI

C

C

C

C

H

G

F

F

E

D

D

D

D

A

D

F

G

D

D

D

D

I

B

A

H

C

D

J

K

E

F

G

Regulation Major Assembling Minor subunits

Subunit


Antagonize the adherence l.jpg
Antagonize the adherence cause infection

  • Bouckaert et al. Mol. Microbiol. (2005) 55: 441

    • D-mannoside exhibits an affinity for FimH (Kd = 0.15 mM)

    • Mannose exhibits an affinity for FimH (Kd = 2.3 mM)

    • Cranberry juice to reduce the recurrence of UTI


Advantages of targeting chaperones l.jpg
Advantages of targeting chaperones cause infection

  • Highly conserved in a multitude of pathogenic bacteria responsible for a variety of diseases such as UTIs, diarrhea, pneumonia and meningitis

  • There is extensive structural knowledge of the interactions that occur between chaperones and pili subunits.


Chaperone inhibitors l.jpg
Chaperone inhibitors cause infection

  • PapD-PapK interaction solved by X-ray crystallography

  • Arg8 and Lys112 play an important role in anchoring PapK and PapG

  • Combinatorial chemistry and docking

  • Three different classes of chaperone inhibitors have been identified:

    • Bicyclic b-lactams

    • Bicyclic 2-pyridinones

    • N-substituted amino acid derivatives

Chembiochem (2001) 12: 915


Cell wall anchoring proteins in gram positive bacteria l.jpg
Cell wall anchoring proteins in cause infectionGram-positive bacteria

  • In addition to the classic signal sequence, the proteins usually have a region of several tandem repeats followed by acarboxyterminal region that contains a proline/glycine-rich segment and a conserved pentapeptide (LPXTG) (100 surface proteins of Gram-positive pathogens)

  • This motif has been found in among others:

    • Fb protein F, protein M and protein G in S. pyogenes

    • Fb protein and protein A in S. aureus

    • Internalin from L. monocytogenes


Cell wall anchoring proteins in gram positive bacteria12 l.jpg
Cell wall anchoring proteins in cause infectionGram-positive bacteria

  • Cleavage between the Thr and the Gly of the LPXTG motif liberates the carboxyl of The to form an amide bond with the amino group of the pentaglycine crossbridge

  • Sortasa is the enzyme catalyzing this reaction (srtA gene). srtA mutants of S. aureus display defects in virulence

    • Cannot bind to cell-matrix proteins such as fibronectin

    • Cannot effect proteinA-mediated binding of the IgG

      PNAS (2000) 97: 5510


Sortase inhibitors l.jpg
Sortase inhibitors cause infection

  • Biochem J (2002) 366: 953-958

    • The Gly in the peptide was replaced by diazoketone or chloromethylketone

    • “In vitro” assay of sortase activity

    • Similar Ki for both derivatives aprox. 0.22 mM


Sortase inhibitors14 l.jpg
Sortase inhibitors cause infection

  • Bioorg. Med. Chem. Lett. (2005) 15: 4927

    • Bis(indol)alkaloid isolated from marine sponge Spongosorites sp.

    • “In vitro” assay of sortase activity

    • IC50 19.44+0.02 mg/ml MIC 100 mg/ml

    • Assay of adhesion to fibronectin: Reduced the capacity of S. aureus to adhere to fibronectin in a dose-dependent manner (0-40 mg/ml)


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COLONIZATION cause infection

HOST DEFENSES AND

CYTOKINE RELEASE

INVASION

RELEASE OF

VIRULENCE FACTORS TISSUE INJURY

QUORUM SENSING

IL-6 TNF-a

IL-8 IL-1b


Invasion of the host cell l.jpg
INVASION OF THE HOST CELL cause infection

  • Once adhered to a host surface, some pathogens gain deeper access into the host (invasion).

  • This process can be divided into two types:

    • Extracellular invasion – bacteria breaks down the barriers of a tissue to disseminate in the host – Secretion of several enzymes that degrade host cell molecules

    • Intracellular invasion – penetrate the cells of a host tissue and survive within this environment.


Yersinia invasion l.jpg
Yersinia cause infectionInvasion

  • Type III secretion system:

  • Type III secretion system is necessary for invasion.

  • Disruption of the type III secretion system renders the pathogens avirulent

    • YopH is a tyrosine phosphatase

    • YopE is an actin cytotoxin

    • YpkA is Ser/Thr kinase


Type iii secretion systems l.jpg
TYPE III SECRETION SYSTEMS cause infection

  • Provide a means for bacteria to target virulence factors directly at host cells where they alter host physiology

  • Conservation of these systems is such that if the appropriate cloned chaperones and secreted proteins are placed in other microorganisms that contain a different type III system, heterologous secretion occurs

  • Components of the type III secretion machinery may represent excellent candidates for drug discovery targets


Inhibitors of type iii secretion system l.jpg
INHIBITORS OF TYPE III SECRETION SYSTEM cause infection

  • Linington et al. (2002) Organic Letters 4: 4089

    • Caminoside A from marine sponge Caminus sphaeroconia

    • IC50 20 mM, MIC of Cam-A for E. coli > 100 mg/L

    • Mechanism of action under investigation


Inhibitors of type iii secretion system20 l.jpg
INHIBITORS OF TYPE III SECRETION SYSTEM cause infection

  • Gauthier et al. (2005) AAC 49: 4101

    • Commercial library of 20,000 small molecules

    • Ability to inhibit TTSS in EPEC

    • Inhibitor: salicylideneaniline derivatives

    • Act on the regulation of the expression of genes associated with TTSS. Does not affect the production of flagella


Inhibitors of type iii secretion system21 l.jpg
INHIBITORS OF TYPE III SECRETION SYSTEM cause infection

  • Nordfelth et al. (2005) Infect. Immun.73: 3104.

    • 9,400 compound collection

    • Acylated hydrazones of different salicylaldehydes

    • Interferes with TTS system from Y. pseudotuberculosis also under in vivo conditions, blocking the microinjection of effector proteins


Slide22 l.jpg

COLONIZATION cause infection

HOST DEFENSES AND

CYTOKINE RELEASE

INVASION

RELEASE OF

VIRULENCE FACTORS TISSUE INJURY

QUORUM SENSING

IL-6 TNF-a

IL-8 IL-1b


Virulence factors that damage the host l.jpg
VIRULENCE FACTORS THAT cause infectionDAMAGE THE HOST

  • EXOTOXINS - Toxic bacterial proteins

    • A-B toxin I.e. Cholera toxin

    • Proteolytic toxins I.e. Clostridium tetani

    • Non A-B toxin, which disorganizing host cell membranes I.e. Hemolysin

    • Other, such as the ST from ETEC

  • ENDOTOXIN - Lipopolysaccharide (Lipid A – toxic portion)


Inhibition of toxins l.jpg
INHIBITION OF TOXINS cause infection

  • C. difficile treatment:

    • Tolevamer which is a soluble high molecular weight linear polymer of styrenesulfonate


Regulation of virulence genes l.jpg
REGULATION OF VIRULENCE GENES cause infection

  • Upon entrance into the host, an invading microorganism encounters a new environment

  • Some parameters that affect virulence factor regulators include temperature, ion concentration, osmolarity, pH, carbon source availability, growth phase and oxygen levels.

  • Some bacteria can have diverse regulatory networks affecting the same virulence factor


Regulation of virulence genes26 l.jpg
REGULATION OF VIRULENCE GENES cause infection

  • Sigma factors –are protein subunits of bacterial RNA polymerases I.e. RpoS regulate the expression of genes in response to stationary phase, nutrient deprivation and oxidative and osmotic stress.

  • Two component systems –Typically these systems are composed of: 1. A sensor protein that is embedded in the bacterial membrane which “senses” different physiological conditions of the bacterial cell and 2. A response regulator which usually binds to the promoter region of a gene to activate or repress transcription.

  • AraC transcriptional activator family –I.e. VirF which is a global regulator of Yops. VirF is also regulated by temperature and Yops are only expressed at 37ºC.


Two component system l.jpg
TWO-COMPONENT SYSTEM cause infection

  • Genomic analysis of S. pneumoniae has identified 13 putative response regulator/kinase pairs, seven of which appear to be important for pathogenicity in a mouse respiratory tract infection model

  • Features making this system attractive for drug development:

    • Significant homology is shared among kinase and response regulator proteins of different bacteria, particularly in aa near the active sites.

    • Pathogenic bacteria use this system to regulate expression of essential virulence factors that are required for survival inside the host

    • Signal transduction in eukaryotic cells takes place by different mechanisms

    • Some two-component systems are involved in the regulation of DNA replication and cell cycle and are essential for viability.


Inhibition of the two component system l.jpg
INHIBITION OF THE TWO-COMPONENT SYSTEM cause infection

  • BARRET et al. (1998) PNAS 95: 5317

    • Family of hydrophobic tyramines

    • Potent bactericial Gram-positive compounds

    • Inhibits the autophosphorylation of kinase A IC50 1.6 mM


Inhibition of the two component system29 l.jpg
INHIBITION OF THE TWO-COMPONENT SYSTEM cause infection

  • MACIELAG et al. (1998) J. Med. Chem. 41: 2939

    • Derivatives of salicylanilide

    • Inhibitors of autophosphorylation of KinA with IC50 of 2.8 mM

    • One compound also inhibited the two-component regulatory systems mediating vancomycin resistance


Two component system30 l.jpg
TWO-COMPONENT SYSTEM cause infection

  • Applicability of this inhibitory approach:

    • Acts on virulence without affecting viability.

    • Acts on viability

    • As a co-drug, I.e. Vancomycin resistance phenotype in enterococci due to the vanA gene is regulated by vanR-vanS. Therefore the inhibition of this component renders bacteria susceptible again to vancomycin.


Slide31 l.jpg

COLONIZATION cause infection

HOST DEFENSES AND

CYTOKINE RELEASE

INVASION

RELEASE OF

VIRULENCE FACTORS TISSUE INJURY

QUORUM SENSING

IL-6 TNF-a

IL-8 IL-1b


Quorum sensing l.jpg
QUORUM SENSING cause infection

  • Some bacteria can “sense” the need to express virulence determinants once bacteria reach a certain concentration

  • It has been shown that knockout of the quorum-sensing genes in P. aeruginosa significantly reduced its virulence


Quorum sensing systems and biofilms l.jpg

Autoinducers: cause infectionHomoserine lactones

lasR/lasI ----- enhances the transcription of genes encoding VF in P. aeruginosa

rhlR/rhlI ----- enhances the transcription of other VF in P. aeruginosa

More than 600 P.aeruginosa genes are QS-controlled.

Quinolone (PQS)

There is an interplay of these three molecules

QUORUM SENSING SYSTEMS AND BIOFILMS


Quorum sensing inhibitors l.jpg

Givskov et al. (1996) J. Bacteriol. 178: 6618. cause infection

Halogenated furanones produced by a marine algae which are structurally similar to the bacterial acylhomoserine lactones

QUORUM SENSING INHIBITORS


Slide35 l.jpg

homoserine - cause infection

lactone

Synthesis of the

polysacharide

“Quorum sensing”

adherence

microcolony

biofilm

Nutrient

Oxygen

  • Bacteria lacking lasI produce a loose and easily disrupted biofilm


Slide36 l.jpg

COLONIZATION cause infection

HOST DEFENSES AND

CYTOKINE RELEASE

INVASION

RELEASE OF

VIRULENCE FACTORS TISSUE INJURY

QUORUM SENSING

IL-6 TNF-a

IL-8 IL-1b


Final questions to be answered l.jpg

Can inhibition of a single individual step or factor in the disease process result in effective therapy?

Would disruption of pathogenesis lead to bacterial death, either directly or by host defense system clearance?

Could a virulence inhibitor be used in combination therapy with current antibiotics or immune system enhancers for therapy?

FINAL QUESTIONS TO BE ANSWERED


In what context could antivirulence agents be used l.jpg

Prevention or prophylaxis disease process result in effective therapy?

Treatment of enteric infections caused by some toxigenic bacteria

In conjunction with conventional therapeutic strategies

IN WHAT CONTEXT COULD ANTIVIRULENCE AGENTS BE USED?


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