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

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

COLONIZATION HOST DEFENSES AND CYTOKINE RELEASE INVASION RELEASE OF VIRULENCE FACTORS TISSUE INJURY QUORUM SENSING IL-6 TNF-a IL-8 IL-1b

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

Long filamentous structures called pili 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

Carbohydrate residues of glycoproteins or glycolipids (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 • 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

G 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 • 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 • 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 • 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 • 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 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 • 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 inhibitors • 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)

INVASION OF THE HOST CELL • 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 • 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 • 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 • 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 SYSTEM • 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 SYSTEM • 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

VIRULENCE FACTORS THAT DAMAGE 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 • C. difficile treatment: • Tolevamer which is a soluble high molecular weight linear polymer of styrenesulfonate

REGULATION OF VIRULENCE GENES • 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 GENES • 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 • 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 • 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 SYSTEM • 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 SYSTEM • 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.

QUORUM SENSING • 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

Autoinducers: Homoserine 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

Givskov et al. (1996) J. Bacteriol. 178: 6618. Halogenated furanones produced by a marine algae which are structurally similar to the bacterial acylhomoserine lactones QUORUM SENSING INHIBITORS

homoserine - lactone Synthesis of the polysacharide “Quorum sensing” adherence microcolony biofilm Nutrient Oxygen • Bacteria lacking lasI produce a loose and easily disrupted biofilm

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

Prevention or prophylaxis Treatment of enteric infections caused by some toxigenic bacteria In conjunction with conventional therapeutic strategies IN WHAT CONTEXT COULD ANTIVIRULENCE AGENTS BE USED?

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