Hos-pathogen Interaction2

Host-pathogen interaction bacterial side of the story Saji George

Facts on host-bacteria interaction Both the host and bacteria have evolved numerous ways in which to communicate their wishes/intensions with each other.

The communication between host and bacteria is essential for the survival of both

Often bacterial side of the story is neglected

Pathogen Extracellular: - S.aureus -

S.pneumoniae B. pertussis N. gonorrhoeae E.coli H.pylori

Intracellular Macrophages: -

L. pneumoniae M. tuberculosis

Macrophage & Epithelial cells -

Salmonella species Shigella species L.monocytogenes Chlamydia species

Disease

Host cell Interaction

Skin/Tissue Pneumonia Whooping cough Gonorrhea UTI, diarrheas, meningitis Ulcers, gastritis

Adherence to ECM Adherence to ECM Adherence to cells Adherence to cells Adherence to cells Adherence to cells

Legionaires’ disease Tuberculosis

Within vacuole Within vacuole

Typhoid fever, gastroenteritis Dysentery, gastroenteritis Listeriosis, meningitis Trachoma, STD, pneumonia

Within vacuole Intracytoplasmic Intracytoplasmic Within vacuole

Stages in host-bacteria interaction-disease condition 1. Adherence

2. Invasion 3. Initial multiplication 4. Evasion of defenses 5. Spread of infection 6. Damaging the host

Prokaryotic and Eukaryotic Interactions Eukaryotic Cell

Pili or adhesins

Prokaryotic Cell

Virulent Bacteria

Receptor

Prokaryotic and Eukaryotic Interactions Eukaryotic Cell

Pili or adhesins

Prokaryotic Cell

Virulent Bacteria

Receptor

COLONIZATION

Prokaryotic and Eukaryotic Interactions Eukaryotic Cell

Pili or adhesins

Prokaryotic Cell

Virulent Bacteria

Receptor

COLONIZATION

INVASION

Chaperone-Usher Pathway A A A A A A

Tip Fibrillum

Usher

C

Pilus Shaft

H Gm(-) Bacterial Periplasm

A

Cytoplasmic membrane

A

H

C

Adhesin: binds Gal(α1-4) Gal

A

D Chaperone

I

B

Regulation

A

Pilus tip Fibrillum subunits

Pilus assembly machinery

Pilus subunit

H

C

D

Anchor

Usher

Chaperone

J

K

E

F

G

Nonpilus Adhesin Intimin (Enteropathogenic E.coli)

Microbial Pathogenesis and the Intestinal Epithelial Cell 2003 Hecht GA (Editor)

Toxins

Listeriolysin O

Superantigens: e.g. Spe, TSST1

Bacteria vs Antibacterial Agents 2003 Mascaretti OA (editor)

Capsule Network of polymers (polysaccharide or protein) covering bacterial surface - S.pyogenes capsule: hyaluronic acid - S.pneumonia capsule: polysaccharide Prevent C3 convertase formation by failing to bind serum protein B (no complement activation) Antibody formation to capsule can be protective (vaccine)

Pathogenicity Islands Different G+C content from host genome Mobile genes associated with tRNA and/or insertion sequence (IS) elements Carry multiple virulence factors Large size

Host Evasion: Adherence sIgA Protease Iron Acquisition mechanisms Intracellular residence: - vacuole - free in cytoplasm Survive phagocytosis Capsule; prevents phagocytosis Evade antibody response: - Antigenic variation (pili, LPS, capsule) - Capsule that mimics host antigens Prevent migration of phagocytes

Adherence to host cell- First step in the host-pathogen interaction Site of bacterial adherence Keratinized or mucosal epithelium

Specialized epithelium (ocular, aural)

Cell membrane components

Why do bacteria adhere? To avoid physical and immunological removal, bacteria must adhere to – cell surfaces and extracellular matrix e.g. in respiratory, gastrointestinal and genitourinary tracts – solid surfaces e.g. teeth, heart valves, prosthetic material – other bacteria

Adherence often combined with manipulation of host cell signalling and cytoskeleton – Invasion – Intimate adherence

Molecular aspects of HostPathogen interaction Mechanisms of Adherence to Cell or Tissue Surfaces 1. nonspecific adherence: reversible attachment of the bacterium to the eukaryotic surface 2. specific adherence: permanent attachment microorganism to the surface

irreversible of the

Nonspecific adherence involves nonspecific attractive forces which allow approach of the bacterium to the eucaryotic cell surface. 1. Hydrophobic interactions 2. Electrostatic attractions 3. Atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies 4. Brownian movement

Specific adherence. 1. 2.

Irreversible attachment of bacteria to host Mediated by bacterial surface appendages such as pilus fimbrae etc

Molecular mechanism of host bacteria interaction PAMPs Pathogen-associated molecular pattern A limited set of conserved molecular patterns that are unique to microbial world and invariant among entire classes of pathogens

Adherence mechanisms General adherence mechanisms – Capsules and slime – Biofilm formation Gram-positive adhesins (PAMPs) – MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), e.g. protein A – Fimbriae Gram-negative adhesins (CHO and protein receptors) (PAMPs) – Fimbriae, Afimbrial adhesins (FHA, Pertactin etc.) – Outer Membrane Proteins – Types III-IV secretion

Molecular mechanism of host bacteria interaction Pattern Recognition Receptors

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-PRR

Molecular mechanism of host bacteria interaction

Toll-Like Receptor 4: first human PRR to be identified Toll: protein from D. melanogaster identified for its role in dorso-ventral embryo patterning and critical for effective immune responses in adult flies against the fungus Aspergillus fumigatus

C-reactive protein is a PRR CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction.

Ng et al. The EMBO Journal (2007), 1–10

TLR detect multiple PAMPs

Annu. Rev. Cell. Dev. Biol. (2006) 22: 409-37

Adhesins In Gram-negative bacteria: Long filamentous structures called pili or fimbriae --. The adhesin may be: Proteins at the tip Pilin subunit itself Afimbrial adhesins – tighter adherence

In Gram-positive bacteria Fibrillar structures Non-fibrillar adhesin, i.e: Protein F in S. pyogenes

Adhesion factors of pathogenic bacteria

adhesion factor Type-I-fimbriae Type-IV-fimbriae Fimbrial adhesins (multi-subunit adhesion organelle)

P-fimbriae S-fimbriae M-protein curli

examples enterobacteria N. gonorrhoeae V. cholerae P. aeruginosa uropathogenic E. coli uropathogenic E. coli S. pyogenes E. coli

Neisseria meningitidis in meningococcal pathogenesis (i) initial adhesion Is mediated by pilus

(ii) intimate adhesion Other bacterial and cellular Structure. Pilus disappear during the intimate adhesion Transcriptional regulation at the initial adhesion is required for the intimate adhesion. crgA gene is up-regulated on initial contact Deghmane et al The EMBO Journal Vol.19 No.5 pp.1068–1078, 2000

Adhesion factors of pathogenic bacteria

Non-fimbrial adhesins (multi-subunit) Polysaccharides Biofilm formation

NFA, AFA

E. coli

LPS LOS EPS

N. gonorrhoeae N. meningitidis P. aeruginosa

PIA (= polysaccharide adhesin; ica) S. epidermidis P. aeruginosa alginate

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.

Invasion- extracellular and intracelular -Urinary tract infection

Invasion-Adhesins Invasin

Integrin receptors

a Yersini

αβ

S. aureus in endothelial cells

αβ

αβ

αβ

αβ αβ

actin

αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ

extracellular matrix αβ αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ αβ

αβ

αβ

αβ

αβ

Yersinia

αβ

αβ

S. aureus invasion depends on: 1. fibronectin 2. Fibronectin binding protein of S. aureus 3. α 5β 1-integrins as receptor on the host cell 4. actin polymerization

actin

αβ

extracellular matrix

Mechanisms used by bacteria to enter cells.

(A) The zipper mechanism used by Yersinia and Listeria.

(B) The trigger mechanism used by Salmonella and Shigella

Zipper mechanism Invasin a Yersini

αβ

αβ

αβ

αβ

actin

αβ

(ii) Phagocytic cup formation. This step is triggered by the transient signals occurring after formation of the first ligand-receptor complexes and propagating around the invading microbe. These signals induce actin polymerization and membrane extension.

αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ

extracellular matrix αβ

actin αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ

αβ αβ

αβ

αβ

αβ

αβ

Yersinia

αβ

αβ

αβ

αβ

(iii) Phagocytic cup closure and retraction, and actin depolymerization.

Integrin receptor αβ

(i) Contact and adherence. This step is independent of the actin cytoskeleton and involves only the bacterial ligand and its receptor. It leads to receptor clustering.

αβ

extracellular matrix

Trigger mechanism Invasion of salmonella Salmonella enters host cells by inducing host cell membrane ruffling membrane ruffles non-specifically wrap around the bacteria and pull them into the cell Salmonella end up in membranebound vesicles called Salmonellacontaining vacuoles (SCV). SCVs are unique environments within the cell defined by the bacteria within them As they mature, SCVs do not follow the defined routes of cellular trafficking of vesicles and differ in their composition from normal phagosomes

Invasion membrane ruffling depends on Spi1 Type III secretion system Spi1 effectors – SopE affects actin cytoskeleton – SipA binds to actin, inhibits depolymerization – SopB inositol phosphate phosphatase – SptP: PTPase, disrupts the actin cytoskeleton

1. A pre-interaction stage. Effector molecules are stored in bacterial cytoplasm Type three secretion systems are properly formed

2. An interaction stage

3. The formation of a macropinocytic pocket.

4. Actin depolymerization and closing of the macropinocytic pocket

Secretion of bacterial products- the secretion systems Type I, II, III, IV, V and VI Type I, III and IV secretions is directly from bacteria to eukaryotic cells Type II and V- two-step processes in which proteins are transported first through the inner membrane (IM) and then through the outer membrane (OM). Type III secretion system is necessary for invasion.

Type VI mechanism of secretion Naturally produced OM vesicles from pathogenic bacteria contain adhesins, toxins, and immunomodulatory compounds. Mediate bacterial binding and invasion, cause cytotoxicity, and modulate the host immune response.

Type III secretion system Type III Secretion Systems are multi-protein complexes connecting bacteria to host cells Mediate protein secretion and translocation from bacterial cytoplasm to host cell interior Effector proteins subvert cellular functions

Modulation of Virulence Factor Expression by Pathogen Target Cell Contact Yop is a virulence factor produced by Y. pseudotuberculosis Yop (Yersinia outermembrane protein, is an inhibitor of NF-kß signaling) Contact with HeLa cells provoke Y. pseudotuberculosis to produce yop proteins into the cytoplasm Yop is secreted by type III mechanism Shigella shows target cell induced production of Ipa proteins Salmonella forms surface structures on contact with target cells

The type III secretion is blocked when bacteria is present with Ca2+ Contact between the target and bacteria opens up the secretion channel at the zone of contact This promotes the bacteria to produce yop proteins.

Salmonella-invasion of epithelial cells

Appendages are required for bacterial entry into epithelial cells

Only salmonella in contact with epithelial cells produced surface appendages

Bacteria that are inside the membrane ruffles lack the surface appendages (second step)

E. faecalis entry into HeLa cells Two mechanisms are involved in the entry of E. faecalis into HeLa cells One, sensitive to amiloride, is most likely a macropinocytic, actin-dependent uptake mechanism, resulting in the production of large smooth membrane vacuoles engulfing enterococci. The other is receptor mediated entry (RME), in which entry is dependent on both MF and MT structural integrity.

Bertuccini et al. Med Microbiol Immunol (2002) 191: 25–31

Ligand-Signaled Upregulation of Enterococcus faecalis ace Transcription, a Mechanism for Modulating Host-E. faecalis Interaction Sreedhar R. Nallapareddy and Barbara E. Murray

mRNA of Ace protein increased in a dose dependent manner as the concentration of collagen was added to growth medium (B) Dose-dependent induction expression by OG1RF.

of

ace

The adherence of E. faecalis (OG1RF) to plastic plates coated with collagen increased once bacteria were grown in presence of collagen and serum.

(iii) Real-time qRT-PCR. Amplification, detection, and real-time analysis were performed by using the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, Calif.). Primers designed to produce amplicons of equivalent length were selected by using Primer Express software (Applied Biosystems). The primer pairs used in qRT-PCR included AceQF1 (5GGAGAGTCAAATCAAGTACGTTGGTT-3)-AceQR1(5-TGTTGACCACTTCCTTGTCGAT-3) and Bacterial strains and growth conditions. The E. faecalis strains used in the present study include OG1RF TX5256 (ace 23S-rRNAF (5-GTGATGGCGTGCCTTTTGTA-3)–23S-rRNAR (5disruption mutant of OG1RF); two endocarditis isolates, TX0052 and MC02152; and a urine isolate, MD9 CGCCCTATTCAGACTCGCTTT-3). For each sample, cDNA synthesis and PCR amplification were performed inand a two-step process. ForBovine cDNA 4 gfrom of total RNA was addedInc.to(Palo 20-l Alto, reaction ECM proteins collagenase digestion. CI synthesis, was purchased Cohesion Technologies, Calif.), human-placenta-derived CIV was from Sigma Chemical Co. (St. Louis, Mo.), fibrinogen was from Enzyme Research solution containing 40 U of RNase OUT, and RT reactions were performed with random primers and Laboratories (South Bend, Ind.), and bovine(Invitrogen). serum albumin (BSA) was from MP Biomedicals, Inc. (Irvine, Calif.). SuperScript II reverse transcriptase Gene expression analysis. (i) Extraction of total RNA. Total RNA was isolated from E. faecalis cultures by using an Neasy minikit (QIAGEN, Valencia, Calif.) according the protocol the supplier some modifications. A lysozyme at 10 Adherence assay. Adherence of E.tofaecalis toofCIVand with LN-precoated six-well plates solution (Becton mg/ml was used instead of 3 mg/ml for the lysis step. RNA (20by to 40 g) was three BX51 times with 20 U of RQ1Each DNase Dickinson Biosciences, Bedford, Mass.) wasTotal determined using antreated Olympus microscope. (Promega Corp., Madison, Wis.) for 30 min at 37°C, and the DNase was removed by using the RNeasy minikit.

well of ECM-precoated plates were blocked with 5 ml of 0.2% BSA in PBS, incubated at 4°C for 2 h, and then washed with PBS three times.

RT-PCR. Total RNA (between 5 ng to 250 ng) was reverse transcribed with ace specific primers (AceMF, 5ACGATTGAAGGAGTGACTAACACA-3; AceMR; 5-AAGTGTAACGGACGATAAAGGAAG-3) using the SuperScript. OneStep RT-PCR with a Platinum Taq kit (Invitrogen Corp., Carlsbad, Calif.) according to the manufacturer’s instructions. As an internal control, a 528-bp fragment of gdh (encoding GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) was amplified by using the gdhF (5-AGTGGCGCACTAAAAGATATG G-3) and gdhR (5-AGTTGTATTGAACCCTTGACCG-3) primers. Reactions without reverse transcriptase were performed as controls to detect DNA contamination in the total RNA preparations.

Stages in host-bacteria interaction-disease condition 1. Adherence

2. Invasion 3. Initial multiplication 4. Evasion of defenses 5. Spread of infection 6. Damaging the host

Summary Bacteria interact with eukaryotic cells via molecular mechanisms The interaction of bacteria with eukaryotic cells could trigger the expression of virulence factors that ultimately determines its persistence or perish

Thank you

Morphology of bacterial adhesins Afimbrial adhesin

Type I fimbriae

Type IV fimbriae (= bundle forming pilus)

Curli

1) Tooth surface with out caries 2) White spot:- 1st sign of demineralization 3) Enamel broken, visible lesion 4) Filling made, but demineralization continue 5) Demineralization continues and undermine tooth 6) Tooth fractured

Virulence Factors of S. mutans Specific adherence to tooth surface using antigen I/II adhesin and GTF Production of extracellular polysaccharides (dextran) Accumulation of intracellular amylopectinlike polysaccharides (carbon/energy reserve)

Dental Carries

Tooth Decay caused by

Perfect example is Strep mutans

Pellicle is protein form from saliva

Cannot attach to a clean teeth

Can attach to a pellicle within minutes

Hours later cariogenic bacteria can occupy this pellicle

Bacteria’s assembles on the dextran to form plaque

Lactic acid break down enamel in the areas of the plaque

Glucosytransferase assembles glucose into dextran

Fructose undergo metabolism resulting in lactic acid

E.g.... S mutans will hydrolyze sucrose into G+F