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MICR570/ZMR/F12 11/12-1

BACTERIAL PATHOGENESIS I & IILectures 11 & 12

LEARNING OBJECTIVES At the end of these lectures, you should be able to:

1. Outline briefly what bacterial factors contribute to the initiation of the infectious diseaseprocess.

2. Explain the role of adhesion in the infectious process by describing the microbial and

host cell components involved.

3. Explain how bacteria are able to enter different types of cells.

4. Explain how some bacteria & fungi avoid host defense mechanisms and be able to

correlate the specific virulence factors to overcome or evade them.

5. Discuss the significance of biofilms with respect to survival of bacteria and fungi within

the host. 6. Characterise the different possible outcomes following exposure to a pathogenic

microbe.

7. Identify the microbial virulence factors that can cause direct damage to host cells.

o Compare endotoxins and exotoxins with respect to their role in infectious

disease.

o Differentiate between the secreted toxins based on their mechanism of action

and effect on the cell.

8. List the various routes by which bacterial and fungal pathogens can be transmitted

from one host to another, giving an example of each.

9. Identify the microbial and environmental factors that might contribute to transmissibility.

INTRODUCTIONThe capacity of a microorganism to cause disease reflects its relative pathogenicity.Pathogenesis refers both to the mechanism of infection initiation and to the mechanisms bywhich disease develops. We will primarily discuss the bacterial (and to a lesser extent thefungal) virulence factors, indicating how they interact with host defence mechanisms bydescribing their role in the pathogenesis of disease. Please recognise that the pathogenicmechanisms of many microbes are poorly understood, while for others it is known at themolecular level (although, we shall not discuss pathogenicity at this level).

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GLOSSARYInfection: multiplication of an infectious agent within the body.Invasion: process whereby microbes enter host cells or tissues and spread in the body.Opportunistic pathogen: agent capable of causing disease only when the host’s resistance isimpaired (immunocompromised individuals).Pathogen: microbe capable of causing disease.Pathogenicity: ability of an infectious agent to cause disease.

Transmissibility: number & period of time organisms are shed for.Virulence: quantitative ability of an agent to cause disease.Virulent: capable of causing disease, accompanied by damage or loss of function.

REMEMBER THE MAJOR STEPS IN A PATHOGENIC PROCESS(See Host-Pathogen Interactions Lecture)

Microbes must complete most or all of the following steps to cause an infectious disease.1. Attachment of the pathogen to the host for colonisation.2. Invasion of the host by the pathogen (from epithelium into deeper tissues).3. Overcoming the host defences against infection.

3. Ability to damage the host. 4. Dissemination of the pathogen to initiate infection in a new host.

Concept: Intracellular vs. Extracellular Pathogens Extracellular pathogens are unable to multiply within mammalian cells. Obligate Intracellular pathogens require mammalian cells for their replication and growth. Facultative Intracellular pathogens can survive both intra- or extracellularly.

Intracellular Extracellular

Obligate Facultative

Bacteria

Chlamydia spp.Mycobacterium lepraeRickettsia spp.

Brucella spp.Bordetella pertussisCampylobacter spp.(Some) E. coliGroup B StreptococcusLegionella spp.Listeria monocytogenes Neisseriagonorrhoeae/meningitidesSalmonella spp.Shigella spp.Yersinia spp.

Bacillus spp.Borrelia spp.Clostridium spp.Corynebacterium diphtheriae(Most) E. coli Group A StreptococcusHaemophilus spp.Klebsiella spp.Helicobacter spp.Proteus spp.Pseudomonas spp.Staphylococcus spp.Treponema spp.Vibrio cholera

Fungi

None Blastomyces dermatitidisCandida albicansCoccidioides immitisHistoplasma capsulatum

Cryptococcus neoformansPneumocystis jirovecii

Adapted from McClane et al., Microbial Pathogenesis p5

Both has its advantages and disadvantages. Think about: evading host defences, protectionagainst antimicrobials, ability to survive intracellularly, etc.



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ATTACHMENT/ADHERENCEPhysical attachment of a microbe to a cell or surface, necessary for both bacteria & fungi tocolonise a particular site. It requires participation of 2 factors: a receptor and a ligand.

Receptors: usually specific CH2O’s or peptide residues on Eukaryotic cell surface.Ligand (Adhesin): macromolecular component of bacterial/fungal cell surface.Both interact in a complementary and specific manner.

See Table 19-2, p195 Murray et al. Examples of Bacterial Adherence Mechanisms

PLEASE NOTE: Bacteria have site specificity attachment (i.e., Tissue Tropism). E.g., S.mutans dental plaque not on tongue epithelial surface; S. salivarius tongue epithelial cells butnot dental plaque.

ADHESINSPili & Fimbriae (Bacteria) (See Introduction to Bacteria Lectures)Expression may be coordinated with other virulence factors. E.g., Cholera toxin & TcpA (ToxinCo regulated Pilus A). Often as a response to favourable environmental conditions.

E.g., Escherichia coli Ligand: Type I pili (Colonization Factor Antigen (CFA))Receptor: GM1 ganglioside intestinal epithelium (See GI Lectures)

Non-Fimbrial (Bacteria & Fungi) Any cell structures: capsule, cell wall (also fungal), flagellae, lipoteichoic acid, etc.

Adhesin Organism Subst ratum

Lipoteichoic acid S. aureusS. pyogenes

EpitheliumBuccal Epithelium

Polysaccharide K. pneumoniaE. coli

MucinBiomaterials

Lipopolysaccharide H. pyloriS. typhimurium

Gastric EpitheliumMacrophages

Mannans C. albicans (fungi) Epithelial Mucosa

E.g., Haemophilus influenzae Ligand: Outer membrane protein P2Receptor: Sialic acid-containing oligosaccharides of mucin

INVASIONIs the entry of the microbe into host cells facilitating its spread.Please Note: NOT all bacteria have to enter a host cell to replicate or cause disease (See Intracellular vs. Extracellular pathogens, p2).

Mechanism of Entry: penetration of epithelium by Endocytosis• Phagocytic cells: via phagocytosis

• Non-phagocytic cells: variety of mechanismso Invasins: bacterial surface proteins (enzymes) that interact with specific

receptors, inducing endocytosis.Fungal secreted proteins (enzymes) cause host cell damage & lysis.

o Host cell cytoskeleton rearrangement: usually triggered by injection of bacterial proteins into host cell, causing membrane ruffling.

END RESULT: bacterium located within a membrane-bound vacuole.



MICR570/ZMR/F12 11/12-4

POST INVASION CONSEQUENCES1. Restricted growth at site of entry (E.g., Shigella spp in the GI tract).2. Spread from epithelium to underlying tissue without further dissemination (E.g., Non-typhoidal Salmonella spp.).3. Spread from epithelium to other body locations (See Dissemination p9 of Handout). Thismay be facilitated in 2 ways:

• Production of spreading factors (enzymes): degrade constituents of connective tissue

underlying epithelium.• Enter the lymph or blood to reach other sites (E.g., S. pneumoniae, L. monocytogenes,

Salmonella enterica serovar Typhi).

SPREADING FACTORSBacterial enzymes affecting the physical properties of tissue matrices and intercellular spaces,promoting spread of the pathogen.

Hyaluronidase: produced by Streptococcus, Staphylococcus and Clostridium spp.Depolymerizes hyaluronic acid of connective tissue.Collagenase: produced by Clostridium histolyticum and Clostridium perfringens. Breaks down

collagen.Streptokinase & Staphylokinase: produced by streptococci and staphylococci respectively.Kinase enzymes convert inactive plasminogen to plasmin which digests fibrin and preventsclotting of the blood.

EVASION OF THE IMMUNE RESPONSE & HOST DEFENCES(See Figure 14-1, p137, Murray et al.)

Overview of Immune System Defences & Mechanisms for Evasion

Component to be evaded Mechanism used

Complement Capsules (bacterial & Fungal)Complement-binding proteinsProteasesHost cell mimicry

Phagocytic killing CapsulesType III secretion systemsIntracellular parasitism

Antigen processing Interfere with MHC function & antigen processing

Antibodies Ig-binding/inactivating proteins Antigenic variation

Host Defence Challenge: Complement & PhagocytesSolution: Bacterial (Fungal) Capsules

Interference with Complement Pathway1. Block complement activation (alternate pathway)2. Inhibit formation of the C3b complex on bacterial surfaces → avoiding C3b opsonizationand formation of C5b and membrane attack complex (MAC) (E.g., Sialic acid capsules of N.gonorrhoeae, N. meningitides and H. influenzae type b.).



MICR570/ZMR/F12 11/12-5

Resist Attachment & Ingestion of PhagocytesVarious methods available:1. Invade/confined to regions inaccessible to phagocytes (E.g., certain internal tissues, i.e.,lumen of glands and surface tissues, i.e., the skin).2. Avoid provoking an overwhelming inflammatory response.3. Inhibit phagocyte chemotaxis (E.g. Streptococcal streptolysin suppresses neutrophilchemotaxis).

4. Hide the antigenic surface of the bacterial cell. “Host-cell mimicry”, microbe not seen asforeign → no phagocytosis/opsonisation (E.g., pathogenic S. aureus: cell-bound coagulase,clots fibrin on the bacterial surface; T. pallidum: binds fibronectin to surface; Group Astreptococci: synthesize a hyaluronic acid capsule).

Some bacteria can resist complement & phagocytosis without using capsulesi. P. aeruginosa: produces extracellular elastase→ inactivates components of complement.ii. S. pyogenes: M-protein binds factor H + protease → cleaves complement protein C5a.iii. Pathogenic S. aureus: produce coagulase → fibrin clots around the organism → resistphagocytosis.iv. Pathogenic Yersinia: proteins → depolymerize actin microfilaments needed for phagocyticengulfment. Another protein degrades C3b and C5a.

Resist Phagocytic Destruction & Serum LysisVarious methods available:i. Exist within an endocytic vacuole, reducing exposure to toxic oxygen compds (E.g., L.pneumophilia and Mycobacterium sp.).ii. Resistant to toxic forms of oxygen and defensins (E.g., Salmonella sp.).iii. Escape from phagosome into cytoplasm prior to phagolysosome formation (E.g., S. flexneri and some Rickettsia sp.)iv. Prevent fusion of phagosome & lysosome (E.g., N. gonorrhoeae: Por protein (protein I),Salmonella, Mycobacterium, Legionella & Chlamydia: block vesicular transport machinery).v. Resist/Inactivate lysosomal enzymes (E.g., Mycobacterium, L. pneumophilia & Histoplasma

capsulatum (fungi) prevent the acidification of the phagosome - the lysosomal enzymesfunction much more effectively at acidic pH.)vi. Kill phagocytes (E.g., S. aureus & S. pyogenes: leukocidin → damages phagocytecytoplasmic membrane or lysosome membranes → phagocyte killed by its own enzymes;Shigella and Salmonella induce macrophage apoptosis, a programmed cell death).

Serum lysis: Lysis of Gram-ve bacteria by pores produced Membrane Attack Complex(MAC). LPS is the principle target.Salmonella sp. lengthen LPS O-polysaccharide side chain, preventing MAC lysis of thebacterium.Some Gram-ves (N. gonorrhoeae, B. pertussis & H. influenza) attach sialic acid to O-antigen,

preventing formation of C3 convertase.

Evasion of Phagocytic Killing may be a multifactoral processExample: Histoplasma capsulatum (fungi)Undergoes phagocytosis but is not killed

Components of fungal cell wall interfere with oxidative burstFactors that contribute to its survival intracellularly:

Modulation of pH of phagolysosome Alteration of yeast cell wall composition



MICR570/ZMR/F12 11/12-6

Host Defence Challenge: Immunological Responses A. Antigen process ing & presentat ionSolution: disrupt MHC function & NK cell recognitionVarious Mechanisms:1. Prevent MHC Class I maturation and assembly2. Express MHC Class I homologue3. Prevent MHC Class II transcription

B. AntibodiesSolution Bi: Ig-binding/inactivating proteinsDirect binding of cell wall protein to Fc domain of Ig preventing opsonization (E.g., S. aureus:protein A; S. pyogenes: protein G).Bacterial IgA proteases (secreted) → inactivate sIgA by proteolysis (E.g., H. influenzae, S.pneumoniae, H. pylori, S. flexneri, N. meningitidis, N. gonorrhoeae & EP E. coli

Solution Bii: Antigenic/phase variation Phase variation: switch protein expression on & off, results in different phenotypes - beneficialfor survival (E.g., Salmonella sp.: phase variation of K & H antigens (previous antibodies nolonger “fit”).

ADDITIONAL CHALLENGIron is an important mineral necessary for microbial growth. BUT has limited availability in the body (i.e., is chelated as transferring, lactoferrin or haem).Challenge: Overcome Iron SequestrationSolution: Siderophores & Surface Iron Receptors

Sythesise surface receptors: “grab” host iron chelators.Synthesise siderophores: low mw compds that have a high affinity for iron.

BIOFILMSBacterial and/or fungal attachment to a tissue/artificial surface and prodn of ExopolymericSubstance (EPS).

Enables attachment, immune response evasion (antiphagocytic & prevents antibody binding)and decreases antibiotic susceptibility.

Types of Surfaces with Bio film Formation

• Tissues (E.g., heart valves)

• Non-shedding surfaces (E.g., teeth, contact lenses)• Artificial joints

• Indwelling medical devices (E.g., catheters, artificial heart valves, sutures)

IMPORTANCE OF BIOFILMSi. Changing medical microbiology

• Important cause of chronic bacterial infections (E.g., cystic fibrosis)

• CDC estimates >60% of bacteria and fungi involved in human infections are growingas a biofilm.



MICR570/ZMR/F12 11/12-8

4. ToxinsFungal Toxins Aspergillus spp.

• A. fumigatus: Gliotoxin - inhibits macrophage phagocytosis, T-cell activation &proliferation and induces apoptosis in macrophages

• A. flavus: Aflatoxin – carcinogenic

Bacterial Toxins Exotoxins: Different types of secreted proteins, very potent.

Comparison of Exotoxins & Endotoxins

Exotoxin Endotoxin

Gram+ve & Gram-ve bacteria Gram-ve only

Released from cell Integral part of cell wall

Protein Lipid A of LPS

Heat Labile Heat Stable

Specific receptors on host target cells Diverse host cells & systems affected

Specific effects in host Diverse range of effects in host

Toxoids can be made (treat with formalin) Toxoids cannot be made

Nomenclature & Classes/Groups

• Target site/cells (E.g., neurotoxins, enterotoxins).

• Producing bacterium (E.g., Staphylococcal α-toxin)

• Mechanism of action (E.g., ADP-ribosylators, pore-forming toxins)

• Associated disease (E.g., cholera toxin, diphtheria toxin)

Classes:

• Class I: Surface acting (E.g., Superantigen toxins)

• Class II: Membrane-acting (E.g., Phospholipases, pore-forming toxins)• Class III: Intracellular (E.g., ADP-ribosylating toxins)

CLASS I EXOTOXINSInduce a pathoglogic host immune response→ T-cell proliferation→ cytokines released (E.g.,TNF).E.g., S. aureus toxic shock syndrome toxin-1 (TSST-1)

Group A β-haemolytic streptococci pyogenic exotoxin A (similar to TSST-1)TSST-1 binds to T-cell receptor → 20% T-cells activated (normal = 0.01% activated)

CLASS II EXOTOXINS2 types:

o Enzymes: degrade membrane constituents (E.g., C. perfringens α toxin(Phospholipase C) active against lecithin and spingomyelin)

o Not enzymes (aka. Pore-forming or CFT’s Channel Forming Toxins): disruptmembrane permeability (E.g., L. monocytogenes listeriolysin O).



MICR570/ZMR/F12 11/12-10

Different Site Movementa) Direct Movement: in surface fluids, by flow of intestinal contents.Facilitated by toxin prodn (E.g., hyaluronidase).Growth through tissues (E.g., Aspergillus).

b) via CSFCross blood-CSF junction (choroid plexus) E.g., H. influenzae (generally requires movement

into bloodstream first).

c) via Blood or Blood ComponentsE.g., Plasma: B. anthracis, Mononuclear cells: L. monocytogenes)

d) via Lymphatics or immune system cellsFrom tissue fluids into lymphatic capillaries (E.g., Y. pestis).

TRANSMISSIONRoute by which microorganisms are transferred from a reservoir to a new host.

Specific routes vary according to the pathogen and reservoir.

Please Note: Understanding route & mechanism of dissemination/transmission enables youto understand how infections are acquired and how to prevent them.

Transmissibility: number & time period organisms are shed for.

Possible Routes & Mechanisms of Transmission:1. 5.2. 6.3. 7.4.

Factors Influencing Microbial Stability/Survival1. Microb ial Factors

• Sensitivity to UV damage

• Ability to resist drying

2. Extrinsic Factors

• Humidity

• Temperature

• Exposure to chemical agents

3. Infectious dose: numbers of a microbe required to infect host (can vary)• With different microorganisms

• Partially dependent on site of entry & route of infection

Via Vehicles & VectorsIt is important for you to recognise the difference between the 2.

Vectors: an agent, usually an insect or other animal, able to carry pathogens from one host toanother.



MICR570/ZMR/F12 11/12-11

Examples of Vector-borne Infections include but are not limited to - Lyme Disease (ticks),Plague (fleas), Rocky Mountain Spotted Fever (RSMF) (ticks)

Vehicles: Nonliving source of pathogens that infect large numbers of individuals.Examples of Vehicle include but are not limited to - Food & water

Shedding Sites

Release of microbes in fluids, on cells or in secretions from a site (usually the site at whichmicrobe has gained entry).

Skin: Directly from lesions or desquamated epithelial cellsRespiratory tract: Aerosols during normal speech, breathing, coughing and sneezingGastrointestinal tract: Shed in faecesBody fluids: Various – blood, saliva, milk, vaginal discharge, etc.

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References in Murray, P.R., Rosenthal, K. S. & Pfaller, M.A. (2005) Medical Microbiology, 5 th Edition (Recommended Course Text)

Chapter 19: Mechanisms of Bacterial Pathogenesis p193-201Chapter 69: Pathogenesis of Fungal Disease p709-718

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