Lecture #14Lecture #14Bio3124Bio3124

Medical MicrobiologyMedical MicrobiologyMicrobial PathogenicityMicrobial Pathogenicity

Pathogens as Parasites

• pathogens are parasites

– organisms that live on or within a host

organism, metabolically dependent on the host

– Parasitism:

• Ectoparasite: parasite lives on the host

• Endoparasite: parasite lives in the host

Parasitism and disease• Infection

– growth and multiplication of parasite on or within host• Infectious disease

– disease resulting from infection• Pathogen: any parasitic organism that causes infectious

disease– primary (frank) pathogen – causes disease by direct

interaction with healthy host– opportunistic pathogen – part of normal flora, causes

disease when gains access to other tissue sites or when host is immunocompromised

• Pathogenicity– ability of a parasite to cause disease

Host-parasite relationship and disease outcome

Disease state depends on:– number of organisms present– degree of virulence of pathogen– virulence factors

• e.g., capsules, pili, toxins– host’s defenses or degree of resistance

Virulence: degree/intensity of pathogenicity• determined by,

– Invasiveness: ability to spread to adjacent tissues– Infectivity: ability to establish focal point of infection– pathogenic potential: degree to which pathogen can cause damage to

host• Toxigenicity: ability to produce toxins• Immunopathogenicity: ability to trigger exaggerated immune

responses

Measuring virulence

• lethal dose 50 (LD50)

– number of pathogens that will kill 50% of an experimental group of hosts in a specified time

• Infectious dose 50 (ID50)

– number of pathogens that will infect 50% of an experimental group of hosts in a specified time

Infection Cycle

• Mode of entry depends on pathogen• Mucosal surfaces, wounds, insect bites

• Infection cycleRoute a pathogen takesto spread

• Spread via direct contact• Indirect contact

– Contact with fomites– Horizontal transmission via vectors

• Mosquitoes—Yellow fever, malaria• Reservoir for disease organism

– May not show disease symptoms

Virulence Factors

• Virulence genes– Help pathogen to invade host

• Toxins, attachment proteins, capsules

• Pathogenicity islands– Section of genome

• Contain multiple virulence genes– Often encode related functions

» protein secretion system, toxin production

– Horizontally transmitted• Often flanked by tRNA genes; phage or plasmid genes• Often have GC content different from rest of genome

Virulence Factors• Several factors contribute

– Protein secretory systems• Examples:Type II, type III and type IV

– Adhesins: host attachement & colonization

– Toxins• Exotoxins

– Membrane active toxins– Protein synthesis inhibitors– Cell signaling inhibitors– Superantigens– proteases

• Endotoxins

– Immune avoidance factors

Role of protein secretory pathways in virulence

• PS Type II (retractable)– Subunits in inner, outer and

periplasmic space– G subunit

polymerize/depolymerize – Extends/retracts past outer

membrane through complex D– like a piston pushes out the

secreted proteins to periplasmic space

– Ex. Cholera toxin

• PS Type II mechanism resemble pili type IV used for twitching motility

Type III protein secretory system• many G- bacteria, live in close

association with their hosts• secrete regulatory proteins via

injectisome directly into host cells– to modulate host cell activities– evolutionary resemblance to

flagellum

• increase virulence potential– Avoids receptor use– Avoids dilution of secreted proteins

outside pathogen

Ken Miller talks about PSIII and flagellum

Salmonella SPI-1 and SPI-2 are type III secretory systems

• 12 pathogenicity islands in S. typhi• SPI-1, a type III secretory system• Injects 13 different toxins (effector

proteins)• Subvert signaling, remodel cytoskeleton• Induce membrane ruffles, take S.typhi

• SPI-2: alter vesicle trafficking– Prevent phogosome-lysosome fusion– Pathogen avoids innate immunity

Injectisome: a type III secretory virulence factor

General SecA dependent secretory system

Toxin secretion by type IV secretory systemToxin secretion by type IV secretory system

• Resemble conjugation apparatus of gram negative bacteria

• Bordetella pertussis toxin secreted through general SecA pathway to periplasm

• Type IV collects toxin in periplamic space

• Exports across outer membrane

Adhesins: Microbial AttachmentAdhesins: Microbial Attachment

• Human body expels invaders– Mucosa, dead skin constantly expelled

– Liquid expelled from bladder

– Coughing, cilia in lungs

– Expulsion of intestinal contents

• Adhesins: surface proteins, glycolipids, glycoproteins– assist in attachment and colonization of host

tissues

• Pili (fimbriae)• Hollow fibrils with tips to bind host cells

Adhesins: Pili type IAdhesins: Pili type I• e.g. Pyelonephritis pili of

uropathogenic E.coli

• attachment to P-blood group antigen

• upper uninary tract infection

• Pili assemble on outer membrane

• First, general SecA dependent secretion to periplasm

• PapG,E,F & major subunit Pilin A

• PapD chaperon sorting/delivery to PapC

• Secretion and pilus formation

• PapG recognizes the digalactoside on P-blood group antigen of host kidney cells

Adhesins: Pili type IVAdhesins: Pili type IV• Found on P. aeruginosa, V. cholera,

pathogenic E. coli & N. meningitidis

• Mediates attachment and twitching motility

• Resemble type II secretory system

• Pil A is major structural pilin

• PilC,Y1 tip attachment proteins

• Assembly: PilA preprotein signal sequence removed by PilD

• PilQ mediates export across outer membrane

• PilF/T mediates energy dependent assembly/disassembly of pilus

Type IV pili: bacterial attachment and motility

Exotoxins

• soluble, heat-labile, proteins

• usually released into the surroundings as bacterial

pathogen grows

• most exotoxin producers are gram-positive

• often travel from site of infection to other tissues or

cells where they exert their effects

More About Exotoxins

• Some toxin genes born on plasmids or prophages

• the most lethal substances known

• highly immunogenic

• can stimulate production of neutralizing antibodies

(antitoxins)

• can be chemically inactivated to form immunogenic

toxoids

– e.g., tetanus toxoid

Membrane-disrupting exotoxinsAlpha toxin of S. aureus• Forms 7-membered oligomeric beta-barrel • Cause cytoplasmic leakage

Phospholipase of Clostridium perfringens

• removes charged head group of phospholipids in host-cell plasma membranes

– membrane destabilized, cell lyses and dies– Also called α-toxin or lecithinase

AB type ExotoxinsComposed of two subunits• “AA” subunit – responsible for toxic effect

– ADP-ribosyltion of target proteins eg. diphtheria toxin

– Cleave 28S rRNA, eg. Shiga toxin

• “BB” subunit – binds to target cell, delivers A subunit

Diphtheria exotoxin• B subunit mediates receptor binding• Endocytosis and fusion membrane

vesicles eg. ER or endosomes• B recycles back to membrane• “A” escapes and enters cytoplasm• In the cytoplasm A catalyses ADP-

ribosylation of EF2, halts translation • Cell death ensues Diphtheria toxin targets EF2Diphtheria toxin targets EF2

disrupts translationdisrupts translation

• Anthrax toxin composed of,– Protective antigen (B

subunit): delivers EF and LF (A subunits)

– Edema factor raises cAMP levels

• Causes fluid secretion, tissue swelling

– Lethal factor cleaves protein kinases

• Blocks immune system from attacking

Anthrax toxin: a deadly proteaseAnthrax toxin: a deadly protease

Bacillus anthracis

Animation: Animation: anthrax toxin mode of actionanthrax toxin mode of action

Superantigens

• Are bacterial and viral proteins that can activate T-cells

• in the absence of a real bacterial antigen mediate the

binding of MHC-II and T-cell receptors (almost 30% of T-cell

population)

• eg. Staphylococcal enterotoxin B (SEB)

• Massive activation results in producing lots of cytokines

• Results in tissue damage and shock and multi-organ failure

Animation: Superantigens

Endotoxins• lipopolysaccharide in gram-negative cell wall can be

toxic to specific hosts

– called endotoxin because it is bound to bacterium and

released when organism lyses and some is also

released during multiplication

– toxic component is the lipid portion, lipid A

• heat stable

• toxic (nanogram amounts)

• weakly immunogenic

• generally similar, despite source

Immune avoidance mechanisms

• Once inside host cell, how to avoid death?– Cell ingests pathogens in phagosome

• Some pathogens use hemolysin to break out– Shigella dysenteriae, Listeria monocytogenes

– Phagosome fuses with acidic lysosome• Some pathogens secrete proteins to prevent

fusion– Salmonella, Chlamydia, Mycobacterium, Legionella

• Some pathogens mature in acidic environment– Coxiella burnetii—Q fever

Survival inside phagocytic cells

• escape from phagosome before fusion

with lysosome

– microbes use actin-based motility to

move within and spread between

mammalian host cells

Burkholderia pseudomallei forming actin tails and protrude through membrane and extend infection to nearby cells

Surviving within the Host

Surviving within the Host

• Outside host cell, how to avoid death?– Complement, antibodies bind pathogen

• Some pathogens secrete thick capsule

– Streptococcus pneumoniae, Neisseria meningitidis

• Some pathogens make proteins to bind antibodies

– Staphylococcus aureus cell wall Protein A

» Binds Fc fragment

» Antibodies attach “upside down”

» Prevents opsonization

• Some pathogens cause apoptosis of phagocytes