chapter 2

Pathogens

Microorganisms that are able to multiply in host and cause disease

4 types of pathogens

Viruses

Bacteria

Fungi

Parasites

2 types of parasites

Protozoan parasites (unicellular)

Helminthes (worms)

What makes a microorganism pathogenic

Virulence factors

Virulence factors role

Facilitate host penetration and invasion

Pathogenicity islands

Virulence factors encoded in blocks of genes found in chromosomal DNA or in plasmids

Adhesins

Virulence factors that allow pathogen to adhere to host tissue

Exotoxins

Soluble virulence factors that are released by bacterial cells and damage host target tissue

Effector proteins

Bacterial virulence factors that interfere with mechanisms of host defense

Koch’s 4 Postulates to Determine Causative Agent of Disease

1. The suspected microbe must be isolated in all cases of disease and must not be found in healthy individuals

2. The isolated microbe must grow in vitro in pure culture.

3. Experimental animals inoculated with the pure culture must show the same symptoms of disease as observed in point 1.

4. Microbes isolated from the inoculated diseased animals must be identical to the original microbial isolate in point 1.

2 Factors Koch’s Postulates do not consider

• Existence of carriers who carry a pathogen and can transmit its disease but who are not sick

• Some pathogens are difficult to grow in culture so might not be possible to fulfill 2nd postulate

Barry Marshall Self Experiment

• Drank pure culture of H. pylori using strain obtained from a patient with acute gastritis

• Symptoms developed (bad breath and vomiting)

• Endoscopy revealed bacteria which were isolated and discovered to be H. pylori

• Cured himself with antibiotics

Relationship between H. pyori and allergy and inflammatory diseases

Inverse relationship; increased incidence of allergy and inflammatory disease in countries with low levels of H. pylori

H. pylori

Part of normal microbiota, present in 50% of population worldwide, causes persistent but silent infection.

True pathogens

Highly virulent pathogens that cause disease in persons with a perfectly functioning immune system

Opportunistic pathogen

Microbes that cause disease only when host is immunocompromised

Nosocomial infections

Infections acquired within hospitals and nursing homes

Reasons why hospitals and nursing homes are reservoirs of pathogens

• Patients/residents are elderly and immunocompromised; they are susceptible to opportunistic infections

• Exposure to pathogens in these facilities is high due to contamination

• Development of antibiotic resistance by bacteria occurs at a very high frequency

Sources of infections in normal commensal microbiota

• Acquisition of virulence factors

• Displacement of commensal bacteria from their natural habitat

Cause of acute infections

Extracellular, pyrogenic bacteria

Pyogenic bacteria

Induce production of pus

Acute infections

• produce acute inflammatory response (heat, pain, redness, swelling)

• rapid onset & rapid resolution when controlled

Chronic infections

• develop slowly, do not resolve completely

Causes of chronic infections

intracellular pathogens, which are resistant to phagocytosis

Granulomas

Structures formed by infected macrophages surrounded by T lymphocytes.

Function of granulomas

physically enclose pathogen to avoid dissemination of infection

Typical feature of mycobacterial infections

Formation of granulomas

Why has death from infectious disease in developed countries declined

Due to identification of the causative microbial agents, diiscovery of antibiotics, creation of vaccines, implementation of vaccination programs

Zoonoses

Animal diseases that are transmitted to humans

Examples of new infectious diseases that have emerged

HIV/AIDS

SARS

Ebola virus

SARS-CoV-2

Ways antibiotic resistance can arise

• DNA mutations

• Transmission to daughter cells

• Horizontal gene transfer

What causes antibiotic resistance

• improper prescriptions

• use of leftover drugs by self-diagnosed

• failing to complete full cycle of prescription(favours survival of more resistant strains that were not immediately killed during first days of therapy)

• abuse of antibiotics in animals farmed for food production

What does low dose of antibiotics do

Favours the rise of resistant bacterial strains

Role of epithelia

Protects body surfaces from the external environment

Examples of epithelia

• skin

• lining of mucosal tissues(ex. respiratory, gastrointestinal and urogenital tracts)

Mucosal

Presence of specialized epithelial cells that produce mucus

Mucus

Viscous fluid with important protective functions

Skin

• physical barrier

• thick

• composed of keratinocytes

Keratinocytes and sebaceous glans secretions

Defensins

Single layered mucosal tissues

• respiratory tract

• gut

Mucociliary escalator

Moves particles trapped in mucus upward and away from epithelial cells

Barriers in gut

• enzymes

• low pH

• peristaltic action

• commensal flora

Antimicrobial peptides

Small molecules that act as natural antibiotics

Antimicrobial peptides sub-families

• defensins

• cathelicidins

• lecticidins

Defensins

Small positively charged peptides folded into amphipathic structure

Amphipathic

Has both hydrophilic and hydrophobic side

Role of defensins in disrupting membrane of microbes

• positively charged side bind negatively charged lipid bilayer/envelope of viruses

• Hydrophobic side facilitates insertion of molecule into lipid bilayer, then formation of holes in membrane and death of microbe

Paneth cells

epithelial cells of the gut that secrete antimicrobial peptides into the gut lumen

Lysozyme

Enzyme that breaks chemical bodn that holds together peptidoglycan

Peptidoglycan

Component of bacterial cell walls

Where are lysozymes found

• tears

• saliva

• neutrophil granules

• secreted by Paneth cells in gut

Cells that recognize pathogens that breach physical/chemical barriers

Sensor/sentinel cells

Where are sentinel cells found

In tissues immediately underneath epithelial linings

Two types of sentinel cells

• macrophages

• dendritic cells

Pathogen recognition receptors

Group of sensing molecules that recognize PAMPs

Pathogen associated molecular patterns

Structural components of microbial origin that are not found on host cells

• do not mutate over evolutionary time

Damage associated molecular patterns

Molecules released by damaged or stressed cells

Types of PAMPs

• components of bacterial cell wall(peptidoglycan and lipopolysaccharide)

• lipoteichoic acid

• flagellin(structural subunit of bacterial flagella)

• genetic material(unmethylated CpG sequences abundant in bacterial DNA)

• Viral RNA (has modifications that allow innate receptors to distinguish it from host RNA)

Cell wall of Gram-positive bacteria

• Contains thick layer of peptidoglycan

• Embedded lipoteichoic acid

• purple(layer of peptidoglycan retains crystal violet dye)

Cell wall of gram negative bacteria

• thin layer of peptidoglycan

• localized between inner and outer mebrane

• composed of LPS which is composed of polysaccharides and lipid A/endotoxin

• pink/red(thin wall cannot retain crystal violet dye)

What releases LPS

Dying gram negative bacteria

What does high doses of LPS in the bloodstream

Septic shock

What does it mean for PRRs to be germline-encoded

PRR genes do not go through gene rearrangement process

Limitation of PRRs

Small number of structurally distinct types, each able to detect broad group of pathogens

each expressed by characteristic subset of innate cells

Complete Freund’s adjuvant

Crude mixture of killed myobacteria emulsified in mineral oil

Function of adjuvants

To alert adaptive immune system of presence of an antigen associated with a microbial threat

First PRRs discovered

Toll like receptor

What transcription factor is activated by engagement of both mammalian IL-1 receptor and drosophilia Toll

NFkB

Transcription factors

DNA-binding proteins that bind to regulatory sequences of genes and regulate their rate of transcription

Role of members of the NFkB family

Induction of inflammatory response

What happens when flies carried loss-of-function mutation of Toll

They were susceptible to fungal infections

Role of Toll

Regulates production of protective antimicrobial peptides

hToll

Through activation of NFkB, lead to production of inflammatory cytokines and induction of the adaptive immunity

What was hToll renamed

TLR-4

What receptor is for endotoxin LPS

TLR-4

Toll like receptor

• Transmembrane proteins that sense PAMPs found in endosomes

• horseshoe-shaped extracellular portion comprises a variable number of copies of a structural domain called a leucine-rich repeat

• cytoplasmic portion has a Toll-IL-1 receptor domain

Ligand binding

Triggers receptor dimerization and modification of the cytoplasmic tail of receptor

Adaptors

relay signal to downstream molecules

Phosphorylation

Addition of phosphate groups by kinase

creates docking sites

Cell signalling general scheme

Ligand & receptor → Adaptor & kinase → intermediate signalling molecules → metabolic change or gene transcription or cell movement

What does NFkB induce

Transcription of inflammatory cytokines (ex. IL-1, TNR)

expression on innate cells of B7

What is B7

Costimulatory molecule that contributes to activation of T lymphocytes

TLR-4 detects

LPS

• can detect infection by gram-negative bacteria

What can LPS do during infection

Can be shed as soluble molecule and released in blood or extracellular fluids

Where is TLR-4 expressed

On surface of innate immune cells in association with MD-2

What does activation through TLR-4 require

• LPS-binding protein

• CD14

LPS-binding protein

Soluble PRR that binds soluble LPS and transfers it to CD14

CD14

Cell surface receptor expressed by macrophages and dendritic cells

What does LPS-bound CD14 do

Interacts with TLR-4/MD-2 complex → binds LPS → dimerization and activation of TLR-4

Which TLR is not used by MyD88

TLR-3

TLR-4 activation results in

MyD88 recruited to cytoplasmic tail of receptor

MyD88 death domain

Kinase IRAK-4

Action of IRAK-4

Assembly of signalling scaffold → activation of IKK → induce activation of NFkB

NFkB inactive form

Found in cytoplasm in resting cell bound to inhibitor IkB

Ubiquitination

Modification of IkB by IKK which marks IkB for degradation → free NFkB moves into nucleus and induces transcription of genes involved in immune response

Signaling through TLR-4 pathway

LPS → LBP → CD14 → TLR-4 & MD-2 → MyD88 → death domain → IRAK-4 → signalling cascade → IKK → IkB → ikB debradation & NFkB → NFkB enters Nucleus → cytokines, antimicrobial peptides, other mediators

Types of endosomal TLRs that recognize viral RNA

TLR-3, TLR-7, TLR-8

Endosomal TLR’s

Nucleic acid sensing receptors

can also activate transcription factor family interferon regulatory factors

Type of endosomal TLR that recognizes DNA from viruses and bacteria

TLR-9

Where do inactive interferon regulatory factors reside

In cytoplasm in non-phosphorylated form