MICROBES, IMMUNE SYSTEMS AND THEIR INTERACTION, TCD BYU22206-202526

Final Prep

Virus size range

0.05-1 micrometer

Bacteria and archaea size range

0.5-5 micrometers

Eukaryotic microorganisms

Yeasts, fungi, moulds, protozoa

Name the types of microorganisms

Viruses, bacteria, archaea, yeasts, fungi, moulds, protozoa

Yeast size range

5-10 micrometers

How many human cells make up the body?

How many cells of bacteria exist in the body?

Around 10^13 human cells

Around 10^13 to 10^14 bacterial cells

Toxins

Alter normal metabolism of host cells with deleterious effects on the host

Where does “toxin” get its name?

“Toxicum” is latin for poison

Toxigenicity

Ability of a pathogen to produce toxins

Intoxication

Disease resulting from specific toxin

Examples of bacteria that infect the intestinal cell by invasion

Salmonella spp.

Campylobacter spp.

Yersina enterocolitica

Escherichia coli (EIEC)

Examples of bacteria that infect intestinal cells by adhesion and production of toxins and effector proteins

Escherichia coli (EPEC, ETEC, EHEC), Vibrio cholera

Examples of toxin productions in the gut causing infection

Bacillus cereus, /clostridium perifringens

Examples of intoxications by toxin production in food

Staphylococcus aureus enterotoxins, Bacillus cereus cereuide, Clostridium Botulinum neurotoxins

When is a toxin classified as an enterotoxin?

If they act in/on the gut

Traits of exotoxin

Secreted by both gram negative and positive bacteria

Usually destroyed at high heat >60 degrees C (except Staphylococcus enterotoxin)

Highly immunogenicity inducing neutralizing antibodies production

Highly toxic (LD50. in microgram range)

Usually binds to specific cell receptors

Examples of exotoxins

S. aureus alpha hemolysin (33 kDa)

AB5 toxins (A:30 kDa, B:11 kDa) - Cholera toxin (Vibrio cholera), Pertussis toxin(Bordetella pertussis), Shiga toxin (Shigella, Shiga-Toxin Producing E. Coli/STEC)

Clostridia Neurotoxins (150 kDa)

Bacillus cereus NHE (HBL) Enterotoxin

S. aureus Enterotoxin (30 kDa)

Shiga toxin

Type of AB5 toxin (A:30 kDa, B:11 kDa)

Exotoxin

Key virulence factor for Hemolytic Uremic Syndrome (HUS)

Types Stx1 and Stx2 share 56% sequence identification and both cause HUS

HUS Located/transmitted on/by prophages on chromosome (similar to cholera) Stx released when phage enters lytic cycle

Antibiotic stress can induce the lytic cycle to further toxin spread

Unknown transport of Six crossing the intestinal epithelium into the blood

Shiga toxin mechanism

Shiga toxin made of A1, A2, and B5 subunits and binds to globotriaosylceramide (Gb3) receptor to enter the endothelial cell membrane

In the Golgi, A1 is cleaved from A2/B5 by furin

A1 binds to ribosomes where biosynthesis of unit A (RNA glycosides) removes an adenine from 28S rRNA causing apoptosis, disrupting the endothelium, and causing inflammation and bloody diarrhea

S. aureus enterotoxin

30 kDa

Secreted from gram positive bacteria with 2 protein domains

Symptoms - >80% vomiting, >65% diarrhea

Min. toxic dose - 0.1-0.4 micrograms

Superantigen is an unspecific stimulation of antigen presenting cells and T-cells

Serotonin production - vomiting?

Local inflammation, nausea, diarrhea, (vomiting?) from uncontrolled secretion of pro-inflammatory cytokines/chemokines (interleukins, interferon gamma, TNF-alpha)

Traits of endotoxins

Part of gram negative cell envelope released upon division or death

Thermostable (2.5 hr at 100 degrees C)

i.e. LPS, toxicity mediated by lipid A

Low immunogenicity, induction of neutralizing antibodies

Low toxicity (LD50 is milligram range)

Binds to CD14 and LPS-binding protein

Chromosomally encoded

Mycotoxins

Secondary metabolite; fungi

Type 6 Secretion System

Used by gram negative bacteria

Allows for horizontal gene transfer (HGT) and transformation

Thought of as an inverted bacteriophage with a phage-like tail poking a hole through membranes of the target bacteria where effector proteins (antibacterial proteins) are transferred

Lesion created is also harmful to the bacteria targeted

DNA uptake channel acquisition

Seen in Acinetobacter baylyi

60 degree angle kill zone

Unit TSSJ/L/M unit on OM and IM of bacteria with a base plate (TSSE/TSSK) and the sword VipA/B which extends and contracts)

Tip of sword is vSrG/PAAR and part going between bacteria is HCP

Steps in bacterial predation

Attack

Attack (5-10 min)

Invade (12-23 min)

Reseal

Early predator growth

Late predator growth

Exhaust prey resources (25-180 min)

Septation

Division

Predator exit (180-210 min)

Bdellovibro (soil) bacteria

Discovered 1962

Naturally kills other bacteria by bacterial predation

Attacks growing and non growing bacteria

Attaches to LPS of gram negative

Doesn’t attack eukaryotes

Modified surface decreases its antigenic profile

Possible use in antimicrobial resistance for medicine?

Bacteriophages

Viruses infecting bacteria

Elongated capsid head enclosing DNA

Protein tail that attaches to host to infect the DNA

Lytic cycle of T4 phage

Attachment

Entry of phage DNA/degradation of host DNA

Synthesis of viral genomes and protein

Self assembly

Release

Lysogenic cycle

Replicate phage genome without destroying host

Viral DNA incorporated into cell chromosome of host

Integrated viral DNA known as prophage

Environmental signal can cause viral DNA to exit and trigger lytic cycle

Temperate phages

Bacteriophages using both lysogenic and lytic cycle

Lambda phage

Temperate phage

How do bacteria fight phages

Natural selection causes mutants with surface proteins not recognized as receptors by a particular phage type

Foreign DNA identified and cut up by restriction enzymes while its own DNA is protected via methylation

Endonucleolytic cleavage protects genome by modification of cognate endonuclease target

Restriction-modification systems

Average is 2 systems per bacterium, >50 in Helicobacter pylori

Tandem systems - DNA methyltransferase and restriction endonuclease are adjacent genes

Orphan - solitary DNA methyltransferase

Restriction enzyme/restriction endonuclease cut dsDNA discovered by Werner Arber, Daniel Nathans and Hamilton Smith in 60s

Mechanism of restriction enzymes

1. Restriction enzyme cuts the sugar-phosphate backbones

2. Base pairing of sticky ends produces various combos using fragments from different DNA molecules cut by the same restriction enzyme

3. DNA ligase seals strands

4. methylation at GATC sites of A or C or transfer of methyl group from S-adenosyl-methionine (SAM) to DNA

5. Dam (DNA methyltransferase) - catalyses addition of methyl to A base, trails DNA replication fork

CRISPR scientists

Jennifer Doudna Berkeley and Emmanuelle Charpentier MPI Berin used Streptococcus pyogenes

Nobel in 2020

What does CRISPR stand for?

Cluster of Regular Interspaced Short Palindromic Repeats

When/in what was CRISPR first identified

1987 in E.Coli

What does Cas stand for and what are they?

CRISPR-associated proteins

Nucleases

Mechanism of CRISPR-Cas system in bacteria

1. phage infects cell with system

2. DNA is integrated between two repeat sequences

3. If cell survives, it blocks repeat attempts of infection by transcription of CRISPR region

4. Resulting RNAs cut into pieces and bound by Cas proteins

5. Cas proteins use phage-related RNA to target invading phage DNA

6. Phage DNA is cut and destroyed

Type II System of CRISPR-Cas in Eukaryotes

Type II system is the precise introduction of dsDNA breaks by Cas9 protein guided by an RNA molecule

Allows for deletions, insertions, and exchanges during repair by endogenous DNA machinery

Components of CRISPR-Cas system

Operon of CRISPR-associated (cas) genes

CRISPR array with leader sequence, short identical repeats with interspersed short unique spacer sequences (Spacer sequences from mobile genetic elements from first infection)

CRISPR spacer acquisition

Fragmentation of invading DNA

Selection of protospacer by recognition of protospacer adjacent motif (PAM)

Nicking of leader-end repeat in CRISPR locus

Integration of new spacer and duplication of flanking repeat

How does bacteria divide?

Binary fission

Generation time

How long it takes for cells to divide

Phases of bacteria growth in lab liquid medium

Lag phase- adapt to environment, uptake of nutrients starts, onset of cell division

Exponential growth - max division rate

Stationary phage - growth ceases, nutrient limitation

Death phage - cell lysis

Why has Big Pharma slowed anti-infective research and development

Economics of antibiotics compared to other drugs

Costs of clinical trials

Pressure to reduce usage

Bacterial resistance evolves quickly

Short life-span of patent protection - augmenting and ciprofloxacin now out of patent

High hopes for and big investment in modern tech not fulfilled

Types of antimicrobial agents

Bactericidal - they kill the microorganism

Bacteriostatic - inhibit the growth or replication of microorganism

Common targets for antibiotics

DNA-directed RNA polymerase - Rifampicin

DNA gyrase - Quinolones

Cell-wall synthesis - Penicillins, Cephalosporins, Glcopeptides, Carbapenems, Monobactams

Folic acid metabolism - Sulfonamides, Trimethoprim

Oxazolidinones

Protein Synthesis - (50S) Macrolides, Chloramphenicol, Clindamycin, (30S) Aminoglycosides, Tetracyclines

How do you test for antibiotic susceptibility

Bacterial lawn on agar plate with each disc soaked in different antibiotic

Antibiotic diffuses into agar

Clearing zone indicates sensitivity to antibiotic

Known as Kirby-Bauer Susceptibility Test,

Another is the E(psilometer) test

Another is the Minimal inhibitory concentration (MIC_

Antibiotics acting on ribosome 30S subunits

Aminoglycosides - misreading of genetic code on mRNA

Tetracycles - block attachment of tRNA

Antibiotics acting on ribosome 50S subunits

Chloramphenicol - misread of genetic code on mRNA

Macrolides - prevent chain elongation

Lincosamides - prevent chain elongation

Streptogramins - premature chain termination

Oxazolidinones - interfere with chain initiation

Beta-lactam antibiotics

Most widely used class

Started in 1920 with Penicillin

Related beta-lactam classes - increase in spectrum activity, address specific resistance mechanisms against targets bacteria

Cephalosporins, cephamycins, monobactams, carbapenems

Core is 2-Axetidinone (cyclic amide)

peptidoglycan transpeptidases are penicillin binding proteins (PBP) , bactericidal