CHIEF KEITH

Question 1

what is virulence?

Answer 1

the ability of a pathogen to cause disease

Question 2

what is colonisation?

Answer 2

multiplication of a microbe on or within host tissues or other surfaces

Question 3

what is a virulence factor?

Answer 3

a microbial product that contributes to the ability to infect and/or cause disease in the host

Question 4

what are stanley falkows molecular koch’s postulates?

Answer 4

1. the suspected virulence gene should be present in strains of bacteria that cause the disease and absent from avirulent bacteria
2. the gene should be isolated by cloning
3. loss of function mutations in the gene should abolish or reduce virulence
4. the gene should be expressed during infection

2 and 3 most important - once a gene isolated through molecular cloning, if you can inactivate it w a mutation that gene is responsible for virulence

Question 5

how do you isolate a gene by cloning (postulate 2)?

Answer 5

take bacterial DNA and chop it using restriction enzymes

cut vector (e.g. plasmid) with same restriction enzyme allowing DNA pieces to go in

add DNA ligase to form recombinant molecules which can replicate copies of that gene

Question 6

how do you show loss of function mutations in a gene reducing/abolishing resistance?

Answer 6

on plasmid with gene present cut out piece of gene and insert drug resistance cassette e.g. a kanamycin cassette

transform into bacteria of interest with wild-type gene X

select for KanR recombinants - these have mutation in gene X

then compare ability of WT bacterium with mutant to cause disease in mouse model

Question 7

what are some limitations of the molecular kochs postulates?

Answer 7

postulate 2 and 3 - in some bacterial pathogens you can’t clone and/or mutate genes (e.g. chlamydia until recently)

postulate 3 and 4 - no animal model that perfectly mimics disease seen in humans (no reduction of virulence from mutant bacteria doesn’t necessarily mean gene isn’t involved in virulence)

Question 8

give some example of animal models for different pathogens?

Answer 8

Mtb - mice, guinea pigs (small rodent models good cause cheap and fast regeneration time = many experiments)

L. monocytogenes - mice, guinea pigs

S. typhi - mice

Shigella flexineri - rhesus monkey (struggle to get disease in rodents)

Neisseria gonorrhoeae - humans (some pathogens only infect humans but allg if ez treatment)

Question 9

how are bacterial infections introduced to animals?

Answer 9

orogastric inoculation

intravenous inoculation

intranasal inoculation

Question 10

what is orogastric inoculation?

Answer 10

way to introduce bacterial infections into animal model

suitable for studying initial stages of food-borne disease e.g listeria monocytogenes, yersinia enterocolitica, salmonella enterica

pretty much just deliver it directly to intestine

Question 11

what is intravenous inoculation?

Answer 11

way to introduce bacterial infections into animal model

useful for analysis of systemic stage of disease caused by various pathogens

systemic = affecting several organs or tissues; involves the spread of pathogens through blood and/or lymph

Question 12

what is intranasal inoculation?

Answer 12

way to introduce bacterial infections into animal model

used to study respiratory infections e.g. streptococcus pneumoniae

Question 13

what is salmonella enterica serovar typhimurium and how would you study it?

Answer 13

causes localised infection of intestinal epithelium but in mice does cause systemic infection

to study infection of intestinal epithelium orogastric inoculation (OG) would be used

you could just let this progress to systemic if you wanna study infection of bone marrow, spleen, liver, gall bladder but better would be to use intravenous inoculation (IV) to bypass the initial steps

Question 14

how is virulence measured?

Answer 14

lethality (survival curve analysis)

infectivity (measurement of colonisation of body sites) - either by analysis of bacterial numbers in organs known to be infected by wild-type strain of pathogen (e.g. liver, spleen) or by real time analysis of bacterial numbers in whole animals (bioluminescense imaging)

Question 15

what is an LD50 analysis?

Answer 15

classic experiment for measuring lethality

LD50 value is point at which 50% of inoculated animals (50% lethal dose)

the more virulent the microbe, the lower the LD50

Question 16

how could you use LD50 analysis to show a mutation has decreased virulence?

Answer 16

LD50 analysis for wildtype bacteria and mutants bacteria and if bacterial mutant has higher LD50 value that mutation in that gene has decreased virulence i.e. its a virulence factor

Question 17

give an example of LD50 analysis being used to demonstrate a role for a bacterial gene in virulence?

Answer 17

hlyA encodes pore-forming enzyme of Listeria that allows pathogen to escape from host vacuole and replicate in cytoplasm

a deletion mutation in hlyA results in a 10^5 fold increase in LD50 compared to WT strain demonstrating hlyA critical for virulence

Question 18

what is a classic method to measure virulence by measuring infectivity?

Answer 18

classic method of analysis of bacterial numbers in organs known to be infected by WT strain of a pathogen

e.g. for listeria hlyA

looking at numbers of viable bacteria in liver over time (high levels of bacteria during these infections) and can see that deletion in hlyA gene greatly reduces these numbers compared to WT strain

problem with this is it involves many mice so expensive and inethical

Question 19

what is a new approach to measure virulence by measuring infectivity?

Answer 19

real time analysis of bacterial numbers in whole animals using bioluminescence imaging

genetically engineer bacteria to express bioluminescence genes from vibrio fischeri - infect these into mouse and put it under imager to study colonisation at multiple points and can get spatial info (sites of infection)

advantages of this compared to classic method is analysis of bacteria in entire animals (can study sites of infection), quantification of bacteria in real time (study kinetics of infection) and fewer animals needed (saves time and money, ethical)

Question 20

outline bacterial bioluminescence in vibrio fischeri?

Answer 20

production of light occurs via production of luciferase enzyme

this is made by two genes; LuxA and LuxB

LuxC, LuxD, and LuxE = enzymes required for production of the aldehyde (RCHO) substrate of luciferase

so 5 genes altogether needed to produce this reaction - can introduce these into bacterium on plasmid

Question 21

how has imaging of mice infected with bioluminescent bacteria been used to measure infectivity of listeria?

Answer 21

introduce 5 luciferase genes and KanR gene into listeria on plasmid so they recombine into it via homologous recombination and can then select for KanR

inoculate mice with these and see high degrees of bioluminescence in regions like liver, spleen and also early in infection the gall bladder which could be site of chronic infection for asymptomatic individuals (confirmed it was through dissection)

shows how key utility of this approach is looking at diff timepoints of infection (kinetic analysis) and ability to spatially assess infection

Question 22

what are tissue culture models of infection?

Answer 22

tissue culture cells can be important for generating hypotheses which can then be tested in intact animal - have been useful for identifying host targets of virulence factors

simplest cell culture system is undifferentiated cells growing on plastic

complex cell culture system using intestinal epithelial tissue is enteroid model; sourced from human biopsies of non-cancerous intestines and contain multiple cell types (enterocytes, goblet, M etc.) which have similar properties to intestinal epithelium (barrier function, selective transport of nutrients, mucus secretion)

can grow enteroids in 3D spherical system embedded in collagen matrix or 2D monolayer

Question 23

what are forward genetics?

Answer 23

asks “what genes are involved in a particular aspect of virulence”

virulence genes are identified through mutational analysis

identify mutants defective in aspects of virulence, clone the mutated genes, characterise them and figure out what their proteins doing

Question 24

what are reverse genetics?

Answer 24

asks “are these genes that i know about involved in a particular aspect of virulence”

potential virulence genes are preliminarily identified through DNA sequence analysis - roles in virulence are tested through mutations

Question 25

what genetic approaches can be used to identify bacterial virulence genes?

Answer 25

forward and reverse genetics

Question 26

give some examples of simple forward genetic screens and what did these identify?

Answer 26

identification of genes in a pathogen that are required for infection or virulence (mutagenesis) e.g. genes requires for entry of salmonella into human cells

isolation of genetic material from a pathogen that is sufficient to allow a non-pathogenic bacterium to infect human cells e.g. genes that mediate entry of salmonella into human cells

these identified salmonella TTSSI and its effectors

Question 27

what is salmonella enterica?

Answer 27

gran-neg species that includes serotypes typhimurium and typhi

cause of gastroenteritis/ diarrhea (typhimurium) or typhoid fever (typhi)

food-borne pathogen, most commonly acquired from contaminated poultry meat of eggs

incidence of salmonellosis in the USA is 1 in 200 people per year

Question 28

how does salmonella induce internalisation into human cells?

Answer 28

s. enterica infects intestinal epithelium by inducing ruffles

ruffles are projections of host plasma membrane that form due to salmonella using TTSS to inject various effect proteins which cause ruffles

Question 29

what are type III secretion systems?

Question 30

how was identification of bacterial genes required for infection done (forward genetic screen)?

Answer 30

use transposon mutagenesis to identify mutants which are unable to enter cultured human cells

analysis of entry in cultured cells using antibiotic protection assay (infect for 1hr, treat w gentamicin (cant act on intracellular bacteria), break open human cells and quantify bacteria - through this they identified InvA mutant which was defective in internalisation (done individually)

this genetic screen very labour intensive as bacterial genome has many genes which all need to be individually tested for ability to enter human cell (required 52x96 well plates)

InvA encodes sorting platform of TTSS and REQUIRED for entry

Question 31

how was isolation of genetic material from a pathogen that is sufficient to allow a non-pathogenic bacterium infect human cells done?

Answer 31

lab strain e coli cant enter human cells

construct DNA library containing pieces of chromosome (45kb regions w many genes cause entry could be multifactorial) from a pathogenic bacteria and introduce this into e coli

analysis of entry in cultured cells using an antibiotic protection assay - positive selection for e coli that entered cell

this approach identified all the genes encoding components of salmonella TTSSI

Question 32

what is transposon sequencing (Tn-seq)?

Answer 32

modern approach to identifying virulence factors as opposed to forward genetic screens which are too much work and two expensive

before this it was not possible to screen directly in animals for mutations that reduce bacterial virulence

essentially just generate a library of bacterial strains mutated in each gene of genome, infect into animal and identify mutants whose presence is reduced in a particular organ

Tn-seq allows screeing of thousands of bacterial mutants simultaneously and can be used to directly identify bacterial genes involved in virulence in animals - has been used to identify VGs of s. enterica, y. pestis, s. aureus…

Question 33

what are the steps in Tn-seq?

Answer 33

use non-specific transpons (e.g. Tn5 or mariner) to generate a collection of bacterial strains w mutation in each gene of genome

infect animals w entire collection of mutants

recover bacterial DNA from organ of interest, PCR to amplify regions bacterial chromosome flanking Tn insertions - DNA sequencing of all amplified genes

identify mutated genes by matching sequenced DNA to data base containing entire genome of bacterial species

the abundance of a particular sequenced gene indicates degree to which a mutant is able to colonise the organ - mutated genes that have DECREASED in abundance in organ contribute to virulence

Question 34

what makes it feasible to use genome sequencing to identify mutations?

Answer 34

DNA sequencing becoming continually cheaper

note that Tn-seq uses genome sequencing

Question 35

what is comparative genomics?

Answer 35

uses WGS to compare genome sequence of pathogenic bacterium with closely related non-pathogenic bacteria

this can indicate genes potentially involved in virulence

proof that gene is involved in virulence requires mutation of that gene in pathogenic bacterium and demonstration that mutation attenuates virulence

Question 36

what are staphylococci?

Answer 36

gram-positive cocci

colonise skin (S. aureus and spidermidis) or anterior nares (aureus)

aureus causes a range of diseases e.g. skin infections, medical implant-associated infections, invasive disease (endocarditis), toxic shock syndrome, necrotizing fascitis

epidermis causes medical implant associated infections

Question 37

discuss the commensal nature of staphylococci and factors determining disease development?

Answer 37

usually are commensals (symnbiosis where one organism benefits other unnaffected)

everyone colonised w epidermidis and 30% with aureus - most people are asymptomatic

factors determining if disease develop include conditions in host (wounds, implants, IC) and the intrinsic virulence of bacterial strain (high heterogeneity; 20% variance)

Question 38

what is endocarditis?

Answer 38

an infection of the heart chambers or valves

occurs via s. aureus or s. epidermidis entering bloodstream via wounds or implants and blood-borne bacteria then attach to damaged or artificial heart valves and grow

possible consequences include heart faliure or clots in the brain or lungs (from bacterial growths breaking off and causing blockage)

Question 39

what is necrotizing fascitis?

Answer 39

caused by certain strains of CA-MRSA or S. pyogenes strains

skin and muscle damaged by bacterial toxins, S. aureus toxins responsible for NF not fully identified although one candidate is leukocidin

greater risk of developing in IC, left untreated leads to 70% mortality - treat with high IV doses of antibiotics, primarily vancomycin

Question 40

what are some virulence factors of S. aureus?

Answer 40

secreted proteins (exotoxins): leukocidin, alpha-toxin (holes in PM of immune cells), TSST (causes TSS)

surface proteins: fibronectin binding protein (FBP) (binds fibronectin promoting attatchment of bacteria to host cells, implants or damaged heart valves), protein A (sequesters IgG helping bacteria evade host immune system

surface proteins anchored to cell wall………

Question 41

discuss the role of fibronectin binding protein (FnBP) in colonisation of medical implants?

Answer 41

involved in the first step of biofilm formation - fibronectin in blood coats the surface of medical implants

FnBP on surface of of s. aureus mediates binding of bacteria to fibronectin coating the medical implant

Question 42

what is protein A?

Answer 42

allows bacteria to avoid phagocytosis

protein A binds Fc portion of IgG meaning phagocytes cannot ingest s. aureus through Fc receptors

Question 43

what is sortase A?

Answer 43

enzyme which anchors a large number of virulence proteins to cell wall in s aureus and many other gram positive species

Question 44

what is peptidoglycan composed of?

Answer 44

carbohydrate chains comprised of alternating units of sugars N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)

peptides that cross-link the different carbohydrate chains together to give a high degree of strength

Question 45

what is the sortase-A mediated pathway of anchoring surface proteins in s aureus and other gram pos bacteria?

Answer 45

about 20 s aureus proteins anchored to cell wall by sortase A

all these have LPXTG in their C terminus

sortase A cuts between T and G and mediates the covalent linkage of a T residue to a peptide in the cell wall thus resulting in an amide bond

in the case of protein A this means mature/M (anchored) protein A is smaller than its precursor (P2/immature) which hasn’t yet been cut between T and G

Question 46

how was the gene encoding sortase A (srtA) identified?

Answer 46

problem with identifying it is that it may be essential gene i.e. hard to identify through transposon mutagenesis so instead they used a conditional lethal screen

steps to identifying gene and its role in linking to cell wall:

isolated conditional lethal (temp sensitive) mutants

identified mutants defective in making mature (processed) protein A

isolated the srtA by molecular cloning through complementation

Question 47

what is a conditional lethal screen?

Answer 47

used to identify essential proteins and enzymes like RNA pol

introduce conditional lethal mutations into organism which allow growth under permissive conditions (e.g. low temp) but not under restrictive conditions (e.g. high temp) i.e. mutation has no effect or has effect

Question 48

how do temp. sensitive (ts) conditional lethal mutations often affect protein folding?

Answer 48

at low temperature (e.g. 22C) mutant protein is folded correctly and functional thus organism is alive

at high temperature (e.g. 36C) mutant protein is unfolded and defective thus organism dies

Question 49

how was isolation of temperature sensitive/conditional lethal mutants done in s aureus?

Answer 49

take culture of mutated s aureus, dilute and plate at 22C (permissive temp)

imprint subsequent colonies onto two new plates and grow one at 22C and one at 36C

temp sensitive mutants grow at 22 but not 36 i.e. an essential gene has undergone a conditional lethal mutation

Question 50

how were s aureus mutants defective in producing mature protein A identified?

Answer 50

mature/M/processed/anchored protein A smaller than precursor/P2/unprocessed protein A

SDS polyacrylamide gel elecrophoresis seperates proteins based on molecular weight and can used to seperate and visualise P2 and M forms of protein A

bacteria were grown at 26C and then incubated w radiolabelled methionine for 20min at 36C

compared to WT s aureus, srtA- mutant produced more P2 and less M forms of protein A

Question 51

how was sortA isolated through cloning by complementation?

Answer 51

complementation is putting WT gene back in cell leading to recovery of phenotype lost from mutation

generate plasmid library with DNA fragments from WT s aureus containing functional srtA

transform plasmids into srtA- mutant and plate bacteria at 36C so only bacteria with srtA gene restored survive

this was then shown to have restored processing of protein A on SDS gel

Question 52

why was the identification of sortase enzymes so important?

Answer 52

they are present in virtually all other gram positive bacteria incl many pathogens

based on amino acid sequence of sortase A they were able to identify other categories of sortases with similar sequences

prevalence means could be effective drug target

Question 53

what are the different categories of sortase enzymes, their roles and what bacteria are they found in?

Answer 53

sortase A (lots of substrates)- adhesion, immune evasion, internalisation, phage recognition - S. aureus, s. pyogenes, B. anthracis, L. monocytogenes

sortase B (only one substrate) - iron acquisition - S. aureus, B. anthracis, L. monocytogenes

sortase C - pilus formation - S. aureus, S. pyogenes, S. pneumoniae, E. faecalis, C. dipthteriae