MICR3001 – Bacteria MICR3001

MICR3001 – Bacteria MICR3001

Print FlashCard
Question Answer
What causes pneumonia? What is the major cause of pneumonia? A variety of factors including viral, fungal and bacterial infections. The most common cause of pneumonia is the bacteria streptococcus pneumoniae.
Is streptococcus pneumonia found in animals? How is it spread? No it is a human pathogen. Via aerosols.
Other than pneumonia which can be fatal, what two other non-fatal infections does streptococcus pneumonia cause? Otitis media and sinusitis.
Can streptococcus pneumonia infections be asymptomatic? Yes!
Describe the pathogenesis of streptococcus pneumoniae. (refer to diagram in lecture 1) adf
What is the role of the capsule in streptococcus pneumoniae. Key virulence factor. They have 92 different, immunologically distinct capsules. It prevents phagocytosis, Those without the capsule are avirulent.
What are the three types of molecules that enable streptococcus pneumoniae to adhere and colonise the lung. Choline binding proteins, pili -> surface adhesins which enable b to bind to epithelial cells. -SA which enable b to bind to extracellular matrix-Enzymes which remove sugars from human glycoconjugates – reveals receptors for further binding.
What are the two types of molecules that cause streptococcus pneumoniae. Part 1 Pneumolysin -> pore forming cytotoxin- Released from cytoplasm of lysed cells-Inhibits activity of ciliated cells- activates complement pathway, induces cytokine release-kills alveolar and endothileal cells
What are the two types of molecules that cause streptococcus pneumoniae. Part 2 Cell wall components such as teichoic acid and peptidoglycan –> activate immune system, activating the complement system and producing cytokines
Overall, the cell wall components of streptococcus pneumoniea and pneumolysin cause what? Damage to the lungs leading to fluid leakage from blood vessels = pneumonia
What types of antibiotics are effective against pneumococcus? Is resistance a problem? Penicilins and beta lactams. HOwever resistance is emerging and is becoming problematic
What vaccines are there against streptococcus pneumoniea? The vaccines use the capsule as an antigen.23V vaccine – protection against 23 serotypes – not very immunogenic7V conjugated vaccine (conjugated to carrier protein)13 V conjugated vaccine
What is a conjugated vaccine? A conjugate vaccine is created by covalently attaching a poor antigen to a strong antigen thereby eliciting a stronger immunological response to the poor antigen.
How do they develop the streptococcus pneumoniea vaccine? They ferment and purify the saccharides of the different capsules. Each type of capsule is separately activated and conjugated to CRM protein carrier. Conjugates are mixed to formulate vaccine
Describe the impact of the 7V conjugated streptoccocus pneumoniea vaccine on pneumococcus disease Greater than 90% reduction in invasive pneumoccocal infection. Has reduced vaccine types in nasopharynx, however has led to increase in potentially invasive non-vaccine types (e.g. increase in otitis media)
Why does the streptococcus pneumoniae vaccine readily lead to the production of new virulent strains. the gene for the capsule is readily transferrable via homologous recombination. Therefore the virulent strains can change their capsule to evade the vaccine
Is there a vaccine for gonorrhoea? No!
are obligate intracellular bacteria easy to target with a vaccine? No!
What type of bacteria is Neisseria gonorrhoeae? Where does it colonise? Is it an exclusively human pathogen? Can it be asymptomatic? Is there a different disease pattern in men and women? Gram negative diplococcus. It colonises mucosal surfaces (cervix, urethra, anus, throat). Exclusivly human. Yes. Females more readily infected than males. Acute symptoms in males, mild in women
What are the symptoms of gonorrhoeae? Urethritis. Infection can lead to ectopic pregnancy, infertility as the scarring narrows the fallopian tube
Is gonorrhoea an immunising infection? No, because it rapidly changes its cell wall components
Know the outcomes of N,. gonnorrhoeae infection (see lecture 2) adf
Why are individuals with a bacterial STD such as gonnorrhoea 5 times more likely to acquire HIV on sexual contact? Due to inflammation caused by bacterial STDs there are more cells that HIV targets
Does N. gonorrhoeae have a capsule? No!
What roles does LPS have in N. gonorrhoeae survival? It is the adhesion for sperm and therefore explains why there is a higher rate of transmission from male to female. LPS has a terminal galactose which binds to galactose receptor in sperm cells
Describe the pathogenesis of N. gonorrhoeae? Bacteria attach to walls of genitourinary epithelium via pili and adhesinsPenetrates into mucosal cells by phagoccytosis and multipliesPasses through cells in sub-epithelial space LPS stimulates inflmmatory reponse leading to symptoms of the disease
How many toxins does N. gonorrhoea have? LPS is the only one
What is another name for pili? Fimbrae
What type of fimbrae/pili does N. gonorrheoea have? What type of molecules are these? What molecule does it have on top? What does it bind to Type 1V fimbriae – part of the adhesion family. It has a pil c adhesion molecule on top which attaches to the CD46 receptor
Other than the 1V fimbriae what other adhesin molecule does N. gonorrheoea have? What receptor does it bind to? Opa – integral outer membrane proteins. Acts as adhesion and binds to CD66 on human epithelial cells.
What is the toxic part of LPS Lipid A
How does LPS help N.gonorrhoeae evade the immune system? The molecules is coated by host derived molecule (N-acetylneuraminic acid). Therefore the bacteria are not recognised as foreign, therefore leading to asymptomatic infection
How does N. gonorrhoeae evade host defences via gene variation Phase variation (on/off expression) of pili and opaAntigenic variation – changes in gene seqiuence of pili (homologous recombination)
Other than LPS and gene sequence variation how else does N.gonorrhoeae evade the immune system? Via glycosylation of the pili. Sugars hange the way protein is presented to immune system. Some are not glycosylated. Therefore the infection is non-immunsing and it is not possible to develop a vaccine
Is there a vaccine for N. gonorrhoeae? Why or why not? No! most surface proteins exhibit phase and or antigenic variation. Therefore there is a high frequency of antigen variation on the surface of the cell
WHat is the treatment for N.gonorrhoea? Is resistance emerging? Treated with single dose of anti-biotics. Yes resistance is rapidly emerging
What type of pathogens cause meningitis? Which is more common and fatal? How do the pathogens affect protein and glucose levels and the opaquness of the CSF Viral and bacteria. Viral more common, almost never fatal. Slight increase in protein, normal glucose, CSF remains clear. B . 10X protein, low glucose, CSF becomes cloudy. Less common, more serious
What are the three main types of bacteria that cause meningitis? Which is the most common Most – neisseria meningitdis2nd – streptococcus pneumoniae3rd – haemophilus influenze (vaccine against this, so only 2%)
What is the main difference between streptococcus pneumoniae and neisseria meningitdis. What do the different serovroups of neisseria meningitdis refer to? Strep – no capsuleNeisseria meningitis – capsuleThe serogroups refer to the different capsules
What serogroup of Neisseria meningitis does Australia vaccinate against? Type C
Why is Neisseria meningitis serogroup B so hard to vaccinate against? Capsule has similar proteins to eukaryotic ones
what age range does Neisseria meningitis affect? Mostly the young
what two diseases do Neisseria meningitidis cause? Meningitis and septicaemia (menginoccocal disease)
Why do some people develop meningococcal disease, whilst other people experience asymptomatic infections DO not know why exactly. Most likely due to host and environmental factors and not due to variation in bacteria
What type of bacteria is Neisseria meningitidis? What are the different capsule types? Can it be asymptomatic? Is it an exclusively human pathogen? What is its reservoir? How does it spread? Gram negative diplococcus. Type A, B. C. Y,. W-13520% of the time it is asymptomticexclusively humanresides in the nasopharynx, spreads by aerosols
What are the two molecules that assist Neisseria meningitidis with adhesion and colonisation. WHat receptors do they bind to and what cell types? Type IV pili (adhesin family). Pil c on top, binds to CD46 receptor – long polymeric proteinsOpa and Opc – integral outer memb prot. Act as adhesions. Opa binds to CD66 on epithelial cells. Opc binds to heparin and induces cytoskeletal changes in e.c.
Describe the variation of Opa and Opc in Neisseria meningitidis They are phase variable and are variable in terms of antigenicity
What are the three other types of molecules (other than Opa, Opc and Pili) present in Neisseria meningitidis? What is their function? Porins (PorA and PorB) – react with antibodies to enhance immune responseIron acquisition proteinsLPS – endotoxin – activates cytokine cascade. Inflammation can lead to organ failure and death
What is the molecular pathogenesis of Meningococcal disease? Colonisation of nasopharynxSpread to mucosal surfacesInvasion of epithelial cells (local invasion)Access to bloodstream (systemic infection – bacteremia)Crossing of blood brain barrier (meningitis)
What is the purpose of Neisseria meningitidis's capsule? Are the capsules immunogenic? Important for its survival in blood – it is anti-phagocytotic. No they are poorly immunogenic – do not usually elicit a protective immune response
Can Neisseria meningitis be effectively treated? Is there a problem with resistance They can be effectively treated if antibiotics are given early enough. Major problem with treatment is difficulty in diagnosing the disease. Low incidence of resistance
Is there a vaccine for Neisseria meningitis? Two effective vaccines available – C and A, C, Y, W-135. New vaccine Bexsero for serogroup B
Describe the vaccine bexsero ( Neisseria meningitis serogroup B). How was it developed. Developed via reverse vaccinology – identification of bacterial surface antigens using genome mining. – The vaccine is based on outer membrane proteins
What are the 7 major components of the Bexsero vaccine and their function? Part 1 fHbp – major antiggen – binds to human factor HNadA – adhesion – major aNHBA – major a – heparin binding moleculeGNA1030 – major a – function unknown
What are the 7 major components of the Bexsero vaccine and their function? Part 2 GNA2091 – major a – function unknown dOMV – por A is a major componentAlum – adjuvant
What demographic exhibit a UTIs incidence? Women and the elderly
What are the four different infections that UTIS cause Urethritis Cystitis – most common UTI – bladder and urethraPyelonephritis – kidney symptomsAsymptomatic
What are the main reservoirs of UTIS Colon, vagina can act as additional reservoir.
Are UTIs often recurrent yes recurrent infections are common (80%) and relapses are common
Describe catheter associated UTIs. What percentage of hospital acquired infections do they account for? Is resistance problem? 40%. Most are multi-drug resistant
How do they diagnose UTIs Cloudiness in urine. INcreased conc. of white blood cells when acccompanied with bacteria. Culture tests. Dipstick test
What are the major causative bacterial agents of community acquired and hospital acquired UTIs E-coli!
What are the virulence factors of uropathogenic E.coli Fimbrae, multi-drug resistant plasmids, flagella, iron acquisition proteins, toxins, polysaccharide shield, autotransporter proteins
What types of fimbrae do uropathogenic E.coli contain? What infections are they associated with and what do they bind to? Type 1 – Bind to D-mannose containing glycoproteins in bladder. Associated with cystitis P – Bind to Gal-Gal dissacharide in upper respiratory tract via PapG tip adhesinAssociated with kidney colonisation (pyelonephritis)
Describe the different genetic components of type 1 fimbrae (uropathogenic E.coli virulence factor) fimB and E – regulationfimA, C, D – major subunitsfimH – adhesion
Do type 1 fimbrae exhibit phase and antigenic variability (uropathogenic E.coli) Only phase variability! Their promotor lies in 314bp invertable element, called fim switch.
Do uropathogenic E.coli form biofilms? Where? What virulence factor is associated with biofilm formation. They form biofilms in the bladder known as intracellular bacterial communities (IBCs). UPEC that form these biofilms express type 1 fimbrae
Describe the genetic components of P fimbrae (uropathogenic E.coli). Do they exhibit phase and or antigenic variability? Comprised of eleven pap genes with a pap G adhesion molecule. Only phase variability mediated by epigenetic modifications
Describe the autotransporter adhesins of uropathogenic E.coli Antigen 43 expressin causes bacterial clumping, biofilm formation, IBC formation and persistence in the bladder.
Describe the three toxins of uropathogenic E.coli Hemolysin – pore forming toxin, stimulates cytokine, cause lysis, Cytoxic necrotising factor 1 (CNF-1) – kills epithelial cells Secreted autotransporter serine protease (Sat) – causes vascuolation tissue damage
Do uropathogenic E.coli have capsules, LPS? Some do
Where are the virulence factors housed in uropathgogenic E.coli. Found on pathogenicity islands in the chromosome. Most strains carry multiple PAIs
Is antibiotic resistance a major problem with UTIs yes
What are the novel treatments for UTIs. Can they treat acute or chronic UTI? Mannosides inhibit fimH, therefore they prevent adhesion. Both!
Describe the vaccine candidates for uropathogenic e.coli. How do they act in vivo and in vitro Part 1 Type 1 fimbrae (fimH adhesion). In vitro -antibodies prevent UPEC adhesion to bladder cells. P fimbrae – pap g – antibodies prevents UPEC adhesion in kidney cells. Immunisation in mice and monkeys prevents UPEC colonisation.
Describe the vaccine candidates for uropathogenic e.coli. How do they act in vivo and in vitro Part 2 Multivalent vaccines – inactivated whole cell vaccine. Probiotics – prophylactic treatment with asymptomatic e.coli prevented catheter colonisation by pathogens and UTIS in people with dysfunctional bladders.
What are the limitations associated with the type 1 fimbrae (fimH) and P fimbrae (pap g) vaccines for UTIs Type 1 fimbrae are present in most e.coli but they are phase variableNot all pyelonephritis strains produce P fimbrae and P fimbrae are phase variable
What is shigellosis? Is it highly contagious? Bacterial infection of the gastrointestinal tract (bacillary dysentery). Yes!
What are the symptoms of shigellosis? How is it transmitted Fever, abdominal craps, watery diarrhoea, mucoid and bloody stool. Complications such as septicaemia, pneumonia and kidney failure may occur. Transmitted commonly via faecal oral route.
Why is shigellosis a disease of impoverished people Poor sanitaiton, close personal contact, lack of clean water, malnutrition, cost of treatment
What type of bacteria are shigella in terms of gram status, the family they come from, are they motile. spore forming, shape, anaeobic or aerobic. Are they an exclusively human pathogen and are they invasive? Gram neg from family Enterobacteriaceae. Non motile (no flagella), non spore forming, rod shaped, faculative anaeorbic bacteria. Invasive and humans and some non primates
How many species of shigella exist? Which is the most virulent? How many serogroups can the most virulent species be divided into? 4 species, s. flexneri is most virulent and can be divided into 13 serogroups
What is the general scheme of entry and spread for shigella bacteria? Shigella infect mucosal absorptive cell. They spread to adjacent cells or are phagocytosed. They escape from phagosome and spread to other M cells or through the layer of mucosal cells.
What encodes the shigella invasive phenotype and what is necessary for their entry into epithelial cells Virulence plasmid encodes invasvive phenotype. Central pathogeniicty island is necessary for their entry as it encodes for the structural components of a Type Three Secretion System
What is a type three secretion system, Molecular syringe -> allows for delivery of a range of effector proteins directly from the bacterial cytoplasm to the host cell cytoplasm
How does a type three secretion system in shigella contribute to virulence/ The effector molecules cause the rearrangment of the host cell cytoskeleton and the uptake of of the bacterium. Causes non-phagocytotic cells to become phagocytotic and to internalise the bacteria
What are the main effector proteins and players in the shigella type three secretion ssytem Invasion proteins – IpaB, IpaD, IpaCMotility – IcsA, host actin
What effector proteins in the shigella type three system mediate the bacteria's escape from phagocytic vesicles? IpaB and IpaC insert themeslves into the membrane of the phagocytic vesicles. Once free the organism multiplies in the cytoplasm of the host cell
What effector protein in the shigella type three secretion system enables the bacteria to use host actin for motility? How does it achieve this? Localisation of IcsA to one bacterial pole elicits accumulation of F-actin. It recruits N-WASp and Arp2/3, which converts g-actin to f-actin
Does shigella contain LPS? Yes!
How is the serotype of S. Flexneri determined? Position of glucose and O-acetyl residues on O-antigen backbone (in LPS)
Are shigella infections immunogenic and are they easy to vaccinate against? Why or why not? Different serotypes (positions of glucose and O-acetyl residues) means that infections are not immunogenic and that they are difficult to treat against
What is the therapeutic treatment for shigella? Is multi-drug resistance a problem? Water and electrolyte balance maintenance + antibioticsMulti-drug resistant strains of S. dysenteriae and S. flexneri are emerging across the world
Why is shigella vaccine development a piority? Resistance, oral rehydration alone is not sufficient for treatment, cost of antibiotics
What are some considerations that need to be made when you are developing a vaccine against shigella? Live or subunitIf life, attenuated but still viable in vivo-Must activate mucosal immune s-immun must be long lasting- cheap- simple to administer
What is the most popular and advanced vaccine type that has been investigated in relation to shigella? Invasive live vaccines
Describe the vaccine candiates for shigella. What are their limitaitions if they have any? Part 1 Invasive live vaccines using shigella with an IscA and an iron transport mutation. Strain is protective nut only safe at low doses. Less immunogenic in children
Describe the vaccine candiates for shigella. What are their limitaitions if they have any? Part 2 Effective subunit vaccinesLPS when conjucated to protein carrierInvaplex – complex of IpaB. IpaC, and LPS.. Increased stability and efficacy compared to previous vaccine
Do the diagnostics for tuberculosis take a long time? Yes!
Is TB isolated to a small region in teh world or is it global? Global! One third of the world's population is infected
How is TB transmitted Infectious aerosols
Describe mycobacterium tuberculosis in terms of whether it is anaerobic or aerobic, motility, growth speed, cell wall, whether it is acid fast Obligate aerobe, non-motile. slow growth, thick cell wall containing mycolic acid, therefore acid fast
Describe the interaction between m. tuberculosis and macrophges from phagocytosis to escape part 1 Macrophage phagocytosis, bacterial replication and mphage infiltration (accumulation), Latency (death of mphages, release of M tuberculosis cells, formation of caseous centre in tubercle)
Describe the interaction between m. tuberculosis and macrophges from phagocytosis to escape part 2 Formation of mature granuloma, rupture of tubercule, M tuberculosis spills into bronchiole and dissemination of the respiratory system
How long can M. Tuberculosis surve in granulomas for? Decades!
Describe the treatment for M. Tuberculosis and drug resistance 4 front line drugs taken for 9 months effective against most strainsHowever multiple resistant TB exists and extremely resistant TB exists, .There have been cases of totally resistant TB
Is M. Tuberculosis opportunistic ? No it is specialised to humans
What are four different aspects of M. tuberculosis pathogenesis? Inhibition of phagosome maturation, escape to cytoplasm, altereration to intracellular conditions and inhibition of macrophage activation
What are the three main molecules that contribute to M. tuberculosis pathogenesis? ESAT-6, LAM and PknG
Describe the role of ESX/ESAT6 in M. Tuberculosis pathogenesis ESX is a secretory system which secretes the molecule ESAT-6, which is a potent T cell antigen. Very immunogenic and therefore is often included as a component in subunit vaccines for TB
What is RD1 in BCG (M. Tuberculosis) Region of deletion. This region of deletion contains the ESX system, which secretes the virulence factor ESAT-6. Therefore RD1 reduces mtb pathogenesis
What are the two molecules in M. Tuberculosis associated with inhibition of macrophage function (prevention of macrophage maturation) . LAM and PknG
What does prevention of macrophage maturation prevent (M. Tuberculosis) Prevents lysosome-macrophage fusion and limits degradation of bacteria by lysosomal enzymes
Describe the role that PknG has in preventing phagosome maturation and preventing phagosome-lysosome fusion (M. Tuberculosis) It resembles a eukaryotic kinase. Inhibits celular signalling to prevent maturaiton. Phosphorylation of host proteins prevents lysosome-phagosome maturation
How does LAM prevent phagosome maturation (M. Tuberculosis pathogenesis) Inhibits PI3P accumulation on phago membrane. PIP3 is inolved in maturaiton of phagosome
What molecule is required for phagosome escape? ESAT-6! Mtb deficient in ESAT-6 do not enter cytoplasm. It perforates the phagosome membrane
What is BCG? Can it be used to protect TB in children and adults Attenuated version of mycobacterium bovis . Protects children against serious compliations of TB. Adults no. ails to stimulate the right kind of immune response (T cell)
What is the new vaccine candidate vaccine for TB. Why is it better than BCG alone .Part 1 New BCG recombinant vaccine called VPM1002. Contains listeriolysin (Hly) and lacks urease. Hly acts like ESAT-6, which BCG lacks. Hly is a pore forming enzyme, that enables the BCG antigens to disseminate into the cytoplasm
What is the new vaccine candidate vaccine for TB. Why is it better than BCG alone .Part 2 Consequently, results in the priming of both CD4 and CD8 T cells through MHC II and I presentation. Hly is pH dependent, hence lack of urease. Hly is degraded after it serves its function
Why don't they just add ESAT-6 to the BCG recombinant vaccine ESAT-6 majorly increases its virulence
Compare speed of T cell recruitment in BCG vaccine vs VPM1002. What does this mean? VPM1002 faster recruitment of Ag specific t cells to lung. Potentially means improved infection containment
What is group A streptococcus known as? Are they gram pos or neg? Streptococcus pyogenes – flesh eating bacteria
What type of diseases do streptococccus pyogenes cause? Most common is pharyngitis Skin infections like cellulitis and impetigoBacteraemia, toxic shock syndrome, necrotising fascitis
What is the main problem with treating streptococcus infections They are sensitive to penicillin but the disease develops to rapidly
since mid 1980s the number of severe GAS infections have been increasing. What has been parallel to this? Emergence of the GAS M1T1 strain, which is prevalent in china
What is inversely related to disease severity among clonal M1T1 isolates? (GAS) Spe8 (cysteine protease) expression!
How did they determine that Spe8 was inversely related to disease severity among clonal M1T1 isolates (GAS) Mice was used to recapitulate disease. Inoculated with Spe8 pos strains. They then measured the Spe8 activity in inoculum, skin and blood. Inoculom and skin had high acitivity, blood low. Therefore, invasive infections are spe8 neg
Loss of speB actiity has been mapped to what frameshift mutation? (GAS) To a frameshift mutation in covRS (control of virulence regulator sensor), which is a two component signal transduction system
Why does the mutation in covRS which leads to Spe8 loss beneifical? (GAS) Results in downregulation of Spe8, but upregulates capsule ( increases resistance to neutrophils), streptokinase and surface plasmin. It is a selection pressure
Why does deletion of the capsule gene abolish the phase shift to a Spe8 negative phenotype? (GAS) There is no selection pressure to induce mutation. Rescue with wild-type capsule gene, results in Spe8 neg phenotype
How do GAS cause serious disease? Streptokinase activates human plasminogen to form plasmin, which is a serine protease involved in fibrinogen degradation and teh degradtion of connective tissue and basement membrane.
How did they determine that human plasminogen was vital for GAS virulence Created transgenic mouse with human plasminogen as mouse plasminogen is not activated by GAS streptokinase. Those with the transgene had a lower survival rate
How does Spe8 interact with the plasminogen system (GAS) The cysteine protease Spe8 degades streptokinase, human plasminogen, fibrinogen binding M1 protein and fibrinogen. Those with Spe* have a higher survival rate
Most GAS clinical isoaltes are not covRS mutatns. Therefore, are hypervirulent covRS mutants M1t1 mutants transmissble? No covRS mutants exhibit impaired colonisation ability as they cannot bind to fibronectin, collagen, human cells well or form biofilms well. It produces too much capsule – its adhesins are covered)
What are host adapted pathogens? Micro-organisms with no other enviornmental niche, which have evolved with the host and usually cause benign (non-life threatening) diseases
What are some examples of host adapted pathogens Neisseria meningitis, neisseria gonorrhea, streptococcus otitis media
What is antigenic variation Is a mechanism which allows alteration of nucleotide sequence of a gene such that the new protein made by this gene has an altered amino acid sequence and may not be recognised by antibodies directed at original sequence
What is phase variation? reversible ,high frequency switching of bacterial surface antigens (genes are switched on or off)
Antigenic and phase variation are particularly present in what type of organisms Host adapted pathogens
Describe bacterial meningitis in terms of prevalence, What are the main causative pathogens? Most common CNS infection. Leading bacterial cause of childhood death. Caused by neisseria meningitis, streptococcus pneumonaia and haemophilius influenzae
How are meningitis causing bacteria typically transmitted Principally transmitted by respiratory droplets from infected individuals. Bacteria can be carried in the respiratory tract without the development of serious disease
Describe the molecular mechanism of bacterial meningitis part 1 Attachment and colonisation of mucosal surfacesCrossing mucosal barriers and invasion of blood systemCrossing BBB and entry into cerebrospinal fluid -> inflammation of meninges
What are the two forms that prevention of bacterial meningitis takes? Prevention of further cases by treating close contacts with antibioticsPrevention of disease by antibiotics (Bexero)
what host adapted pathogen virulence factors are essentialfor colonisation of a host Bacterial surface virulence factors such as pili
Provide an example of a pathogen that undergoes antigenic variation The type iV fimbrae (pili) of pathogenic neisseria species undergo antigenic variation.
Describe the molecular mechanism of type IV fimbrae antigenic variation in pathogenic neisseria species part 1 Pilin is expressed from the pilE gene. In addition to the pilE gene there are upt o 11 copies of a silent gene called pilS. Pil S cannot express pilin, but recombination with pilE alters the pilin sequence.
Describe the molecular mechanism of type IV fimbrae antigenic variation in pathogenic neisseria species part 2 Pil S serves as a repository of alternative sequence possibilities. Homologous recombination between pilE and the multiple copies of pilS can theoretically generate 10^7 different versions of pili
How did the antigenic variation of IV fimbrae in pathogenic neisseria species affect vaccine development. Pili were an ideal candidate for vaccines as they are immunogenic, essential for colonisation and expressed by all strains. Found not to be an effective vaccine due to high degree of antigenic variation
Provide an example of phase variation Phase variation occurs in OPA (outer membrane adhesins) of Neisseria gonorrhoaea
What is the significance of phase variation Reversible – can revert back to original (virulent) phenotype As a no. of different surface factors are randomly and independnetly switching on and off, a large repertoire of different proteins is presented on surface
What are the four molecular mechanisms of phase variation and which is the most common Gene conversion (homolgous recombination)Site specific insertionMethylationSimple tandem repeats <- most common
Describe how random tandem repeats contribute to phase variation During DNA replication tandem repeats cause frame shift mutations in open reading frame
What tandem repeat underlies opa phasic variation in neisseria gonorrhoeae 5'CTCTT-3' repeat causes frameshift mutations in opa reading frame
What type of genes commonly undergo phasic variation LPS biosynthesis genesIron acquisition genesPili glycosylation genescapsule biosynthesisOuter membrane proteins(adhesins and pili)
What are the main implications from antigenic and phase variation? Immune evasion – vary structures targeted by immune systemNiche adaption- variation allows the expression of ideal bacterial phenotype for a particular microenvironment
What are the four types of vaccines? Inactivated (killed) vaccinesSubunit vaccineDNA vaccineLive attenuated vaccine
What are inactivated (killed vaccines) Produced by chemical treatment (e.g. formaldehyde) or heat inactivation
What are subunit vaccines Purified single antigen or group of antigens used as vaccine (avoids toxins)
What are DNA vaccines Eukaryotic expression plasmid containing antigen gene
What are live attenuated vaccines Produced by growth of the micro-organism in unusual conditions (e.g. animal passage), chemical mutagenesis and genetic modification
Define vaccination Is a process which aims to prime the adaptive immune system to the antigens of a particular microbe so that a first infection induces a secondary response
What are the characteristics of a good vaccine Stimulates strong immune response and right kind of response (correct position)elicits protective effectCost effectiveSafety (no side effects)
What are examples of inactivated vaccines Rabies, influenza, polio, hepatitis A, bordetella pertussis
Why did they switch from inactivated bordetella pertussis vaccine to a subunit (recombinant one) Because the whole cell one had side affects (or alleged ones anyway)
What is bordetella pertussis Bacteria. Causative agent of whooping cough
How did they genetically modify bordetella pertussis for a subunit vaccine? Knocked out vital amino acids (Arg9 and Glu129) in enzymatic portion of pertussis toxin (S1). Which converts it to a toxoid
Describe the process through which they converted the pertussis toxin into a toxoid (part 1) – subunit vaccine Process: Site directed or oligonucleotide mutagenesisDNA fragment cloned into plasmid vector, isolate single stranded DNA template, anneal with oligonucleotide containing mutation
Describe the process through which they converted the pertussis toxin into a toxoid (part 2) – subunit vaccine Anneal with oligonucleotide containing mutation. DNA polymerase I synthesises mutated DNA strand. Transofrm e.coli and transfect into b pertussis (incorportated into genome via homologous recombination)
What are the benefits and disadvantages of a subunit vaccine? Less protective, less reactogenic
Describe how they created the recombinant hepatitis vaccine? Part 1. Yeast cell stripped of cell wall, yeast sphaeorplast treated with Ca2+ and polyethylene glycol DNA. Hepatitis B surface antigen gene clones into e.coli then transferred to yeast expression vectors for expression studies.
Describe how they created the recombinant hepatitis vaccine? Part 2. Transformed yeast with construct yeast expression vector and purified hepatitis B surface antigen (which encodes for envelope) to be used for recombinant vaccine.
What are the benefits of DNA vaccines? Encoded protein is expressed where you inject the geneInduces antibody, cellular immunity and memorySingle plasmid can be tailored to make a variety of vaccinesRefrigeration not needed for DNAcan be delivered via gene gun on microscopic beads
WHat are the disadvantge sof DNA vaccines So far work well in mice. ,not so well in humans
Describe how DNA vaccines can activate the immune system The expressed gene product of the injected antigen is recognised as foreign asn is presented by MHC class 1. Therefore it mimics a viral antigen
What are the three ways you can deliver DNA vaccines? Liposomes, carrier proteins which target specific cell types. DNA injection and gene guns
What is vaccina virus? What can it be used for and why? Attenuated co-pox virus. Could be used for recombinant antigen delivery because it does not insert into the eukaryote genome and therefore does not produce mutations.
What are the advantages of using vaccina as a recombinant antigen delivery system Part 1 It replicated within the cytoplasm therefore not inserting into the host chromosome causing mutations. Has a wide host range and infects a variety of cells. Can be used as a human or animal vaccine vector
What are the advantages of using vaccina as a recombinant antigen delivery system Part 2 Has large genome and lacks packing contraints -> can insert multiple recombinant genesHigh stability as a freeze dried preparation, administered by dermal abrasionTechnology for large scale production of VV is available
What are the disadvantages of using vaccina as a recombinant antigen delivery system Causes disease in immunocompromised individualsCan cause side effects in a small proportion of individuals (encephalitis)
Describe how they made the recombinant vaccina virus delicery system? Part 1 Cloned gene encoding antigenTransfered antigen to ecoli shuttle vector inside of thymidine kinase geneIsolate plasmid and transfect into human cells, which are infected with WT VV
Describe how they made the recombinant vaccina virus delicery system? Part 2 Recombination between genetically engineered tymidine kinase gene and WT gene occursCell line is administered BUdr. TK converted BUdr into toxin, therefore recombinant cells survive
What is a shuttle vector A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species [1]. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types.
What has vaccina virus been used to deliver? Rabies (for the fox population)Herpes simplexHepatitis BHIV
How long does reverse technology take 4-5 years ish
What is reverse technology, What does it involve The application of a genome based approach to vaccine development. Identification of potential antigens (all proteins) through gene sequencing and cloning, then perform in vitro and in vivo assays to identify vaccine candidates
What is proteomics? Identifiction and analysis of proteins expressed by an organism
Surfaceome proteins expressed on surface of a cell
secreteome proteins secreted by a cell
Describe chlamydia trachymatis? Is it gram neg or pos, describe its life cycle, what disease it causes Gram negative bacteria. Obligate intracellular pathogen. Takes two forms elementary body (infectious, non replicative) or reticulate body (non-infectious, replicative). Pneumonia, conjunctivitis, pelvic inflammatory disease, ectopic pregnancy
Using a diagram, describe the strategy used by Novartis to identify Chlamydia trachomatis antigens that promote CD4-th1 immune response. Part 1 CT elementary bodies have been isolated and used to infect mice. CD4 cells are harvested from spleen once infection has progressed. The CD4 cells are stimulated with elementary bodies. If they recognise EB, they will proliferate
Using a diagram, describe the strategy used by Novartis to identify Chlamydia trachomatis antigens that promote CD4-th1 immune response. Part 2 They transfect tye cell lines that recognised the EB bodies, into a mouse undergoing an allergen challenge. If the mice survive the antibodies confer protection. Ccell lines are stimulated with chlamydia proteins to see which ones they proliferate to
How long did the chlamydia reverse vaccinology protocol take. How many vaccine candidates did they find 2-3 years. four antigens (COMBO5) were selected and tested to see whether they conferred immunity
All bacterial subunit vaccines are comprised of what type of proteins? What implication does this have for reverse vaccinology? Surface or secreted proteins. These type of proteins induce protective anti-body responses. Therefore, reverse vaccinology should focus on the secreteome and surfaceome
How do you mine the bacterial surfaceome for antigens (gram pos) Gram pos – use trypsin protease to shave off surface proteins, then use LC/MS/MS for identification.
How do you mine the bacterial surfaceome for antigens (gram neg) Gram neg produce outer membrane vesicles, you can make mutants that produce lots of these vesicles. You can recover these vesicles and undertake protein digestion and identify proteins using LCM/MS/MS
How do you validate the existence of surface proteins Flow cytometry – involves using antibodies raised against each recombinant surface protein
Using a diagram describe the three step approach, employed by Novartis to develop an experimental Group A streptococcal vaccine part 1 1. Proteomic analysis of the bacterial surface to identify proteins (trypsin or vesicle)
Using a diagram describe the three step approach, employed by Novartis to develop an experimental Group A streptococcal vaccine part 2 Once proteins are identified, make polyclonal sera against proteins checking that they are found on cell surface using FACs analysis (flow cytometry). To do so you express protein and make antibodies against them in mice.
Using a diagram describe the three step approach, employed by Novartis to develop an experimental Group A streptococcal vaccine part 3 Construct protein chip. Express all proteins separately, put them on chip and take antibodies from human sera from convalescent patient (recovered from disease), and see which of the antibodies reacts with. Perform allergen challenge
How much quicker is the surface proteome method than typical reverse vaccinology 1 year vs 4 years

Leave a Reply

Your email address will not be published. Required fields are marked *