Finals: Midterm 1 Content Flashcards
pathogen
- a microbe that is capable of causing host damage
pathogenicity
- the ability to produce disease
virulence (2)
- degree of pathogenicity
- only expressed in a susceptible host
gram negative bacteria (2)
- thin peptidoglycan cell wall, surrounded by outer membrane containing lipopolysaccharide
- double membrane
gram positive bacteria (2)
- lack an outer membrane
- surrounded by thick peptidoglycan cell wall
Koch’s Postulate: First Postulate
- the microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms
Koch’s Postulates: Second Postulate
- the microorganism must be isolated from a diseased organism and grown in pure culture
Koch’s Postulate: Third Postulate
- the cultured microorganism should cause disease when introduced into a healthy host
Koch’s Postulates: Fourth Postulate
- microorganism must be re-isolated from the inoculated diseased experimental host and identified as being identical to the specific causative agent
Challenges to Koch’s Postulates: First (3)
- host susceptibility varies in human and animal populations due to a variety of factors
- opportunistic bacteria can be found in healthy hosts too
- may be difficult to isolate bacteria from certain areas, such as brain and bones
Challenges to Koch’s Postulates: Second Postulate (2)
- not all bacteria can be cultured in pure form
- many bacteria undergo genetic and phenotypic changes in culture environments
Challenges to Koch’s Postulates: Third Postulate (2)
- not all bacterial species are equally virulent
- closely related bacterial strains can be very different
Challenges to Koch’s Postulates: Fourth Postulate
- re-inoculation of a host may not be possible or ethical
Modern Approaches to Satisfy Koch’s Postulates (4)
- PCR
- immunohistochemistry and immunofluorescence
- ELISA
- eliminate the pathogen and prevent/cure the disease
Modern Approaches to Satisfy Koch’s Postulates: PCR
- PCR and 16S sequencing can be used to amplify and detect bacteria during culturing
Modern Approaches to Satisfy Koch’s Postulates: PCR/16S Sequencing steps (2)
- 16S RNA primers are designed to detect conserved regions, with unique regions in-between that act as barcodes to identify the bacteria
- PCR is done to amplify the bacterial nucleic acid for detection
Modern Approaches to Satisfy Koch’s Postulates: Immunohistochemistry (2)
- microscopic images of immunohistochemical staining can be used to visualize the pathogen in the tissue directly
- uses of antibodies against the bacteria with attached reporter enzymes
Modern Approaches to Satisfy Koch’s Postulates: Immunofluorescence (2)
- micrographs of cells with fluorescently labelled antibodies against cell markers and bacterial
- cells infected with bacteria will exhibit colocalization of fluorescent markers, while healthy cells will not
Modern Approaches to Satisfy Koch’s Postulates: ELISA (2)
- use of fluorescently labelled antibodies against bacteria to detect pathogens in infection site without culturing
- Western Blot/SDS-PAGE can be used subsequently
ELISA methods (3)
- direct ELISA
- indirect ELISA
- capture/sandwich ELISA
Modern Approaches to Satisfy Koch’s Postulates: Eliminate the Pathogen and Prevent/Cure the Disease; Why didn’t Koch try this?
- antibiotics did not exist at the time, so treatment was a lot more complicated and time-consuming
virulence factors
- bacterial product or strategy that contributes to the ability of the bacterium to survive in the host/cause infection
Molecular Version of Koch’s Postulates: First Postulate
- gene for virulence should be present in the strain of bacteria that cause disease and absent in avirulent strains
Molecular Version of Koch’s Postulates: Second Postulate
- (i) knocking out or disruption the gene should reduce virulence, and (ii) introduction of the cloned gene into an avirulent strain should render the avirulent strain virulent
Molecular Version of Koch’s Postulates: Third Postulate
- expression of the gene should be demonstrated in human or a relevant model
Molecular Version of Koch’s Postulates: Fourth Postulate
- antibodies or a cell-mediated immune response to a virulence factor should be protective
what must a pathogenic microbe do to survive in a host and establish infection (5)
- attach to host cells for colonization
- evade host’s innate and adaptive immune defenses and persist
- obtain iron and other nutrients needed to multiply
- disseminate or spread within a host and to other hosts
- produce symptoms of disease (to be considered pathogenic)
what is adherence mediated by (2)
- bacterial appendages or surface structures
- interactions can occur directly or indirectly
adherence: direct interactions
- “adhesin” on bacterium binds directly to a specific receptor
adherence: indirect interactions (2)
- adhesin binding first to the protein in the extracellular matrix (ECM)
- then, the ECM proteins binds to specific receptors on the host cell, acting as a bridge
types of adhesins (2)
- fimbrial adhesins
- afimbrial adhesins
fimbrial adhesins (3)
- fimbriae or pili
- several types that form polymers
- Pap pilus is heavily characterized and studied
afimbrial adhesins (2)
- several types
- a single protein
why do bacteria use long fragile structures to bind to host cells (3)
- outreach
- protection and survivability of core bacterial body from host immune system
- bacterial surface and host cell are negative; allows for connection despite repulsive forces
can bacterium have multiple adhesins
- yes, they can be expressed all at once or sequentially during infection at different stages of pathogenesis
are all adhesins virulence factors (2)
- as bacteria can have multiple adhesins, it is difficult to prove necessity of any one adhesin
- not all adhesins are virulence factors
host receptors for adhesins (4)
- often carbohydrate-based
- glycolipids or glycoproteins
- often integrins
- receptors determine the tropism of the disease
tropism
- tissue specificity
pathogen invasion (3)
- pathogens can enter and survive in host cells
- allows them to breach host epithelial barrier
- provides them with protected niche for replication and persistence
pathogen mechanism of invasion (2)
- can actively direct their entry into host cells
- usually done by a protein called “invasin”
invasins (2)
- activate a receptor
- leads to a signaling event that enables uptake through cytoskeletal rearrangements
mechanisms of particle uptake into cells (2)
- zippers
- triggers
mechanisms of particle update into cells: zippers (3)
- sequential engagement of phagocytic membrane with particle surface
- pseudopod advances no further than receptor-ligand interaction permits
- partial engulfment where receptor-ligand interactions are
mechanisms of particle update into cells: triggers (2)
- all-or-nothing effect
- complete phagocytosis
zipper uptake steps (4)
- initial contact (adherence)
- receptor clustering
- phagocytic cup formation involving actin polymerization and membrane extension
- closure of phagocytic cup and retraction involving actin depolymerization
what does trigger uptake involve (3)
- mechanism similar to micropinocytosis
- macropinosomes
- surface ruffling
trigger uptake: mechanism similar to micropinocytosis
- a form of endocytosis that is accompanies by cell surface ruffling
trigger uptake: macropinosomes
- large vesicles that serve as a route for cells to take up macromolecules non-selectively
trigger uptake: membrane ruffling
- ruffles fall back onto bacteria randomly and this leads to formation of large endosomes, which eventually become smaller
trigger: distinct steps (3)
- filopode formation: membrane ruffles
- lamellipode formation: membrane ruffles filled with actin support through polymerization
- formation of phagocytic cup made of actin or through adherence
zipper vs trigger (2)
- zipper results in localized contact-dependent receptor-medicated particle engulfment
- trigger results in generalized engulfment event that could include neighbouring particles
anti-phagocytosis (2)
- some bacteria actively inhibit their uptake
- secrete protein YopH through T3SS
can one pathogen exhibit both zipper and trigger mediated invasion
- yes, for instance, salmonella can invade host cell using different routes and mechanisms
phagocytosis: what is involved in uptake (2)
- microfilament rearrangements
- lead to formation of a phagosome membrane
what does phagocytosis activate
- activation of respiratory/oxidative burst
normal endocytic events (4)
- early endosome
- late endosome
- fusion with lysosome to create the phagolysosome
- destruction of the bacteria
Rabs
- small GTPases
Rab5
- early endosome marker
Rab7
- late endosome marker
LAMP1 (2)
- lysosomal associated membrane protein
- phagolysosome fusion marker
what tools can we use to see where the phagosome is located in the pathway (3)
- western blot
- IH
- IF
bacterial phagosome survival mechanisms (4)
- failure to trigger oxidative burst by inhibiting phagosome acidification
- inhibit/stall the fusion of the phagosome with the lysosome
- survival within the phagolysosome
- escape from the phagosome
autophagy (2)
- induced when cell undergoes starvation
- mechanism for recycling proteins
