Micro - E3 Flashcards
Clostridium difficile is a ____-shaped, _____-forming, Gram-______ bacterium
It is also a strict ______.
Clostridium difficile is a rod-shaped, spore-forming, Gram-positive bacterium.
It is also a strict anaerobe.
C. diff synthesizes two large toxic proteins. What is their function?
These two large toxin proteins (TcdA, TcdB) disrupt the intestinal epithelium
Name 5 microorganisms
Bacteria Viruses Yeast and Fungi Protozoa Archaea: commensals, but no known pathogens
What are the 4 key differences between prokaryotes and eukaryotes?
Nuclear membrane
- eukaryotes –> YES
- prokaryotes –> NO
Membrane-bound organelles - eukaryotes –> YES
- prokaryotes –> NO
Endocytosis
- eukaryotes –> YES
- prokaryotes –> NO
Volume
- eukaryotes –> 1 µm3
- prokaryotes –> 1000 µm3
Rod shaped bacteria –> “_____”
Sphere shaped bacteria –> “_____”
Rod shaped bacteria –> “bacilli”
Sphere shaped bacteria –> “cocci”
Describe gram positive bacterial envelopes
Thick peptidoglycan layer (bacterial cell wall)
Describe gram negative bacterial envelopes
Thinner peptidoglycan wall + lipopolysaccharides + endotoxin
The makeup of polysaccharide chains of peptidoglycan
Repeating disaccharide units –> -M-G-M-G-M-G-M-G-
M = N-acetylmuramic acid G = N-acetylglucosamine
Peptide side-chains are attached to _______________ of peptidoglycan, and are unique in that they contain _______________.
Peptide side-chains are attached to N-acetylmuramic acid of peptidoglycan, and are unique in that they contain amino acids in the D conformation.
What determines bacterial cell shape?
Pattern of cross-links between peptide side chains of peptidoglycan of cell wall
How does penicillin harm growing bacterial cells?
Penicillin mimics structure of D-Ala-D-Ala Chains –> prevents cross links from being formed on growing bacterial cells –> lysis
- does not do anything to pre-existing cross-links
How to prokaryotes undergo cell division?
Binary fission
*Much faster than mammalian cell growth in culture
Describe the bacterial growth curve
Growth is exponential –> during each doubling time the population increases by a factor of 2.
Except for very short periods, exponential growth does not occur in nature.
*growth rate depends on nutrient composition of medium.
Lag phase
Time it takes for a bacterial cell that has not been growing to…
- sense that it has entered a nutrient-rich environment
- use nutrients to repair any cellular damage, make the cellular compounds for unrestricted growth
Stationary phase
- Secretion of degradative enzymes
- Transport of secondary nutrients
- Intracellular catabolic pathways
- Motility and chemotaxis
- Genetic competence
- Antibiotic and toxin production
- spore formation
Prototrophs
Bacteria that have no requirements for organic compounds other than a simple carbon source (e.g., a sugar).
Auxotrophs
Bacteria that have more complex nutritional requirements, typically requiring amino acids and/or vitamins and/or pyrimidines and/or purines.
- C. difficile is a natural auxotroph
What are the two ways that bacteria may satisfy their energy needs?
- Fermentation: (“substrate-level phosphorylation”) synthesize ATP through enzymatic rxns of glycolysis
- Respiration: synthesize ATP through electron transport chain and ATP synthase
Difference between aerobic respiration and anaerobic respiration
Aerobic respiration: electron acceptor = oxygen; produces H2O
Anaerobic respiration: electron acceptor = fumarate, nitrate, other
Strict aerobes
- terminal e- acceptor for respiration –>_________
exp. _________________
Strict aerobes
- terminal e- acceptor for respiration –> oxygen
exp. Mycobacterium tuberculosis (lungs)
Strict anaerobes
- terminal e- acceptor for respiration –>_________
exp. _________________
Strict anaerobes
- terminal e- acceptor for respiration –> organic molecule
exp. Bacteroides, Clostridium (colon)
Facultative anaerobes
Can carry out aerobic respiration if oxygen is present, or anaerobic respiration or fermentation if no oxygen.
Exps.
- Escherichia coli (small intestine),
- Vibrio cholerae (small intestine)
- Streptococcus pneumoniae (nasopharynx, lungs),
- Pseudomonas aeruginosa (lungs)
Spores
- triggered via nutritional limitation
- hardy, non-growing structures
produced by some Gram-positive bacteria (Bacillus and
Clostridium) - low water content allows them to survive harsh conditions
Examples of spore-forming pathogens
Clostridium difficile –> pseudomembranous colitis
Clostridium botulinum –> botulism (flaccid paralysis)
Clostridium tetani –> tetanus (spastic paralysis)
Bacillus anthracis –> anthrax
Bacillus cereus –> food poisoning
Organization of Toxin Genes - Pathogenicity Locus (PaLoc)
Pathogenicity Locus (PaLoc) is composed of several transcription units. The genes (involved in production of toxins) are transcribed from multiple promoter sites.
Operon
In bacteria –> multiple functional related genes often organized into transcriptional units called operons.
Genes are typically co-transcribed from common promoter
Bacterial DNA-Dependent RNA polymerase
Template dependent –> needs a DNA primer
Have β, β’, α, and σ subunits
Inhibitors of RNA synthesis bind the __ subunit of RNAP.
Inhibitors of RNA synthesis bind the β subunit of RNAP.
Inhibitors of RNAP (bind __ subunit)
Rifamycins: Blocks ________
Example: ________
________: Blocks ______
Example: fidaxomicin
Inhibitors of RNAP (bind β subunit)
Rifamycins: Blocks elongation
Example: rifampicin
Lipiarmycins: Blocks initiation
Example: fidaxomicin
Role of σ subunit of bacterial DNA-Dependent RNAP
σ subunit
- causes stable binding at promoter regions
- reduces binding at non-promoter regions of DNA
- removal allows escape from the promoter (translocation) and elongation of the transcript
There is one type of RNAP core enzyme.
Many kinds of __ subunits (recognize promoters with different sequences)
One type of RNAP core enzyme.
Many kinds of σ subunits (recognize promoters with different sequences)
How does factor-Independent termination stop transcription?
A Factor-Independent termination site is encoded in the DNA sequence appearing as a region of dyad symmetry.
Results in an RNA sequence that is able to form a stem-loop structure.
How does factor-dependent termination stop transcription?
Rho (ρ) factor binds directly to RNAP and causes RNAP to stop and release when reaching the factor-dependent terminator.
Describe how transcription can be positively regulated
X
- positive regulator
- binds UPSTREAM of promoter
- has affinity for RNAP, stabilizes RNAP binding at the promoter and helps recruit promoter
Describe how transcription can be negatively regulated
Y
- negative regulator (repressor)
- can overlap promoter, or be downstream of promoter
- If active, gene not turned on
TcdR is a _______ factor for toxin gene expression.
During which phase it made?
Sigma factor. Recognizes the promoter sites for the tcdA and tcdB genes.
tcdR made during stationary phase
How is CodY a repressor?
CodY binds to the tcdR promoter region and prevents RNA polymerase from binding to the promoter.
Able to do this by sensing presence of amino acids like isoleucine (nutrient xs (more ile) –> active CodY)
TcdC
An anti-sigma factor.
Binds and inactivates TcdR. Deals with “leaky expression” of TcdR during exponential phase
Deficiency in TcdC
Results in more active TcdR –> could cause high toxin production because
Commensal relationship
microbe benefits, host unaffected
Which areas of the body have been shown to NOT be colonized?
CNS/Spinal Fluid
Blood
Which areas of body are unlikely to be colonized and have few if any bacteria?
Lungs
Uterus
Bladder
What is the ratio of human and microbial cells?
Roughly equivalent # human and microbial cells
weight of microbiota = 2-6lbs
How do humans deal with undigested polysaccharides and host-derived glycans?
Bacteria ferment them into short chain FAs
What do paneth cells do?
Located at bottom of intestinal crypts. Make specific antimicrobial peptides
Functions of our symbiotic gut microbiome
Metabolic (vit K, fermentation, gut epithelial cell differentiation/proliferation)
Immune/Structural (immune system development, induction of IgA Abs, intestinal barrier integrity)
Protective (competition for niche and nutrients, production of antimicrobial factors)
What is dysbiosis of the gut microbiota?
Which conditions are associated with this?
Imbalances, skewed #s, low diversity of the microbiota.
IBS, colon cancer, alzeimers, autism, allergies, asthma, obesity
*what microbiota is making gets into our serum, some are neuroprotective
Describe experiment that showed that an altered gut microbiota does call disease.
Germ-free mice had affected OR unaffected human microbiota.
(lean human twin vs. obese human twin)
Germ-free mouse w/ lean twin’s microbiota became lean
Germ-free mouse w/ obese twin’s microbiota became fat.
Infants are first exposed to microbes in birth canal.
Give an example of a bacteria infant comes into contact with during breast feeding.
Breastfeeding –> Bfidobacteria
What are the two main proposed notions of how dysbiosis of the gut microbiome occurs?
- Ideal bacteria never introduced into gut in the first place
- formula feeding
- hyperintensive sanitation - Beneficial bacteria is reduced/lost entirely from the gut.
- western lifestyle
- abx usage
Distinguish between exotoxins and endotoxins
Exotoxin –> classical toxins secreted by bacteria
Endotoxin –> LPS complexes, secreted
Endotoxin is the non-proteinaceous lipopolysaccharide of gram-negative bacteria . Molecule is a PAMP (Pathogen-Associated Molecular Pattern) which is recognized by the Pattern Recognition Receptors (PRR) on cells of the innate immune system (neutrophils, dendritic cells, macrophages, etc)
How are exotoxins classified?
Based on their site of action
- target cell surface
- target cell cytosol
Explain type of experiment to show particular part of bacterium is causing toxicity
Protein isolated out of bacterium is associated with symptoms and disease.
Exp. injecting tetanus toxin in lizard.
C. difficile toxins
Have enzymatically active domain, cysteine protease domain, hydrophobic region, receptor binding carbohydrates
Mechanism:
- Binds to carb on host, endocytosed, early endosome is acidified via H+ pumps, vacule is acidified.
- Protein changes conformation, exposure of N-terminus into cytosol.
- Auto-cleavage via protease, enzymatic part of protein is released into cytosol
- Releases active site
- leads to cell death
What are the functions of the different domains of A-B toxins?
B Domain –> Binding
- RME
- acidification –> membrane insertion
A Domain –> Active (enzymatically)
- domain translocated by B domain
- cleaves proteins involved in synaptic transmission
Clostridum tetani
Tetanus
- obligate anaerobic
- gram-positive
- spore forming (can enter wound, germinate, produce toxin)
Symptomology of Clostridum tetani (tetanus)
trismus (lockjaw), which is spastic paralysis
How is tetanus toxin similar to botulinum?
Tetanus toxin is similar to botulinum neurotoxin: poisons NT release by cleavage of proteins involved in vesicle fusion
Mechanism of flaccid (botulinum) vs spastic paralysis (tetanus)?
Cell-binding specificities of the B subunits
- tetanus toxin –> translocated to CNS (prevents inhibitory NTs like GABA)
- botulinum toxin –> translocated to peripheral nerves (prevents stimulatory Its Its like acetylcholine)
Clostridum botulinum
- obligate anaerobic, gram-positive spore-forming bacillus.
- Toxin causes flaccid paralysis.
- Rare.
- Intoxication rather than a true infection.
- Abs directed against the toxin can alleviate symptoms
Cholera
Vibrio cholerae
- gram-negative
- comma shaped
- facultative anaerobe
- noninvasive (all sx due to actions of single toxin)
- A-B model toxin
- toxin causes secretion of fluids into lumen of small intestine (generates alkaline pH –> inhibits competing bacteria)
- causes watery “rice water” diarrhea (entirely due to toxin)
- mild to severe disease, can result in extreme dehydration/hypotension, can be rapidly fatal.
Mechanism of cholera toxin
G proteins act as “molecular switches”
Binding of GTP (on-state, adenylate cyclase is active –> cAMP goes up –> less Na+ absorption –> more Cl- secreted –> more water secreted)
or
GDP (off-state, adenylate cyclase is inactive)
Cholera toxin is an ADP ribosylating toxin, what role does this play in its pathogenicity?
Transfers the ADP-ribose group to G-protein.
With ADP-ribose bound, cannot shed GTP (turned on constitutively –> less Na+ absorption, –> increased Cl- secretion –> more water secretion)
Examples of colonized parts of body
Nares, oral cavity, skin, small intestine, Proximal colon, distal colon, MAYBE stomach
Factors contributing to the decrease of death rate for ID
- Discovery of microorganisms as the cause of many serious diseases
- Better hygiene and sanitation
- Implementation of childhood vaccination programs
- Discovery of antibiotics
Antibiotics (bactericidal vs bacteriostatic?)
Give exps.
Agents that either kill (bactericidal) or inhibit growth (bacteriostatic) of different species of bacteria.
Exps of natural vs synthetic abx
Natural secondary metabolic products from microbes (e.g., penicillin)
Semi-synthetic or synthetic (e.g., Ciprofloxacin)
6 strategies for abx to target bacteria
- Cell wall synthesis (penicillin)
- DNA replication (fluoroquinolones)
- RNA synthesis (rifampicin)
- Protein synthesis (tetracyclines, chloramphenicol)
- Folic acid synthesis (sulfonamides)
- Membrane disruption (daptomycin, Gram-positives, colistin for Gram-negatives)
What makes peptidoglycan layer is rigid?
cross-linkages
Mechanism of penicillin
Penicillin is a chemical mimic of D-ala-D-ala
Transpeptidase cannot see D-ala D- ala
How do abx target DNA replication. (exp. via fluoroquinolones)
- DNA is supercoiled (needs to be relaxed during DNA replication)
- DNA gyrase –> enzyme that relaxes supercoiling
- Gyrase makes a break and rejoins the strands
- Fluoroquinolones (cipro) covalently link to gyrase, and cause lethal double stranded DNA break
How do abx target RNA transcription.
(exp. via Rifampicin)
Rifampicin binds to RNAP and blocks transcription (elongation)
How do abx target protein synthesis.
Tetracycline - Preventing binding of charged DNA to ribosome
Chloramphenicol - Preventing peptide bond formation
How do abx target folic acid synthesis.
Exp. via sulfonamides –> similar to structure of PABA (enzyme that makes folate in bacteria)
(Folic acid is a precursor of DNA and is impermeable to bacterial cell, so bacteria must synthesize folic acid from p-aminobenzoic acid (PABA))
How do abx target membrane disruption?
Daptomycin –> Gram-positives
^ BIG molecule, can’t get through membrane of gram-negative
Colistin –> Gram-negatives
^ only targeting LPS, outer membrane
Horizontal gene transfer
transfer of genetic material between different organisms
Transformation
Transduction
Conjugation (direct contact)
Transposons
Mobile genetic elements that carry drug resistance gene and are capable to move within and between replicons
HGT can lead to rapid spread of __________
Drug resistance
Plasmids
Mobile genetic elements that mediate their own transfer between bacterial cells
Transposons
mobile genetic elements carry drug resistance gene and are capable to move within and between replicons
In transformation, imported pieces are typically _____ than a bacterial chromosome.
In transformation, imported pieces are typically smaller than a bacterial chromosome, usually they carry 1-10 genes.
Two types of transduction
Specialized (lysogenic phage)
Generalized (lytic phage)
Specialized (lysogenic phage)
Imprecise. Part of the bacterial chromosome adjacent to the attachment site is aberrantly packaged during prophage excision
Generalized (lytic phage)
Randomized. Any parts of the host chromosome can be transduced by phages
Restriction modification systems
Bacteria defense mechanisms to prevent foreign DNA invasion, cuts unmethylated DNA sequences
CRISPR-Cas systems
Bacteria defense mechanisms to prevent foreign DNA invasion.
These prokaryotic “acquired immune” systems, recognize and cut previously “seen” genetic materials
homologous recombination
Process by which some DNA that displays homology to the bacterial chromosome can integrate into the host genome
Requires RecA ATPase which facilitates strand exchange during homologous recombination
Result: Recipient chromosomal DNA acquired new gene (e.g., antibiotic resistance, virulence factor, new enzyme)
Steps of Conjugation
- Donor bacterial cell makes a special sex pilus (via Tra genes, found on plasmid)
- The donor’s sex pilus attaches to the recipient cell
- Relaxase binds to the oriT site of the plasmid –> generates a single-stranded DNA substrate
- Single stranded copy circulates and replication completes complementary strand
- Recipient MAY become new donor cell after completion of conjugation
Replicons
DNA elements capable of self replication (bacterial chromosome and plasmids)
Require rep and origin of replication (oriC, V, R) to be replicated
Requirement to carry out another round of conjugation
Need tra operon (encodes for transfer apparatus/proteins req for conjugation) and orIT (must be on same replicon being transferred)
Transposons
- mobile genetic elements
- no self-replication
- require transposase to jump
- recognize inverted repeats at ends of transposon
- usually have drug resistance genes on plasmids
Insertion vs simple vs composite transposon
insertion: transposase gene only
simple: transposase gene + drug resistance gene
composite: two matching IS elements flanking a central region
How do bacteria resist action of abx via inactivating the abx?
Beta-lactamase is secreted from the cytoplasm, cleaves beta-lactam ring of ampicillin
The gene is often encoded on a plasmid DNA (transferable to other bacteria) and inducible
Vancomycin mechanism of action
Vancomycin binds to the D-Ala-D-Ala motif in the PG, thus prevents transpeptidase access to the substrate
How do bacteria resist action of abx via modifying or replacing the target?
- D-Ala-D-Ala can be changed to D-Ala-D-Lactate (by a new enzyme)
- Vancomycin can’t bind D-Ala-D-Lactate
- Transpeptidase is able to able access its substrate
How do bacteria resist action of abx via removal the antibiotic from the cell?
Efflux pumps (multi drug resistance)
How do bacteria resist action of abx via prevention of abx uptake?
- Decrease outer membrane porin (OMP) gene expression in Gram negatives
- (example: regulation of OprD in Pseudomonas aeruginosa)
- increased thickness of peptidoglycan cell wall (Gram +)
- capsule formation
- biofilm production
How do bacteria resist action of abx via persistence?
Development of persister populations (cells neither grow nor die during antibiotic exposure)
- not genetic mutants, the majority of their offspring is still sensitive to antibiotics
Intrinsic vs. Acquired Resistance
• Intrinsic resistance - innate resistance
– Gram-negative resistance to vancomycin (the drug is too big to pass through the outer membrane porins)
– Gram-positive resistance to colistin (lack of outer membrane LPS)
• Acquired resistance - developed resistance through acquiring mutations or new genes via horizontal gene transfer (HGT).
Rep proteins
initiate chromosome or plasmid replication by binding directly to the plasmid-ori. Act by helping recruit DNAP to recognize orientation and initiate replication.
ori
origin of replication – a DNA binding site for Rep protein
- oriC- chromosome
- oriV- vegetative plasmid
- oriR- R factor plasmid
R factors
plasmids with abx resistant genes
Integrative and Conjugative elements (ICEs)
- Possess properties of phages/transposons (integration & excision) and plasmids (conjugal transfer)
- Cannot maintain extra-chromosomally
