T1 Flashcards
Genera of Gram + bacteria
Actinomyces Bacillus Bifidobacterium Clostridium Corynebacterium, Propionibacterium (& other diphtheroids) Enterococcus Gardnerella Lactobacillus Listeria Mobiluncus Peptostreptococcus Staphylococcus Streptococcus
med-important G+ cocci
Strep
Staph
Enterococcus
general of bacterial endospore-formers
Clostridium
Bacillus
Genera of acid-fast + pink bacteria
Mycobacteria
Nocardia (usually)
cell shaped determined by
murein sacculus and cytoskeleton
cocci arrangements
diplococcus (pairs)
G- : (kidney bean)
G+: (lancet)
chains (strep)
clusters (staph)bacillus/rod
v. short: G-
short, thin: G-
short, thin, needle-like: fusiforms, G-
long, thick: G+
short, thick: G+
clubbed-shaped: G+
-thin, branching filamentous rods w. club ends
*arranged as single cells, pairs (doublet) or chainshelicoidal
curved, comma: (G-)
curved, comma: (G+)
spirochetes
*arranged as single cells, pairs, chains
pleomorphic
can vary in size/shape:
flagella
H-Ag, protein composition
plain: extend out from cell surface
endoflagella: internal structure (spirochetes)
motility, sensory system (chemotactic), surface translocation, aids in identification, virulence factor (chemotactic)
Pili (Fimbriae)
protein composition
normal (I-IV) and sex pili extend out into environment
adherence (virulence factor), antiphagocytic, surface translocation (v.f.)
sex pilus: conjugation
capsule
glycocalyx, exopolysaccharides (EPS)
mucoid-like coat around cell, slime layer
polysacch. polymers: K-Ags: single, complex, D-glutamic acid repeating units OR O-Ag: LPS (G-)
biofilms
communal, protected, complex, 3-D structure (bac/yeast)
in exopolysacch. film, sometimes protein amyloid fibers
CDC: 50% human bac inf. involved biofilms
phenotypic changes: become sessile in biofilm, MDR, quorum sensing–>activation?
can revert to normal/planktonic form–>recurrent inf.
D-A.A. can disrupt amyloid protein connections–>release bad
*are a capsule
clin sig of EPS/biofilms
adherence, anti-phagocytic, anti-antibiotic, anti-dessication, Ag used to ID some human pathogen
HKO Ag
H: flagella K: capsular O: LPS (acts as capsule) G+: capsule or no capsule G-: O+K-, O+K+, O+,K- (again)
S-layers
A- or T-layers, (glyco)protein composed, 10-20% cell mass
present on some human normal flora AND pathogens
on cell wall, rigid layer w. pores of fixed diameter
virulence factors: anti-complement (C3b), anti-phago(PMN)
translocation
thru human cells to new site
paracellular, sliding motility (biofilms)
cell membrane contains no sterols except
Mycoplasma
Helicobacter
Ehrlichia
Anaplasma spp.
Type III secretion system
injectosome; conserved multiprotein system used by G- bac to insert protein toxins into human cells
cell membrane contains respiratory system
ETC, ATP synthase, PMF/ion current
cell membrane systems
perm/transport, respiration, cell wall synthesis components, cellular replication components, osmoreg/sensory (chemotactic) mechanisms (hypertonic to ext. environ.)
G+ or G-: polysacchs are covalently linked to peptidoglycan layer in cell wall and lipoteichoic acid polymers are anchored in cell membrane
G+
G+ or G-: contains an outer membrane with LPS, peptidoglycan layer located in periplasm
G-
cell wall functions
sieve, prevents bursting, mech. strength, vir/tox factors, Ag-comps, rec. for Abs, sex pilus, bacteriophages; anchors external bac structures (flag, pili, caps)
peptidoglycan layer
“fabric” of crosslinking, covalently bound threads: N-acetylmuramic acid (NAM) and N-actelyglucosamine (NAG)
- cleavage by lysozyme (human tears/saliva)–>bac lysis
- all peptide stems possess some D-a.a.s (i.e. D-alanine)
lytic transglycosylases
cell wall enzyme, causes cell wall turnover during exponential phase growth
product recognized by TLRs–>SIRS!
pathogenic bacteria WITHOUT peptidoglycan
Mycoplasma
Rickettsia
Ehrlichia
Anaplasma
G+ or G-: contain periplasm and an outer membrane
G- (thin pep. layer)
*confers resistance to dyes, hydrolytic enzymes, detergents
periplasm functions
osmotic protection (for thin pep. layer), nutrient uptake from OM–>CM, chemotactic sens. mech, degradative enzyms, osmoreg
OM functions
bac-environ interaction site, vir/tox factors, Ag-comps, ref for Abs, sex pilus, bacteriaphages, anchors ext. structures of bac, shield against dyes, hydrolytic enzymes, detergents
OM comp
lipopolysacc, phoslip, proteins (OMP)
OM structure
lipid bilayer (NOT phoslip bilayer) LPS as outer leaflet, phoslip inner porins: OMP, allows hydrophilics to pass thru
LPS
aka: endotoxin, exogenous pyrogen
Lipid A + core + O-Ag
Lipid A
disacch, phos grps, fatty acids, toxic factor
Core oligosacch of LPS
sugars, aminosugars, sugar acids, or sugar alcohols
-ketodeoxyoctulonate (KDO): common Ag in enteric bac
terminal polysacch.
aka O-Ag, repeating unit, contains sugars (like core), highly specific btw genera AND species
Lipooligiosaccharide (LOS)
lipid A + extended core, NO O-Ag
syn. instead of LPS, assoc. w/ Neisseria meningitidis and N. gonorrhoeae; Haemophilus influenza and H. ducreyi
props of LPS/LOS
part of OM, chromosome encoded, broad sp.(effects many org sys in susc. host) i.e. induction of IL-1 (endo. pyro), also acts directly on hypothalamus itself (exogenous pyrogen)
act. alt compl pathway, activator of Hageman factor (XII), induction release of endo mediators, heat stable, does not form toxoids
endogenous mediators induced/released by LPS/LOS
TNF-a, IL-1, IL-6
arch acid metals, bradykinin, histamine, NO, free radicals
only ways to disrupt primary structure of LPS/LOS
burning, oxidation –>detoxifies the endotoxin
Endotoxin/LPS/Lipid A is a potent immunomodulatory substance
SIRS–>distributive shock (DS) (G-)
systemic w. macros, PMNs, endothelial cells
SIRS activated by
50%: infection (usually G- (contain LPS))
50%: non-inf. etiology
initiation of SIRS
LPS binds LBP–>LPS-LBP complex interacts with mem-bound CD14 rec on PMS, macros/monos–>LPS-CD14 binds TLR–>signal transduction–>cytoplasm–>nucleus
–>induction/release of endogenous mediators
soluble CD14 rec in serum–>binds ECs–>dysfunction/leakiness>hypotension/vascular leak syndrome (ARDS: feature of DS, not SIRS)
why endotoxin (LPS) is not a true toxin
human response to presence is what causes fatality, not toxin itself
SIRS classification
2+:
temp >38 or =90 bpm
tachypnea >=20
leukocytosis: >12000 or
3 pathophys processes of sepsis etc.
systemic inflammation (“hyperinflammatory state”)
coagulation activation
fibrinolysis inhibition
Disseminated Intravascular Coagulation (DIC)
LPS activates Hageman factor (XII)–>activation of coag (fibrin deposition)–>fibrinolysis activated by this but inhibited by plasminogen activator inhibitor–>accumulation of undiss. thrombin in microcirculation
DIC–>MOF +/- purpura fulminans
if survived hyperinflammatory state of sepsis, pt enters
“immunoparalysis”; hypoinflammatory state:
loss of type IV hypersn, failure to clear primary infection, dev. of new secondary infections, dormant viruses may “awaken” (HSV-1, EBV, CMV, HHV-7)
sepsis classification
2+ of SIRS criteria PLUS proven infection: i.e. pneumonia, UTI, bacteremia
severe sepsis
sepsis criteria PLUS organ failure (OD, MOD, MOF) most: heart, lungs, liver, kidneys
septic shock
severe sepsis criteria PLUS refractory hypotension
shock
inadequate profusion of tissues; 3 forms:
cardiogenic, vascular obstructiv, hypovolemic (DS)
distributive shock (DS)
“warm shock” (dilation)
- EC dysfunction/leakiness–>loss of plasma into tissues–>hypotension
- loss of vasc resistance–>hypotension
- coagulopathy–>DIC
- septic cardiomyopathy: rev., does not damage heart structure–>reduces CO (LPS, C5A, IL-1B, TNF-a, IL-6)
clinical manifestations of DS
fever or hypothermia, chills, leukopenia/cytosis tachy x2 DIC hypotnsn, shock, DIC-->OD, MOD, MOF
Early Goal-Directed Therapy (EGDT) for SIRS/DS
- sepsis resuscitation bundle: measure serum lactate, obtain blood spec for culture, admin broad spec Ab, if hypotnsv: admin fluids: iconic crystalloid or iso-onccotic colloid (4% albumin) or vasopressin, achieve O2 sat goals
- sepsis management bundle: corticosteroids, tight glycemic control
failed SIRS/DS tx
eritoran tetrasodium: anti-TLR-4 compound
Drotecogin alfa: act. recombinant protein C, approved, no proof of benefit
detection of LPS in pharm industry
Limulus Amebocyte lysate test and/or monoclonal Abs (MoAb) against LPS (detects nanogram amounts of endotoxin)
G+ peptidoglycan layer
larger (50% cell wall) and more cross linked than G-
can induce TNF-a, IL-6
can lead to SIRS/shock/DS (not as much as LPS)
Lipoteichoic acid structure
polymer of glycerol-PO4 or ribitol-PO4, covalently bound to glycolipid, integrated/NON-COVALENTLY bonded into outer leaflet of CM, extends thru cell wall/pep layer into environ
*adhesion of Strep. pyogenes to fibronectin on surface of pharyngeal epithelial cells
Teichoic acids (and other polymers)
polymer of glycerol-PO4 or ribitol-PO4, covalently bound to peptidoglycan, extends thru cell wall/pep layer into environ
- peptidoglycan + teichoic acids–>can produce endotoxin-like shock in pts with Staph aureus infections
- interacts with CRP–>activates alternative comp pathway–>inflammatory response
PAMPs (pathogen assoc. molecular patterns)
on pathogen, recognized by PARs/PRRs–>can initiate release of endogenous mediators that cause SIRS/DIC/DS
*caused DIC/DS to occur in absence of endotoxin: G+ bac inf., fungal inf., viral inf.
PARs (PRRs)
NOD1/2: internal/cytoplasmic *NOD2 def. related to Crohn’s
TLR rec: extracellular, 11 total, bind to sp. PAMPs: peptidoglycan, teichoic acid polymers, N-f-met-leu-phe, CpG nucs, LPS
bacterial spores
-most bac do not form spores, help survive adverse conditions, forms inside mother cell (dies), complete but inactive cell in protective shell, germination when conditions are favorable
bacterial endospores have increased
longevity, resistances to heat/temp, desiccation, chem agents *req special disinfectants/sterilization
medically important spore-forming bacteria
Bacillus
Clostridium
small colony variants (SCV)
growth-def variants, form colonies 1/10 normal size
enhanced resistance to Ab (*aminoglycosides)
formed by both G+/G-: Staph aureus, Pseudomonas aeruginosa, E.coli, UTIs
phenotype switching: able to revert to normal size
SCVs are associated with chronic, recurrent infections of
bones, heart, lungs, urinary tract
bac are good metabolizers b/c
large surface:volume ratio, close contact w. environ, accumulate nutrients quickly, grow rapidly
autotrophs
“fix CO2”, cell energy from redox of inorganic ions (chemoautotroph) OR harvesting light energy (photoautotroph)
heterotrophs
oxidize organic molecules for cellular energy
*all bac which cause disease in humans
heterotrophs utilize..
carbs, proteins, then lipids
fastidious bacteria
will not grow on blood agar (complex growth req)
non-fastidious do
optimal growth occurs at temps..
closer to maximum
min determine primarily by reduced enzyme activity and red. mem fluidity
max: protein denaturation
mesophile growth occurs btw
20-50 degrees C
*most pathogens are mesophiles: 35-36 C
thermophiles
(obl. or fac) >55 C
psychrophiles/cryophiles
(obl, fac)
microaerophilic organisms
grow in presence of red. O2 conc.
facultative anaerobe
*most pathogens
aerobic respiration when O2, otherwise fermentation
-early on use aerobic, consume all O2, switch to anerobic
aerotolerant anaerobe
grow best in absence of O2 (only fermentation) but can grow in O2
obligate anaerobe
only grow in absence of O2 (only fermentation)
bacteria become O2 tolerant when..
they produce enzymes (SOD, catalase) to detoxify the byproducts of O2 metab (O2-, H2O2, respectively)
if bac are O2 intolerant
they lack nec. enzymes, so toxic agents (O2-, H2O2) kill them
special ionic requirements
non-halophile vs halophiles (need Na+) eg: low Fe+++: C. diph-->produces diphtheria toxin low Ca++: plague bac-->produces exotoxin low Mg++: S. aureus strains-->TSST-1
glycolysis
catabolic pathway; partially oxidizes organic matter–>end products are substrates for other pathways; phosphorylation generates ATP
- additional energy if end-products enter:
- TCA(gen red power (NADH2)–>respiration (gen ATP, recyc. NADH2)
- fermentation pathways
TCA
completes oxidization of organic carbon into CO2
gen. intermed. for anabolic pathways
gen. reducing power (NADH2) for ana/catabolic pathways
FADH2 and/or NADH2 is recycled in resp/anabolism
fermentation pathways
produce shorter C chain-org comps +/-CO2
recycle NADH2 for ana/catabolic pathways
may gen ATP via substrate level phosphorylation* sole source energy
respiration must occur
in mem vesicle/sack, generates energy: ion current (PMF) for ATP synthesis, occurs during recycling of NADH2–>NAD+(oxidized) *Abs exist that collapse the gradient
1st part of respiration: ETC..
transfers e-s and H+ from NADH2 to TEA–>generates both PMF and reduced TEA
oxidase test
determines presence of ETC comp (cytochrome C) in some bac that can oxidize derivatives of P-phenylenediamine to a colored product
-used by lab to ID bac: enterobacteria (oxidase -)
other G- rods (oxidase +)
2nd part or respiration: ATP synthetase…
uses PMF to synthesize ATP from ADP and inorg. phos
aerobic respiration
(oxidative phosphorylation)
TEA is O2–>red. to H2O by ETS
*common pathway among pathogens and humans
anaerobic respiration
TEAs are inorganic comps
med sig:
-methemoglobinemia (MetHb) happens when elevated levels of NO3 are in drinking water
-GI tract NF convert NO3–>NO2–>if absorbed in blood–>MetHb (risk esp to fetus)
fermentation
simpler than respiration, incomplete oxidation of C substrate, utilizes substrates less efficiently (NO respiration), but still allows growth, occurs in cytosol (NOT vesicle), does NOT directly produce PMF
fermentation substrates are..
partially oxidized to 1-4 C compounds and some CO2, these serve as TEA (accept e- from NADH2, H+) during recycling of NADH2–>NAD+
–>then excreted from cell (pyruvate–>ethanol +CO2)
microbial end products of fermentation cause..
dental caries; end prod. is lactic acid from homolactic fermentation (like hum. musc.)
microbial end products of fermentation lead to…
acidic pH of vagina and skin (lactic acid, again, and propionic acid, acetic acid, CO2)
microbial end products of fermentation cause abscesses..
that are acidic and anaerobic
- many Abs not effective at low pH
- many Abs bind free NAs and render them unavailable
- low pH kills surrounding viable human cells –>rel. compounds that bac req for growth (para-aminobenzoid acid) –>Abs like sulfas are ineffective
microbial end products of fermentation help..
ID bac
- mixed acids: lactic, acetic, formic, succinic, etOH, CO2, H2
- typical of enteric bac of human gut (coliforms)
certain bac can only grow fermentatively
- lack cytochrome/ETC OR cannot use it for energy prod.
- do not use NAD+ or NADP+ as e- and H+ ion carrier, rather ferredoxin is used–>must be recycled to oxidized form
aerotolerant anaerobes
(Strep and Lactobacillus)
-produce lactic acid and H2O2 (from ferredoxin recycle)
H2O2 detoxed by human host’s peroxidase (otherwise can’t grow)
fermentation by Clostridium
end products include H2, CO2 and 4 C compounds; recycling of ferredoxin catalyzed by hydrogen lyase (not aerotolerant?) –>H2 byproduct
H2 gas produced by Clostridium’s fermentation can cause..
Gas gangrene! (myonecrosis)
H2 is insoluble in tissues, tracks along fascial planes (sep muscles, collapses blood vessels, impeding perfusion (anaerobic)
alkaline end products are NOT
fermentative or oxidative phosphorylation end products
i.e. Proteus spp. (cause UTIs, kidney stones) rel. urease–>hydrolyzes urea in urine–>ammonia and CO2–>raises pH to above 7, allows Proteus to grow–>Ca++ and NH4+ ions form salts, precip. at alkaline pH–>renal calculi
renal calculi are composed of
triphosphate: Struvite: Mg ammon phos)and poorly crystalline form of apatite (hydroxylated Ca phos (some repl by carbonated)
H. pylori cause..
type b and duodenal ulcers
produce urease–>cleaves urea to CO2, NH4+–>raises microenvironment pH (stomach mucous lining) so bac can grow
primase
synthesizes short ssRNA primers for DNA synthesis
DNA gyrase (topoisomerase II)
negatively supercoils bac genome and plasmid DNA
relieves torsional stress cause by helicase: “unwinding”
topoisomerase IV
required for decatenation (separation of 2 daughter chromosomes (rings))
*both topo. II and IV are essential for bac DNA sun
DNA synthesis originates..
at one origin of replication in prokaryotes is bidirectional (as is eukaryotic)
DNA synthesis must be primed with..
RNA
primes synthesizes primers
partitioning of daughter strands
- in prok: req membrane attachment
- in euk: utilize spindle fibers and centromere to sep. each chromosome
DNA synthesis occurs when
prok: lag and exponential phase
euk: S-phase
RNA synthesis produces
mRNA, tRNA, rRNA
why antimicrobial protein synthesis inhibitors are so effective
mRNA in bac has a v. short 1/2 life
some gene transcription req. DNA gyrase
to neg. supercoil DNA
protein synthesis occurs in the
cytoplasm
bac ribosomes
free, 70S (30S + 50S)
rRNA + proteins + accessory comps
charged tRNAs accomplished by
aminoacyl-tRNA synthetase: covalently bond sp. aa’s to appropriate tRNA (mRNA is template)
mRNA is codon, tRNA is anticodon (base-pair)
what catalyzes peptide bond formation during which ATP is consumed
rRNA (not proteins)
–>polypeptides are product
protein turnover is v. short, so
Abs that inhib port sun are effective
biosynthesis of peptidoglycan(PTG) backbone of cell wall:
1. synthesis of amino sugars/PTG subunits
uracil diphosphate (UDP) is tag for directed synthesis of amino sugars/PTG subunits
NAG and NAM syn. occurs while cov. bonded to UDP
peptide side chain of NAM syn. by ind. enzymes
biosynthesis of peptidoglycan backbone of cell wall:
2. assembly and translocation of PTG subunit thru CM
PTG subunit formed by transfer of NAM and then NAG from UDP–>bactoprenol (Lipid P (carrier)) w/ release of UMPs
PTG subunit then shuttled thru CM to growing end of PTG chain
addition of subunit to PTG chain occurs via..
transglycolase enzyme (transglycosylation)
biosynthesis of PTG backbone of cell wall:
final cross linking (transpeptidation)
done by transpeptidases (penicillin binding proteins (PBPs)
2 aa subunits from each peptide side chain covalently bonded–>”fabric shell”: mech. strength and rigidity to PTG
G+ bacteria use unique enzymatic pathway to make heme
HemQ, used in final step
MRSA, enterococcus, listeria, Mtb
*selective toxicity
lag phase
cell volume and mass increase, chromosome replication (DNA syn) begins
*NO cell division/change in #
exponential/log phase
balanced growth occurs
cell number, mass, volume, and cell comp. amounts increase by same exponential factor
exp./log phase expressed as
generation (doubling/replication) time (GT): time req for one bacterium to divide into 2 cells (replicate)
stationary phase
no net increase in cell numbers occur
death phase
cell death occurs at logarithmic rate
most bac autolyse (everything is gone)
rapidly growing organism produces
acute disease (fulminant) -in general short mean GT high Ag dose, strong IR anti-infective tx usually 8-10 days
slow growing organism produces
chronic disease (insidious) -in general long mean GT low Ag dose, weak IR anti-infective tx prolonged
chemotherapy that inhibits protein and/or PTG synthesis is most effective against
fast growing organisms
what DOES NOT determine GT/growth rate
Gram stain, metabolism, ext. cell structures, spore production
genome
chromosomes PLUS any extrachromosomal elements (plasmids) deemed crucial to the organism
-in bac: sing or doub stranded, covalently closed, circular
replicon
DNA or RNA molecule that controls its own replication, can self-duplicate
extra chromosomal elements (ECE)
replicons in cell, except host DNA
genomic islands are
horizontally transferred?…
plasmids
typ. ds, much smaller, code for ancillary genes, replicons
control their own DNA replication and copy number
plasmids DO NOT code for
housekeeping genes (req for viability)
plasmids replicate in ..
the cytoplasm, utilize host bacterial cell DNA replication machinery
conjugative plasmids
encode for mech to transfer a copy of itself from donor cell to recipient cell
resistance plasmids
possess Ab resistance determinants
plasmids can be..
acquired or lost from bac cells (non essential info) and are a metabolic burden on the host
bacteriophage
virus replicons (DNA, RNA) which infect bac cells *can exist latently in bac cells as a prophage
prophage can be..
- a plasmid in bac cytoplasm (ECE)
- integrated into bac cell chromosome
- can encode genes that confer a new phenotype to their host bac
* most abundant bio entity on earth!
recombination
exchange of recipient DNA w/ donor DNA
- breakage and joining of replicon DNA molecules to form hybrid, recombinant molecules
- can only occur in cell
homologous/legitimant recombination (prok)
- donor DNA integrated into rec. chrom. and excised rec. DNA fragment is degraded
- allows gene transfer/exchange, esp. w/ transformation or abortive transduction
homologous/legitimant recombination requires
RecA, an enzyme which func. when donor and rec. DNA segments share sig. homologous sequences
site-specific/DNA seq specific
AKA illegitimate/non-homologous recombination
insertion of or replication of genetic elements in DNA w/ recombination restricted to identical sites at 2 locations on one replicon or at identical locations on 2 replicons
site-specific/DNA seq specific does NOT require
RecA
several types of site-sp recombination mediated by mobile genetic elements
insertion sequences
transposons
since genetic elements cannot move themselves from donor to recipient in site-sp recombination, this must occur
Horizontal gene transfer (HGT) w. genetic element carried on transferred DNA
genetic apparatus of bacteria
chromosome: structure, size, number
ECEs: bacteriophages (prophages in bac cells), plasmids
phenotypic variation
an event in which all cells in a population respond to environmental stimulus in the same what which produces a new/altered phenotype via gene expression, without change in genotype**no genotypic variation is involved!
the total potential phenotype is limited by
the organism’s genotype
*expressed phenotype is usually LESS than the full genotype potential (genotype can encode for more traits then the phenotypic traits currently express)
phenotypic variation occurs b/c
microorgs are exposed to radically diff environments, req different phenotypes
microorg does not express its entire phenotype at one time b/c
it would require enormous energy expenditure and cell mass (would be out-competed by more reserving, environ-appropriate cells)
i.e. fungus switches from minimal capsule–>max capsule when in human host–>cause meningoencephalitis
phenotypic variation occurs via
-reg proteins controlling transcr/translation of sp. operons
-2 component signal transduction via sensor kinase and response regulator
-quorum sensing
-Ag variation
(and other)
genotypic variation
event in which genome of 1+ cells in population is altered
acquire new genetic information
2 mechanisms of genotypic variation
-internal by mutation (NO foreign/donated DNA involved)
or
-external: trasformation, conjugation, transduction
–>transduction: generalized/abortive or lysogenic conver
mutation (internal alteration of genotype)
-internal alteration of genotype
rare, but happens due to so many bacteria
mutations create new genes, but do not transfer them; require this mechanism to transfer
Horizontal gene transfer
dynamic duo!
horizontal gene transfer
transformation, transduction, conjugation
transformation
DNA fragments released by donor cell autolysis and accumulated by recipient cell (donor dies!)
transduction
abortive phages carrying donor cell chromosomal fragments transfer their DNA to the recipient cell
conjugation
donor cell plasmid encodes for mechanism to transfer a self-copy of itself to recipient cell which lacks the plasmid
genetic info is either..
carried/contained: -plasmid: conjugation, transformation -gen/abortive bacteriophage: transduction -lysogenic bacteriophage: transduction OR "naked" :transformation
external genetic information exchanged when..
- highly homologous genes form donor replace (recomb) the corresp. chromosomal or plasmid genes in the rec cell
- plasmid carrying new/altered genes makes a home in the rec. cell
- latent bacteriophage/prophage (carrying new/altered genes) makes a home in the rec cell by 2 mechanism..
2 ways a latent bacteriophage/prophage can make a home in the rec cell
- integrating into rec cell’s chromosome
- functioning as a plasmid in the rec. cell’s cytoplasm
clin sig of HGT
can transfer Ab resistance
-Acinetobacter baumannii: major cause of hops-acq inf
transformation
uptake of “naked” EC DNA (fragment) by rec cell by mech encoded by rec cell’s chromosomal genes
- donor cell autolyses
- can be accomplished in vitro (HSSN) and imp. to certain genera: Strep
only what types of cells can acquire “naked” extracellular DNA
competent:
- naturally competent: chromosome of rec. bac cell contains genes that encode for acquisition of extracellular DNA
- “forced” competent: chem/phys tx which “force”/induce bac cell to acquire EC DNA by unknown mech*
- used in recomb DNA technology (G- enteric bac E. coli)
transformation part 1
rec accumulates EC DNA of any origin (bac chromosome or plasmid)
transformation part 2
integration of acquired DNA requires RecA (i.e. mediates homologous recombination)
any accum. DNA fragments not integrated into host (rec) cell replicon (host genome/plasmid) cannot replicate and is degraded and consumed for carbon and energy
HGT mechs that DO require RecA
transformation
generalized/abortive transduction (SOMETIMES-if homolog.)
HGT mechs that do NOT require RecA
conjugation
generalized/abortive transduction (SOMETIMES)
lysogenic conversion
conjugation
donor cell plasmid encodes for mechanism to transfer copy of itself (the plasmids) to rec cell
- incidental transfer of donor cell chrom DNA v. rare, may not be possible
- involves ECE (plasmid)
1st mechanism of conjugation: in G- bac
ex: transfer of resistance (R-factors)
- “mating”: cell to cell contact is essential- sex pili functions to make contact in some, not all conditions
- ssDNA copy of R factor transferred through mem pore from donor to recipient cell during replication of R factor
- transferred DNA made double stranded in rec cell
- NO RecA OR site sp. recombination involved
limitation of conjugation
limited bacterial host range
limitation of transformation
not all bac cells are naturally competent
transduction
horizontal transfer of information (chromosomal, plasmid DNA, latent virus bearing a particular gene) btw bacteria mediated by bacteriophage
lytic phage
infects bacterial host cell to generate a productive infection(new virions) by binding to a sp. comp. on cell surface–>penetration of phage NA (DNA, RNA) into cytoplasm–>replication of NA–>transcription & translation of phage core and coat genes–>phage components self-assemble into infectious particles–>host cell releases new inf. particles (virus)
new infectious particles produced in lytic phage transduction are release into medium via
virus encoded cell wall hydrolase(PTG hydrolase)->cell lysis
OR slow release without lysis
temperate phage
infects host cell by binding to sp comp on cell surface–>penetration of phage NA into cytoplasm–> then 2 options for phage..
temperate phage after penetration
- phage can undergo normal lytic cycle as above OR
2. phage can become latent
latency
involves repression of phage genes which code for lytic (productive) cycle of phage replication–>will reside as plasmid or is integrated into host cell DNA–>replicates in synchrony with host cell DNA & passed on to daughter cells–>are now “lysogenized”–>latency ends when phage genes become productive again: replicate and lyse host cell
lysogenized
bac strains with prophage DNA
2 options for temperate bacteriophage replication (again):
- lytic cycle will result in a productive viral infection with lysis of host cell
- temperate cycle viral reproduction habbpens via replication of lysogenic virus genome and distribution to each daughter cell
mechanisms of transduction (HGT)
generalized/abortive transduction
lysogenic conversion
which form of transduction occurs is determined by
the vector (bacteriophage)
generalized (abortive) transduction
occurs with defective phage particles of both lytic and temperate phages and may require RecA
gen (abort) transduction: during viral lytic replication cycle..
the donor genome is sheared into fragments–>v. rarely; the DNA or intact plasmid is randomly “packaged” into virus particles: pseudovirions/abortive phages–>function as normal infectious virion: attach to rec on uninf. rec cell–>pkgd DNA is introduced into the rec cell cytoplasm–>any DNA frag not integrated or unable to replicate is degraded and consumed
in generalized transduction, the pseudovirion/abortive phage DOES NOT
carry viral genome, so no lytic or temperate life cycle can occur in rec cell
*NO viral reproduction can occur!
unlike a typical virus, the packaged DNA of a pseudovirion is a..
- plasmid that can replicate in rec cell
- chromosomal fragment that shares homology with a portion of the recipient chromosome and is integrated into that chromosome via RecA
- plasmid fragment that shares homology w. portion of rec plasmid and is integrated via RecA
- no site sp. recombination is involved!
lysogenic conversion is mediated ONLY by
a temperate bacteriophage (rec. is inf by temperate virus)
lysogenic conversion
when rec cell possess a new phenotype/trait due to acquisition of a prophage (latent temp. phage) which encodes for the new phenotype can be expressed without activation of the temp. virus genome!
does lysogenic require RecA
No
lysogenic conversion process
temp. phage encodes for an exotoxin gene- not req for viral replication or any part of viral infectious cycle
the exotoxin gene
is reg sep&ind of the viral genes
can be expressed without altering repression of the phages genes which code for the lytic cycle (does not affect latency)
-changes the cell phenotype from exotoxin negative to exotoxin positive
clin sig of transduction
transfer of drug resistance
lysogenic conversion: prophages can carry genes for toxin production
bacterial genomes consists of a conserved core gene pool
most encode “housekeeping” proteins
exhibit homogen. G+C contents/codon usage
bacterial genomes also consist of flexible gene pools
the functional (flexible) gene pool consists of
genomic islands (GEIs, >10kb) related to mobile genetic elements genomic islets (
genomic islands (aka fitness islands) traits
presence of:
-residual material from mobile genetic elements
-flanking direct repeats
-genes that aid in an org’s adaptation (put/virulence funcs)
carry fragments (transfer genes) of other mobile elements (phages, plasmids)
large: >10Kb up to 100Kb
genomic islands are inserted in
the vicinity of tRNA sequences or other small RNA genes, which leads to instability, risk for excisement
genomic islands can be differentiated from native genome
has evidence of horizontal/lateral origins:
-abnormal %GC index, dinuc frequency diff, codon usage bias
GEIs help an org survive a certain ecological niche, i.e.
PAIs: pathogenicity islands carry type 3 and 4 sec sys
Ab resistance islands
DNA polymorphism answers
what/whom is the SOURCE of the infection, does NOT identify the agent present
ex: plasmid analysis (GE), RFLP/ribotyping, PFGE
in plasmid analysis, plasmid DNA is
isolated, purified, and may be treated with restriction endonucleases
gel electrophoresis
separates plasmid DNA/fragments by size–>stain, take pic
*bac must possess plasmids!
restriction fragment length polymorphism (RFLP) and Ribotyping
bac chromosome (DNA) is isolated to fragments and purified–>treated with restriction endonucleases to fragment (again?)–>gel e-phoresis used to sep DNA fragments–>compare genomes (not used for banding patterns-too many!)
RFLPs and Ribotyping uses this as detection
Southern or Northern blots–>labeled probe is hybridized to sep. DNA fragments–>detection
in RFLP analysis, the probe detects
a particular sequence
*used for cellular life-forms: prok and euk
in ribotyping, the probe detects
DNA encoding for rRNA; which is unique for each genera/spp
*used only for cellular life-forms: bacteria
pulse field gel electrophoresis
variation of RFLP BUT inf. agent’s cells are gently lysed to rel. their chrome. DNA INTACT (vs. fragments)–>dig w/ rester endonuclease reg rare sites so large DNA frags–>sep by special agarose GE where the e- field orientation is changed often (“pulsing”)
PFGE produces restriction fragments profile of
5–>20 bands, 10–>800kb, visualized by staining
*restricted to cellular life forms: prok and euk
clin sig of DNA polymorph technologies
epidemiological; compare strains of partic pathogens
- typically multiple strains for one infectious agent in a host population
- determine if there is a common source
detection of etiologic agents in human specimens:
detection of target NA AND amplification of target NA genome answer..
what AGENT is making the patient ill?
NOT used to answer what/whom is the source (DNA polymorphisms)
detection of target DNA
Southern blot
detection of target RNA
Northern blot
to detect target, use
a single probe for each nat. occurring in vivo target, labeled with either radioactive or non radioactive marker
tissue sample prep (w/ target NA)
in situ prep: specimen treated so NA is single stranded and accessible to probe
blotting
NA extracted–>dig by restriction endonuclease–>electrophoresed–>transferred to support (blotted to membrane)–>denatured (made ss)
*may or may not occur
RNA is often chosen as target NA b/c
1000x more (r)RNA than DNA in bac cells possess unique signature of sequences sp. for genus/spp
hybridization
ss, labeled (DNA/RNA) prob anneals w/ ss NA in sample
wash off non-spec. bound probe
detection
label probe–>anneals to target NA–>detection!
since target DNA may be rare in certain human infections this helps detection
amplification: signal or target
signal amplification
produces multiple signals vs. one signal per in vivo target
- mult. non-radioactive marker bind to each target so SIGNAL not target is amplified
- ->developed so no need to employ heat stable DNA polymerase/PCR, save $
PCR
on form of in vitro or in situ amplification of target DNA or RNA
amplifies NA sequences unable to be detect. by N/S blots directly (10-100x more sensitive, more in vitro targets!)
PCR: 3 step cycling process
(if RNA, amp then converted to DNA (cDNA) by reverse transcriptase)
1. denature (heat) ds–>ss DNA
2. anneal primers to ss DNA (cDNA) after cooling
3. extension of primer: amp w/ heat stable DNA polymerase
then detect with Northern or Southern blot
PCR clinical applications:
- screen blood
- detect viruses, bac, genetic defect, genetic marker (neoplasm), infectious agent (not successfully cultured)
other amplification techniques
LCR: ligase chain reaction
TMA: transcription mediated amplification
*do NOT employ heat stable DNA polymerase
do viruses require living cells
YES (some bacteria do not)
do viruses divide by binary fission
NO (bac do)
do viruses have both DNA and RNA
NO (bac do)
are viruses susceptible to Ab
NO (bac are)
exception: mimivirus and CMV
contain BOTH RNA and DNA
viruses are
submicroscopic entities
obligate intracellular parasites
infect specific living cells and reproduce in said cells
consist of DNA OR RNA and protein, often an envelope
have a definite structure
although relatively simple replicons, viruses..
can take over host cell, utilize for own replication and self-assembly, do not always kill the host they infect
viruses are small
15–>300 nm, observed by e- microscope not light
virion
complete viral particle, able to infect another host cell and repeat the replicative cycle
for any one virus, each infectious virion possess the same NA..
- dsDNA (w/ ss regions)
- ssDNA:
- +ssRNA (positive sense polarity)
- -ssRNA (negative) (seg/nonseg genome)
- dsRNA: 2 identical or complimentary strands
the NA genome is either…
linear or circular
usually condensed by histones or histone-like proteins
protomer
protein subunit of capsid, repetitive polypep subunits arranged in symm patterns, either:
- protomers–>capsomers–>capsid (i.e. icosahedral capsid)
- protomers–>capsid (i.e. helical capsid)
capsomers
oligomeric clusters of protomers
form the capsid
(in orgs w/ icosahedral symmetry)
capsid
protein shell or coat of protomers that self-assemble by non covalent bonds to enclose the core of NA genome + associated proteins
3 architectural structures of capsids
complex, cubic/icosahedral, helical
complex symmetry
viruses w/ unresolved structures b/c too complex
cubic symmetry of all animal viruses is icosahedral which
consists of 20 faces, each an equilat triangle, in most protomers are arranged into capsomers
helical symmetry
capsid consists of multiple copies of single species of protomer that bind to each other and ssRNA genome
helical structure of non-enveloped virus
individual protomers assembled in helical structure around NA genome of virus–>forming the nucleocapsid
- RNA genome extends the entire length
- the core is hollow
- each helical virus has sp diameter/length
nucleocapsid
capsid + NA genome + associated proteins
*stable structure; resistant to drying, mild acids, detergents
some virus consist solely of a..
nucleocapsid
envelope
viral membrane that covers/encloses the nucleocapsid (not always present)
envelop composition
virus-encoded proteins
host-derived lipids and carbs
2 important proteins in viral envelope:
peplomer= spikes: viral glycoproteins, play a role in virion-rec attachment
layer btw envelope and nucleocapsid that mediates interaction btw capsid and envelope:
-tegument: amorphous layer, complex funds
-matrix protein: structure-providing lattice
viral membrane derived from
nuclear membrane, sub-cellular organelle membranes, cytoplasmic membrane
-obtained from host during “budding” of some viruses
enveloped viruses are…
inherently fragile, sn/inac by drying, detergents
the NUCLEOCAPSID is more stable
disruption of envelope of enveloped virus leads to
virus inactivation, no longer infectious
nearly all enveloped viruses are transmitted via
arthropods
respiratory droplets
bodily fluids: semen, saliva, secretions
*non-env. viruses may also be transmitted these ways
how to classify animal viruses
type of nucleic acid with polarity
type of capsid symmetry
+/- envelope
quantitative: # of capsomeres for icosahedrals, diameter of helix for helicals
clade
group containing all descendants from a given common ancestor, grouped based on seq similarity among members and seq diversity w.in total viral population
- NOT a serotype
- *the clinical importance is immune evasion
serotypes are based on
antigenic diversity
all human viruses that successfully replicate in humans result in ? infections but only some also produce ? infections
productive
persistent
non-productive
no infectious virions:
- abortive (lack infectious viral synthesis post-absorption?)
- interference: virus interferes w. growth of other viruses in same cell
productive infections comprised of..
- non-lethal alteration of cell & functions
- cell damage/death: lytic infections: via viral replication
- persistent infection without cell death
persistent infections without cell death..
may persist for years
- latent: intermittent acute episodes of disease, periods of NO infectious particles (HSV, varicella-zoster)
- chronic: continued viral presence, disease may be absent (CMV, HHV, HBV) or is assoc. with late immunopathologic disease: HBV–>cirrhosis of liver or primary hepatocellular cancer
- slow: long incubation period, slowly progressive, lethal (SSPE, measles) no infectious virion may be detected!
the most common outcome of viral infection is
asymptomatic infection with seroconversion
mechanisms which cause injury in viral infections
- immunopathology
- autoimmune induction
- cell damage/death (lytic infections)
lytic infections cause cell death
viral replication modifies and damages host cell:
- inhibits/shuts down macromolecular sun
- cytopathic effect (CPR); toxic
- inclusion bodies and cell fusion (syncytia formation-multinuc giant cells)
- induce apoptosis
- chromosomal alterations–>oncogenesis
characteristics of tumors
- transformation to unrestricted cell growth
- loss of contact inhibition/senescence (unreg growth)
- appearance of new Ags (tumor sp Ags)
- other changes: metabolic, genetic
viral agents assoc with malig neoplasms
RNA tumor viruses HTLV
DNA tumor viruses: HPV, EBV, HBV
viral agents of benign neoplasms
(somewhat organized growth, does not invade)
human wart viruses: verruca lesions, condyloma acuminatum
poxvirus: molluscum contagiosum
6 stages of replication
attachement–>penetration–>uncoating–>macromolecular synthesis–>viral genome replication–>translation of viral transcripts–>synthesis of other/non-protein comps
virus-rec interactions are major determinant of
infectivity
-specificity determined by:
host range (low affinity rec)
tissue tropism (high affinity rec)
HIV therapy target (Fusion inhibitors)
inhibit HIV virion fusion w/ human cell surface receptors–>prevent infection of individual cells
penetration
direct
surface eclipse
rec-mediated endocytosis
direct penetration
non-env. viruses, nucleocapsid attaches cell surface–>release viral NA genome into host cytoplasm
surface eclipse penetration
pH-independent cell entry
enveloped virus membrane fuses with cell membrane releasing viral genome into cytoplasm (*penetration and uncoating happen in 1 mechanism)
rec-mediated endocytosis
viroplexis: pH-DEPENDENT cell entry
attachment of enveloped virion to cell rec–>endocytosis of virion–>enclosed in endosome–>acidification–>virus env. fuses with vesicle membrane–>releasing viral genome into cytoplasm
uncoating
removal of protective coats with release of NA
- infectivity lost here!!*
- some virus AND host encoded mechanism to uncoat
- target of antiviral therapy
macromolecular synthesis
need to make more genome and more viral proteins:
1. DNA viruses replicate in nuc and RNA in cytoplasm
-exp. pox (DNA) virus in cytoplasm, influenze (RNA) in
nuc
2. +ssRNA, -ssRNA, and dsRNA virions all must encode for an RNA-dep RNA polymerase (RNA POL)
-exp. retroviruses: reverse transcriptase: RNA
dependent-DNA polymerase
3. both -ssRNA and dsRNA infectious virus must also carry function RNA POL (the protein) to make the mRNA along with possessing the gene
-+ssRNA viruses DON’T carry RNA POL
DNA viruses replicate genome utilizing
host cell DNA replication machinery
RNA viruses
+ sense or - sense RNA genome replication produces many new viral genomes and produces RI (req. RNA POL, virus encoded)
retroviruses are the exception
-encode for reverse transcriptase (RT; RNA dep-DNA POL)
transcribes +ssRNA–>DNA–>incorp. into host genome–>viral DNA transcribed into mRNa
RT is target for many anti-HIV drugs
-nucleoside analogue RT inhibitors
-non-nucleoside RT inhibitors
integrase enzyme also target (integrate inhibitor prevents insertion of HIV DNA genome into human genome)
primary replication
site or sites in host where 1st replication occurs
-is it near the portal of entry? (POE), related to incubation
period
secondary replication
where replication occurs after spread from primary site (not present in all viruses)
some not all viruses spread from
the POE, primary site
local spread
always occurs
when does spread occur?
before, during, after entry, or only local
before, during or after primary replication
spread/replication within host
cell to cell within tissues
in bloodstream/lymphatics
in nerves: peripheral–>CNS
transplacentally
tissue tropism
specificity of a virus for a particular host tissue
*major determinant of infectivity!
signs and symptoms may only occur..
during the primary and/or secondary replication
immune response to viral infection
outcome related to capacity of virus to damage host and disrupt host defense mechs
