Module 3

antibiotic discovery

* Alexander Fleming accidentally discovered penicillin in 1928 (modern antibiotic revolution)
* mold that inhibited S. aureus colonies on a plate by releasing substance that killed the bacteria = __(fungal) agents can produce compounds to inhibit bacteria__

selective toxicity

* toxic damage to bacteria while limiting side effects on host
* *ability of an antibiotic to attack unique bacterial physiology*

what do antibiotic drugs target that are unique to bacterial physiology?

* peptidoglycan
* differences in ribosome structure
* biochemical pathway missing in humans
* cell wall syn, cell mem integ, DNA/RNA syn, protein syn, metabolism

how are antibiotics classified?

broad spectrum

* effective against many species
* higher potential for side effects on normal flora

narrow spectrum

* against few or a single species
* intensive research on physio of the target

bactericidal

* kill target organisms
* bacterial cell death can release cellular constituents

bacteriostatic

* prevent growth of organisms (stop binary fission)
* allow time for immune system to clear microbe (or “flush”)

minimal inhibitory concentration (MIC)

* lowest conc that prevents microbial growth (no bacterial growth)
* varies for diff bacterial species
* test by diluting antibiotic
* no growth = conc of antibiotic can impair

how would you use MIC to determine whether an antibiotic is bactericidal or bacteriostatic?

* place on plate or broth with no antibiotic
* living bacteria, grows = bacteriostatic
* no growth = bactericidal

peptidoglycan synthesis

1. precursors made in cytoplasm
2. carried across cell mem by lipid carrier, bactoprenol
3. precursors polymerized to existing cell wall structure by transglycosylases
4. peptide side chains are cross-linked by transpeptidases

bacitracin

* peptidoglycan synthesis halted at step 2
* precursors made but not transported across mem

soft tissue inflammation

* caused by S. aureus
* usually responds to penicillin
* MRSA a concern

MRSA

* S. aureus responds to penicillin-like drugs
* MRSA = methicillin-resistant
* encodes gene “mecA” (penicillin binding protein - allows resistance); difficult to treat clinically

Vancomycin

* inhibits cell wall syn of Gram (+) bacteria only by inhibiting transpeptidase enzymatic activity by binding to __peptidoglycan precursors__ (NAG, NAM)
* peptidoglycan synthesis halted at step 3
* large molec that cannot transmit across intestinal epithelium (usually IV administration)

VRSA (Vancomycin resistance)

* life threatening infection
* treated with Daptomycin; if in-vitro susceptibility testing is promising

what are the 4 forms of antibiotic resistance?

1. modify target so it no longer binds antibiotic


1. mutations in ribosomal proteins confer resistance to streptomycin
2. destroy antibiotic before it gets into cell


1. beta-lactamase enzyme specifically destroys penicillin
3. add modifying groups to inactivate antibiotic
4. pump antibiotic out of cell


1. multidrug resistance (MDR) efflux pumps
2. using specific transporters and transport complexes; similar strategy used in cancer cells

what are the 2 phylogenetic branches gram-positive bacteria can be divided into?

phylum __Firmicutes__ (“low-GC” species)

phylum __Actinobacteria__ (“high-GC” species)

phylum Firmicutes

* low-GC bases
* many form endospores
* many are pathogens
* *Clostridium difficile*

genus Clostridium

* rods, obligate anaerobes
* Firmicute, bacillus (rod), strict anaerobes
* spore forming (some have terminal drumstick)

*Clostridium botulium*

* agent of foodborne botulism
* common in environment/soil
* spores allow dormant survival until ideal conditions are met (anaerobic)

botulism

* improperly preserved foods common source of infection
* bacteria produce a powerful toxin that blocks nerve function, causes double vision, drooping eyelids, paralysis

botulism in wild animal

* growth of *Clostridium* in decaying material
* toxin consumed by unaffected organisms (maggots)
* maggots can transmit toxin to sens organisms
* organismal death perpetuates cycle

botulism in humans

Infant

* exposed to endospores or toxin
* consumption of honey before age 1 (immature gut microflora)
* 65% of botulism cases
* treated by intensive care, antitoxin

Adult

* exposure to toxin
* many routes of exposure
* food-borne botulism (home canned foods - ingested)
* treated by intensive care, antitoxin

how do the different mechanisms of human botulism compare and contrast?

infant

* endospore itself → establishment and replication in GI → produce toxin
* gut flora not mature

adult

* consuming toxin itself (germinated from outside body)
* normal flora in GI not enough to compete with toxin

how can Clostridium botulinum toxin be used cosmetically?

* active ingredient in Botox; low dose
* temporarily paralyze nervous sys at small locations
* Bell’s palsy symptoms can be managed with botox
* wrinkles, sweating, headaches

genus Bacillus

* one of first bacterial genera to be classified
* large rod-shaped cells (B. subtilis = “model sys” for gram-(+))
* vegetative cells develop inert endospores in times of starvation and stress (released spores germinate in favorable cond → restart veg growth)

Bacillus thuringienesis

* most successful biological control agent yet produced
* insecticide - spores applied against gypsy moth caterpillar
* sporulating cell produces crystal that contains an insecticidal protein (delta endotoxin)

phylum actinobacteria

* high-GC
* form complex multicellular filaments
* some are acid-fast

genus Streptomyces

* aerobic, non motile, nonpathogenic
* inhabit soil (1-20% of culturable soil microbes)
* produce **geosmin** (moist earth odor)
* grow onto and into their substratum
* raised/rigid/flat areas (on agar)
* linear chromosomes with telomeres
* has medically useful 2ndary metabolites (antibiotics/anticancer)

life cycle of *Streptomyces*

* vegetative cells form dense substrate mycelium
* nutrient limit/stress induces growth up into air (aerial mycelium)
* aerial mycelium “cannibalize” substrate mycelium for nutrients
* aerial mycelium form spores (arthrospores) that can disperse in wind

\
vegetative growth (substrate mycelium) → stress → erection of aerial hyphae → older cells lyse → sporulation septation & chromosome segregation → spore maturation (arthrospore production) → spore dispersal → spore germination into substrate mycelium

how do Streptomyces form aerial mycelium?

* use of aerial mycelium and its ability to use spores that disperse into wind
* energy to do this comes from consuming substrate mycelium

traditional microbial taxonomy

* not rooted in evol relatedness
* naming referenced diseases they caused or processes they performed
* Mycobacterium tuberculosis

polyphasic taxonomy

* genus, phenotype, evolutionary (rRNA)

major bacterial groups

* __deep-branching thermophiles (aquificae: thermocrini/ thermotogae:maritima/chloroflexi: mats)__
* __cyanobacteria (heterocysts)__
* __gram-postive bacteria (frimicutes: clostridium, bacillus/ actinobact: Streptomyces)__
* proteobacteria
* deep-brancing gram-negative bacteria
* spirochetes
* chlamydiae, planctomycetes, verrumicrobia

deep-branching thermophiles

* diverged earliest from ancestral archaea and eukaryotes
* fastest doubling rates of all bacteria
* high mutation rate

phylum Aquificae

* “water maker”
* oxidize hydrogen gas with molecular oxygen to make water
* ether linked membrane lipids (usually found in archaea)

Thermocrinis ruber

* genus and species in phylum Aquificae
* on/in standard **lab** media grows as a **rod**
* 82-88 deg C temp preference
* when exposed to **water currents**, grows as long “streamers”
* Yellowstone’s octopus spring: grows as mat of “pink streamers”

phylum Thermotogae

* “Toga”
* loosely bound sheath (abs of “classical” outer mem)
* mosaic genomes (bacterial - archaeal)

Thermotoga maritima

* one of highest recorded growth temps (90 deg C)
* during growth, “sheath” extends from poles
* outer envelope “grows”
* cytoplasmic growth “stalls”

phylum Chloroflexi

* deep branching thermophiles that are participants in microbial mats (no EPS; layers)
* layers of bacterial growth
* gram (-); atypical

Chloroflexus aurantiacus

* lower layers of microbial mats (under cyanobacteria)
* gram neg (atypical)
* no outer mem
* Yellowstone’s octopus spring: grows as mat at 50-65 deg C

phylum Cyanobacteria

* largest, most diverse group of photosynthetic bacteria
* only ones oxygenic
* thick peptidoglycan (almost as thick as Gram +)
* appear green bc of predom blue and red abs by chlorophylls
* filamentous or colonial appearance
* may form “multicellular-like” communities
* Chroococcus with layer of mucus

how are cyanobacteria unique in cellular structure?

* may have thylakoids (photosynthesis)
* may have carboxysomes (fix CO2)

cyanobacterial mutualism

* participate in multilayered microbial mats
* mutualistic associations (require each other)
* cyanobacteria + chloroflexi

heterocysts

* specialized cells used for N fixation
* produced when organism is N deprived
* thick heterocyst will prevent o2 diffusion into heterocyst which would inactivate nitrogenase

heterocyst formation

* plenty of nitrogen → dont make
* nitrogen starved/depleted → form heterocysts → fix into more useful form

organisms in Yellowstone’s Octopus Spring

Thermocrinis ruber

* “pink streamers” at 82-88

Chloroflexus aurantiacus

* mat 50-65

Synechococcus elongatus

* ≤ 75 deg C

viruses

* acellular agents (cannot replicate independently w/o host)
* few naturally “beneficial” viruses
* most cause harm, nuisance, or some form of problems for host

what viruses are an exception?

viruses that kill pathogens or gene therapy

Dmitri Ivanovsky (1892)

studied Tobacco Mosaic Disease

* mottling of leaves, stunted leaves, wrinkles
* observed agent was not removed by filters
* nearby plants infected
* failed to trap bacteria in filter (liquid soln)

Beijerinck (1898)

* made conceptual leap on TMD → agent must be so small it passes thru filters

Loeffler and Frosch (1898)

* former students of Koch
* studied foot and mouth disease of livestock
* high fever, blisters, weight loss
* agent NOT removed by filter
* thought chemical or infectious protein

Peyton Rous (1911)

* studied sarcomas (tumors) in chickens
* cell free “filtrate” from diseased chickens could transmit tumors to healthy chickens
* cancer transmitted by a virus

how are viruses grouped by their shared properties?

* nature of nucleic acid (DNA or RNA)
* symmetry of their protein shell
* presence of absence of a lipid membrane
* nucleic acid comparisons

general properties of viruses

* ≥1 molecule of DNA or RNA enclosed in coat of protein
* may have additional layers
* cannot reproduce independent of living cells nor carry out cell div
* can exist extracellularly

structure of viruses

* \~10-400 nm in dia
* all contain a nucleocapsid (nucleic acid + capsid)
* some have **envelopes** (plasma mem comp derived from host)
* some have **spikes** (proteins used for attachment to host)

capsids

* protect viral genetic material and aids in its transfer btwn host cells
* made of *protomers* (protein subunits) that aggregate to form *capsomers*
* helical, icosahedral, or complex (symmetry)

helical/filament capsids

* shaped like hollow tubes with protein walls
* may be bent or twisted
* tobacco mosaic = helical, influenza = twisted

icosahedral capsid

* polyhedral with 20 identical triangular faces
* structure exhibits rotational symmetry
* adenovirus

complex capsid

* do not fit into either categories; no consistent pattern (not 100%)
* “tailed viruses” = multipart structures

viral genome types

DNA

* ds stranded DNA
* ss DNA (+/”sense”)

RNA

* ss RNA (+/”sense”)
* ss RNA (-/”antisense”)
* ds RNA

virus replication

1. host recognition and attachment
2. genome entry
3. assembly of virions
4. exit and transmission

bacteriophages

viruses that attack bacteria

complex capsid shape

how do bacteriophages attach to host cells?

cell-surface receptors (proteins used for important func and specific to host species)

* recognize specific things on surface; consistently expressed and high abundance

phage reproduction within host cells

* most only *inject* their genome into a cell
* capsid remains outside on cell surface (“ghost”)
* ds DNA genome enters to take over

lytic cycle

* bacteriophage quickly and actively replicates, killing host cell
* immediately makes more virus particles

lytic cycle steps

1. attachment
2. entry of phage DNA and degradation of host DNA
3. synthesis of viral genomes and proteins
4. (phage) assembly
5. release (induction of lysis of host)

lytic burst

* lysis: exit from host; virus kills host
* make enzyme that breaks down cell wall
* host cell bursts to release progeny phage

lysogenic cycle

* bacteriophage is quiescent
* integrates into cell chromosome (**prophage**)
* can reactivate to become lytic to make more virus
* dormant = ensures DNA passed

lysogenic cycle steps

1. phage DNA integrates into chromosome (prophage)
2. reproduces, copying prophage and transmit to daughter cells
3. pop of bacteria infected with prophage
4. phage DNA circularizes
5. can switch to lytic or enter lysogenic again

how is it decided which replication cycle a bacteriophage will undergo?

* environmental cues
* events that threaten host cell survival trigger a lytic burst
* lytic = sick or stressed
* lysogenic = healthy
* can be both or strictly one (usually lytic)

how can bacteria protect themselves from phage attack?

* restriction enzymes chop DNA that is palindromic
* modify surface proteins/features (cannot attach)

how do viruses get around such defense mechanisms?

* attach to something else on surface of host
* modify DNA so it is not recognized
* modify DNA so more familiar to bacteria
* ex: bacteriophage T4 (encodes HMC nucleotide)

animal virus replication

* greater complexity and diversity due to complex eukaryotic cell structure
* bind specific receptor proteins that determine *tropism*

tropism

range of cells or tissues that virus will infect

* cellular: HIV = macrophages not neurons
* tissue: flu = lung not brain
* host: myxoma = rabbits not humans

how do animal viruses enter?

* endocytosis (vesicle surrounds virus)
* membrane fusion
* virus passes thru mem → mem lipids surround capsid to fuse envelope

types of animal virus genome replication

DNA viruses

* utilize some or all of host replication machinery

RNA viruses

* use viral RNA-dependent *RNA-polymerase* to generate RNA template

retroviruses

* use viral **reverse transcriptase** to copy their genomic sequence into DNA for insertion in the host chromosome
* make RNA → DNA (against central dogma)

animal virus protein production

* make proteins w/ host ribosomes in rough ER
* assembly of new virions occur in cytoplasm or nucleus

animal virus release

* lysis of cell
* exocytosis
* budding
* virus passes thru mem → mem lipids surround capsid to form envelope

herpesviruses

* DNA viruses
* productive (lytic) or latent (lysogeny) infections that last for life
* 3 families: alpha/beta/gamma
* distinguished from ea other by type of cells they exhibit latency in
* **icosahedral, enveloped, spiked,** ***tegment*** **(layer of proteins), dsDNA**

herpesvirus infections

* productive infections: 50-200k virions produced per cell
* host cell may die due to degraded DNA

herpes simplex virus type 1 and 2 (HSV1/HSV2)

* cold and genital sores
* **alpha** family-hallmark characteristic: establish **latency in neurons** (replicating in 1 cell type and exhibit latency in a diff)

latency

virus is dormant and does not make any more active virus

* absence of virion prod; DNA remains in host cell
* capable of reactivation to virion prod if given the appropriate stimuli
* dont know exact reactivation stimuli (host cell stress?)

HSV1/HSV2 attachment

* virions “surf” host cell surfaces
* initially attach to host Heparan Sulfate
* full attachment requires several other tissue specific receptors (ex: Nectin on epithelial cells and neurons)

HSV1/HSV2 entry

* virions enter host thru **fusion** or **endocytosis**

HSV1/HSV2 genome replication

* nucleocapsid finds its way to nucleus to replicate its DNA

HSV1/HSV2 protein synthesis and assembly

* proteins synthesized w/ host ribo then shuttled back to nucleus to assemble nucleocapsid
* nucleocapsid leaves nucleus (w/ help from nuclear envelope)
* travels to Golgi for re-envelopment

HSV1/HSV2 release/exit

* mature virions release from host via exocytosis
* upregulate host Heparanase for their release

productive infection (lytic)

* events describes
* may exhibit:
* flu-like symptoms (initial infection)
* red, fluid fill lesion(s)
* tingling, pain at site of lesions

HSV1/HSV2 latency

During latency, host exhibits no symptoms

* virus enters sensory neurons near site of productive infection
* remains in neurons for lifetime of host

During reactivation, host may exhibit

* virus leaves sensory neurons
* copy of viral DNA remains in nucleus
* virus returns to site of initial infection and undergoes productive replication

can you contract HSV-1 infection in the genital area? and vice versa?

yes, though HSV-2 less likely to infect well outside the genital tract

* bc of cellular/tissue tropism; won’t replicate as well

human immunodeficiency virus (HIV)

* virus that exhibits latency
* can become latent in T cells (immune cells)
* react/rep in T cells leads to T cell death (immune suppression)

HIV illness

symptoms

* virus spreads via bodily fluids
* flu-like symp
* swollen lymph nodes
* sores that wont heal
* fatigue
* rash
* night sweats

complications

* virus can persist and lead to AIDS

how may HIV develop into AIDS?

* some patients rapidly develop AIDS within 2-3 years (no treatment)
* some remain healthy for at least 10-20 yrs post infection
* T cell count reduces and *opportunistic infections* begin
* AIDS patients don’t usually become seriously ill directly from HIV itself (complications from other infections)

HIV

* (+) ssRNA
* carries reverse transcriptase (reverse transcribed into dsDNA, which integrates into host genome)
* can remain latent/reactivate
* new virions cause host cell lysis (T cell death)

quasispecies of HIV

* genetically variant versions of HIV virus
* chronically infected patients have diverse HIV populations in their blood
* hard to target with antiviral drugs

transmission of HIV illness

* certain variants “seed” tissues of genital tract; same variants are seen in fluid from genital tract
* fast rep variants are transmitted to others; seed blood of newly infected
* pop becomes diverse in new patient (hard to target)

why are there so few antiviral agents available?

* selective toxicity harder for viruses than bacteria
* few targets are unique

HSV1

* cold sores; blister at lips, mouth, gums - can gain access to eye
* lifetime latency with periodic reactivation in times of stress
* treatment (not cure):
* Acyclovir (antiviral - seems like NTs, incorp into viral DNA, stops polymerization)

OTC treatment for cold sores

* Docosanol (fatty acid): changes host cell mem which surrounds healthy cells so that virus can’t enter cells (blocks entry)
* not an antiviral, effective if applied at earliest signs of outbreak (tingling; spreads quickly - targets oral epithelial cells

HIV drug “cocktail”

targets diff areas of HIV replication to limit amount of virus

* AZT (reverse transcription inhibitor): prevents HIV reverse transcription
* Indinavir (protease inhibitor): prevents HIV protein cleavage
* Enfuvirtide (fusion inhibitor): prevents entry of HIV into cells

HIV protease

* normally = virus makes proteases to cleave and assemble its proteins
* inhibitor prevents

virus therapy

* phage therapy may be able to be used to target antibiotic resistant bacteria
* otherwise would not target human cells; selectively target bacteria causing problems