Bio 350 Exam 3

Macrolides, chloramphenicols, lincosamides

(Protein synthesis inhibitor)
Binds large subunit, block transfer of peptides. Erythromycin, azithromycin

Aminoglycosides

(Protein synthesis inhibitor)
Prevents 30s and 50s subunits from binding to each other. Streptomycin

Tetracyclines

(Protein synthesis inhibitor)
Binds small subunit, blocks binding of
aminoacyl-tRNA

Penicillins

(Cell wall synthesis inhibitor)
• b-lactam and thiazolidine rings
• Properties determined by side chains groups

Penicillin G

(Cell wall synthesis inhibitor)
• Sensitive to b-lactamases
• Ineffective against Gram- bacteria

Ampillicin, amoxicillin

(Cell wall synthesis inhibitor)
Resistant to acid hydrolysis
• Broader spectrum, including Gram-
• Higher serum levels

Oxacillin, dicloxacillin, methicillin

(Cell wall synthesis inhibitor)
• Resistant to b-lactamases due to bulky side chain
• Not effective against Gram-

Antibiotic Resistance Mechanism: Destroying the antibiotic

• Many bacteria make b-lactamase, which destroys penicillin analogues, makes strain resistant & lowers penicillin concentration, protecting nearby cells

Antibiotic Resistance Mechanism: Modifying the antibiotic

Enzymes modify aminoglycosides (acetylation, phosphorylation, adenylation); can no longer interfere with ribosome

Antibiotic Resistance Mechanism: Altering the target

• Modify target enzyme of pencillin, or ribosome
• Most common streptomycin-resistance mechanism

Antibiotic Resistance Mechanism: Drug efflux

Multidrug resistance (MDR) exporter
Removes drugs from cell
Confers resistance to several classes of antibiotics at once

Antibiotic Resistance Acquisition: Spontaneous mutations

• Modification of antibiotic targets

Antibiotic Resistance Acquisition: Enzymes to disable antibiotics

• Modification of antibiotic synthesis enzymes

Antibiotic Resistance Acquisition: Horizontal gene transfer

• Conjugation
• Phage transduction
• Plasmids, transposons carry drug-resistance

Staphylococcus aureus MRSA

-First noted ~1970 in USA; now -35%, mostly nosocomial infections.
-Gene mecA encodes a new Transpeptidase (PBP2a) with low affinity for b-lactam antibiotics including Methicillin.
-Vancomycin only predictably active drug.
-mecA on transposon acquired via horizontal gene transfer.

The Future of Drug Discovery

•Evolutionary pressure is constant
•Requires constant search for new antibiotics
•Use genomics—identify new targets
•Design compounds to inhibit targets
•Alter compound structure to optimize MIC
•Determine spectrum of compound
•Narrow or broad?
•Determine pharmaceutical properties
•Not toxic to animals; persistence in body
•Phage Therapy?

Virulence genes

encode factors allowing pathogen to invade host:
toxins, attachment proteins, capsules

Pathogenicity islands

Section of genome that contains multiple virulence genes, encoding related functions such as protein secretion or toxin production.
Transferred as a block from other organisms,
with distinguishable genetic characteristics
(GC content, flanking genes...)

exotoxin

soluble protein released into the surrounding by a microorganism during growth and metabolism

endotoxin

lipopolysaccharide portion of the outer membrane of Gram- (lipid A is responsible for toxicity)

protein export

translocation out of the cytoplasm

protein secretion

translocation into the external environment

translocation

transfer or movement from one location to another

AB exotoxins

composed of A and B subunits

Specific host site exotoxins

categorized on the basis of the affected site
-neurotoxins (nerve tissue)
-enterotoxins (intestinal mucosa)
-cytotoxins (general tissues)

bacteriocin

small protein or peptide produced by bacteria and toxic to other bacteria!

Protein export system: Type I

ABC exporter
ABC exporter \= ATP-binding cassette transporters (ABC transporters)

Protein export system: Type II

Two-step secretion
the Sec system

Protein export system: Type III

Contact-dependent, based on FLAGELLA
Flagellum synthesis is homologous to type III secretion!
injects proteins directly into host cell
SALMONELLA uses Type 3 Secretion System for Host Entry

Protein export system: Type IV

Conjugal transfer system
PILI are homologous to type IV secretion
DIPHTERIA toxin (AB toxin) delivered via Type IV Secretion

Protein export system: Type VI

phage-derived

Signs of Disease

Objective and measurable
(i.e body temperature ; blood pressure).

Symptoms of Disease

Subjective
(i.e Pain, Loss of Appetite).

Iatrogenic Disease

Contracted as a result of a medical procedure.

Nosocomial Disease

Acquired in hospital settings.

Zoonosis

Infectious disease transmitted from animals to humans. i.e. rabies

Periods of disease

incubation --\> prodromal --\> illness --\> decline --\> convalescence

Robert Koch (1843-1910)

German physician and microbiologist.
Founder of modern bacteriology.
Identified specific causative agent of tuberculosis (Nobel Prize), cholera and anthrax.
1884: Published four postulates that summarized method for determining whether a particular microorganism was the cause of a particular disease.

Koch's postulates

1. causative agent must be present in all diseased organisms and absent in all healthy organisms
2. must be isolated and grown in pure culture
3. agent must cause disease when inoculated into a healthy organism
4. the same causative agent must then be reisolated from the inoculated diseased organism

Limitations of Koch's postulates

•the microbe cannot be "grown in pure culture"
•there is no animal model of infection with that pathogen

•not all individuals infected by a pathogen develop disease; subclinical infection is often more common than symptomatic infection (eg. for many viruses)
And "harmless" microbes may cause disease if they:

•acquire virulence factors
•gain access to deep tissues (eg. trauma, surgery, IV line, etc)
•infect an immunocompromised host

Pathogenicity

Ability of a microbial agent to cause disease.

Virulence

Degree to which an organism is virulent.

Median Infectious Dose (ID50)

\# of pathogen cells or virions required to cause active infection in 50% of inoculated animals.

Median Lethal Dose (LD50)

\# of pathogen cells, virions or toxin required to kill 50% of infected animals.

Stages of Pathogenesis

1. Exposure (Contact) - major portals of entry for pathogens
2. Adhesion
3. Invasion: Dissemination of a pathogen throughout local tissues or the body.
4. Infection
5. Transmission: Pathogens leave infected host through a portal of exit.

Adhesins

Molecules (either proteins or carbohydrates) found on the surface of certain pathogens that bind to specific receptors (glycoproteins) on host cells.

Glycocalyces (slime layers and capsules)

Bacterial adhesion to host cells.

Biofilm

Community of bacteria that produce glycocalyx, allowing attachment to surface

Exoenzymes

Extracellular enzymes produced by pathogens (Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens.)

Localized Infection

Confined to small area of the body, typically near portal of entry. (Staphylococcus aureus: boil)

Systemic Infection

Whole-body dissemination. (Varicella-zoster virus: chickenpox)

Capsules

Bacterial virulence factors that aid in immune evasion

Antigenic Variation: Influenza Viruses

Antigenic variation refers to the mechanism by which an infectious agent such as a protozoan, bacterium or virus alters its surface proteins in order to evade a host immune response.
Antigenic Drift: Result of point mutations causing slight changes in spike proteins (hemagglutinin (H) and neuraminidase (N))
Antigenic Shift: Major changes in spike proteins due to gene reassortment.

symbiosis

intimate association between organisms of different species
organisms or populations of different species living together

commensalism

one organism is benefited and the other is unaffected

parasitic

one organism (parasite) is benefited and the other (host) is harmed ex: Agrobacterium tumefaciens

mutualistic

organisms benefit one another
ex: Lichen- mutualistic association between fungus and algae or cyanobacterium
ex: nitrogen fixing microbes in legumes

The Endosymbiotic Theory

1. In a first endosymbiotic event, the ancestral eukaryote consumed an aerobic bacteria that evolved into mitochondria (cellular respitation, found in most eukaryotes)
2. In a second endosymbiotic event, a descendant consumed a photosynthetic bacteria that evolved into chloroplasts (photosynthesis, found in most photosynthetic eukaryotes)

mitochondria

cellular aerobic respiration, found in most eukaryotes; proteobacteria

chloroplasts

photosynthesis, found in most photosynthetic eukaryotes; cyanobacteria

Evidence for the endosymbiotic theory

Chloroplasts + mitochondria similar to bacteria;
-Division: binary fission
-Ring formation: FtsZ, min proteins (chloroplasts and some mitochondria)
-Protein synthesis: formyl-methionine
-Ribosomes: smaller than eukaryotic
-Inhibition of protein synthesis: by antibiotics as in bacteria
-DNA structure: circular, no histones (in mitochondria)

endosymbiont

organism that lives within the body or cells of
another organism
some cyanobacteria are endosymbiont in
lichens, plants, various protists, or sponges,
and provide energy for the host

richettsiae and chlamidiae

strict intracellular parasites
still kept genes for transfer from host to host
adapted genes for ADP and ATP transport
in many invertebrates (insects, worms, clams)
in many vertebrates intracellular microbes become parasites

Apicoplasts

DNA-containing organelle present in some protists
synthesis of fatty acids, DNA replication, transcription and repair

Kinetoplasts

DNA-containing organelle present in some protists (trypanosomes), with highly specialized mitochondrion features

Bacteriocysts

Bacteria cell or mycetocyte - specialized adipocyte
Found in some insects, such as aphids and german cockroaches
Maternally-transmitted in the egg as in Buchnera or via milk, as in Wigglesworthia

Rhizobium-legume interaction

mutualistic;
legumes secrete flavonoid attractants
Rhizobium expresses nod genes
Rhizobium enters cortical cells
differentiates into bacteroids (no cell wall)
bacteroids do not reproduce, specialize in Nitrogen fixation

Hydrothermal vent tube-worms

mutualistic;
Chemoautotrophic symbiosis
Adaptation to exploit sulfide-rich environments
(Riftia pachytila)

Ruminants

Mutualistic relationship between bacteria in cow gut and the cow;
additional mechanical digestion by chewing food multiples times after mixing it with enzymes

what we know about microbes

are ubiquitous
shape environment
vast majority has never been cultured

Microbial dark matter

Comprises the vast majority of microbial organisms (usually bacteria and archaea) that biologists are unable to culture in lab due to lack of knowledge or ability to supply the required growth conditions.

Producers

Algae, Phytoplankton, Bacteria

Consumers

Bacteria, Archaea, Eukaryotes

Decomposers

Fungi, Bacteria, Archaea

Grazers/Predators

Fungi, Protists, Viruses, Bacteria

key environmental factors

¨ Availability of nutrients
Energy sources
Electron donors
Carbon source
Electron acceptors
Other minerals: phosphorus, sulfur, nitrogen
minerals
¨ Temperature
¨ Salinity
¨ pH
¨ Pressure
¨ Moisture (water)

electron acceptors

O2, Fe3+, NO3, Mn4+, SO4, CO2, As5+, minerals, organic compounds
differ in their tendency to accept electrons (reduction/redox potential)
the more electropositive the potential, the higher the energy yield

Redox tower: standard reduction potential

more electropositive potential (E) \= higher the energy yield \= faster growth
Growth slows in anaerobic environment

Symbiotic relationships

both positive and negative associations
• persist over the majority of the lifespan of the organisms
• obligate or facultative
• endosymbiosis / ectosymbiosis

Mutualism

Both partners benefit, specific association

Synergism

Both partners benefit, non-specific association

Commensalism

Only one partner benefits

Amensalism

One partner harmed by non-specific association, the other unaffected

Parasitism

One partner harmed by specific association
More parasite species than any other kind of creature
All disease organisms, viruses

Human microbiome (fxns)

• Breakdown of complex polysaccharides ("dietary fiber")
• Production of vitamins, amino acids, short-chain fatty acids
• nutrient sources for both the microbiota and its mammalian host.
• Microbiota-associated metabolites shape immune responses
• Microbial production/consumption of GABA might affect the personality and the stress management

Great oxidation event

2.5 bya, atmosphere has more O2 suddenly!
-the presence of oxygen in the atmosphere corresponds with evolution of diverse life!

How do we know about first life on earth?

~3.8 bya \= first life. Carbon12/Carbon13 ratio indicates photoautotrophy. Sulfur34/Sulfur32 ratio indicates sulfate reduction by microbes

Chemical biosignatures
§ Oxidative state of metals
§ Isotopes and Isotope ratios (δ13C or δ34S)
§ Molecular fossils (such as hopanoids)

Morphological biosignatures
§ Microfossils
§ Stromatolites

3.8 bya to 0.6 bya \=only microbial life! plants and animals after .6 bya

Oxidative state of metals

A chemical signature
ex: Banded iron formations. Banded iron formation in ancient sedimentary rock. Its main component is chert, a form of quartz (silicon dioxide, SiO2) with layers colored red by iron oxide (Fe2O3) and iron oxyhydroxides [FeOx(OH)].

Phototrophic iron oxidation

Stromatolites

a morphological signature
an ancient life-form
ex: Cyanobacteria form colonial stromatolites, the present-day structures believed to most closely resemble the earliest forms of life on Earth.

The Anthropocene

August 2016 - scientists declare the human influenced age
§ "recent age of man"
§ most recent geologic time
§ global evidence
§ atmospheric, geologic, hydrologic and biosphere processes
§ Begins in the year 1950

CO2 levels today

405 ppm

CO2 Pools (gigatonnes)

Sedimentary rocks (terrestrial): 100,000,000 Gt
Oceans: 38,000 Gt
Atmosphere: 830 Gt
CH4 pools: Vents, seeps, methane hydrates 2,500 Gt

N-Pools (gigatonnes)

Soil: 220 Gt
Oceans: 1500 Gt
Atmosphere: 3,700,000 Gt

Nitrogen fixation...

Nitrogen fixation by humans greatly exceeds that of all microbes
The result - emission of N2O, a patent greenhouse gas (310 x CO2)

Haber process

Nitrogen fixation

ammonification

sources of nitrogen, carbon and energy. lead to accumulation of NH3 (ammonia) and methylamines
volatile, have neurotoxic effects
can accumulate in areas with dense animal population (cattle feedlots)
trimethylaminuria (also known as Fish odour syndrome)

assimilatory nitrate reduction

nitrate as a nitrogen source
§ mostly cytosolic (soluble) enzymes
§ convert nitrate/nitrite to ammonium
§ require energy in form of NADH or ferredoxin

N2 fixation

catalyzed by nitrogenase
occurs in all ecosystems
Rhizobium within legumes
cyanobacteria
many microbes
anaerobic
aerobic
N2 --\> NH4+
Requires a lot of energy

nitrification

NH3 --\> NO2\- --\> NO3-
¨Oxidation of NH4+ provides electrons and energy
¨In soil, one species oxidizes NH3 to NO2-
Nitrosomonas (beta-proteobacterium)
¨2nd species oxidizes NO2- to NO3-
Nitrobacter (alpha-proteobacterium)
¨Nitrospira - both steps in one microbe!

Excessive fertilizer use causes nitrate runoff
Eutrophication of water streams
Danger to water supplies

Denitrification

Dissimilatory nitrate reduction
NO3\- --\> NO2\- --\> NO --\> N2O --\> N2
¨ Anaerobic respiration
Nitrate/Nitrite are anaerobic electron acceptors
Catabolic pathways
¨ N2O (nitrous oxide) buildup if much NO3- present
prevalent in hypoxic ocean waters
greenhouse gas