EXAM 3

Pyrodinium bahamense

• microbial eukaryote (domain: eukaryota)

• produces saxitoxin

• bioluminescent !! (in water)

• phylum: Dinoflagellata

• class: dinophyceae

• order: Gonyaulacales

Vibrio vulnificus

• gram negative

• motile

• rod, bacillus

• present in marine environments

• LPS (Lipopolysaccharides) — endotoxin, multiple exotoxins that cause the diseases

• 3 types of infection:

  • gastroenteritis (oysters)

  • severe wound infections (swimming in contaminated water)

  • sepsis (bloodstream; oysters/shellfish)

ATCV-1 Virus

• Acanthocystis turfacea chlorella virus 1

• giant dsDNA virus

• type of Chlorovirus → infects green algae, common in fresh water like lakes and ponds

• virus associated with reduced cognitive functioning

Cutibacterium acnes

• slow-growing

• typically aerotolerant anaerobic

• Gram Positive

• rod shaped

• part of normal skin flora also found deep in hair follicles and pores → can cause acne

• use sebum, cellular debris, and metabolic byproducts form surrounding skin tissue as primary sources of energy and nutrients

Saxitoxin

• a highly potent natural neurotoxin

• 200 micrograms lethal for average-weight human

• primarily known as the causative agent of paralytic shellfish poisoning (PSP) and pufferfish poisoning

• mid-2000s FL saxitoxin outbreak from pufferfish caused by Pyrodinium bahamense

sxtA4

• gene that appears to be specific for saxitoxin synthesis = proxy for toxin production

• phylogenetic analysis indicates it is lost in nontoxic strains and species

Microbial Competition

• success in environment based on ability to multiple and withstand adverse conditions

• other factors in success → chemical warfare & streptomyces (many microbial compounds)

Microbial Communities

• microbial mats

• biofilm (distinct layers)

Biofilm Layers

• cyanobacteria (top) → green photosynthetic pigments

• purple sulfur bacteria → use light-harvesting pigments of dif. wavelengths than cyanos

• sulfate-reducers (bottom)

Sulfate reducers

• bottom layer of microbial mats

• iron-reacting hydrogen sulfide

• sulfate as terminal e- acceptor

• Desulfovibrio oxyclinae

• Desulfovibrio halophytica

• Desulfonema & Desulfococcus species

Methods for Microbial Ecology

• metagenomics (taxonomy & genetic/functional capabilities)

• FISH (fluorescent in situ-hybridization)

• DNA microarrays

Metagenomics

• Who’s there and what are they capable of?

• method of DNA extraction

• overall numerous short sequencing reads that are software-analyzed

• method of microbial ecology

Metatranscriptomics

• What are they doing?

• RNA sequences

• nucleic extraction method

• short half-life

• presence of Rnases

• lots of rRNA

Taxonomic profile comparison at genus rank

FISH

• fluorescent in-situ hybridization

• method for microbial ecology

• can be used to distinguish subsets of prokaryotes that contain a specific nucleotide sequence

Which of the following is false?

a) culture techniques are an accurate way of determining which members in a microbial community are most common

Oligotrophic

nutrient poor

Eutrophic

nutrient rich

Eutrophication

excessive enrichment of water bodies with nutrients—primarily nitrogen and phosphorus—leading to dense algal blooms, severe oxygen depletion (hypoxia), and ecological degradation

Aquatic Habitats: Marine

• oceans cover 70%+ of the Earth

• oligotrophic = nutrient poor

• eutrophic = nutrient rich

• eutrophication can lead to hypoxic conditions (too little O2)

• heterotrophs in lower layers → consumption of organic compounds + dissolved O2

Dead Zones

• Eutrophication in coastal areas

• Gulf of Mexico Dead Zone

Harmful Algal Blooms

• many difference species and toxins

• Dinoflagellates (microbial eukaryotes) & cyanobacteria

• Pyrodinium bahamense

Lake Stratification — Freshwater Systems

the separation of lakes into three distinct horizontal layers based on water density differences caused by temperature variations

Epilimnion

the top-most, warmest, and least dense layer of water in a thermally stratified lake

Hypolimnion

the dense, cold, and dark bottom layer of a thermally stratified lake, located below the thermocline

Metalimnion

the middle layer of a thermally stratified lake, located between the warm surface water (epilimnion) and cold deep water (hypolimnion)

Which of the following pairs that relate to aquatic environments does not match?

Epilimnion — O2 poor

Adding high levels of nutrients to a lake or inshore area would have all of the following effects in that environment except…

increased levels of dissolved O2

Soil Microbiome

• great diversity

• Rhizosphere = important component of soil microbiome → plant roots (secret organic compounds)

• Streptomyces (soil bacteria) = antimicrobials, geosmins (soil smell)

• Bacillus = endospores for survival

• microbes in soil are good source of bioremediation potentials

Which of the following is NOT a matching pair?

soil — minimal biodiversity

Biogeochemical Cycles

1. carbon cycle

2. nitrogen cycle

Carbon Cycle

• carbon fixation — autotrophs convert CO2 into an organic form

• respiration & fermentation — CO2 usually produced via activities of heterotrophs

• methanogenesis & methane oxidation

  • methanogens — archaea oxidize hydrogen gas, CO2 as terminal e- acceptor, make methane

  • methyltrophs — methane as energy source

• the carbon cycle revolves around carbon dioxide !!

Nitrogen Cycle

• nitrogen fixation → nitrogenase, diazotroph (azotobacter, clostridium)

• ammonification

• nitrification

• denitrification

• anammox

Food Poisoning

when a pathogen or its toxin is consumed

Exotoxin

highly potent, protein-based toxins secreted by living bacteria into their environment

Foodborne Intoxication

• illness that results form consuming exotoxin

• Staphylococcus aureus

• Clostridium botulinum

Staphylococcus aureus

• many toxins

• gastroenteritis — enterotoxin = protein exotoxin

• strain detection: Qpcr, MLST, PFGE

Clostridium botulinum

• anaerobic

• gram positive

• lab isolation — in tryptose sulfite cycloserine (TSC) in anaerobic (<2%) oxygen

• endospores

• neurotoxin produced by vegetative cells = botulism

• 8 neurotoxins = A-G; A, B, E, F, H cause human illness

• Toxin binds to nerve endings, so nerves cannot signal to heart

• Qpcr for toxin genes A, B, E, F

Which of the following organisms cause foodborne intoxication?

Staphylococcus aureus

Virus

• requires host cells

• most only visible with electron microscopy

• prokaryotic viruses (bacteriophages) vs eukaryotic viruses

• ~10-800 nm in size

Virus structures

• Virion

• Nucleocapsid

• Capsomeres

• Enveloped vs non-enveloped/naked viruses

• tail fibers (phages) and spikes (animal) for specific receptors

Virion

• complete extracellular viral particle

• capsid = protection + infection

• DNA/RNA — linear or circular, DS or SS, type of nucleic acid used in classification

Nucleocapsid

nucleic acid + capsid

Capsomeres

protein subunits

Enveloped vs Naked Viruses

• lipid bilayer is unique to enveloped viruses

• phage = naked and non-enveloped

• enveloped viruses are more susceptible to disinfectants

Capsids are composed of…

protein

The tail fibers on phages are associated with…

attachment

Shape of viruses

• determined by capsid

• icosahedral

• helical

• complex (most phages)

Virus Taxonomy

• key characteristics for classification:

  • genome structure (nucleic acid, strandedness)

  • host

  • shape and disease

• families: -viridae

  • geographic location

• genera (many within a family): -virus

• species: based on disease

• no bacterial nomenclature

• route of transmission rather than taxonomical relatedness

Zoonotic

disease transmitted from animal to human

Arboviruses

virus spread by arthropods, can infect many different species (Wes Nile; yellow fever)

Bacteriophages

viruses that infect bacteria

Phage Replication

• similar mechanisms as for viruses that infect animal cells

1. productive infection

2. latent state

Lytic Phage

• lytic/virulent phage (productive infection)

• productive infections → new viral particles

• 30 min cycle

• T4 model — dsDNA; E. coli

Lytic/virulent Phage

• productive infection

• exit host at end of infection via cell lysis

Temperature Phage Infections

2 choices = Lytic vs Lysogenic

Lysogenic Phage Infection

• incorporation of phage genome into host genome (no damage in host cell)

• Lysogen

• prophage — integrated phage DNA

• prophage passed to bacterial daughter cells

• metabolic state of cell can influence which cycle occurs

Lambda as Temperate Model

• linear chromosome that circularizes upon entry

• integration via phage integrase

• repressor protein regulars excision gene and maintains lysogenic state

• role of DNA-damaging agents in phage induction

Filamentous Phage

M13

• productive infections but do NOT kill host

• M13 = F pilus of E.coli

• single stranded DNA upon entry

• dsDNA by host DNA polymerase = “replicative form”

• extrusion

Extrusion

process by which virus exits cell

Filamentous phages…

are extruded (formed) from the host cell

All phages must have the ability to…

1. have their nucleic acid enter the host cell

5. have their nucleic acid replicate in the host cell

Animal Virus Replication

• viral enzymes can serve as targets for antiviral drugs

• infection cycle similar to that of phages:

  • attachment

  • penetration and uncoating (Genome Entry)

  • synthesis of proteins & genome replication

  • assembly

  • release

Attachment of Viruses

spikes bind to specific receptor on cytoplasmic membrane with receptor specificity

Tissue Tropism

viruses can only enter specific cell types

Host Range

the variety of species (hosts) a virus can infect

Measles

• 2 cell surface receptors

• Cd46 = widely expressed in all human nucleated cells

• SLAM — signaling lymphocyte activation molecule

Rabies

• mammalian nervous system = nicotinic acetylcholine receptor

• humans, dogs, horses, skunks, foxes, etc.

Penetration & Uncoating (Genome Entry)

• entry dependent on envelope vs non-enveloped

• different from phage — entire virion enters cell

• enveloped viruses can enter via (1) Membrane Fusion or (2) Endocytosis

• non-enveloped viruses = endocytosis only

• all viruses = uncoating after entry

Membrane Fusion

mechanism of penetration in which viral envelope fuses with cytoplasmic membrane and the nucleocapsid is released into the cytoplasm

Endocytosis

• mechanism of penetration in which viruses capitalize on receptor-mediated endocytosis, a normal cellular process

• viruses bind to receptors that trigger endocytosis

DNA Virus Replication

• in nucleus

• host cell separates and carries own DNA polymerase

• recall (-) DNA strand is the template for mRNA

• ssDNA → 2nd strand must be synthesized

RNA Virus Replication

• in the cytoplasm

• requires viral RNA polymerase

• replicase = RNA-dependent RNA polymerase

• antigenic variation due to lack of proofreading

• influence and antigenic drift

• antigenic shift in segmented genome

Reverse Transcribing Virus (Retrovirus) Replication

• retroviruses encode reverse transcriptase = RNA-dependent DNA polymerase

• DNA from RNA template

• HIV = +RNA = carries RTase in virion

• permanent integration into host chromosome

Assembly & Maturation of Viruses

• packaging nucleic acid with capsid proteins

• spontaneous self-assembly

• different sites of assembly and maturation based on virus type

  • non-enveloped = cytoplasm

  • enveloped = part of maturation can occur as virion exits cell

  • DNA viruses = nucleocapsid in host cell nucleus

Release of Viruses

• mechanism dependent on whether virus enveloped or not

• enveloped viruses → budding

• non-enveloped → apoptosis

Viral Spread

• influenza = coughing and sneezing

• norovirus & rotavirus = fecal-oral (contaminated food & water)

• HIV = sexual contact

• vectors = organisms that carry the virus

Host Range

• specificity determines the host range

• receptor-binding is major determinant of host range (specific binding btwn viral capsid proteins & specific host cell receptors)

• receptor recognition by viruses = 1st and essential step of viral infections of host cells

Narrow Host Range

• HIV — humans

• surface protein gp120 interacts with CD4 molecule on human leucocytes

Broad Host Range

• cucumber mosaic virus — 1000 plant species

• influence A virus — mammals and birds

• Sialic Acids (SAs) of cell surface glycoproteins and glycolipids are the receptors for the influenza virus, recognized by the viral glycoprotein hemagglutinin

Coronavirus

• large family of viruses with broad host range

• cause respiratory, gastrointestinal, and central nervous system diseases

• contain and envelope, helical capsid, and single-stranded, positive-sense RNA genome

• 3 major genera = a, B, y (alpha beta gamma)

  • a = human coronavirus NL63, porcine respiratory coronavirus

  • B = severe acute respiratory syndrome coronavirus (SARs-CoV), Middle East respiratory syndrome (MERS-CoV), bovine (BCoV)

  • y = avian infectious bronchitis virus (IBV)

• MERS-CoV, SARS-CoV, and SARS-CoV-2 are the exceptions of animal to human infection

Coronavirus Specificity

• coronaviruses recognize variety of host receptors

• receptor binding domains = S1 subunit of spike proteins both N-term (S1-NTD) and C-term (S1-CTD)

• recognize 4 protein receptors & 3 sugar receptors

• coronavirus spike protein binds to receptor on host cell surface then fuses viral and host membranes

  • spike has 3 parts = ectodomain, transmembrane anchor, tail

SARS-CoV-2 Virus Characteristics

• S protein protrude from the viral surface resembling a crown or corona

• an enveloped virus with an unsegmented single-stranded positive-sense RNA genome

• 4 main structural proteins

  • spike (S) glycoproteins

  • envelope (E) glycoproteins

  • membrane (M) glycoproteins

  • nucleocapsid (N) proteins

• genome codes for 16 nonconstructural proteins involved in viral replication, maturation, and release

Ectodomain

• S1 subunit binds to receptor

  • 2 domains in which either or both function as receptor-binding domain

  • S1-N/CTD (N-term vs C-term)

• S2 subunit for membrane fusion

• receptor-binding domain can serve as vaccines against coronavirus infections

Virus Entry into Hosts

• portals of entry = nasal, oral, conjunctiva of eyes passing through nasolacrimal duct, esophagus

• once transmitted, viral S-proteins bind to host cells via angiotensin-converting enzyme 2 (ACE2) (viral tropism)

• S-proteins embedded in viral envelope bind to ACE2 on host cells triggering viral endocytosis, or membrane fusion and viral genome entry

Horizontal Gene Transfer via Bacteriophages

• generalized transduction

• specialized transduction

• differences based on type of error

Generalized Transduction (Horizontal Gene Transfer via Bacteriophages)

• packaging error during assembly = bacterial rather than phage

• homologous recombination

• any bacterial gene can be transferred (“generalized transduction”)

Specialized Transduction (Horizontal Gene Transfer via Bacteriophages)

• excision error by prophage

• bacterial + phage DNA genes

• “specialized” = only bacterial genes adjacent to phage genes transferred

Plaque Assay

• method of detecting and quantifying phages

• bacterial host + phage sample

• based on zone of clearing

• used to determine titer within sample

  • titer = laboratory measurement that determines the concentration of a specific substance, usually antibodies, in a blood sample

Innate Immunity

immunity at birth, germ-line encoded

Adaptive Immunity

immunity vis antigens and antibodies

First Line (Immune) Defenses

• physical barriers, skin

• mucous membrane

• antimicrobial substances

• normal microbiota

Epidermis

many layers of epithelial cells, keratin

Sensory Systems (Immune Defense)

• sentinel cells = pattern recognition receptors, other PRRs, cytoplasm

• complement system = circulates in blood and tissue in inactive form

Innate Effector Actions (Immune Defense)

• interferon response

• phagocytosis

• complement activation

• inflammatory response

• fever

Phagocytes

cells that engulf and digest microbes and other foreign debris via phagocytosis

Bodily Antimicrobial Substances

• lysozyme

• lactoferrin/transferrin

• antimicrobial peptides

  • defensins = + charge

  • AMPs and defensins → destroy microbes, other roles in immune system, vitamin D / AMP regulation

Hematopoietic Stem Cell

• from which all blood cells arise

• bone marrow

Immune System Cells

• Hematopoietic stem cells

• red blood cells (erythrocytes) → O2

• platelets → clotting

• white blood cells (leukocytes) → defense

Granulocytes

• type of leukocyte

• cytoplasmic granules containing compounds important for cell protection

• neutrophils, eosinophils, basophils