Bisc303 Final

How much in losses did the tomato industry face as a result of reported Salmonella poisoning?

$100M

What is Salmonella and what does it cause?

A pathogenic Gram-negative bacteria of the Enterobacteriaceae family

Causes: Salmonellosis and Typhoid fever

Transmission: person-to-person, animal-to-person, contaminated food

Infective dose: 100-1,000,000,000 organisms

What is Salmonellosis?

Caused by Salmonella Typhimurium (not italicized); varies from country to country

Occupies GI tract

Symptoms: Headache, cramps, nausea, vomiting, loss of appetite, diarrhea, high fever

Duration: 4-7 days

What is Typhoid Fever?

Caused by Salmonella typhi (italicized)

Only lives in humans, can become a chronic infection -

a systemic disease; occupies the bloodstream and GI tract

Symptoms: Abdominal pain, weakness, headache, loss of appetite, sustained high fever of 39-40°C

Duration: 3-4 weeks without treatment

Salmonella Characterisitcs

Gram negative

Intracellular pathogen

Actively invasive

Controls the host cell cytoskeleton

T3SS

Where does the Salmonella go once it invades its host cell?

It lives in Salmonella-containing vacuoles (SCV), that protects it from host enzymes, and allows a location for it to replicate

How does Salmonella infect its host?

Injects effector proteins into the host by using a T3SS

Gains entry into the cell by controlling the actin dynamics of the cell

Causes massive amounts of membrane ruffling

Enters the cell and is housed in an SCV

Injects a separate set of effector proteins once in the cell, again using a T3SS

Blocks lysozymes

Survives and replicates in the SCV

Eventually kills the host cell

Who was Mary Mallon?

A cook for wealthy families in New York from 1900-1907. The first person in the US identified as a chronic carrier of Salmonella.

What happened when it was discovered she was causing Typhoid fever in those that ate her food/came in contact with her?

She was put into quarantine, but was eventually let out and found work in a laundry.

It didnt pay enough so she changed her name to Mary Brown and she was re-employed as a cook in a hospital, where 25 people contracted Salmonella, and 1 person died.

She was put back into quarantine until she died at age 69.

What are the two species of Shigella, and what disease do they cause? Where do they localize and how are they transmitted?

Shigella dysenteriae, Shigella flexneri → both cause Bacillary Dysentry (AKA. Shigellosis)

Occupies: GI tract

Transmission: person-to-person, animal-to-person contaminated food or water

Infectious dose: 10-100 organisms (highly infectious)

What happens during a Shigella infection?

Symptoms: painful bowel movements, cramps, nausea, vomiting, loss of appetite, diarrhea (often bloody) and high fever

In highly infected people: kidney failure, seizures in children under 2

Duration: 5-7 days

Summarize Shigella traits.

Gram negative

Anaerobic

Intracellular pathogen

Actively invasive (gets in the same way as Salmonella, releases the same toxin as E. coli)

Controls the host cytoskeleton (twice; one to get in, another to move around)

What does Shigella have features common to?

Pathogenic E. coli, Salmonella, Listeria (nightmare pathogen!!)

What happens in a Shigella invasion?

The pathogen uses a T3SS to inject effector proteins into the host cell, causing massive amounts of membrane ruffling seen in Salmonella.

The bacteria enters the cell and is found in a vacuole, which is later lysed.

Shigella then uses its effector IcsA at one pole (homologous to ActA in Listeria) to recruit host actin and ultimately propel the bacteria around the cell.

Unlike Salmonella, Shigella replicates in the host cytoplasm rather than the vacuole.

What, besides from the effector proteins, does Shigella use for pathogenicity?

Shiga toxin (originally identified in Shigella dysenteriae) → generates diarrhea

How much in loses did California face from the E. coli outbreak in 2005?

$74M. (Spinach crop was valued at an estimated $258.3M)

How many different types of pathogenic E. coli are there and what types of diseases do they cause?

Over 700 serotypes.

Can cause intestinal diseases (gastroenteritis), urinary tract infections (UTIs) and neonatal meningitis

What are the 5 main types of pathogenic E. coli that can cause intestinal disease?

Enterohaemorrhagic E. coli (EHEC)

Enteropathogenic E. coli (EPEC)

Enterotoxigenic E. coli (ETEC)

Enteroinvasive E. coli (EIEC)

Enteroaggregative E. coli (EAggEC)

How is pathogenic E. coli transmitted? About how long is the duration of infection?

Contaminated food, person-to-person.

Usually lasts 7 days (without the use of antibiotics)

What is EHEC O157:H7

A serotype of enterohaemmorhagic E. coli that was first isolated in Argentina in 1977.

Infective dose = 10-100 organisms (highly infective).

Symptoms: bloody diarrhea, abdominal cramps.

Can also lead to haemolytic-uremic syndrome (HUS) —> Can cause kidney failure.

When the bacteria is killed, a toxin is released (hard to eliminate/cure)

How to G- pathogens infect cells?

They use bacterially-derived effectors.

Method of delivery: On the bacterial surface, directly delivered into the host cytoplasm, secreted (toxins)

Modify: cytoskeleton, organelles functions, cellular structures, channels, signalling pathways

How are pathogenic effectors delivered directly into the host cytoplasm?

Secretion systems that connect the pathogen to the host (7 different types) → pathogenic E. coli uses the Type 3 Secretion System (T3SS)

What is the T3SS?

A proteinaceous needle extending from the bacterial cell membrane into the host cell membrane (eukaryotic for pathogenic E. coli), allows the injection of effectors into the host via a membrane pore.

Invisible/ undetected by the host immune system.

What are some general traits of pathogenic E. coli?

Extracellular pathogen

Injects effector molecules into cells

Collapses host cell microvilli

Generates 'pedestals'

Causes disease (diarrhea)

What happens if the T3SS of EHEC is mutated so that it does not function properly?

Disease symptoms are greatly decreased (or non-existent). The T3SS is required for EHEC pathogenicity.

What are E. coli pedestals and what are they for?

Once E. coli translocates its effectors into the host cell, actin localizes to the site of bacterial contact and forms a pedestal-like structure.

The formation of pedestals has been shown to foster intestinal colonization by EHEC.

Although their function is unknown, we do know that they are apart of the EHEC invasion process (→ they are a hallmark of pathogenic E. coli disease).

They also allow bacteria to surf atop of infected cells, allowing bacteria to infect neighbouring cells.

What else does EHEC release besides effector proteins?

Shiga toxin, a potent toxin

What is the Shiga toxin?

a specific host site exotoxin;

targets the intestine which results in diarrhea and loosened blood vessels (blood release)

First identified by Kiyoshi Shiga in a pathogen called Shigella dysenteriae.

Thus, E. coli O157 - H7 is a "shigatoxigenic" E. coli

What are E. coli O156 H7's two strategies to create diarrhea?

Inject effectors, release Shiga toxin

Why would a pathogen want to cause diarrhea, and how is this a problem to us? How may this be beneficial to us?

To disseminate into the environment.

problem for us: Results in dehydration, loss of ions, haemorrhage (with O157 H7)

Diarrhea may also help us quickly rid the bacteria from our system.

What are the mechanisms of diarrhea generation by pathogenic E. coli?

Breach of tight junctions,

alteration of H2O channels,

alteration of serotonin functions,

alteration of ion channels

How does pathogenic E. coli cause a breach in tight junctions?

Use of bacterial effector proteins EspF, EspG, Map

How are H2O channels (aquaporins) altered by pathogenic E. coli?

EspG and EspF are used;

AQP2 and AQP3 are removed from the membrane

How does alteration of serotonin functions contribute to diarrhea?

It works on the nervous stem of the intestine (enteric nervous system)

How are ion channels disrupted by pathogenic E. coli?

Shiga toxins attach to receptors, causing a cascade of intracellular signalling that alters the function of many ion channels.

Ions are released from channels, followed by an efflux of water, resulting in diarrhea.

Define bioterrorism.

The intentional or threatened use of viruses, bacteria, fungi or toxins from living organisms to produce death or disease to humans, animals and plants.

What are the features of Category A Select agents?

Easily disseminated or transmitted from person to person

High mortality rate

Potential for major public health impact

Causes public panic

Causes social disruption

Requires special action for public preparedness

What are the features of Category B Select agents?

Moderately easy to disseminate

Moderate morbidity and mortality rate

e.g. Salmonella spp., E. coli

What are Category C Select agents?

Emerging pathogens that could be engineered for mass dissemination

Potential for high morbidity and mortality rates

Major impact potential

e.g. Hantavirus, multi drug-resistant mycobacterium tuberculosis

Name the 6 category A select agents and the disease that they cause.

Bacillus anthracis → Anthrax

Clostridium botulinum toxin → Botulism

Variola major virus → Smallpox

Filoviruses and arenaviruses → Viral hemorrhagic fever

Yersinia pestis → Plague

Francisella tularensis → Tularemia

What is Viral hemorrhagic fever?

A severe, multisystem syndrome caused by Ebola virus and Marburg virus

What type of viruses are Ebola and Marburg virus? What do they do and how are they transmitted?

Single-stranded antisense (-) RNA

Can cause damage to the vascular system

results in hemorrhaging

Primarily transmitted by bats (bats and rodents thought to be the microbial reservoir)

Which of the 5 ebolaviruses cause human disease, and which do not?

Zaire ebolavirus, Sudan ebolavirus, Tai forest ebolavirus, Bundibugyo ebolavirus → Infects humans, spread by aerosol and through bodily fluid contact

Reston ebolavirus → infects non-human primates and pigs, spread by aerosol and through bodily fluid contact

What are some traits of the Ebola virus?

Incubation time: 2-21 days (usually 5-7)

Infectious dose: unknown (estimated to be 1-10)

Symptoms: fever, headache, joint and muscle aches, sore throat, weakness, diarrhea, vomiting, hiccups, stomach pain, rash, red eyes, bleeding

Infects: endothelial cells, mononuclear phagocytes, hepatocytes

Zaire ebolavirus = 80-90% fatal

How is the Ebola viral invasion similar to Salmonella and Shigella?

Uses membrane ruffling (macropinocytosis) and requires actin and actin-associated proteins of the host cells for internalization.

BUT: the Zaire ebolavirus uses clathrin-mediated mechanisms to enter the host cell

Summarize the Marburg virus

Also causes Viral hemorrhagic fever

Only 1 species

Closely related to, but generally distinct from the Ebola virus (i.e. if you are infected with one, there is no cross protection from the other)

Genome: 19.1 kB in length = the longest genome of (-) strand RNA viruses

Mortality rate ~ 25%

What is Yersinia pestis?

A pathogenic Gram-negative rod that causes Plague (Black Death), due to black hemorrhages beneath the skin

Transmitted through infected fleas on rodents to humans

Transmitted from human to human through inhalation

Infectious dose: 100 - 20,000 (airborne)

Where does Yersinia pestis replicate and what are the symptoms of infection?

They replicate in the blood or lymph

Symptoms: hemorrhages, fever, chills, headache, exhaustion, appearance of buboes (enlarged lymph nodes)

How does infection by Y. pestis result in Bubonic plague?

The pathogen enters/infects the skin.

50-60% death rate, results in death in 3-5 days if left untreated from toxic effects.

What happens if Y. pestis enters the blood?

= Septicemic plague = 100% death rate

What is Pneumonic plague?

Results from the inhalation of Y. pestis; 100% mortality rate

Where does Y. pestis replicate and what does it use for pathogenicity?

Replicates in phagocytes

Uses T3SS and effectors called YOPs (Yersinia Outer Membrane Proteins)

What is thought the be the first use of bioterrorism?

Development of Y. pestis as an aerosol in 1346

What is Francisella tularensis?

A highly infectious invasive pathogen that enters epithelial cells and phagocytic cells

~10 bacteria are required to infect

The Schu S4 strain: up to 60% fatal

Can infect through any opening: Ingestion, inhalation, bites from arthropods, eyes, urethra

How does F. tularensis enter its host?

Uses clathrin-mediated endocytosis and cholesterol for epithelial cell invasion

Enters phagocytes by general phagocytosis

What happens once F. tularensis enters the cell?

It is briefly found within a Francisella-containing vacuole (FCV, probably for 30-60 min) It then breaks the vacuole and replicates in the cytoplasm of the host cell, eventually occupying the whole cytoplasm

What does F. tularensis use to deliver its effector proteins?

Type 6 Secretion System (T6SS)

What does infection by F. tularensis cause and what are the symptoms?

Tularemia (AKA. rabbit fever)

Incubation time: 2-10 days

Symptoms: Flu-like (fever, chills, headache, diarrhea, muscle pain, lesions)

If tularaemia is treatable with antibiotics, why is it such a concern?

If F. tularensis is disseminated over a large enough area, it could overrun the medical system. The bacteria has been weaponized and made drug resistant.

What are the requirements to handling BSL1 (biological safety level 1) microbes?

@ Lab bench, sink No barriers needed, personal protective equipment e.g. Cultured cells, non-pathogenic microbes

What are the requirements to handling BSL2 microbes?

In BSL1 facilities, autoclave if available, negative air pressure BSL1 equipment, biological safety cabinets e.g. Pathogenic E. coli spp. (excluding O157), Salmonella spp.

What are the requirements to handling BSL3 microbes?

In BSL2 facilities w/ self-closing doors away from access corridors, separate exhaust systems, everything is autoclaved (lab clothing, all waste, serum testing) BSL2 equipment + stricter procedures e.g. Francisella tularensis

What are the requirements to handling BSL4 microbes?

In BSL3 facilities, shower to exit, change clothes to enter, separate building or zone, decontamination systems, separate ventilation Pressurized suits, glove boxes e.g. Zaire ebolavirus

What does the term "virus" originate from?

Comes from latin meaning: "toxin or poison"

What are the general features of viruses?

1) Simple acellular structure

2) DNA or RNA genome (not both)

3) Obligate intracellular parasites

NOTE: contrarily, there are bacteria that are obligate intracellular parasites that possess both DNA and RNA (e.g. Chlamydia, Rickettsia)

How did Chamberland contribute to the history of viruses?

(1885-1900) Developed a porcelain chamber that eliminated bacteria

What did Iwanowski and Beijerincki find?

(1885-1900) Found that filtered extracts from tobacco could transmit disease to another plant even after filtering the extract through pores that trap even the smallest bacteria → discovered the Tobacco Mosaic Virus (TMV) (The beginning of virology)

Who and how did they contribute to the finding that some viruses were linked to the development of cancer?

(1900-1935) Ellerman & Bang → found chicken leukaemia virus

Peyton Rouse → discovered Sarcoma virus (purified virus caused disease in chickens; fulfilled by Koch's postulates)

How did Twort and D'Herelle find that some viruses could infect bacteria?

(1900-1935) Twort→ found infectious agents that lysed bacteria (discovered bacteriophages)

D'Herelle → discovered bacteriophage plaque assay

Who discovered viral compositions and in which virus?

(1900-1935) Stanley Bowen & Pirie → found that TMV is mostly protein & contains nucleic acids

What is the basic structure of viruses?

Size: 10 nm - 400 nm (0.4 um)

# of genes: Few to several hundred

Structural elements: nucleocapsid core (= DNA or RNA enclosed in protein coat (capsid);

some viruses are enveloped with a membrane

What other special features do only some viruses have?

Lipid membrane (envelope) from host

Virally-encoded enzymes/proteins

Whats the difference between the terms "virus" and "virion"?

virus = replicative intracellular form

virion = non-replicative, extracellular infectious form (non-replicating b/c it's outside the host cell)

How does a virus differ from a plasmid?

A plasmid is a small, circular extrachromosomal DNA. Plasmids do not package into phage particles like viruses do, so the plasmid outside of the cell would be "naked DNA". Unlike viruses, plasmids don't kill the host cell after they have replicated

What are virus protomers?

Protein monomers that self-associate to form the final capsid

How do viral capsid proteins assemble?

They associate and are held together by non-covalent bonds (making the capsid easy to break apart).

Capsids are comprised of one or only a few polypeptides, as using a small genome saves space (viruses are very small!!)

Explain the 3 types of capsid symmetries.

1) Helical: protomers form a helix that wraps around the nucleic acid genome (e.g. TMV)

2) Icosahedral: protomers associate to form a 20-sided polygon with 12 vertices, a structure that is the most efficient way to enclose a space (e.g. Adenovirus)

3) Complex: often asymmetrical or symmetrical with other structures (tail) (e.g. Vaccinia virus - pleomorphic structure; Bacteriophage = a combination of capsid symmetries)

What is the viral envelope?

The lipid bilayer that surrounds the nucleocapsid of some viruses. It is found on most animal viruses, some plant viruses and very few bacteriophages.

The lipid comes from the host (the plasma membrane, ER, Golgi, nuclear membrane)

What do the host and virus contribute to the final virion?

Host: provides lipids, carbohydrates, & amino acids

Virus: encodes protein (& genome template)

What are naked viruses?

Viruses without lipid envelopes → influences their susceptibility to disinfectants

What are spike proteins?

Virally encoded proteins embedded in the envelope

Spike proteins are immunogenic:

In Influenza, the spike protein genes encode:

• Hemagglutinin (HA): can crosslink & clump RBCs

• Neuraminidase (NA): cleaves host sialic acid (involved in viral entry and release of virions upon binding)

• NOTE: HA & NA are the H and N in H5N1

How do viruses bud from host cells?

Viral proteins are inserted into the host plasma membrane, allowing the nucleocapsid to bind to the inner surface of the host plasma membrane.

Viral proteins collect at the site of viral contact, while host proteins are excluded.

The plasma membrane envelops the nucleocapsid, forming and releasing the enveloped final virion.

What are some features of viral genomes?

Genome size: 4000 to 5000 nucleotides

# proteins encoded by viral genome: few to a hundred (or more)

Overlapping genes, and the exclusion of introns allow the virus to save space on the genome

What are the 2 types of RNA strands that RNA viruses can have?

"+" or "sense" strand = approximately equal to mRNA, can be immediately translated into viral proteins

"-" or "antisense" strand = complimentary "+" strand must be synthesized before the viral proteins can be synthesized

Why is HIV classed as a retrovirus?

Once HIV gets into the host cell, the virus is transcribed (only once), then the synthesized viral ssDNA is duplicated to make viral dsDNA, then integrated into the host genome.

The virus requires virally encoded reverse transcriptase that transcribes mRNA to DNA, as well as degrades the viral RNA.

HIV also uses integrase: clips ends of viral dsDNA (makes sticky ends), allowing the DNA to be placed into the host genome

HIV protease is used to break down proteins and peptides

What is required for RNA viruses? For retroviruses?

For +, -, +/- RNA viruses, RNA polymerase is needed

Retroviruses need reverse transcriptase to copy +RNA to DNA

How many viruses have been identified by the International Committee for the Taxonomy of Virus es (ICTV)?

~2000 different viruses (there are probably more)

How are viruses named (suffixes)?

Order: -virales (e.g. monogenevirales)

Family: -viridae (e.g. paramyxoviridae)

Subfamily: -virinae (e.g. paramyxovirinae)

Genus: -virus (e.g. rubalavirus)

Type species: -virus (e.g. mumps virus)

What is the Baltimore Classification System?

Developed by David Baltimore (discovered reverse transcriptase, won the nobel prize)

Viral classification that is based on the nature (makeup) of the genome and how the virus makes mRNA

There are 7 groups based on the Baltimore Classification System

What are the 7 groups of viruses?

Group. genome type/ genome replication/ mRNA synthesis: I. double-stranded DNA genome/ dsDNA → dsDNA/ dsDNA → mRNA II. single-stranded DNA genome/ ssDNA → dsDNA → ssDNA/ ssDNA → dsDNA → mRNA III. double-stranded RNA genome/ dsRNA → ssRNA → dsRNA/ dsRNA → mRNA IV. plus-stranded RNA genome/ +RNA → -RNA → +RNA/ +RNA = mRNA V. negative-stranded RNA genome/ -RNA → +RNA → -RNA/ -RNA → mRNA VI. single-stranded RNA genome/ ssRNA → dsDNA → ssRNA/ ssRNA → dsDNA → mRNA VII. double-stranded gapped DNA genome/ gapped dsDNA → dsDNA → +RNA → -DNA → gapped dsDNA/ gapped dsDNA →dsDNA → mRNA

How can viruses be cultivated?

They require a host cell (cannot be gown in complex media)

Animal viruses can be cultivated in host animals (if host is suitable),

embryonic chicken eggs (egg is fertilized, then the virus is injected),

or cultured animal cells (monolayer of cells is plated, then virus is plated)

Define: Pocks, Plaques, and Cytopathic effect.

Pocks = virally-induced lesions

Plaques = zone of clearing on plates caused by virally-induced lysis

Cytopathic effect = observable changes to host cells due to viral replication (changes depend on the virus and host cell used)

How are bacteriophages grown?

Plate a lawn of susceptible bacterial cells, then add viral culture → growth of bacteriophages will result in plaques; different phages create different plaques

How are plant viruses spread, and how are they often cultivated?

Whole plant: plant viruses are transmitted by insects and applied to the plant surface

Plant tissue culture: often used to cultivate viruses in specific cell types on a plant.

What limits the host range of viruses?

Viruses will recognize the most cells because they bind to specific host cell receptors

Provide examples of host cell surface proteins that act as viral receptors.

HIV: recognizes CD4 + co-receptors on specific WBCs; causes AIDs

Epstein-Barr Virus (EBV): recognizes complement C3b on lymphocytes; causes mononucleosis

Human Rhinovirus: recognizes VLDL or ICAM-1; causes colds

How do viruses usually enter the cell?

By endocytosis or membrane fusion

How do enveloped and naked viruses enter the cell via endocytosis?

The virus docks to receptors, which initiates the invagination of the host plasma membrane.

Enveloped viruses are contained in a clathrin-coated endosome.

The endosome is then acidified and the nucleocapsid is released from its envelope

Endosomes with naked viruses are not acidified, rather the nucleic acid is excreted out of the endosome

What are viral fusion proteins?

Specific virally-encoded proteins that force envelope fusion with the plasma membrane