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Lecture 1 8/23: Prior to late 19th century = pre-germ era (1870-1880s) Girolamo Fracastoro (1546)- recognized that some diseases were transmittable and they could pass by direct contact, through the air, and through inanimate objects J. Henle (1840)- theorized that microbes cause diseased and about how to prove it (never actually did the experiment) Louis Pasteur – first gained fame as a chemist in 1840s and 50s Showed experimentally that alcoholic fermentation results from yeast growth (Germ theory of fermentation) Anthrax  Caused by Bacillus anthracis  Primarily in herbivorous animals  Devastated European agriculture in the 19th century  Pasteur isolated organism from infected animal’s blood and cultured them and then used that to infect other animals  Used serial passaging to prove that it wasn’t a chemical Robert Koch  Directly observed w/ a microscope that anthrax bacterium alternates between a growing bacilli and a heat resistant spore  Wrote about how to obtain pure cultures  Proved that microbes retain their characteristics such as shape  Discovered the microbe that causes TB Falco  Yersinia appeared to invade host cells  He isolated DNA from Yersinia and transferred it into E. coil  Found out that there was a Yersinia gene that made a protein called Invasion Diphtheria  Killed large number of children  Discovered that symptoms were caused by a soluble toxin  Isolated antibodies from immunized g pigs and injected them into kids

Lecture 2 8/28:

 Most microbes are about 1 micron or 2 1884- Hans Gram came up with gram stain Function of Peptidoglycan- gives the cell strength (strength comes from covalent cross links) It is proposed that gram negatives come from an event of endosymbiosis of two single membrane bacteria LPS- on outer leaflet of outer membrane = endotoxin

The Challenges of Getting Large Molecules through Membranes The inside of a folded globular protein is hydrophobic, if you somehow put it into a greasy bilayer it will unfold/turn inside out  Protein transporters- solves the chemistry problem  Transport unfolded proteins- solves the size problem  Signal sequences- allow for selectivity  Chaperon proteins- keep the protein in an unfolded or partially folded position while it comes though Virulence factors can… (fate depends on the protein’s “signature”) 1. Be secreted 2. Be modified with a lipid and become part of the membrane 3. Become non-covalently associated with the peptidoglycan 4. Can become covalently associated with the peptidoglycan (must stronger interaction) Sortase – protein attached to outer surface of membrane that recognizes LPXTG sequence and attaches the protein with that sequence on its surface. Used to make something have anything you like on its surface (becomes covalently bonded to the glycine bridge of peptidoglycan) 

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Bacteria have proteins that act as sensors that will degrade unfolded proteins

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To make peptidoglycan you need a transglycolase (puts sugar polymer together) this enzyme is associated with the plasma membrane and transpeptidase

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Listeria degrades its own peptidoglycan

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Controlled peptidoglycan synthesis and degradation occurs on the outside of the cell

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SEC pathway (how things are exported in gram-negatives)

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Also have other types of pathways that are “protein injection” machines that inject virulence factors (Types 3, 4, and 6 secretion systems) see articles and get general idea of how they work

Lecture 4 8/30:   

T2SS how most things exit the cell of a gram negative bacteria T3SS inject proteins directly into other cells “Get a feel” for how the different secretion systems work

Signaling of Bacteria  Use signaling pathways similar to eukaryotes  Serine, tyrosine, threonine (A.As that are phosphorylated)  Signal causes phosphorylation  Often the things that are phosphorylated are transcription factors  What does the sensor kinase respond to and what does the response regulator activate?  How does the signal get turned down? -The RR can inhibit the activity of itself -A target gene of an RR can express a protein that can negatively regulate the response regulator or the sensor kinase Things that can activate signaling  LPS can be altered to have a net positive charge (instead of negative)  Mg2+ levels drop and the signal turns on expression of a magnesium importer  If misfolded proteins are in the periplasm they have to be degraded, there is a sensor kinase that is activated by the hydrophobic regions of misfolded proteins Vibrio cholerae  We know most about proteobacteria because of E. coil  Vibrio is a gram-negative  Modifies its LPS by putting glycine onto one of the lipids at a particular place  This modification makes them more resistant to antibiotics  Has to adhere to the walls of the intestine once ingested, then secrete cholera toxin  Makes flagellum for motility when outside host (inter-host)  Pili are required for it to stick (certain signals tell it to make pili once it is inside host) knows because of signals such as low pH, raised temperature The Toxin  Composed of two different proteins (made in cytosol)  The proteins has to be exported by T2SS  The toxin is AB-5 (interaction between toxin CTX and GM1 on surface of intestinal cell)  After that interaction the toxin is internalized  Now the toxin is inside an endosome inside the cell (still “topologically” outside the cell)

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Toxin from endosome goes to Golgi and then to ER and then into cytosol (retrograde transport, because backwards and this is normal) NaCl is then exported out of cell, gut gets salty and the water follows the salt (diarrhea) Virulence factor: microbial products or strategies that contribute to the ability of the microbe to be transmitted to a new host (why it wants you to have diarrhea) Not all vibrio have genes that encode for CTX Bioassay- inject into something and see if there is a biological response How does Elisa work? The genes for CTX are part of the genome of a phage The genome of vibrio contains two circular chromosomes (small and large)

Comparing environmental and laboratory strains for infectivity  Human shed vibrio is isolated from stool  Co infect infant mouse with this and a lab grown strain  Infection started at 1:1 ratio, determine ratio after 24 hours  If they were still equal the competition index would be 1  If it is greater than 1 then a lot more of the stool derived vibrio was recovered  Then they compare the pattern of gene expression between these two strains...