MICRO420- exam 3

Fungi structure

membrane with ergosterol, chitin layer, B1,3-glucan layer, mannan layer

Ergosterol

fungal equivalent of cholesterol, adds rigidity to cell membrane

B 1,3-glucan

very immunogenic

Candida albicans

dimorphic yeast, causes localized infections like thrush, natural commensal of oral, digestive, and vaginal tracts

Risk for Candida albicans

diabetics and those on steroids, neutropenic people, low immune system

Candida albicans cell wall

masks potential antigens from adaptive immune system but hard for adherence, must have anchor proteins within cell wall

Yeast form of Candida albicans

single celled, commensal

Hyphae form of Candida albicans

extended filaments, invasive form, can penetrate tissues, if phagocytosed it could grow w/in phagosome and puncture cell wall, harder to kill

both forms required for virulence

yeast and hyphae

Als3

Candida albicans, in Als family of protein adhesins, found on hyphae, attach to N-cadherin protein on epithelial cells and trigger endocytosis of hyphae

Sap5p Protease

important for invasion, cleaves E-cadherin when secreted by hyphae, disrupts tight junctions between cells, hyphae can grow deeper into tissues

Candida albicans virulence

secrete degradative enzymes to destroy tissues and gain food
proteases, lipases, phospholipases, etc

Dectin-1

neutrophil, recognizes B1,3-glucan, turns on immune response

AIDS patients and Candida albicans

have no T cells but do have neutrophils, can respond to infection

Tyrosol

tyrosine derivative used by yeast as signal to drive to hyphae form

Farnesol

inhibit yeast transition to hyphae, similar structure to homoserine lactones which can be sensed by candida as farnesol, increased gram neg bacteria prevents hyphae

Ergosterol as a Candida drug target for Azoles

first line drug fluconazole preferred, target step in ergosterol synthesis, 50% or more of hospital isolates were resistant due to drug pump or point mutation

Ergosterol as a Candida drug target for Polyenes

bind ergosterol in membrane and destabilize membrane, amphotericin B, toxic and can bind cholesterol

Echinocandins

target B1,3-glucan formation by inhibiting linkage enzyme to form polymer, cas profungin: "fungal penicillin", resistance is rare but due to point mutation

Candida glabrate

not as common as albicans but more mortaility, yeast form only, naturally resistant to azoles, CDr1 drug pump

Cdr1

C. glabrata drug pump, controlled by transcription factor Pdr1

Pdr1

C. glabrata, activator of Cdr1, mutations cause increase of Pdr1

A toxin portion

enzymatic/biochemical function

B toxin portion

receptor binding, dictates cellular specificity, made in cytoplasm and must be transferred outside of membrane completely

Simplest AB toxin

AB is single protein, protein gets cleaved after translocation, AB portion held together by disulfide bonds

Other AB toxin

A and B are encoded by separate genes, still held by disulfide bonds, often 5B subunits to 1A

AB toxin mechanism

B binds specific receptor (glycolipid or glycoprotein), receptor bound with toxin is endocytosed, drops pH of vesicle, dissociation of A from B, A somehow leaves vesicle and acts on its target inside host cytoplasm

Function of A toxin portion

often ADP ribosyltransferase activity, sometimes protease

C. diptheriae physiology and symptoms

gram pos, non motile, non spore, rod, aerobic, makes DT, human reservoir, colonizes throat

sore throat, fever, loss of apetite

HB-EGF

c. diptheriae target, heparin-binding epidermal growth factor precursor, on lots of cell types

DT toxin

B portion is specific to HB-EGF, bacteria very localized in throat, limits toxin spread
binds HB-EGF, endocytosed, acidification of vacuole, partial unfolding and A leaves, A enters cytoplasm, ADP ribsylation activity, targets EF-2, irreversible halt of protein synthesis, cell death

EF-2

target of DT A portion, elongation factor 2, involved in translation

DT and Heart/neurons

have lots of HB-EFG, if toxin reaches here: human death

C. diphtheriae and pseudomembrane

toxin kills host cells and triggers inflammatory response, innate cells kill bacteria

pseudomembrane forms and adheres to underlying tissue, if lifted: bleeding, often used as diagnosis

DT toxin genes

single protein encoded by 1 gene, AB portion cleaved after translation, gene encoded by prophage and must have prophage to get pseudomembrane

Diphtheria vaccine

DTaP or DT, antibodies are only to the toxin, can still be colonized by bacteria

Balto

Nome, Alaska in 1920
diphtheria outbreak among kids, antitoxin was 100 miles away in Anchorage
sled dog relay, Iditarod

Vibrio cholerae physiology

gram neg, rod, polar flagella
associated with invertebrates and crustaceans, form biofilms
has 2 chromosomes
1 is for house keeping and other has metabolic enzymes and virulence traits
transmission by contaminated water

Cholera disease risk

malnourishment and infrastructure issues, children under 5
if malnourished, stomach pH not low enough to kill cholera
70% mortality rate
must drink 1L of water per hour or death by dehydration

Cholera history

similar disease description in ancient texts, first disease reported to US government

Cholera serotypes

O1 antigen, Classical and El Tor, O139 not associated with disease until 1930s

El Tor cholera

less severe than Classical but greater dissemination, spread during Haitian earthquake

How you get cholera

consume contaminated food or water, colonizes small intestine, adheres to intestinal cells via TCP

TCP

toxin coregulated pilus, made of TcpA subunits, TcpA encoded on Vibrio Pathogenicity Island-1

Cholera toxin mechanism

toxin encoded on ctxAB operon, RBS is closer to consensus for B than A so 5B:1A, CTA is cleaved (CTA1 is enzymatic), CTA2 binds B and is held together by disulfide bonds, CTB binds GMA1, endocytosis of receptor with toxin, dissociation of CTA, CTA1 has ADP ribosyltransferase activity, targets Gs alpha, CTA1 adds ADP-ribose to GS alpha in GTP bounds form, Gs alpha stays bound to GTP which activates adenylate cyclase and converts ATP to cAMP, ions flow out and so does water

Where is CtxAB operon/genes found?

CTX prophage, El Tor variants can go lytic, CTX receptor is TCP

ToxT

Cholera, transcriptional activator for ctxAB and tcpA expression, encoded on VPI-1
highly regulated, only translated at 37C, RBS is in 4U thermometer

Weber paper

Cholera, translational fusion, converted U to G, stem couldn't melt at 37C, avirulence after 2 nt change

Cholera vaccines

live but attenuated, oral, whole cell
fading immunity

Clostridium difficile

gram pos, anaerobic, spore former, intestinal colonizer
recognized as disease causing pseudomembranous colitis

C. diff risk factors

hospitals and nursing homes, outgrowth due to antibiotics or spore ingested and bile acids cause germination

C. diff toxin mechanism

Produce 1 or both toxins TcdA or TcdB, bind receptor endocytosed, transfers glucose from UDP-glucose to small G proteins, when glucose attached, G can't bind nucleotides, no actin polymerization, cells round and detach from tissues, cell death, neutrophils respond and water follows

PaLoC

pathogenicity island where c. diff toxin genes are located

TcdA receptor

sulfated glycolipids

TcdR

encodes alternative sigma factor needed for toxin gene expression

TcdC

encodes anti sigma factor, binds TcdR so no toxin transcription

hypervirulent C. diff

produce lots of toxin, very severe disease, mutation in TcdC

Metronidazole

for C.diff, works for anaerobes only, prodrug, organism enzymes activate drug and forms nitrogen radicals, build up of toxin metabolites that damage DNA

Vancomycin

for C. diff, resistance is an issue in the clinic, prone to relapse

Fecal transplants

effective treatment for patients with relapsing C. diff, microbiome initially looks like donor but reverts to recipient over time

Sec system

general secretatory system, in gram neg and gram pos
transports from cytoplasm to periplasm
sec signal sequence on n-terminus of protein
exports unfolded cytoplasmic proteins

Sec mechanism

as protein emerges from ribosome, SecB binds signal sequence, SecB transfers protein to SecA, SecA hydrolyzes ATP and uses energy to drive protein through SecYEG membrane complex channel

Gram pos Sec protein folding

protein folds either as it goes through complex channel or when it reaches surface

Gram neg Sec protein folding

some proteins fold in channel, most fold in periplasm

SRP

signal recognition particle, bind protein as it emerges from ribosome, pulls protein and ribosome to the membrane, SRP transfers protein to Ftsy, hydrolyzes GTP, inserts protein into SecYEG

TAT transport

twin arginine transport, found in some gram pos and gram neg, fully folded proteins transported across cytoplasmic membrane and into periplasm, can have cofactors bound
polar n-terminus with SRR motif and serine-2-arginine hydrophobic residue

TatBC

recognizes signal sequence, interacts with TatA

TatA

must form complex with TatBC for transport, energized by PMF

Sec/TAT Dependent SS

type II, V, IX
periplasm through outer membrane

Sec/TAT independent SS

I, III, IV, VI
cytoplasm through outer membrane

Type II SS

Sec dependent, most common way to get across outer membrane
ATP hydrolysis in cytoplasm energizes transport
cholera toxin transported this way

Type V SS

Sec dependent, autotransporter, BAM or SKP as chaperone in periplasm

BAM complex

Beta-barrel assembly machinery, help orient autotransporter so that its Beta-barrel inserts properly
sIgA protease from N. gonorrhoeae is an autotransporter

Type IX SS

Sec dependent, proteins and degradative enzymes are anchored to LPS

Type I SS

Sec independent, powered by ATP hydrolysis, exported proteins have glycine rich C-terminus signal sequence

Type I SS structure

Inner membrane has ABC transporter, atp hydrolysis happens there
Periplasm has membrane fusion protein
Outer membrane has 3 monomers forming a beta-barrel
unfolded proteins enter and are folded during and post transport

Arc-Tol Complex

Inner membrane: ArcB
MFP: ArcA
Outer membrane: TolC
can transport proteins and antibiotics, are drug efflux pumps

Type III SS

Sec independent, molecular syringe, crosses IM OM and Euk membrane, adhesin must bind host cell receptor to be in close proximity, found on PAI or plasmids, species specific and core proteins, effectors are proteins transported via type III SS

Type IV SS

Sec independent, transports DNA and proteins, some can also cross eukaryotic membrane

Argobacterium tumefaciens

uses type IV SS, injects oncogenic plasmid DNA and proteins into plant cells, DNA encodes altered metabolites, results in crown gall disease and plant tumor

Type VI SS

used to kill other bacteria, phage-like tail fibers, effectors can be a part of secretion apparatus, often used in cell competition, producer must have immunity proteins, can influence invasion and adhesion

SecA2

in gram positives, second copy of SecA but is usually shorter, results in proteins with altered secretion patterns

aSec

in gram positives, accessory Sec system, additional copies of Sec, used for heavily glycosylated proteins

Sortase

in gram positives, attach protein to gram pos cell wall, recognizes LpxTG motif in secreted proteins

SrtA

housekeeping sortase, cleaves between T and G and LPxTG and attaches to lipid 2 peptidoglycan precursor

Type VII

mycobacterium and some gram positives, important for growth in macrophages, BCG vaccine targets this

Yersinia

gram neg, facultative anaerobe, found outside humans

Y. pestis

capsulated at 37C, spread by fleas, 200 inactivated genes and 2 plasmid

Y. pseudotuberculosis

no lung disease but GI issues, 99% similar rRNA to y. pestis, spread fecal-oral

Y. enterocolitica

GI issues, spread fecal-oral

Justinian plague

first great yersinia plague, DNA extracted from teeth showed microbe with unique nt differences

Black death

1300-1400, first time quarantining ships, TarTars besieged Kaffa and catapulted dead soldiers, 1/3 of Europe died

Alexander Yersin

took fluid from buboes and injected into guinea pig, saw infection

Simond

found fleas were cause of plague spread, found yersinia within fleas

Y. pestis and fleas

pestis replicates via nutrients in blood meal and form biofilm (has 3 genes inactivated that block biofilm formation), biofilm blocks flea from eating, regurgitates biofilm into lymphatics where yersinia settles in lymph nodes and turns off immune response using T3SS
humans die due to bacterial burden

pYV/pCD1

plasmid in all 3 strains, encodes T3SS, genes expressed at 37C, Ysc, ATP dependent transport, Yops are effectors that prevent cytoskeletal rearrangement and phagocytosis

Ysc

on pYV/pCD1, needle system, remains closed until across eukaryotic membrane

YopE

inactivates Rho GTPases by maintaining it in the inactive GDP bound state, blocks actin polymerization and phagocytosis

YopH

phosphotase, removes phosphates from proteins involved in phagocytosis

YopJ/P

targets NfkB, acetyltransferase, reduces expression and release of proinflammatory cytokines