microbio 3

bacteria

host interactions
- symbiosis
- commensalism
- mutualism
- parasitism
- normal microbiota and hosts are usually mutually beneficial (rely on healthy microbiota)
- normal microbiota often prevent colonization by pathogens
- bacterial products are beneficial to the host

symbiosis

living together

commensalism

one benefits, the other is not affected

mutualism

both organisms benefit

parasitism

one benefits, the other is harmed
- usually a microbe is affecting a much larger organism

infection

a pathogen grows and multiplies within or on another organism
- infection doesn't always cause disease
- most infections removed by immune system

infectious disease

due to the presence and multiplication of pathogens, part or all of the host is not capable of performing its normal function
- normal function is comprimised

steps of bacterial infections

- attach and invade host tissue
- suppress host defenses
- acquire nutrients from the host
- propagate in the hosts
- transmit to a new host

host-pathogen interaction

- pathogensis
- parasite
- pathogen
- pathogens and parasites enter and infect their animal and plant hosts in different ways
- some microbes enter into a latent state during infection
- pathogenicity

infection

entry of pathogen or parasite
- infection doesn't always cause disease
- most infections removed by immune system

primary pathogens

have ability to penetrate host (health) defenses

opportunistic pathogens

cause disease only in compromised hosts
- immune system defective
- break in tissue allows organisms access to new site
- loss of other microflora allows organism to bloom

some microbes enter into a latent state during infection

- organism can't be found by culturing
- may remain dormant for years, then suddenly emerge to cause disease (ex: herpes virus)
- they're there but don't come out

pathogenicity

- ability to be a pathogen
- the ability of an organism to cause disease
- defined in terms of infectivity and virulence
- infectivity
- virulence
- you either are a pathogen or you're not

infectivity

how easily an organism causes disease

virulence

measure of how severe the disease is
- ebola virus: highly virulent: fatal (70%)
- rhinovirus: highly infective but low virulence: not fatal
- all pathogens but varies in degree of virulence (low or high)
- usually additive

virulence of organism

- we are quanitfying this
- measure of the degree or severity of disease
- determined by genetic makeup of organism
- infectious does = ID50
- rate of lethal infections
- lethal dose

infectious dose = ID50

# of organisms needed to colonize 50% of hosts
- determine number of microbes required to cause symptoms in half of an experimental group of hosts

lethal dose = LD50

different because asks the amount needed to kill the plant instead of just infecting it
- number of organisms to kill 50% of hosts

microbe with low LD50

few microbes needed to kill organisms
- highly virulent --> more virulent than high LD50

microbe with high LD50

more microbes needed to kill organism
- need more microbes to kill

infection cycle

- for disease to spread, pathogen must pass from one animal (organism) to another
- route an organism takes to spread disease is known as the infection cycle
- must go from one host to anotherin

infect spread via

- direct transmission
- indirect contact

indirect contact

- contact with formites
- horizontal

contact with formites

- inanimate object through which pathogens can be relayed to host
- ex) spraying groceries during covid - "left behind" bacteria/germs

horizontal transmission via vectors

- carried by others
- mosquitoes- yellow fever, malaria
- reservoir for disease organisms
- may not show disease symptoms
- ie. animal, bird, insect that harbors the pathogen
- rats were harboring bacteria --> bubonic plague

pathogens portals of entry

- pathogens use portals of entry best suited to their mechanism
- mode of entry depends on pathogen
- food-borne pathogens
- airborne pathogens
- mucosal surfaces
- wounds (skin)
- parenteral route

food-borne pathogens

ingested through mouth --> colonize intestine

airborne organisms

infect through the respiratory tract

mucosal surfaces

mouth or other mucosal area

parenteral route

- needle use / dwelling IV
- injected into the organism bloodstream

virulence factors

- individual characteristics (genes) of a pathogen that allow the pathogen to invade hosts and cause disease
- all virulence factors enhance the disease-producing ability of the pathogen
- pathogens can be distinguished from avirulent organism by the presence of virulence factors
- help establish the organism in the host and alter host functions to cause disease
- encoded by virulence genes
- include toxins, attachment protein, capsules, other factors (avoid host innate & adaptive immune systems)

virulence factors enhance the disease-producing ability of pathogen

- facilitate bacterial attachment and invasion
- promote bacteria growth
- promote disease symptoms
- evade host defense mechanisms
- allows better survival for bacteria

virulence genes

- encode factors allowing pathogen to invade host
- include toxins, attachment protein, capsules, other factors (avoid host innate & adaptive immune systems)
- virulence factors

pathogenicity islands

- section of genome
- contain multiple virulence genes
- often encode related function
- eg. protein secretion system, toxin production
- transferred as a block from other organisms (HGT)
- often flanked by phage or plasmid genes
- a lot of these are moved around by viruses
- often have GC content different from rest of genome

microbial attachment

- human body expels invaders
- flush out invaders before they effect
- mucosa, dead skin constantly expelled
- liquid expelled from bladder
- coughing, cilia in lungs
- expulsion of intestinal contents
- bacteria must adhere to host tissue
- pili (fimbriae)
- adhesins
- capsules
-biofilm

pili (fimbriae)

- hollow fibrils with tips to bind host cells
- hairlike surface appendages
- protein at the tip binding to host receptor

adhesins

surface proteins bind host cells
- any microbial factor that promotes attachment
- anything that helps bacteria attach

afimbriate adhesins

bacteria surface proteins that promote attachment, but are not pili (bacterial hemaggulutinnin)

types of pili

both involved in attachment
- different pili from different bacterial species have been classified based on phenotypes
- type I pili
- type IV pili

type i pili

adhere to mannose residues on host cell surfaces (static)
- doesn't extend/retract
- made of repeats of the pilA subunits (focus)

type iv pili

- assemble on cell surface, dynamic (continuously assemble and disassemble)
- motor allows bacteria motility
1) Pilin PilA is made as a preprotein and inserted into inner membrane
2) PilD is a peptidase that removes a leader sequence from a PilA preproteins prior to pilus assembly
3) PilT and PilF are NTP-binding proteins that provide energy for retraction and assembly
4) The secretin PilQ is required for the type IV pils to cross the outer membrane

type IV pili and motility

- involved in gliding or twitching motility
- spider man motility
- bacteria make type IV pilus --> depolymerize --> pulls it back in through the cell
- pilus has become shorter
- bacteria has moved through the cell

twitching motility

- other bacteria use non-pilus adhesins that mediate binding to host tissue:
- streptococcus pyogenes: M protein
- bordetella pertussis: pertactin

biofilms and infection

- bacteria can attach to surfaces, forming a biofilm
- make it more difficult to expel
- biofilms play important roles in chronic infections (cystic fibrosis, dental plaque, chronic otitis media, osteomyelitis, chronic wound infections)
- biofilms are often resistant to antibiotics
- incomparsion to their platonic bacteria

toxins

- exotoxins
- endotoxins
- cause damage to host cell
- goal: release nutrients so they can consume

exotoxins

- secreted out the cell --> impacts another cell (host cell)
- kills host cells --> releasing nutrients
- many microbes secrete exotoxins after attachment
- bacteria secrete exotoxins (cytotoxin)
- cytotoxin kills host's cells

endotoxins

- only found in and made by gram- bacteria
- non-protein toxic compound
- hyper-activate host immune systems to harmful levels
- cause endotoxic shock
- present in gram neg. bacteria
- part of LPS; released when gram neg. bacterial cell is broken down
- dead gram- bacteria release endotoxin (lipid A) which includes effects such as fever, inflammation, diarrhea, shock, and blood coagulation
- present in lippolysaccharide of outermembrane
- lipid A released as bacteria die - causes massive release of cytokine from host cells
- triggers fever, shock, and death
-

bacterial toxins

- following attachment, many bacteria can secrete protein toxins called exotoxins
- exotoxins fall into five broad categories

toxins subvert host function

5 categories of protein exotoxins
1) cell membrane disruption
- cause host cell membrane leakage
- bacterium pokes hole in membrane --> leakage
- hemotoxin
2) block protein synthesis
- target eukaryotic ribosomes --> not prokaryotic
3) block 2nd messenger pathways
4) superantigens over-activate the immune system
5) proteases cleave host proteins

cell membrane disruption

alpha toxin, cap and rim, stem

inhibit protein synthesis

shiga toxin, receptor mediated endocytosis via Gb3, mRNA, ribosome, NH3

activate second messenger pathways

stable toxin, Gtp --> cGMP, Na+, Cl-, H2O

alpha toxin

- hemolysin
- category 1 of toxin
- the hemolytic alpha toxin is produced by straphylococcus aureus
- forms transmembrane, seven-member pore in target cell membranes
- can determine if patient has strain through blood plating
- disrupts cells (red blood cells) by forming pores in the membranes

shiga toxin

- disable ribosome
- fits into category 2. of exotoxin
- produced by shigella flexneri and e. coli O157:H7 (jack in the box microbe)
- O157:H7 gastrotestional disease that was found in jack in the box
- disrupt protein synthesis by destroying 28S rRNA found in ribosomes
- disables ribosomes from translating proteinsA

AB toxin

- b subunit
- a subunitb

B subunit

binds to host cell
- looks the same outside but can contain whatever
- delivers A subunit to cytoplsm
- delivery module (not toxic)
- often 5 B subunits form a pore for A entry

A subunit

has toxic activity
- ADP-ribosyltransferase
- toxin to eukaryotic cells

ADP-ribosylating toxins

- diphtheria toxin - category 2
- cholera toxin - category 3
1) the 5B:1A toxin complex binds the ganglioside GM1 on host membrane lipid rafts
2) toxin is endocytosed
3) the phagosome containing CT is taken to the ER
4) the A1 subunit is removed from the B subunits and exported into the cytoplasm
5) the A1peptide attaches an ADP ribose to an amino acid within the host G protein that regulates adenlate cyclase
6) cyclic AMP levels rise and activate ion transport systems causing an electrolyte imablance
- water from the cell follows the ions, causing diarrhea

diphtheria toxin - category 2

- disrupts protein synthesis
- made by corynebacterium diphtheriae
- ribosylates elongation factor 2
- blocks ribosome function; cell dies
- forms pseudomembrane over trachea

chlorea toxin - category 3

- get it from dirty, unfiltered water
- made by vibrio cholerae
- quorum sensing system
- ribosylates to overactivate adenylate cyclase
- adds a ribose
- cAMP activates ion transport; water follows
- water goes into gut
- uncontrollable diarrhea
- very water --> dehydration

anthrax toxin

- made by Baccilus anthracis
- good spore formers
- has tried to be used as bioweapon
- two active toxins
- edema factor
- lethal factor

edema factor

raises cAMP levels
- cause fluid secretion and tissue swelling

lethal factor

cleaves protein kinase
- blocks immune system from attacking

bacillus nathracis and anthrax toxin

1. protective antigen subunit PA is made as a single peptide
2. PA binds to host cell surface where a human protease cleaves off the orange part showing in part B
3. seven PA fragments autoassemble in the membrane to form a pore
4. the other two components of anthrax toxin- EF and LF- bind to the ring and are carried into the cell by endocytosis
5. EF and LF are expelled through the PA pore into the cytoplasm

Endotoxin - LPS

lipopolysaccharide LPS
- only made by gram- bacteria
- has LPS on outer layer
- component of outer membrane
- LPS contains endotoxing
- activates inflammatory response
- imefmune system recognizes it as foreign
- can cause toxic shock (endotoxic)
- barrier of outside and inside

effect of neisseria meningitidis endotoxin

some antibiotics lower bacteria # but this causes large release of LPS
- little pools of blood under skin

protein secretion pathways

- many pathogens use specific protein secretion pathways to deliver toxins
- close to host cell or in host cell
- proteins may not kill cell but redirect host signaling pathways in ways that benefit microbe
- many secretory systems have structural resemblance to other innocuous systems
- type 1, 2, ,3, and 4

type 1 secretion pathway (sec)

- general secretion pathway
- one step process of secretion
- consists of 3 protions
- ATP binding cassette protein
- periplasmic protein: membrane fusion protein
- outer membrane channel protein
- hemolysin (alpha toxin) secreted through type 1 secretion system

type 2 secretion system

- use pilus like extraction/retraction mechanism to push proteins out of cell (similar to type 4 pili)
- modified for secreting proteins
- can extend and retract

type 3 secretion system T3SS

- injects proteins directly into host cell
- hypodermic needle
- one step injection (similar aspects to flagella motor)
- injected proteins (type 3 effectors)
- essential for gram- pathogens
- e. coli, salmonella, yersinia pestis, pseudomonas aeruginosa and syringae

type 4 secretion system

- type 4 similar to conjugation pilus
- secrete both DNA and proteins
- can extend and retract - motility
- transport proteins or DNA either:
- outside of cell or into target cell

type 3 effectors T3SE

salmonella injects over 13 toxins
- induce tight attachment to host
- cause host to engulf bacteria
- induces actin rearrangement in cell
- suppress host immunity
- causes diarrhea in host
- if we disable it would allow for many diseases to be controlled

crown gall

plant cancer
- uncontrolled cell growth implemented by bacteria

T4SS - T-DNA

- crown gall disease: creates a home for bacteria that they can only live in and has food production
- T-DNA: T region
- oncogenic genes
- opine synthesis (specialized amino acids)
- Ti plasmid
- opine catabolism
- virulence region: T4SS

beneficial uses of A. tumefaciens T-DNA integration

- basis of how to make transgenic plants
- agro-mediated plant transformation
- efficient way of getting transformed plants
- agro-mediated transformation is used for many different species including plants and fungi
- disarmed Ti plasmids are used to introduce genes of interest into plant host chromosome

helper ti plasmid

- need functions of helpers to help clone into smaller plasmid
- divides plasmid into 2 because its really big

other virulence factors

siderophores, hormones, and ice nucleation

siderophores

- bacteria produced iron catchers
- steals iron from host

ice nucleation

- certain infected crops fell more to frost damage
- economic loss
- plant pathogenic bacteria can catalyze ice formation by serving as biological ice nuclei
- water remains in supercooled state to temps as low as
-40c in absence of ice nuclei
- need for ice to form around
- presence of certain bacteria, freezing can occur at much higher temps like -2 to 10c

applications of ice nucleation

- snowmax
- freeze dried formulation that serves as source of ice nuclei for snow making (ski resorts)
- frost ban
- ice- p. syringae mutant thats sprayed on frost sensitive plants to out compete ice+ strains
- niche displacement prevents damage

antibiotics

- infections considered minor now killed most patients 60 years ago
- importance of treating disease using antibiotics was recognized early 1940
- what are they and where do they come from?
- compounds produced by one species of microbe can inhibit growth or kill other microbes
- soil dwellers and other bacterias in soil biome

discovery of antibiotics

- usefullness of molds known to ancients
- fleming found penicillin in 1929
- drug forgotten --> thought to be unstable
- penicillin: bread mold paste
- put paste on patients
- howard florey rediscovered penicillin 1940
- purified penicillium

gerhard domagk - sulfa drugs
(antibiotic discovery)

- drug taken (prontosil) doesn't affect bacteria
- drug metabolized by body into sulfanilamide
- sulfanilamide is an analog of para-aminobenzoic acid (PABA)
- drug stops bacterial growth by inhibiting the conversion of PABA --> folic acid

prontosil

- screened for on agar plate didn't affect bacteria
- found to be successful in animal trials

sulfanilamide

- infects with synthesis
- some antimicrobial agents are initially inactive until converted by the body to an active agent

PABA --> folic acid

- paba is precursor to folic acid
- folic acid not synthesized by humans, dietary supplement
- bacteria don't transport folic acid
- need to make from precursor
- if they can't make it, they can't grow it

normal folic acid formation

P, PABA, G components go into enzyme --> synthesis --> folic acid

folic acid formation blocked

P, SFA, G components go into enzyme --> P doesn't fit properly --> no synthesis --> components go back out

selective toxicity of antibiotics

- must affect target organisms
- shouldnt affect humans or other animals
- many have side effects at high concentration
- drug should affect microbial physiology

side effects at high concentration

- chloramphenicol interferes with ribosomes
- high levels interferes with red blood cell making
- can cause allergic response
- antibiotics foreign substances in our bodies

drug affects microbial physiology

- doesn't exist or is greatly modified in humans
- peptidoglycan
- differences in ribosome structure
- prokaryotes vs eukaryotes
- biochemical pathway missing in humans/animals

spectrum of activity

- broad spectrum
- narrow spectrum
- source of antibiotics

broad spectrum

effective against many species
- can affect your native bacteria
- doctor doesn't need to know specific bacteria

narrow spectrum

effective against few or a single species
- need to know what patients infected with (time cost)

source of antibiotics

- most discovered as natural product
- some modified by artificial means
- increase efficacy, decrease toxicity to humans
- synthetic chemotherapeutic agents
- clinically useful but chemically synthesized

types of bacterial compounds

- bactericidal
- bacteriostatic antibiotics

batericidal

antibiotics kill target organism

bacteriostatic antibiotics

- prevent growth of organism
- cant kill organism
- immune system removes infection
- many drugs only affect growing cells
- inhibitors of cell wall synthesis
- only effective if organism is building new cell wall (penicillin)
- have to take antibiotic until all cells leave stationary phase

minimal inhibitory concentration

- lowest concentration that prevents growth
-varies for diff bacterial species
- test by diluting antibiotics
- lowest concentration wit no growht
- may have some living but non-growing organisms
- plate liquid without antibiotic
- no colonices = minimal lethal concentration