Bacterial Pathogenesis Final

Gram positive properties

thick peptidoglycan, retain 1st crystal violet gram stain (purple), contain teichoic acids, NAG-NAM cross links, NO outer membrane

gram negative properties

thin peptidoglycan, retain safranin (pink), has an inner leaflet made of phospholipids and an outer leaflet containing the lipopolysaccharide (LPS), periplasm

LPS (lipopolysaccharide)

Lipid A → endotoxin, core polysaccharide, O antigen (variable)

acid fast properties

arabinogalactan layer, mycolic acid, cytoplasmic membrane, thin peptidoglycan, no outer membrane, thick and waxy coat

Be able to provide 2 similarities and 2 differences between Prokaryotic and Eukaryotic cells

Similarities:DNA, Cell membrane

Differences: Prokaryotes don't have a nucleus, Eukaryotes have a nucleus, no plasmids, Eukaryotes have bigger ribosomes

Cytosol

fluid inside the cell; soluble gel-like network enclosed by cell membrane

Cytoplasm

cytosol + organelle structures

Organelle

any subcellular specialized structure

Cell membrane

phospholipid bilayer; encloses cytoplasm

Outer membrane

extra layer in gram negative

Cell wall

rigid peptidoglycan; covers cell membrane

Nucleoid

DNA region in prokaryotes, contains chromosome in form of looped coils, nonmembrane bound

Flagellum

Mobility structure, random run/tumble, extracellular organelle

Pili

Attachment structures

building blocks

monomers such as amino acids, sugars, nucleotides

macromolecules

polymers such as proteins, lipids, DNA

simple diffusion

no energy input, high → low concentration gradient, involves small, nonpolar molecules, no channel required

facilitated diffusion

no energy input, high → low concentration gradient, involves a channel or carrier proteins, polar or charged molecules like Na+, K+, glucose

primary active transport

requires ATP as energy, travels against gradient low → high, uses pumps, ex. Na+/K+ ATPase

secondary active transport

coupled, requires energy made by primary transport, one substance goes down gradient and another goes up, either symport or antiport

structural components of phospholipid

-hydrophilic head

-Hydrophobic tails

-Glycerol backbone

-Ester bonds between glycerol and fatty acid, phosphoester between glycerol and phosphate head

-Spontaneously form a bilayer with heads outward and tails inward

3 roles of membrane proteins

- Transport (channels, pumps):Simple diffusion: no protein, down gradient, Facilitated diffusion: uses protein, down gradient, Active transport: against gradient, requires energy (ATP or Ion Gradient)

- Signal detection (receptors)

- Energy (ATP generation)

- structural support

How do bacteria alter their membrane composition when exposed to low vs high temperatures?

- Cold: more unsaturated fatty acids, shorten fatty acid chains, decrease hopanoids, increase cyclic structures

- Hot: more saturated fatty acids, lengthen chains, increase hopanoids

Cell wall composition and antibiotics that target cell wall

- Peptidoglycan: NAG-NAM crosslinks

- Antibiotics (B-lactams) like penicillin target this by binding penicillin-binding proteins (PBPs) and blocking transpeptidation.

- Vancomycin binds D-Ala-D-Ala and blocks it from adding new subunits

chemotaxis

- Chemotaxis is the movement of bacteria in response to chemical gradients.

- CCW rotation → Run (straight) - flagella bundle together toward chemoattractant

- CW rotation →Tumble (change direction) - flagella fly apart and reorient

- Biased random walk

correlation vs causation

- Correlation does not equal causation

- Correlation indicates that two variables move together, suggesting a relationship (implies 2 variables are not independent)

- Causation means one variable directly produces a change in the other

Chain of Infection

explains how pathogen is transmitted from infected individual to naïve host; infectious agent, reservoir, portal of exit, mode of transmission, portal of entry, susceptible host

Koch's First postulate

The microbe is found in all diseased individuals, but not in healthy ones

Koch's 2nd postulate

The microorganism can be isolated from the diseased host and grown in a pure culture

Koch's 3rd postulate

The cultured microorganism causes the same disease when introduced into a healthy, but susceptible, host (causation!)

Koch's 4th postulate

The same microorganism is re-isolated from the newly infected host

Koch's 5th postulate

Prevent exposure of the pathogen or eliminate it, you prevent disease

Koch's first molecular postulate

1a. pathophenotypic trait should be observed only in virulent strains not avirulent strains

1b. gene that contributes to virulence should be present in virulent strains not avirulent

Koch's 2nd molecular postulate

2a. Inactivation (knockout) of the gene reduces or eliminates virulence

2b. Pathogen is isolated by cloning and put in a avirulent strain (wild-type)

Kochs 3rd molecular postulate

3a. Replace wild type with the mutant, than virulence is restored

3b. Disruption of cloned gene in new virulent bacterium should attenuate it

koch's 4th molecular postulate

The gene should be expressed by the pathogen at some point during the infectious process

pathogen

cellular disease causing agent

what are all pathogens

parasites

pathogenicity

ability of a pathogen to cause infection, qualitative

virulence

degree to which a pathogen can cause disease, quantitative concept, continuium,

colonization

ability to inhabit a niche

infection

pathogen colonized and starts to harm, no signs or symptoms

disease

symptoms and signs, normal processes are impaired

outbreak

rapid increase in number of cases in a limited area

endemic

baseline levels of disease/infection

epidemic

increase in cases above endemic levels over a larger area

pandemic

epidemic that has crossed continents

viroids

infectious nucleic acids

prion

infectious protein

infectious dose

dosage of pathogen needed to cause an infection

What does the ID and # mean in ID50

Infectious dosage, 50% of host infected

parasite

benefits at expense of host

are all parasites pathogens?

no

primary infection

initial infection the host encounters

secondary infection

pathogen colonizes immunocompromised host (primary), the direct result of the primary infection

prevalence

total number of cases of an infection or disease, Sum of all preexisting and new cases

Incidence

refers to only new cases of a disesase

morbidity rate

rate at which it occurs

mortality rate

death rate

mechanisms that the immune system uses to ward off pathogens

- Mucus is a secretion that traps bacteria and viruses, preventing them from reaching the underlying tissues

- Lysozyme is an enzyme found in secretions which break B1,4 linkages between NAG-NAM of the bacteria cell wall

- Lactoferrin binds available iron to deprive bacteria of nutrients

- Cilia use ATP hydrolysis to beat upward and expel pathogen

- sIgA modifies carbohydrates

mechanisms that pathogens use to counteract the immune system

- Modify peptidoglycan by adding acetyl groups → resist lyozyme

- Produce mucinase → break mucus, microbes are no longer trapped

- IgA protease → evade antibodies, pathogens produce IgA proteases that cleave sIgA antibodies at the hinge region which prevents destroys antibodies and blocks attachment

- Use siderophores to steal iron

Type I pili

- pili extends from bacterial surface, binds to mannose receptors, form tight, stable attachment (static), chaperone-usher mediated

- Protein comes in the inner membrane unfolded at its N terminus

- The unfolded protein goes through the Sec pathway and attaches to papD

- PapD is the chaperone that brings the protein to the site of papC

- PapC is located on the outer membrane and is the usher. It enables protein subunits to build on each other via the papC porin channel

- Once complete, papA (major subunit) begins to assemble a ring like structure and assembles the pilus

papH finishes the assembly, marking termination

Type IV pili

- twitching motility, pili continuously retract, this pulls the bacterium forward

- Pilin pilA is made of a preprotein and inserted into the membrane as unfolded

- It goes through the sec pathway and pilD peptidase removes the leader sequence from pilA preproteins prior to pilus assembly

- pilT and pilF are NTP binding proteins that provide energy for retraction and assembly. pilF is an extension ATPase and aids in fiber formation. pilT is a retraction ATPase.

- Fiber goes through pilQ pore which the pilus can exit

formation of biofilms

- Structured bacterial communities which start out as reversible attachment

- Protected by matrix- if it becomes irreversible it will form an EPS containing polysaccharides, enzymes, structural proteins, etc

- Harder to treat- reversible it can be broken apart, however, if it becomes irreversible, the only ways is due to environmental signals and lack of nutrients

- Quorum sensing- small chemical signals, when signal reaches a threshold all the cells together either turn on and off a gene expression

mechanisms used by intracellular pathogens to survive and proliferate

- Escape phagosome - pathogen break out of the phagosome and then move throughout the cytoplasm into adjacent cells by forming actin tails

- Prevent lysosome fusion - pathogen remains in the phagosome and prevents fusion with the lysosome. The pathogen gets expelled into the extracellular space and is engulfed by a macrophage and survives within the phagosome. The macrophage travels to regional lymph nodes and disseminates through the circulatory system.

- Survive inside lysosome - (phagosome-lysosome fusion and replicate resulting in inclusion bodies)

Gastrointestinal pathogens

- Pathogens in the mouth can damage teeth, gums, and disseminate to other tissues and organs

- Oral microbiota was discovered by Antoine van Leeuwenhoek who examined his own dental plaque

- Dental caries - caused by lack of good hygiene, genetics, tooth decay / cavities

Streptococcus mutans

- Dental cavities are most frequently caused by streptococcus mutans

- Gram positive cocci, opportunistic pathogen

Facultative anaerobe

- Natural resident of oral microbiota and mutualistic relationship with C. albicans.

- C. albicans → farnesol → stimulates S. mutans hyperproliferation → This forms biofilm, accelerates tooth decay, and ferments to produce lactic acid.

- Lactic acid lowers pH and demineralizes enamel, resulting in cavities.

- A biofilm can form once glucosyltransferases convert sucrose → glucose + fructose and polymerize into a biofilm.

gingivitis

Porphyromonas gingivalis, Gram negative, anaerobic bacteria

Virulence factors used in gingivitis

- collagenase (breaks down connective tissue to weaken matrix and loosen tooth)

- gingipain (endopeptidase that cuts internal peptide bonds) inhibit immune response

- Uses blood agar with RBCs and bacterium appears black

- Blood agar - extracts heme

H. pylori

- survives in stomach acid

- Gram negative, spiral shaped, tuft of flagella at one pole, colonizes highly acidic environment of the stomach

- Flagella propels bacteria through the mucus lining of the stomach, urease (urea → NH3) increases local pH so that pathogen doesn't degrade, mucinase degrades the mucin proteins, fimbriae allow attachment to host integrins, CagA protein injects itself and is involved in cancer

- Releases CagA carcinogen to cause stomach cancer, gastritis, and stomach ulcers

- Barry Marshall and Robin Warren

Staphylococcus aureus (Food poisoning, 2nd most reported food-borne disease)

- Gram positive coccus, opportunistic pathogen

- Facultative anaerobe

- Secrete enterotoxins into tainted foods such as pies, turkey dressing, or potato salad

- Genes encoded on plasmids, bacteriophages, and pathogenicity islands

- Resistant to heat and acid

- Pyrogenic, induce emesis, gastroenteritis, resistant to inactivation by proteases

periosteum

2 distinct layers - inner is fibrous with no cells, outer is cellular with osteoclasts and osteoblasts

CSF

provides buoyancy, shock absorber, nutrient exchange

Blood brain barrier

endothelial cells have tight junctions

layers of meninges

dura mater, pia mater, arachnoid mater

bacteria that can cross the blood brain barrier

- Neisseria meningitidis

- Haemophilus influenzae

- Streptococcus pneumoniae

Neisseria meningitidis

- Gram negative, diplococci, human pathogen, saliva, respiratory secretions

- Lipoologiosaccharide, capsule, type 4 pili, opa proteins (phase variation)

the overall process of transcription

RNA polymerase makes RNA from DNA, reads DNA 3'→5'. Synthesizes RNA 5'→3'

What enzyme transcribed RNA in bacteria?

RNA polymerase

role of sigma factor

to cause RNA polymerase to bind to the promoter, promoter recognition

In what direction does RNA Polymerase read the template strand and in what direction does it synthesize RNA.

Reads DNA, reads DNA 3'→5', Synthesizes RNA 5'→3'

the overall process of translation

Ribosome reads mRNA to make protein and reads from mRNA 5'→3'. Builds protein N → C terminus

What happens at the ribosomal "E", "P", "A" sites

A: incoming tRNA, P: growing chain, E: exit

In what direction does the ribosome read mRNA and what direction does it synthesize polypeptides

mRNA 5'→3'. Builds protein N → C terminus

transforamtion

naked DNA from environment

transdution

use of phage

conjugation

cell to cell transfer

Transformation vs transduction vs conjugation

- All transfer DNA

- All increase genetic diversity

- Different mechanisms

- They require contact (only conjugation does)

does positive stain stain the background or cell

stains cell

does negative stain stain the background or cell

stains background

Describe Gram-staining (principle)

- Based on cell wall thickness

- Gram + = purple

- Gram - = pink

Describe Acid-fast staining (principle)

Mycolic acid retains dye

Spread plate method (you must be able to calculate bacterial cfu)

- Quantifies CFU

- Formula

- CFU = colonies / dilution

what does Streak plate method do

- Isolate colonies

Broth culture method

Grow bacteria in liquid