week 10

pathogenesis caused by

disruption of homeostasis of microbes that live within us; disruption of normal balance; need to understand microbiome to understand pathogenesis

skin disease outbreak caused by

dominant species of bacteria is removed by rival species

parasite

consumes host resources at expense of the host

pathogenesis

disease

pathogenicity

ability to cause disease

virulence

quantitative measure of pathogenicity (degree and efficiency); how pathogenic something is; how well it causes disease

primary pathogen

causes disease in a healthy individual; not opportunistic; can cause disease at any time

opportunistic pathogen

causes disease when opportunity presents itself; takes advantage of opportunity; always has access but won't cause disease; eg: yeast infection; toxoplasma gondia

pros of infecting a human host

features good for microbial growth (nutrients, pH, temperature); varied microenvironments for diverse metabolisms; microbes ofen in exposed area (skin and mucousal)

exposed areas

microbes present; skin, mouth, respiratory tract, GI tract, urogenital tract; host defenses protect these areas with physical barriers, and the immune system (innate and adaptive)

disease occurs when

new pathogen invades (gets through defenses); balance of normal flora shifts and bad guys get a foothold; normal flora move to tissue where they don't belong

host defenses

skin, mucous membranes, oral cavity

skin

line of defense against bacterial invasion; most skin bacteria are gram +; warm moist env. support growth (eg: underarms) - especially sweat glands; fewer microbes where there are no sweat glands

propionibacterium acnes

causes acnese

sebaceous glands

secrete lipids and that prevent drying

apocrine glands

secrete lactic acid and lysozyme

musous membranes

eg: airway, gut, mouth, GI, and urogenital tracts; tightly packed epithelial cells line these tracts; mucus consists of secretions with antimicrobrial activity (physical and enzymatic barrier against pathogens); tight junctions and mucus help prevent infection of epithelial cells and underlying tissue

pathogens in the mucus layer

pathogens have proteins that help with adhesion and invasion; pathogens break through barrier, loosely associate with cells, tightly bind to cells, then invade by breaking through barriers

oral cavity

hundreds of bacteria species; has nutrients; physical arrangement of teeth protects tissues; saliva contains antimicrobials (lysozyme - cleaves peptidoglycan, lactoperoxidase - toxic); has plaque

plaque

mixed-culture biofilm; colonization develops in a stepwise fashion; with increasing complexity different bacteria species grow; species are interacting with eachother

plaque development

1. streptococcus colonization causes plaque
2. filamentous fusobacterium thickens plaque
3. anaerobic actinomyces grow in thick plaque

tooth decay

occurs when balance shifts towards bad guys; occurs in crevices and gum line (physical barriers break down); sugars cause tooth decay because they are fermented to lactic acid (causes tissue damage and shifts the microbe balance); often caused by S. mutans

streptococcus mutants

can cause cavities; adheres with a polysaccharide made from sucrose; opportunistic; 90% of people in US has teeth colonized by S. mutans but not everyone has tooth decay

antimicrobial smart bombs

links an antimicrobial peptide with a targeting protein for S. mutans, so it will get rid of only S. mutans and not all microbiome

H. pylori

causes ulcers; present in more than 80% gastri ulcer patients; under the right conditions in breaks down tissue and causes ulcers; uses flagella and S-shape to invade mucus, secretes urease to alter local pH (change in environment allows in to grow better and damage tissue); disruption of mucus layer and destruction of epithelial cells result in ulcers; resulting immune response can also damage tissue; is associated with gastric cancer; can be cleared with antibiotic treatment

Barry Marshal

showed that H. pylori causes stomach ulcers; performed an endoscopy with a normal stomach, drank H pylori, had nausea and vomiting, endoscopy and biopsy showed inflammation and colonization, was cleared with antibiotics

successful pathogens have

1. access to host and the appropriate tissue site
2. evade host defenses (barrier and immune system)
3. establish a replication-permissive niche (adjust env)
4. replication - acquisition of nutrients and the molecular mechanisms to achieve this; get to other areas
5. damage to host (pathogenesis) - host damage is collateral damage of growing; keeps host alive long enough to ensure replication, dissemination and transmission to new host

virulence factors

induce host cell damage and/or promote pathogen survival (eg: protiens, lipids)

7 features of pathogens

exposure, adherence, invasion, growth, dissemination, evade host defenses, host cell damages; virulence factors can help with any of these

adhesion

infections begin at breaks, wounds, exposed epithelia; pathogens infect after adherance to epithelial cells using molecular interactions and/or surface structures; proteins bind to the surface of host cells (tissue specificity); structures of pili and capsule

adherence structures

pili and glycocalyxpi

pili

longer, a few per cell; help with conjugation; bind to host glycoproteins for adherance

glycocalyx

secreted polysaccharide (capsule or slime layer); also has protein; prevents uptake by host immune cells

Streptococcus pneumoniae

has rough or smooth appearance (slime or capsule layer - rough vs smooth)

invasion, growth & and dissemination

penetrate epithelia to initiate pathogenicity; invasions begins at site of adherence; disruption of host mucous, tissue, and cellular barriers; can include pathogen and host factors

colonization and growth

pathogen multiplies to be able to cause damage; once at an appropriate location, uses special systems to enable growth eg: obtain limiting nutrient, move location (through epithelia to blood)

iron

limiting nutrient; host has lactoferrin and transferrin that chelate iron to limit availability; bacteria have siderophores - iron-binding proteins to compete with host iron binding

siderophores

iron-binding proteins to compete with host iron binding

septicemia

pathogens in blood

evolution of virulence

virulence of a pathogen increases when new hosts are readily available; decreases when new hosts are limiting and pathogen-host interactions move to equilibrium

attenuation

decreased virulence; due to repeating passaging in the lab or being in an environment long-term; adaptation to one growth condition results in change to another; these strains can be used to make vaccines

measuring virulence

use LD50; use multiple groups of mice and infect each group with a different amount of bacteria; see what % of mice in each group die; after extensive culturing is attenuated and LD50 decreases

LD50

dose required to cause death in 50% of the test population

why virulence is reduced

less efficient step of pathogenic process (adherence, invasion, lysis, host cell damage); associated with loss of virulence factors

finding virulence genes

virulence assay (mutant screen or test specific candidates); genome comparisons

virulence assay with mutant screen

use transposon mutagenesis mutant screen with NeoR gene then selct for NeoR; assay each NeoR mutant and WT for virulence feature eg: invasion by adding to culture of host cell; incubate then add Kan (kills bacteria but doesn't get in host cell); wash, lyse, and assay for viable bacteria; then isolate the Tn tagged gene

limitations for virulence assay

need a good assay for virulence feature

negative control for virulence assay

need to make sure it is not just growing slower and that is why it doesn't have as many bacteria

genome comparisons

genomes that are compared need to be closely related and pathogenic vs not; can also look for evidence of HGT

identified clusters of pathogen-specific genes

HGT; pathogenicity islands; altered GC content and flanked by inverted repeats

limitations for genome comparisons

must be closely related; need genome sequence availability; gives candidates which then need to be tested by virulence assay

endotoxin

often lipopolysaccharide (LPS); cell associated; part of gram - outer membrane; causes systemic fever and inflammation

exotoxins

secreted extracellularly; 3 main types: cytolytic toxins, AB toxins, superantigen toxins

cytolytic toxins

form pores, degrade cell membrane; enzymatically attack cell components and cause lysis; can use hemolysis assay

hemolysis assay

have RBCs on agar; add bacteria; where there is a halo is where RBCs are lysed - loss of red color)

superantigen toxins

stimulate a significant immune response and causes extensive inflammation

AB toxins

2 subunits- A (active) and B (binding); eg: cholera toxin, diptheria toxin

cholera toxin

B binds to host receptor, A is delivered to cytoplasm where it is toxic and interferes with endogenous cAMP pathway; alters adenylate cyclase which disrupts ion transport which causes water efflux which causes diarrhea and dehydration

viral origins of pathogenic cholera

pathogenic V. cholera have AB toxin; located of phage genome so pathogenicity from infection of phage with AB gene; also has toxin coregulated pili (TCP)

TCP

toxin coregulated pili; coregulated with AB toxin; acts as a receptor for the bacteriophage; found on pathogenicity island that was later discovered to be a phage

African trypanosome

protozoan extracellular parasite; Trypanosoma brucei; causes African sleeping sickness; eg: T.b. rhodesiense, T.b. gambiense, T.b. brucei (doesn't infect humans)

African sleeping sickness

fatal human disease; endemic to sub-saharan africa; found in wild and domestic animals; transmitted by tsetse flies; different strains have different host range specificity

African trypanosome lifecycle

different in insect and humans; developmental changes (gene expression) within each host and from one host to another; adaptations to different environments

T. brucei in insects

infects gut and salvary gland

T. brucei in humans

infects blood stream then moves to the central nervous system

host defenses

1. adaptive immunity - makes antibodies that are specific to the pathogen and help destroy it
2. innate immunity - lytic factor in serum

parasite defenses against immune system

1. against adaptive - antigenic variation
2. against innate - serum resistance

T. brucei infection

fever and # of parasites cycle; waves of fever correspond to peaks of parasitemia - they keep coming back

T. brucei immunofluorescence assay

isolate parasites from each peak; get antibodies against parasite surface proteins in another animal and tag them; incubate Abs with T.b., wash, fluorescence microscopy; results show that each Ab only recognize Ab from when they were isolated

antigenic variation

parasites varying their surface antigens exposed to host immune system; immune system destroys first peak but some parasites express a different SA which then generates a new peak; allows parasite population to avoid destruction by adaptive immune system

VSG

variant surface glycoprotein

mechanism for antigenic variation

VSG in telomere expression sites; each VSG has different gene; 1000s of VSGs; 10 expression sites at telomeres but 1 is active per cell; can recombine VSGs and can switch promoters on and off; controls position and promoter elements

human sera and Tbb/Tbr

Human sera cured mice infected with T.b. brucei but not Tbr; there is a factor in human sera that lyses Tbb

TLF

trypanosome lytic factor; lyses Tbb; serum lypoprotein particle; specific to primates and therefore causes host range specificity

T. brucei infectivity assay

add trypanolytic sera (primate) and non-lytic sera to parasite to see if they are infectious or noninfectious

test for serum resistance/host specificity

Tbr has serum resistance antigen (SRA) not expressed by Tbb or Tbr* (mutant that lost virulence factor); identify gene in Tbr and not in Tbr*; use microarray or RNAseq

Tbr microarray

isolate Tbr DNA; spot 1 gene onto each microarray; probe with labeled Tbr and Tbr* RNA; if there is a signal then that RNA is present

test if SRA confers serum resistance

either transform Tbb with SRA (GOF) or knock out SRA from Tbr (LOF); confirms it gives resistance; it is a surface protein that inhibits TLF lysis activity

serum resistance and evolution of human infectivity

Tbb is serum sensitive; Tbr is serum resistant but unstable and can lose resistance (acute disease) - recently emerged pathogen; Tbg is stable in its resistance (chronic disease)

treated LA water

waste water is pumped into ocean, used for irrigation; new projects want to recycle into the ground water for future use

catabolism of benzene derivatives

TCA helps breakdown aromatic carbon sources; eg: lignin from wood; benzene; polycyclic aromatic hydrocarbons; microbes can remove aromatic pollutants from water and soil;

bacteria and fungi recycle lignin and degrade toxic pollutants;

benzoate undergoes aerobic catabolism to catechol which is then degraded through TCA alternative pathways

microbial syntrophy

couples metabolism between 2 organisms such that the overall reaction yeilds energy; usually anaerobic conditions; aeromitc waste products of petroleum are stable so breakdown gives + dG; better than autoclaving

syntrophic partners

usually thermodynamic extremophiles
eg 1: benzoate catabolized to acetate; H2 is used by sulfate-reducing bacteria; by conjugating the process both benzoate and sulfate are broken down
eg2: bacteria release H2 and methanogens consume it and produce methane (we can harness as a biogas)

water treatment

1. pump water from reservoirs (collect it)
2. purify the water - slowest step so has most revision)
3. store and distribute clean water

water purification

coagulation (clumping); flocculation (separating clumped products); sedimentation (separation of heaviest products); polymer and filter (separates what we don't want); fluoride and disinfectant; corrosion control (removes metals)

coliform bacteria

lactose-fermenting, rod shaped, gram -, non spre forming; don't usually cause illness but their presence indicates pathogenic organisms are present due to fecal matter contamination; how they test drinking water

total coliform test

microbe test that reports recent fecal contamination; present in all human fecal matter (1 million/gram); easy to culture; sample, serial dilute, plate

EMB plate

eosin methylene blue; methylene blue interacts with gram + cell wall and binds DNA/RNA; eosin responds to pH changes; forms complex w methylene blue; turns to purple/black under acidic conditions

drinking water standards

must be below 1 cell per 100 mL drinking water; different purposes have different standards

clean water assays

EPA and most probable number

EPA clean water assay

collect sample, filter sample, grow on plate

most probable number clean water assay

collect sample, serial dilute sample, grow on plate

water treatment plants use

lakes and natural water reservoirs, drinking water purification facilities, primary and secondary treatment methods

primary treatment

screening and sedimentations

secondary treatment

anaerobic digestion (anaerobic microbes - digested sludge is incinerated, used as fertilizer, etc.) and aerobic oxidation (syntropy to create O2, for disinfection)

microbial communities in aeration tank

trillions of cells, 1000s of species, metabolic needs vary (different substrates and nutrients available), different microbes in different conditions due to available substrates; often coliforms overpower other communities

aerobic biofilms

cells synthesize EPS (exopolysaccharide - cell wall/capsule) to form biofilm; all cells together degrade most carbon compounds; biofilms allow them to stay in reactors for a long time

anaerobic sludge digestor (bioreactor)

releases biogas; anaerobic microbes (fermenting bacteria and methanogenic archaea) form methane, CO2, and water from bio-organic molecules

biogas

valuable biproduct; methane can generate electrical energy; CO2 and water are waste products

water contaminants

can't purify everything; drugs and medicines, xenobiotic chemicals (plastics and dyes); heavy metals; haven't found microbes that can degrade them or degrade them fast enough; need to find the right syntropy