MCB 100 E2

What are the 5 Kingdom classifications?

1) plants

2) animals

3) fungi

4) protists

5) monera

Who determined the 5 kingdom classifications?

Robert Harding Whittaker

Animalia in Eucarya

worms, flies, fish, mice, and humans

E.coli belong to…?

proteobacteria

Yeast belong to…?

fungi

• go back over the gram pos and gram - bacteria

review

Coccus shape, description

round, single coccus

Bacillus shape, description

rod, single rod

Vibrio shape

curved rod

Coccobacillus

short rod

Spirillum

spiral shape

Spirochete

long, loose, helical spiral

Diplococcus description

pair of two cocci

Tetrad Description

grouping of four cells arranged in a square

Streptococcus description

chain of cocci

staphylococcus description

cluster of cocci

streptobacillus description

chain of rods

corynebacterium description

palisades

Universal Tree of Life

based on sequence comparisons of ribosomal small subunits 16s rRNA genes

Bacteria that form Biofilms Stages

1) free swimming, swarming planktonic stage, motility by flagella

2) attachment and aggregation into biofilm, colony stage

3) spore/endospore stage

Gram Positive Bacillus and Clostridium species

form endospores to protect them against environmental extremes (temp, desiccation, UV, pH, chemicals)

2 Examples of Complex Microbial Cycles

1) Myxobacteria

2) sporulation in Actinomycetes

Sporulation in Actinomycetes Steps

1) under favorable conditions, 1-2 germ tubes emerge from a spore and produce substrate mycelium (10 hrs)

2) after 48-72 hours, bld genes cause production of aerial hyphae, tips of hyphae form spiral compartment with multiple copies of the genome

3) when growth stops, the compartment segments, each segment changes shape and wall thickens to become desiccation resistant spore (4-10 days)

Actinomycetes

spore forming gram pos. bacteria (often mistaken for fungi)

Sporulation in myxobacteria

over the course of 72 hours, myxobacteria (gram negative) form fruiting bodies and sporulate under starvation conditions

Visualizing biofilms

bacteria forming biofilms attach to surface and to each other through slimy matrix layer of extracellular polysach. coatins that holds commuty together, biofilm comp.. can be washed away

Bacteria in Biofilm visualization, GFP

express GFP (green) and secrete extracellular polysaccharide capsule and matrix (dyed red in images)

Biofilm Formation Detection

detected and measured by staining with crystal violet dye (binds extracellular polysaccharides)

Bacterial biofilms resistant to?

antibiotics and contribute to many medical problems

4 Problems Biofilms contribute to

1) medical device complications

2) legionella pneumophila

3) pseudomonas aeruginosa

4) dental plaque

Medical Devices

catheters that can breach skin or mucosal surfaces and plastic implants (heart valves, joint replacements), many nosocomial (hospital) infections from this

Legionella Pneumophilia

biofilms on walls of AC system were source of aerosolized bacteria that cause new lung disease called Legionnaire’s disease

Pseudomonas aeruginosa

forms biofilms on mucosal surfaces such as on lung of cystic fibrosis patients, or on burn wound areas, forms biofilms in rocks, waterways, plants, soil, jacuzzi environments

Dental plaque

biofilm made of many bacterial species that are mineralized over time to make calculus (tartar) and can cause dental caries, gingivitis (inflamed gums), or periodontal disease

Vibrio species found in…?

marine environments attached as biofilms to marine snow (tiny particles of sand and seashells or zooplankton)

Vibrio Bacteria Length

1-3 micro m long, with flagella and/or pilli they are longer or can be trapped

Vibrio, cholera, and sari filters

rita colwell’s lab reported 48% reduction of cholera in 65 rural Bangladeshi villages by simple filtration using saris

Motility

movement with flagella, twitching, or gliding

Flagellin

polymerized subunits of a protein, long helical structures of the flagella

TLR-5

evolved immune recognition molecules in vertebrate immune system which bind and sense flagellin

Flagellin genes are often…?

strongly regulated in invasive pathogens and not expressed inside the host body

Vibrio Cholerae Flagella

single polar flagellum helps to move through low viscosity fluids (water) and high viscosity of the host (mucus)

Non motle mutants of V. Cholerae

can’t reach the intestinal epithelium, less virulent than WT strains

Treponema Pallidum

spirochetes, single flagellum in their periplasm (to avoid immune detection)

Chemotaxis in E.Coli

have 4-10 flagella and can rotate:

1) CCW (default) aligns flagella into single rotating helical bundle (same direction), causes bacteria to swim in straight line

2) CW, breaks flagella helical bundle apart, causes bacteria to tumble in place

When E.coli senses it’s going in the wrong direction…?

it tumbles sooner and more often to move in the other direction

Taxes

movement involves ability of bacteria to sense a substance

Chemotaxis

sensing chemicals, towards attractants and away from repellents, key role in virulence of many pathogens

Aerotaxis

ability of bacterium to sense oxygen

Phototaxis

ability of bacterium to sense light

Magnetotaxis

ability of bacterium to sense magnetic fields

Osmotaxis

ability of bacterium to sense salinity/osmotic strength

Hydrotaxes

ability of bacterium to sense water

Measuring Chemotaxis

measured by inserting capillary tube with attractant or repellent in a culture medium of motile bacteria, or can also be seen under a microscope

Helicobacter Pylori Chemotaxis

uses chemotaxis to target and penetrate deep into gastric glands (pits) of stomach mucosal epithelium

Listeria monocytogenes

food-borne pathhogens

Listerosis

severe human and animal disease caused by gram positive pathogens (Listeria monocytogenes most common in humans) and Listeria ivanov

Most common listeria in humans is…?

listeria monocytogenes

Listeria

food borne pathogen responsible for 28% of food borne deaths in USA

Listerosis Susceptibility

occurs mainly in immunocompromised individuals (fetus, neontae, immunospressed elderly adults

Listeria and Spontaneous Abortion

can transit placenta and lead to infection which can cause spontaneous abortion

Untreated Listeria

if not stopped by antibiotics, it can cause CNS infections, meningitis, and fatal bacteremia (sepsis)

Listeria and Temperature

psychrotropic/ psychrotolerant, can grow in low temps (optimal of 37C but still grows in 2-3C), refrigeration won’t stop the growth of this bacteria

Listeria Monocytogenes Motility

uses actin, bacteria use host cell to propel through the cytosol, comet tail propulsion from cell to cell

Listeria monocytogenes

An intracellular bacterium that invades host cells and spreads directly from cell to cell using actin-based motility

Entry of Listeria to host cell

1) internalin A (In1A) - A Listeria surface protein that triggers entry into host cells by binding E-cadherin

2) Internalin B (In1B)- A Listeria surface protein that triggers entry into host cells by binding C1qR

Lysis of the Vacuole in Host Cell

1) Listeriolysin O (LLO)- pore-forming toxin that lyses the phagosome so the bacterium can escape into the cytosol. It also helps lyse the two-membrane vacuole after spread

2) PlcA- A phospholipase C that works with LLO to lyse the single-membrane phagosome and the two-membrane vacuole

Lysis of two-membrane Vacule in Host Cell

LLO and PIcA also PIcB:

A phospholipase C that works with LLO and PlcA to lyse the two-membrane vacuole formed after cell-to-cell spread

Cell-to-cell spread by Listeria

Act A: A Listeria surface protein that recruits host actin machinery to build actin comet tails, driving intracellular movement and cell-to-cell spread

Cell to Cell Spready by Actin Based Motility

rapid spread without exposure to humoral (antibody) immune system

Listeria movement

actin based motility, comet-tail propulsion from cell to cell

Bacterial Adhesion/Attachment

important for colonization of new environemnt or host, contribute to bacterial pathogenesis (virulence)

Adhesins

attachment proteins, bacterial cell surface components that facilitate adhesion or attachment to surface or proteins/sugars on other bacterial/ eukaryotic cells

Pilli and Fimbriae have…?

adhesins at tips that mediate the attachment to surfaces

Types of Attachments

1) nonspecific

2) highly specific

Nonspecific Attachment

mostly based on hydrophobic interactions or charge interactions

Highly Specific Attachment

dependent on tight and specific ligand-binding to its cognate receptor

Cell or Tissue Tropism

bacteria preference for growth at location in body or surface type, due to adhesion to specific cells or tissues

Adherent Bacteria Daughter cells

form one stationary and one mobile, holdfast (binds to surface)

ex) in the bacterium Caulobacter crescentus

Attachment Process

1) loose attachment mediated by pili and fimbriae or fibrillar adhesins (long protruding adhesins)

2) tight attachment usually mediated by surface protein afimbrial adhesins

Gram Negative Adhesins

pili aka fimbriae, long rod-like surface adhesins that are ordered, helical arrays or chains of single protein called pilin, pili have tip protein at end that is the adhesin to bind to a host cell receptor (usually a sugar group)

Gram Positive Adhesins

fibrillar adhesins, short, thin rod-like structures on surface that bind to host CT and extracellular matrix proteins (fibronectin, collagen, fibrinogen, laminin)

Afimbrial Adhesins

proteins embedded on surface of Gram neg or pos bacteria, not organized to rod-like structures, closer to surface, mediate tight binding interaction of bacteria in host cell, bind to surface proteins rather than recognize sugar groups

Adherence is…?

the essential first step for bacteria to colonize

Bacteria Adhering to Mucosal cells

mucosal surfaces constantly washed by fluids to reduce number of microbes, bacteria capable to adhering to mucosal cells have advantage

Modification of Adherence

some pathogens modify adherence by injecting additional adhesin proteins into host cell that mediate tight binding and effector proteins that modulate host cell

Extracellular Pathogens

microbes that reside on outside or on surface of host cells or in medium

ex) Vibrio, E.coli, Yersinia, Pseudomonas

How do extracellular pathogens survive?

produce toxins and enzymes that fight off the host immune system

Intracellular Pathogens

microbes can enter and reside in host cells, can evade host cell immune system and other killing mechanisms

4 Bacteria on the Host Cell Surface

1) propionibacterium

2) staphyloccosus

3) streptococcus

4) neisseria

Mechanism of Entry

bacteria injects signals to host cell that causes membrane to ruffle up and engulf it

ex) salmonella, shigella

Zipper Mechanism of Entry

bacterial surface protein binds a host receptor and membrane zips around the bacterium

ex) listeria, yersinia

Phagosome

membrane-bound vacuole that encloses a bacterium after it’s taken into host cell

Bacteria that escape from phagosome

1) listeria

2) shigella

3) rickettsia

Bacteria Staying in the endosome

mycobacterium and chlamydia, live inside an endosome-like vacuole

Legionella Bacteria Survival

sends its vacuole to the ER and lives in an ER associated compartment

Lysosome

organelle full of digestive enzymes (hydrolases) that fuse with phagosome to destroy bacteria inside

Clearance by degradation

normal outcome when phagosome fuses with lysosome, bacterium broken down and killed

2 Extracellular Bacterial Pathogens

1) P acnes

2) S epidermidis

P. acnes

gram positive anaerobic bacillus colonizes sebaceous fat glands and eat oils in sebum, generate low pH environment that protects against other microbes