MICB 212 Chapter 10 Notes

Community Lifestyle of A Biofilm

Bacteria and Biofilms

Biofilms

-organized communities of bacteria that are adhered to a surface and surrounded by (enmeshed in) matrix of extracellular polymeric substances (EPS)

-composed of single bacterial species

-biofilm formation occurs as a result of coordinated chemical signaling between cells (quorum sensing)

-when increased amounts of bacteria are present in close proximity to each other → autoinducer concentration increases

• once binding of autoinducer molecules has exceeded a certain threshold → signaling cascade initiated that modulates gene expression → modulates bacterial physiology to encourage community development

-bacteria in biofilm behave as a group

• sense and respond to stimuli in a coordinated manner

-some biofilm structures include network of pores, held together by EPS

• provides a primitive circulatory system

• O₂ and nutrients, bacteria deep inside biofilm have different physiology than bacteria closer to surface

-physical biofilm formation process

1. adherence of bacteria to a surface

2. growth & production of EPS, resulting in much stronger (often irreversible) attachment leading to biofilm maturation

3. dispersion of single cells (or parts of the biofilm) from the site of mature biofilm

Extracellular Polymeric Substances (EPS)

• slimy, film-like substance produced by bacteria in biofilm

• composed of polysaccharides, proteins, nucleic acids

Quorum Sensing

• uses signaling molecules (autoinducers) produced by bacteria

• bacteria has receptors for these signaling molecules

Planktonic

• single bacterial cells that are growing in liquid culture

• single bacterial cells swimming freely above a film

• can be recruited to a biofilm or may have seeded off an existing biofilm

Biofilms in Nature and in Human Disease

-commonly found environmentally and with biological hosts

• classified as benign or pathogenic

Benign Environmental Biofilms

• don’t cause human disease

• responsible for significant industrial problems (corrosion)

  ex: slipper coating on rocks in streams, coatings on ship hulls

• may also impact human health

  ex:

  • colonization of hot water systems in hospitals by mycobacterium avium

  • drinking water reservoirs contaminated by salmonella typhimurium

• can attract, recruit and concentrate pathogenic bacteria that might not form a biofilm on their own

Pathogenic Biofilms on Medical Implants

• Gram (-) and Gram (+) readily form biofilms on many foreign devices implanted into human bodies

• account for significant human infection & disease

  ex: affected medical devices (urinary catheters, heart valve implants, hemodialysis equipment, dental implants)

• may also form on human body surfaces

  • ideal environment for formation of different classes of biofilms

• many of these infections caused by opportunistic pathogens that are human commensals

• biofilm infections result in chronic disease difficult to treat with antibiotics

  ex:

  • cystic fibrosis (CF) infections in lung caused by pseudomonas aeruginosa

  • tuberculosis infections caused by mycobacterium tuberculosis

  • urinary tract infection caused by escherichia coli

  • ear infections (variety of bacteria)

  • tonsillitis (variety of bacteria)

  • oral films

    • primary causative agents of dental caries and gingivitis

Treating Biofilms

Antimicrobial Resistance and Persistence of Biofilms

-biofilm communities significantly more resistant to antibiotics and antimicrobial stressors

• including those mounted by natural host responses (immune system) compared to planktonic bacteria of same species

Biofilm Properties that Contribute to Antimicrobial Resistance

EPS Matrix

• complex EPS layer enmeshing the biofilm can impede penetration of antimicrobial agents to bacteria buried in depths of biofilm

Nutrient and O₂

• bacteria within a biofilm are subject to nutrient and O₂ gradients

• bacteria closer to surface have better access to nutrients and O₂ than interior bacteria

• bacteria in biofilm interior (while alive) are metabolically inactive

• antibiotics most effective against metabolically active cells

  • bacteria in interior protected

Persister Cells

• small percentage of population remains viable despite prolonged exposure to antimicrobial or even increased dosage of antibiotics

• confer no heritable resistance to progeny once selective pressure is removed

• able to survive for extremely long periods of time

New Pharmaceutical Approaches to Anti-Biofilm Therapy

-interfering with EPS synthesis

• coating medical devices with chemicals that hinder matrix formation

-inhibiting adherence of biofilms to their surface substrate

• identifying chemicals that bind to bacterial cell surfaces prevent biofilm formation

-targeting autoinducers

• bacteria unable to signal to each other → unable to form/maintain a biofilm

Biofilm Regulatory Systems

-bacteria rely on signal transduction systems in order to form biofilms

• senses environmental changes

-during biofilm formation, two core signal transduction systems play collective role in mediating biofilm formation

• quorum sensing

• chemotaxis

Quorum Sensing

-prokaryotes able to sense presence of others of their own kid that are nearby in environment

-type of environmentally-regulated gene expression where it’s regulated by population density

• prokaryotes sense presence of their own kind and work together

• large changes in gene expression change a cell’s phenotype from individualistic growth to community-based growth

-used to ensure sufficient numbers of a given species are present before initiating a response that requires certain population density to have an effect

-several different quorum sensing mechanisms of varying complexity based on either one-component or two-component systems have evolved in prokaryotes to regulate gene expression in response to population density

-involves a communication chemical autoinducer (AI)

Quorum Sensing in E.Coli

-Lsr quorum sensing system

-LsrR

• global regulator of system in an inhibitor of its own operon and lsrABCD operon

• keeps quorum sensing system shut off when cell concentration is low

  • represses expression of both operons which includes its own expression

-one operon contains lsrK and lsrR genes that encode 2 regulators in the system

-second operon consists of several genes lsrABCD that encode proteins involved in sensing and transporting autoinducer into cell when it reaches the threshold

Low Population Density

-LuxS enzyme responsible for synthesizing autoinducer (A12)

-converts a molecule (DPD) into AI2 which is transported out of cell by membrane-bound protein (YdgG)

-even when population density is low → cell continues to secrete AI2 to indicate to other cells that it’s present

High Population Density

-as population density increases → concentration of AI2 increases as well

-once concentration reaches threshold → LsrABCD complex (membrane bound) starts to import AI2 from environment into cell where LsrB senses external AI2 levels

-after AI2 is imported into cell → AI2 phosphorylated by LsrK → AI2 + P binds to LsrR and relieves repression of 2 lsr operons to allow further production of quorum sensing proteins

-LsrR

• global regulator

• regulates its own operon and other genes in cell including other regulators

• able to activate a cascade of changes in gene expression in cell

• changes often result in cell undergoing some form of community based phenotype

• can act as transcriptional repressor or activator

  • binds to AI2 + P to change how it binds to certain promoters

Bacterial Motility and Chemotaxis

Chemotaxis vs Biofilm Formation

-biofilms are bacteria’s response to persistent environmental changes leading individual bacteria to aggregate in large microbial communities of immobile cells

-requires transformation from planktonic bacterial lifestyle

• requires motility to respond to rapid fluctuations in their microenvironments

• responsive and motile lifestyle dependent on chemotaxis

-chemotaxis is a change in motile behaviour as a result of sensing a chemical concentration difference (concentration gradient) in the environment

• immediate change in behaviour doesn’t require synthesis of new proteins

Positive Chemotaxis

• movement toward higher concentrations of chemical substances (attractants)

Negative Chemotaxis

• movement away from higher concentrations of chemical substances (repellents)

Sensing

-in order to exhibit chemotaxis, organisms must be able to sense when environmental concentration of an attractant or repellant is increasing or decreasing and be able to respond to the environmental sensation appropriately

Spatial Sensing

• concentration difference sensed by comparing concentration of chemical at two points in space at time using receptors at the front and back of the cell

Temporal Sensing

• concentration difference sensed by comparing concentration of chemical at two points in time

Responding

Run

• smooth forward motion of swimming organism

• typically associated with flagellar rotation in counter-clockwise rotation

• relate flagellum to tail-like structure

Monotrichous Organisms

• single flagellum organisms

• change direction by reversing flagellar rotation (clockwise)

• pulls cell backward in erratic way causing random change in direction for subsequent run to twitching motion (tumbling)

• others change direction by briefly stopping flagellar rotation

• random motion points them in random direction for a subsequent run

Peritrichous/Lophotrichous Organisms

• multi flagella scattered over cell surface

• lophotrichous organisms possess a tuft of flagella at a single location

• rotation of flagella in one direction causes them to come together in a bundle and act in coordinated manner → running motion

• tumble results from a switch in direction of rotation by at least one flagellum

  • bundle “flys apart” → run disrupted → bacterium undergoes chaotic 3D whirling motions (tumble)

  • subsequent run occurs in random direction

Chemotaxis in E. coli

Default Non-Chemotactic Conditions

-in absence of chemical concentration gradient → motility rarely results in net movement in any particular direction

-cells follow random pattern (Brownian motion)

• no set end point or net directionality

-depending ont he chemical and chemical gradient present → chemoattractant if promotes movement

• cells to move away from it → chemorepellent

-relative position and type of chemical may promote either tumbling or running

-can’t say that a single type of chemical only promotes one type of reaction

• dependent on the location

-if chemoattractant behind cell → may tumble first to change direction then run towards it

-if senses chemoattractant in front of cell → run towards it

Tumbling Motion

Chemoreceptors

• proteins clustered at the front of the cell

• bundled in high concentrations in order to be more sensitive to signals and promote signal amplification

-if chemorepellent (ex. antibiotic) is sensed in front of the cell → cell will tumble and change direction

1. a class of chemoreceptors will sense signal and activate CheA (response regulators in pathway)

2. once activated by chemoreceptors → CheA phosphorylates CheB to activate enzymatic activity

   • CheB demethylates CheA (protein that just activated it)

3. when CheA is demethylated → able to phosphorylate second protein CheY

4. when CheY phosphorylated → changes conformation so it actively binds to flagella motor switching its default, unbound rotation from counter-clockwise (CCW) to clockwise (CW) causing cell to tumble and change direction

CheA

• kinase; enzyme capable of phosphorylating other proteins

CheB

• demethylase; enzyme that removes methyl groups from other proteins

Running Motion

-cell has changed directions and now wants to put repellent behind it and run

-resetting system means going back to default rotation of flagella when it’s not bound to CheY-P which is counter clockwise (CCW)

1. cell senses it’s facing direction where antibiotic concentration is lower

   • CheA stops phosphorylating CheB

   • CheR (methylase) starts to methylate CheA to CheA-CH₃

2. CheA-CH₃’s kinase activity is turned off → can’t phosphorylate CheY

   • CheZ dephosphorylates CheY to change its conformation → can’t bind flagellar motor

3. free, unbound motor rotates in CCW direction to promote running