Bacterial Sensing and Communication

• Midterm Exam 1 grades are available on onQ.
• The class average for Midterm Exam 1 was approximately 75%.
• Students can review their midterm today(Feb. 10) from 12-1 PM in Botterell Room 449.
• Students can review their midterm on Feb. 11 during office hours, 12-1 PM in Botterell Room 449.
• The lecture is on Sensing and Communication, Feb 10, 2025.
• Lecture 15 examines Sensing and Communication.

Lecture Learning Outcomes

• The lecture will cover how two-component signal transduction systems (TCSs) and phosphorelays control transcription.
• The lecture will cover how vancomycin-resistant enterococci and Agrobacterium tumefaciens use TCSs.
• The lecture will cover how bacteria use quorum sensing (QS) to sense population density.
• The lecture will cover how QS regulates Vibrio fischeri bioluminescence, EHEC virulence, and the formation of dental plaque.

Bacterial Sensing

• Bacteria sense environmental conditions such as nutrient levels, antibiotics, and pH.
• Bacteria can sense each other and work together or kill each other.
• Bacteria can sense host organisms whether they are commensals or pathogens.
• Bacteria often respond by changing gene expression.

Direct Interactions with Regulators

• Stimuli can directly impact transcription.
• The lac operon is an example.
• Allolactose forms when lactose is present.
• Allolactose binds to LacI.
• LacI is prevented from binding to the operator.

Two-Component Signal Transduction Systems (TCSs)

• Sensing and response can be carried out by separate proteins in TCSs.
• There are over 200 TCSs in some species.
• Sensor kinases are membrane proteins that detect stimuli.
• Response regulators are cytoplasmic DNA-binding proteins.
• TCSs sense external stimuli such as nutrients and antibiotics.
• TCSs sense the host environment, for example, hormones and high osmolarity.
• Stimulus activates the sensor kinase.
• The kinase domain is autophosphorylated.
• Phosphate group is transferred to response regulator.
• Phosphorylation changes the response regulator structure.
• The regulators can now bind to DNA and change transcription as an activator or repressor.

Example: Enterococci and Vancomycin

• Enterococcus faecalis and E. faecium are common gut bacteria.
• Enterococci are major nosocomial pathogens, particularly healthcare-associated infections.
• Enterococci persist in hospital environments.
• Enterococci cause infections in immunosuppressed patients.
• Examples are bacteremia, endocarditis and urinary tract infections.
• Enterococci have high genome plasticity and can easily acquire new DNA like genomic islands.
• Enterococci accumulate antibiotic resistance genes.
• Vancomycin is used for resistant Gram-positive infections.
• Vancomycin-resistant enterococci (VRE) is increasing in prevalence.
  • In 2017, there were approximately 54,500 cases in hospitalized patients.
  • In 2017, there were approximately 5,400 deaths.
  • In 2017, the costs associated with VRE were approximately $539M.
• Changes to PG(peptidoglycan) structure confer vancomycin resistance.
• van genes make PG with D-lactate, not D-alanine increasing resistance.
• There is a major fitness cost for Enterococci (impairs growth).
• TCS regulates van genes.
• They only make PG with D-lactate if vancomycin is present.
• PG(peptidoglycan) is normal and the growth isn't impaired if vancomycin is not present.

Agrobacterium tumefaciens

• Agrobacterium tumefaciens are plant pathogens forming tumorlike growths or a crown gall disease.
• Gall-causing strains carry a tumour-inducing (Ti) plasmid.
• Agrobacterium tumefaciens transfers part of Ti plasmid (T DNA) to plant cells.
• Genes in T DNA of Agrobacterium tumefaciens are transcribed in the plant cell.
• Transcription leads to tumour formation.
• Transcription encodes enzymes that make nutrients for bacteria.
• Agrobacterium tumefaciens enters plant through surface wound and senses the plant using a TCS.
• The Agrobacterium tumefaciens sensor kinase VirA detects phenolics such as plant metabolites.
• The Agrobacterium tumefaciens response regulator VirG is then phosphorylated.
• The Agrobacterium tumefaciens vir (virulence) genes on Ti plasmid are transcribed.
• vir genes encode type IV secretion system (T4SS).
• Agrobacterium tumefaciens T4SS is similar to that used for conjugation.
• Agrobacterium tumefaciens forms a bridge between bacterium and plant.
• Agrobacterium tumefaciensVir proteins excise T DNA from Ti plasmid.
• Agrobacterium tumefaciens T DNA is secreted into plant by T4SS.
• Agrobacterium tumefaciens T DNA enters the plant nucleus.
• Agrobacterium tumefaciens integrates T DNA into the plant’s genome.
• Plant cells produce enzymes that are encoded by T DNA.
• This causes the plant to make phytohormones casing cell proliferation
• Which leads to the formation of tumors.
• Opines are made.
• Opines are nutrients made with a source of energy, carbon, nitrogen.
• Then A. tumefaciens consumes the opines.
• A. tumefaciens is used to genetically engineer plants.
• The process begins with a recombinant Ti plasmid prepared in the lab.
• Then desired gene(s) is added to the engineered strands.
• The tumor-inducing genes are removed to ensure little damage.
• T DNA (and desired genes) are transferred to plant to add the new properties.
• A. tumefaciens is used because they change plant properties after bioengineering.
• This can assist with Resistance to insects/viruses.
• Engineering them increases plant nutrition.

Phosphorelays

• Phosphorelays are a more complex version of TCS.
• Phosphate is transferred from protein to protein in the system.
• The system has one or more proteins between the sensor kinase and response regulator.
• Regulation occurs with more opportunities because of more proteins in the system.

Sporulation Phosphorelay

• Endospore formation in Bacillus spp. is very highly regulated.
• Each step of phosphorelay is controlled by different factors.
• Factors can be positive and negative signals.
• This set up ensures that endospores are only formed when absolutely necessary.
• The response regulator Spo0A controls >500 genes.
• Endospore generation is irreversible once started.

Chemotaxis and Sensing

• Sensing can impact processes other than transcription.
• E.g., methyl-accepting chemotaxis proteins (MCPs).
• Senses: attractants, repellants.
• Response: change in flagellum rotation.

Quorum Sensing (QS)

• Bacteria live in communities with lots of other bacterial cells.
• It can be helpful to their collective survival to work together.
• Bacteria use quorum sensing (QS) to sense population density.
• QS causes cell-cell communication in the bacterial communities.
• The QS effects can be seen within intraspecies and between interspecies.
• If density is high enough because of QS, it leads to changes in gene expression.
• QS regulates functions that work best when carried out by multiple cells.
• Quorum sensing is used to help bacteria with Virulence factor production.
• QS will increase it until toxins are effective because toxins are ineffective if amount is too low.
• In Biofilm formation needs a coordinated production of EPS which QS provides
• QS is used by competing cultures to determine competence..
• This is useful if other bacteria (and their DNA) are present.
• Vibrio fischeri lives in light organs of fish and squid.
• At a high cell density, V. fischeri emits light.
  • Benefits the host (confuses predators, lures prey).
  • Benefits the bacterium (safe, nutrients).
• At a low cell density, there is no bioluminescence because the light wouldn't be bright enough, so it might be a waste of energy.
• Bacteria constantly make autoinducers (AIs).
• Autoinducers are signaling molecules.
• E.g., N-acylhomoserine lactones (AHLs).
• AI level has a direct relationship to number of cells.
• At certain level, autoinducers impacts gene expression through transcriptional regulators or Sensor kinases (TCS).
• Proteins are encoded by lux operon and these signals produce light.
• LuxAB is the code for luciferase.
• LuxCDEG codes for enzymes that make luciferase substrate.
• lux operon is regulated by LuxI (makes autoinducers) and LuxR (transcriptional activator).
• High cell density leads to high concentration of autoinducers.
• Autoinducers bind to LuxR which binds near promoter this recruits RNAP, creating light.
• Then Lux proteins are produced leading to light.
• Low cell density means that low concentration of autoinducers exist.
• Autoinducers are not abundant enough to bind to LuxR.
• LuxR doesn't bind near promoter.
• The bacterial colonies have no transcription therefore there are no lux proteins and no luminescence.
• Enterohemorrhagic E. coli (EHEC)
• Hemorrhagic colitis, hemolytic uremic syndrome is a by product of enterotoxins.
• The toxins produce virulence factors if the cell density is high enough.
• The pathogens have a genome which contains a pathogenicity island.
• The pathogenicity island encodes type 3 secretion system
• The resulting T3SS effectors help the pathogen during infection.

QS and EHEC Virulence

• Tir (effector) secreted into host epithelial cells
• Tir then binds to intimin protein on EHEC surface which attaches EHEC to host.
• Tir remodels host cell cytoskeleton and forms pedestal-like structures ("attaching-effacing lesions").
• It Helps EHEC to colonize the gut.
• EHEC releases Shiga toxin which infects the gut.
• EHEC virulence factors are regulated by QS through a TCS.
• QseC (sensor kinase) senses autoinducers which activates QseB (response regulator).
• The Qsb genes upregulates genes for motility, toxins, lesion formation.
• QseC is also activated by adrenaline a stress hormone.
• Quorum sensing is as a result a potential therapeutic target with antivirulence (cf. antimicrobials).
• Lactobacillus spp. is commonly used as probiotics in the fight against virulence of pathogens.
• Lactobacillus Live microbes which offer health benefit.
• Lactobacillus strains disrupt QS in EHEC.
• The effect will cause it will prevent EHEC from transcribing virulence factor genes preventing can't attach, GI tract colonization.

Dental Plaque

• Plaque: biofilm on surface of tooth
• Dental hard enamel is covered with a soft pellicle.
• Inside the soft pellicle, mucin glycoproteins from saliva which Negative charge limits the process colonization.
• Commensal bacteria can attach to the soft pellicle.
• Then, the pathogens can attach to the commensals.
• Oral microbiota must be attachable or must be attachable or risk of being swallowed.
• Commensals (e.g., Streptococcus mitis) have surface proteins that bind to mucins in pellicle.
• Initial colonizers release autoinducers (Als)
• Commensals are sensitive with detection and regulation when very low levels of Als.
• Shift to biofilm mode.
• As biofilm grows, Al concentration increases.
• Pathogens can detect/respond to Als made by commensals.
• pathogens are responsive with less sensitive interactions.
• The Inter-species communication that happens helps in infections.
• Pathogens attach to commensals in this system.
• Eventually Becoming part of biofilm structures.
• Pathogens release insoluble extracellular polymeric substances (EPS)
• Tooth surface then becomes anoxic.
• Pathogens ferment sugars which make acids.
• As a result plaque impermeable, saliva doesn't dilute acid.
• The Acid demineralizes the enamel and leads to Dental caries
• Biofilm results in inflammation causing it is how periodontal disease occurs.

Reminders

• Midterm 2 Review will occur in class on Feb. 11
• Midterm Exam 2 will is held Feb. 13, 9:30 – 10:20 AM
• Midterm 2 is made of 15 multiple choice and 5 short answer questions.
• Testable lectures: 9 – 15
• Students must bring a pencil
• Accommodated exams are administered by Exam's Office during the midterm period(check Ventus).
• Lab Assignment 2 is due at Feb. 13 at 2:30 PM for section 004.
• Lab Assignment 2 is due Feb. 14 at 2:30 PM for sections 003 and 005.

Lecture 15 reviews bacterial sensing and communication. It examines two-component signal transduction systems and quorum sensing. It also covers how these systems regulate bacterial processes.