CMMB 343 - Lecture 20 Communication

protein secretion

need to be secreted - either embedded in membrane or outside the cell

translocase systems

moderate protein transport using ATP, GTP, or PMF to power movement

universal translocases

in all bacterial cells

= Sec and Tat

general secretion system (sec)

• Co-translation of membrane-associated proteins into cytoplasmic membrane 

  • Signal recognition (SRP) mediated - binds to N-end of polypeptide

  • Uses GTP to power translocation

• Transports unfolded extracellular proteins out of the cytosol - exported then folded into 3D shape 

  • SecA-mediated - binds to N-end 

  • Uses ATP to power translocation

signal recognition protein (srp)

mediates co-translation of membrane-associated proteins into cytoplasmic membrane

binds to N-end of polypeptide, GTP powers translocation

secA

mediates transportation of unfolded extracellular proteins out of cytosol

binds to N-end of polypeptide, ATP powers translocation

twin arginine system (tat)

• Transports folded extracellular proteins out of the cytosol

  • RR-mediated (leader peptide has twin Arg) - N-end

Uses PMF to power translocation

Gram negative secretion systems

• May needs proteins to be embedded in the outer membrane or released on the outside of the cell (past the periplasm and OM)

• Two-step translocases - Types II and V

• One-step translocases - Types I, III, IV, VI

two-step translocases

Types II and V - Move proteins out of the cell one membrane at a time

one-step translocases

Types I, III, IV, VI - Move proteins across both membranes at the same time OR move proteins across both membranes and into a recipient cell at the same time

type II secretion system (T2SS)

two-step

1. Sec or Tat move proteins across inner membrane (into periplasm)

2. Second translocase moves folded proteins across the OM 

3. Proteins attach to secretion pore and pushed out of cell using an ATP-mediated pseudopilin extension

• Common for AB toxins secretion 

type V secretion system (T5SS)

two-step

1. Sec moves (unfolded) autotransporter across inner membrane into periplasm

2. Transporter domain (amino half) of the autotransporter forms a pore in the OM for the passenger domain (carboxyl half) to exit the cell

• Autoproteolysis separates the two domains - leaves pore/transporter domain so passenger domain can be secreted

• Common for secretion of exoenzymes (IgA protease)

type I secretion system (T1SS)

One-Step

• ABC transporters move proteins across both bilayers in one step

1. Inner membrane ABC transporter delivers proteins to a periplasmic membrane fusion protein via ATP hydrolysis → pushes the protein out an OM pore

• Common for secretion of bacteriocins and RTX toxins (repeat intoxin)

type III secretion system (T3SS)

One-Step

• Crosses three membranes 

  • If recipient is G+, directly into cytosol

  • If recipient is G-, directly into periplasm

• “Injectisomes”

• Proteins directly from bacterial cytosol → recipient cell cytosol (prokaryote or eukaryote)

• Pathogens and symbionts

1. Contact with recipient cell

2. Tip fuses with host cell membrane

3. Proteins transported using PMF

• Used to transport cytotoxins or Nod factors 

type IV secretion system (T4SS)

One-Step

• Transports proteins/DNA directly from bacterial cytosol → recipient cell cytosol (prok. or euk.)

• Most common type of secretion system 

• ATP-mediated

• Conjugation systems: responsibly for majority of HGT, used to transfer F plasmids and Ti plasmids (often coded in tra region of plasmids)

• Protein transport systems: transfers proteins/DNA into other cells or into the extracellular space 

• Common for pertussis toxin (whooping cough)

type VI secretion system (T6SS)

One-Step

• Transports proteins directly from bacterial cytosol → recipient cell cytosol (prok. or euk.)

• ATP-mediated T4 phage-like injection system 

1. Close to target

2. Contractile sheath proteins undergo conformational change → extends a spike out of donor cell into recipient

3. Delivers exoproteins 

• Primarily used in microbial warfare (toxins)

responding to signals

Prokaryotic cells - need to respond to environment

• Changes in temp, pH, nutrient concentration, cell density

• Presence of antibiotics, enzymes, immune components

signals

Can act directly as effector molecules (inducers or corepressors)

Can act indirectly by binding to cell surface receptors 

inducers

bind to transcription factors (activators or repressors) and turn transcription ON

corepressors

bind to repressors and turn transcription OFF

two component regulatory systems

sensor kinase + response regulator

sensor kinase

• Transmembrane histidine kinase - inner component 

• Outer component recognizes signal (receptor)

• Histidine component autophosphorylates (converts ATP → ADP) when bound to a signal and transfers a phosphoryl group to a response regulator 

response regulator

Usually a transcription factor that is active when phosphorylated (acts as an effector molecule)

phosphatase

• Sort of a third-component…to the two component regulatory system

• Removes the phosphate and turns off the response regulator 

• Resets 

quorum sensing

density-dependent mechanism for cellular communication to induce a population/community response 

• For biofilm formation, sporulation, competence (ability to transfer DNA), bioluminescence, production of virulence factors 

• Facilitated by extracellular peptide or non-peptide signaling molecules - autoinducers 

• When a high enough concentration of a signal is reached → “quorum is reached” → coordinated gene regulation occurs (all or nothing)

• For related/close species

autoinducers

Molecules accumulate as population densities increase 

Produced by bacteria - float in environment to reach other bacteria

acyl homoserine lactones (AHLs)

gram negative autoinducers, species-specific

• Only closely-related species produce AHLs that can communicate - if too far related, AHLs won’t be recognized 

• AHLs diffuse out of the cell into surrounding environment 

• High cell densities → AHL concentrations increase both outside and then inside the cells 

• AHLs binds transcription factors - some bind sensor kinases that regulate Tc factors 

• Results in coordinated up-regulation of quorum-specific genes 

  • Some down-regulate gene expression too

oligopeptide autoinducers (AIPs)

Gram-positive autoinducers

• Peptides, not lipid soluble 

  • Can’t diffuse in and out of cells in active form like G -ve autoinducers 

• Pre-AIPs are transported out of the cell via ABC transporters into the surrounding environment (inactive form) 

  • final processing by peptidases

• High cell density → AIP concentration increases outside the cells 

• AIPs bind sensor kinases (2CRS)

• Results in coordinated up-regulation of quorum-specific genes 

  • Some down-regulate gene expression too

how autoinducers vary

all have a sugar group and an acyl chain

• Autoinducers can vary in the length of the acyl chain (4-18C) and R group (-OH, =O, -H), this is how they vary between species!

universal autoinducers

interspecific quorum sensing, best studied is AI-2 (autoinducer-2), results in coordinated up-regulation of quorum-specific genes across multiple species 

• Biofilm formation, HGT events, bacterial warfare

bacterial bioluminescence

• Some species can produce light via bioluminescence

• An example of quorum sensing

• E.g. Vibrio, Aliivibrio, Photobacterium

bioluminescence symbiosis

• Bioluminescent bacteria can colonize the light organs of marine creatures such as squid and flashlight fish 

• Mutually beneficial relationship (symbiosis)

  • Bacteria provide light for defense and predation (can hide from predators by mimicking light in surroundings or find prey)

  • Bacteria receive nutrients from the host 

A. fischeri and bobtail squid

• Bobtail squid have a special light organ 

  • Sterile at birth, but colonized by A. fischeri within hours 

  • Ciliated ducts trap bacteria in mucous layer (sweep bacteria toward a pore)

  • Chemotactic sugars lure A. fischeri inside 

  • Also kill bacteria that’s not A. fischeri so it’s easier for it to colonize 

• Presence of A. fischeri stimulates maturation of light organ 

  • Once colonized, A. fischeri lose their flagella and grow exponentially 

    • Don’t need to swim around anymore once inside 

    • Reproduce inside light organ 

• At high concentrations (i.e. at quorum), A. fischeri is bioluminescent and protects the squid by providing counter-illumination at night when the squid is hunting 

  • Some prey attracted to glow 

  • Also to avoid predators

• Squid burrows in sand in the morning (glowing can be detrimental during the day) and vents ~90% of the A. fischeri and the process begins again 

  • A. fischeri expelled and can find another host 

  • Reach quorum again by sunset 

luciferase

produces light in the presence of a luciferin and oxygen 

• A. fischeri uses RCHO (aldehyde) + FMNH₂ as a 2-component luciferin 

• At night, bobtail squid directs O₂ into the light organ 

• Chemical reaction generates light

• RCHO + FMNH₂ + O₂ → RCOOH + FMN + light + H₂O (via luciferase enzyme)

  • FMNH₂ is an e-carrier, also used in electron transport chains - donates e- to reduce O₂

• At night, blue-green light can mask itself and shadow from predators

  • Augmentation of its ink sac and an iris helps regulate the light intensity (so not so bright)

quorum sensing in A. fischeri

• A. fischeri expresses LuxI at basal levels (24/7)

• LuxI: an AHL (Gram -ve) synthase, produces an AI-1-type AHL autoinducer (3-oxohexanoyl-homoserine lactone/3OC6-HSL)

• 3OC6-HSL diffuses throughout the squid light organ to reach quorum 

• At high concentrations, 3OC6-HSL binds LuxR

  • LuxR regulated by an independent quorum sensing system

LuxI

• an AHL (Gram -ve) synthase, produces an AI-1-type AHL autoinducer (3-oxohexanoyl-homoserine lactone/3OC6-HSL)

• expression generates a positive-feedback loop, expressed at basal levels

LuxR

an activator protein (transcription factor), which binds to activator binding sites and induces the Lux operon

• regulated by an independent quorum sensing system

has a different promoter than LuxI/C/D/A/B/E/G

the lux operon

one promoter - luxI/luxC/luxD/luxA/luxB/luxE/luxG

another promoter - luxR

LuxC/D/E

produce a multicomponent fatty acid reductase

• Synthesizes RCHO (a long-chain fatty aldehyde)

LuxA/B

• produce a heterodimeric enzyme (luciferase)

  • Luciferase - the light-generating enzyme!

LuxG

produced FMN reductase

Synthesizes FMNH₂ (a reduced electron carrier)

symbiosis between S. aureus and humans

• S. aureus is ubiquitous on skin and nasal passages and once thought to be commensal (GRAM-POSITIVE)

  • Now known as an opportunistic pathogen - can bypass the innate immune defense

• Produces an impressive variety of virulence factors that lead to variety of infections

  • The pyogenic infections (pus-forming)

    • Can cause acne, boils, meningitis, etc. - many virulence factors (adhesins, exoenzymes, toxins, protein A, antimicrobial resistance)

S. aureus global control system

an inducible set of regulons (group of operons under control of same inducer) controlled by quorum sensing, all turned on at the same time

AgrD, AgrB, AgrC, AgrA

AgrD

synthesizes an autoinducing peptide (AIP)

AgrB

cell membrane transporter - exports the pre-AIP

AgrC

bound to AIP at high concentrations of AIP, a histidine kinase, will phosphorylate AgrA

AgrA

• response regulator/activator protein

  • Once phosphorylated, AgrA-P binds at activator binding sites (operators) with multiple regulons

  • Upregulates the production of multiple virulence factors (many gene products)

inhibiting S. aureus global control system

microbial warfare by GI microbiota inhibits via competitive inhibition

• Blocks pre-AIP from binding to ArgC kinase 

• Yay for bacillus spp.!