MICR 221 Lecture 14 PDF: Bacterial Secretion and Efflux

Lecture 14: Secretion and Efflux Feb. 6, 2025 1 Lecture Learning Outcomes After this lecture, students will be able to describe… How AB toxins, pore-forming toxins, and superantigens lead to damage to host tissues How proteins are translocated through (a...

Lecture 14: Secretion and Efflux Feb. 6, 2025 1 Lecture Learning Outcomes After this lecture, students will be able to describe… How AB toxins, pore-forming toxins, and superantigens lead to damage to host tissues How proteins are translocated through (and into) the cytoplasmic membrane by the Sec system, Tat system, and ABC transporters How proteins are targeted to the outer membrane How bacteria secrete substances using secretion systems, efflux pumps, and extracellular vesicles How Gram-positives attach proteins to their surfaces 2 Exotoxins Proteins secreted by pathogens Disrupt host cells Contribute to many bacterial infections E.g., pertussis toxin, cholera toxin Can travel from site of infection to other tissues Grouped by mechanism, structure AB toxins Pore-forming toxins Superantigens 3  AB Toxins Interfere with processes inside host cells A and B components form a complex A: active component B: binding component B component binds to A receptor on host cell Triggers endocytosis Helps A component enter B A component is an enzyme Cause of toxic effect cholera toxin 4 Image from: doi: 10.3390/toxins2071612  Diphtheria Toxin (AB Toxin) Diphtheria: Corynebacterium diphtheriae infection Diphtheria toxin damages cardiac, nervous tissue B component activates endocytosis A component released from vacuole Modifies elongation factor Blocks translation One toxin can modify all EF-2 in cell Image from: Prescott’s Microbiology, 11th Edn 5  Shiga Toxin (Stx) Enterohemorrhagic E. coli Severe inflammation, gastrointestinal bleeding Carries prophage Encodes Shiga toxins (e.g., Stx-1) Toxin damages vascular endothelium AB toxin A component removes a nucleobase from rRNA Disables ribosome 6 Image from: https://doi.org/10.1007/978-1-4939-7349-1_14  Pore-Forming Toxins Insert into host cell membrane, forming channels Disrupts ion gradients, and cytoplasmic contents leak out Water enters, leading to swelling Cell is lysed 7 Image from: Prescott’s Microbiology, 11th Edn  Pore-Forming Toxins Lysis of host cells releases nutrients Hemolysins: toxins that lyse red blood cells Bacteria scavenge released heme/iron Help bacteria escape from phagosomes E.g., Listeria monocytogenes Intracellular pathogen After escape, replicates in immune cells Spreads through blood Bacterial meningitis 8 Image from: https://doi.org/10.1038/nrmicro1413  Superantigens Macrophages, DCs present antigens to T cells Superantigens bind these cells together even if no antigen is present Triggers overproduction of proinflammatory cytokines Leads to fever, low blood pressure, organ failure E.g., Staphylococcus aureus TSS toxin Toxic shock syndrome 9 Image from: Prescott’s Microbiology, 11th Edn Protein Translocation and Secretion ~1 in 3 bacterial proteins leave the cytoplasm Targeted to every part of cell Each target requires a distinct transport strategy Translocation: transport across membrane Also: insertion into membrane Secretion: release into external environment Requires translocation through one or two membranes 10  Crossing the Cytoplasmic Membrane Proteins produced in cytoplasm Chaperones may help folding E.g., GroES/GroEL Translocated/secreted proteins often have signal peptides Specific amino acid sequence at N-terminus Recognized by transport signal systems peptide Proteins translocated to periplasm by: Sec system Tat system ribosome ABC exporters 11 Image from: https://doi.org/10.3389/fphys.2022.933153  Sec System: Post-Translational Translocation Sec system translocates unfolded proteins into periplasm Chaperones stabilize protein before translocation periplasm SecA binds to signal peptide, escorts protein to SecYEG SecA drives translocation of protein through SecYEG Uses ATP Post-translational translocation Occurs after translation is complete cytoplasm 12 Image from: Prescott’s Microbiology, 12th Edn  Sec System: Periplasmic Proteins Signal peptidase (SPase) recognizes, cuts signal peptide Occurs during translocation Translocated protein folds in periplasm Periplasmic chaperones Periplasm is an oxidizing signal peptide environment Promotes disulfide bond formation Periplasmic proteins ensure correct disulfide formation E.g., protein disulfide isomerase 13 Image from: https://doi.org/10.1016/j.bmcl.2015.07.072  Tat System Not all proteins can be folded in the periplasm Some may require: Co-factor insertion E.g., iron-sulfur clusters need >10 proteins Cytoplasmic chaperones Assembly into complexes Tat system translocates folded proteins into periplasm Tat = twin arginine translocase Different signal peptide than Sec system 14 Images from: https://doi.org/10.3389/fcell.2021.735678 , https://www.chemistry.nat.fau.eu/span-group/research/  Tat System Protein folds in cytoplasm Targeted to TatABC by signal peptide (SP) No targeting factor needed (unlike Sec system with SecA) TatABC translocates protein to periplasm Proton motive force (PMF) SP is cleaved off 15 Image from: https://doi.org/10.1111/mmi.14461  ABC Exporters ATP-binding cassette (ABC) transporters Primary active transport ATP Specific Lecture 11: many ABC transporters import substances Figure of ABC importer Others act as exporters Waste, antibiotics Also translocate proteins Recognize signal peptide Signal peptide cleaved during export 16 Image from: Prescott’s Microbiology, 11th Edn  Sec System and Membrane Proteins Sec system can also insert proteins into cytoplasmic membrane Signal peptide translated first Very hydrophobic for membrane proteins Signal peptide recognized by signal recognition particle Halts translation Escorts ribosome to SecYEG Translation continues, inserting protein into membrane Co-translational translocation 17 Image from: Prescott’s Microbiology, 11th Edn  Gram-Negative BAM Complex β-Barrel proteins targeted to outer membrane Outer membrane proteins (OMPs) Unfolded protein translocated by Sec system Stabilized by periplasmic chaperones (SurA, Skp) Delivered to β-barrel assembly machinery (BAM) complex Inserts into membrane 18 Image from: DOI 10.1038/nrmicro.2016.191  Gram-Negative Secretion Systems Secretion systems transport proteins out of cell Secreted proteins can be: Bound to cell surface Released into external environment Injected into other cells 19 Image from: Prescott’s Microbiology, 12th Edn  Type V Secretion System (T5SS) Tat or Sec translocates protein to periplasm external Protein is translocated through β-barrel in outer membrane β-Barrel can be: periplasm Separate protein Part of protein being translocated Protein remains tethered to cell cytoplasm surface 20 Image from: Prescott’s Microbiology, 11th Edn  Type I Secretion System (T1SS) Three components: ABC transporter Membrane fusion external protein (MFP) β-barrel protein ABC transporter: Powers transport Determines specificity β-barrel forms channel through outer membrane cytoplasm MFP connects ABC transporter, β-barrel 21 Image from: Prescott’s Microbiology, 11th Edn  Type III Secretion Systems (T3SSs) T3SSs inject proteins into eukaryotic cells T3SS substrates called effector proteins Effectors travel from bacterial cytoplasm to cytoplasm of host cell Effectors manipulate host cell structure and function, facilitating colonization 22 Image from: Prescott’s Microbiology, 11th Edn  T3SS Structure and Assembly Made of >20 proteins tip needle Related to flagella Basal body, needle/filament Export functions During assembly, subunits (needle, tip) travel through hollow central channel After assembly complete, plug blocks T3SS flagellum channel 23 Image from: https://doi.org/10.1016/j.tibs.2015.09.005  T3SS Effectors Once T3SS binds to host cell, channel opens Chaperones deliver unfolded effectors to T3SS Effector travels through T3SS, folds in target Transport powered by ATP, proton motive force chaperone A single T3SS can secrete many different effectors Recognizes conserved N-terminal secretion signal 24 Image from: https://doi.org/10.3390/biom11020316  T3SS Effector Proteins Effectors target host cytoskeleton, signal transduction Help bacteria attach and invade Salmonella: intracellular pathogen Gastroenteritis Systemic illnesses (e.g., typhoid fever) Salmonella T3SS effectors rearrange cytoskeleton of epithelial cells Forms surface “ruffles” Allows bacteria to enter non- phagocytic cells 25 Image from: https://doi.org/10.1073/pnas.97.16.8754  Efflux Pumps Transport molecules to periplasm or outside cell (e.g., antibiotics) Several classes, e.g., ABC transporters RND tripartite system Resembles T1SS 26 Image from: https://doi.org/10.1016/B978-0-12-818480-6.00010-2 DOI 10.1038/s41579-018-0048-6  Efflux Pumps and Antibiotic Resistance Major contributor to multidrug resistance Single pump can pump out different antibiotics Mutations increase number of pumps produced 27 Image from: https://doi.org/10.1128/CMR.00117-14  Extracellular Vesicles (EVs) Bacteria export substances in extracellular vesicles Spherical buds released from cell surface Released by Gram-positives and Gram-negatives Gram-negatives: outer membrane vesicles (OMVs) Contain cargo (e.g., proteins, DNA) Contribute to: Horizontal gene transfer Virulence Antibiotic resistance 28 Image from: https://doi.org/10.1038/nrmicro3525  Extracellular Vesicles (EVs) and Virulence EVs deliver cargo to other cells Taken up by fusion, endocytosis Can be used by bacteria to deliver toxins to host cells E.g., enterotoxigenic E. coli (ETEC) Travelers’ diarrhea EVs deliver enterotoxin to intestinal cells Toxin causes secretion of water, diarrhea 29 Image from: https://doi.org/10.1038/nrmicro3525  EVs and Antibiotic Resistance EVs can help protect against antibiotics E.g., Gram-negative EVs containing β- lactamases β-lactam enters EV through porins β-lactamases in EV degrade β-lactam Keeps β-lactams away from PBPs 30 Image from: https://doi.org/10.3390/ijms21082822  Gram-Positive Bacteria and Secretion Protein secretion in Gram-positives is simpler No outer membrane Elaborate secretion systems (usually) not needed Gram-positives have Sec and Tat systems Some secreted proteins get attached to cell surface Sortase attaches proteins that have cell wall sorting signal 31 Image from: https://doi.org/10.1038/nrmicro2520  Gram-Positive Surface Proteins Surface proteins often contribute to virulence Attachment to host cells, extracellular matrix Pili, other adhesins Nutrient acquisition in host E.g., heme binding (iron) Immune evasion Proteases Degrade antibodies Protein A Binds to antibody Fc region, preventing opsonization 32 Image from: https://doi.org/10.1016/j.tips.2015.11.008 Reminders Bacteriology Quiz 4 opens Feb. 6 at 3 PM Lectures 12 - 14 Lab 2 Assignment Section 002: due Feb. 6, 2:30 PM Lab 3 Section 002: Feb. 6 at 2:30 PM Section 004: Feb. 6 at 4:00 PM Section 003, 005: Feb. 7 at 2:30 PM Complete Lab 3 Pre-Lab Quiz before 33