Endospore Formation in Bacteria PDF

Endospore formation  A typical bacterial spore A spore is a dormant cell Resistance to: Protective layers: Heat Desiccation...

Endospore formation  A typical bacterial spore A spore is a dormant cell Resistance to: Protective layers: Heat Desiccation Cortex Chemicals (e.g., bleach, chloroform) Enzymes (e.g., lysozyme) Multi-layered coat Antibiotics (~80 proteins) UV radiation 0.5 m McKenney et al. (Current Biol. 2010) McKenney, Driks, Eichenberger (2013)  First publication on endospore formation (1876) B. subtilis Ferdinand Cohn B. anthracis Robert Koch Beiträge zur Biologie der Pflanzen, 1876  Firmicutes and the ability to sporulate B. subtilis model organism B. licheniformis B. anthracis pathogen (human and cattle) Bacillus B. cereus food poisoning Aerobic, B. thuringiensis insecticidal toxin Spores B. halodurans G. kaustophilus O. iheyensis L. monocytogenes food poisoning Listeria L. innocua Aerobic, No spores S. aureus pathogen Staphylococcus S. epidermidis pathogen S. haemolyticus pathogen C. difficile colitis and diarrhea Clostridium C. perfringens necrosis (gangrene) Anaerobic, C. tetani neurotoxin (tetanus) Spores  Diversity among species of spore-forming bacteria B. amylo- B. subtilis B. subtilis natto B. pumilus liquefaciens Model organism B. pseudo- B. anthracis B. flexus B. megaterium B. clausii mycoides B. pychnus Br. laterosporus P. alvei C. difficile Colitis and diarrhea Nosocomial infections Proliferates after antibiotic treatment Driks and Eichenberger 2016 Microbiol. Spectrum Spore-forming bacteria and human health Anthrax Bacillus anthracis Category A bioterror agent Colitis and diarrhea Clostridiodes difficile Half a million infections (29,000 deaths) in 2011 (CDC) Nosocomial infections (proliferation after antibiotic treatment) Botulism Clostridium botulinum Neurotoxin (Botox) Tetanus Clostridium tetani Neurotoxin (TeTx)  Sporulation cycle Vegetative cell Sporulation Germination -nutrients Spore +nutrients  Anthrax Note: the spore is the delivery vehicle, the vegetative cell is causing the disease Mock and Fouet (2001) Annu Rev Microbiol Sporulation cycle McKenney, Driks, Eichenberger (2013)  Spo0A is the master regulator of sporulation Spo0A is a response regulator N-terminal regulatory domain C-terminal effector domain (Asp phosphorylation site) (DNA binding) Lewis et al. (2002) Zhao et al. (2002)  Spo0A is activated by a phosphorelay Classic 2-component: HK RR KinA HK KinB RR KinC Phosphorelay: Intermediates between HK and RR Stephenson & Hoch Mol Micro 46: 297-304  Regulation of the phosphorelay Redox state DNA damage Sda Cell density (quorum sensing) KinA KinB KinC PhrA RapA Spo0F Spo0B Spo0E (phosphatase) Spo0A Piggot & Hilbert (2004) Asymmetric division Driks and Losick  B. subtilis has 18 factors 1 primary factor and 17 alternative factors A primary factor B general stress response D motility, chemotaxis, autolysis E spore formation (mother cell-early) F spore formation (forespore-early) G spore formation (forespore-late) H stationary phase (pre-divisional sporulating cell) Sporulation K spore formation (mother cell-late) L enhancer-dependent factor ( 54-like) M antimicrobial resistance and cell envelope stress (ECF) V antimicrobial resistance and cell wall stress (ECF) W antimicrobial resistance and cell wall stress (ECF) X antimicrobial resistance and cell envelope stress (ECF) Y antimicrobial resistance and cell envelope stress (ECF) Z unknown (ECF) I(ykoZ) unknown ylaC unknown (ECF) yvrI-yvrHa unknown (composite factor) Compartmentalization of gene expression F activity H F E E activity G K  F inhibition: the anti- factor SpoIIAB H IIAA IIAB sigF spoIIA operon F is expressed before asymmetric division but is held inactive Complex with anti- factor (SpoIIAB or AB) AB2: F Stragier & Losick (1996)  F activation: the anti-anti- factor SpoIIAA H IIAA IIAB sigF spoIIA operon AB is also a kinase AA is inactivated by phosphorylation Ho, Carniol, Losick (2003)  F activation: the anti-anti- factor SpoIIAA H IIAA IIAB sigF Release of F is induced spoIIA operon by binding of the anti-anti- factor (SpoIIAA or AA) Ho, Carniol, Losick (2003) F activation: dephosphorylation of SpoIIAA by SpoIIE For F activation, AA must be dephosphorylated SpoIIE is the corresponding phosphatase Phosphatase domain Stragier & Losick (1996) Bradshaw & Losick (2015)  F activation: SpoIIE is a membrane protein SpoIIE is localized at the polar septum (interacts with FtsZ) Membrane domain (10 TMs) Stragier & Losick (1996) Bradshaw & Losick (2015)  F activation if SpoIIE is equally distributed on both sides Given that the forespore is much smaller than the mother cell, the concentration of dephosphorylated AA is expected to be higher in the forespore Roush (1995)  E activation A F IIGA sigE spoIIR spoIIG operon E is synthesized as pro- E (inactive) spoIIR operon E is activated by proteolysis (SpoIIGA is the protease) Proteolysis is in response to a signal from the forespore F-dependent Rudner and Losick (2001)  E and K activation E and K are activated by proteolysis in response to a signal from the forespore Rudner and Losick (2001) Crisscross regulation of factors H F E G K Stragier & Losick (1996) Article#8: Preferential localization of SpoIIE in the forespore Regulatory domain? Bradshaw & Losick (2015) Article#8: Preferential localization of SpoIIE in the forespore Capture at the cell pole (interaction with FtsZ) Bradshaw & Losick (2015) Article#8: Preferential localization of SpoIIE in the forespore Capture at the cell pole (interaction with FtsZ) Proteolysis (dependent on FtsH and Tag) Spatially restricted proteolysis (only in the mother cell) Degradation signal Bradshaw & Losick (2015) Article #8: Stabilization and multimerization of SpoIIE in the forespore Residues involved in stabilization Bradshaw & Losick (2015) Article #8: Preferential localization of SpoIIE in the forespore Capture at the pole, proteolytic stabilization and stimulation of the phosphatase all depend on oligomerization of SpoIIE Bradshaw & Losick (2015)  Systems Biology: Gene regulation networks The B. subtilis gene regulation network TF nodes gene edges Arrieta-Ortiz et al. (2015, Mol. Syst. Biol.)  Organization of the protein interaction network Connectivity distribution of yeast protein interaction network follows a power-law distribution Many genes with few interactions, few genes with many interactions Systems are buffered from random mutations Gene regulation network in yeast: mapping regulons with ChIP-chip ChIP-chip analysis of 106 transcriptional factors Lee et al. (2002) Science 298, 799-804 Regulatory network motifs Lee et al. (2002) Science 298, 799-804  Regulatory network inference Transcriptomics data Network Inference Algorithm Network model= Genes connected to their regulators  The Inferelator Using RNA expression levels to find potentially causative relationships Transcriptomics data expression steady state TFn data genem (correlation) Experimental conditions expression time-series genem Richard Bonneau data (causation) TFn experiments (in time series) TFn Genem Bonneau et al. (2006, Genome Biol.)  Modulation of transcription factor activity Hetero Effectors Modification -dimerization Inactive TF (cAMP, Phosphorylation, GTP, proteolysis, etc. etc.) Active TF PDB Levdikov et al (2017) Lorca et al (2014) Fu, Y., Jarboe, L. R., & Dickerson, J. A. (2011), BMC Bioinformatics  TF transcription vs. TF activity CodY activity is modulated by GTP and branched chain amino acids (BCAA) CodY Low DNA binding affinity R2=0.767 of known CodY targets Average transcription GTP BCAA CodY High DNA binding affinity codY CodY 134 genes transcription activity Levdikov et al (2017) Arrieta-Ortiz et al. (2015, Mol. Syst. Biol.)  The B. subtilis transcription network 3,040 regulatory interactions (manually curated-SubtiWiki, 2013) 1,874 genes 153 transcription factors (TFs) Regulation for 1/3 of predicted TFs ~half of the genes without known target is unknown TF gene interaction Michna et al. (NAR, 2014)  Using TFA in network inference Gene Expression Prior Network Network Inference X P Activity Estimates Gene TF Expression activities Estimate TF Activities Inferred Network  Gene Expression Known TF-gene Regulatory interactions Arrieta-Ortiz et al. (2015, Mol. Syst. Biol.)  Prior 3,040 interactions 1,874 genes network 153 TFs TF nodes gene edges Inferred 4,516 interactions 3,086 genes network 215 TFs Sporulation genes Arrieta-Ortiz et al. (2015, Mol. Syst. Biol.)  Sporulation cycle Vegetative cell Sporulation Germination -nutrients Spore +nutrients  Germinants Germinants are species-specific B. subtilis germinants L-Alanine: GerA receptor L-Asparagine, glucose, fructose and KCl (AGFK): GerB and GerK receptors Spore germination and outgrowth in B. subtilis Germination Outgrowth Loss of optical density Sinai et al (2015) Germination receptors are nutrient-gated ion channels Gao et al (2023)  Stage I of spore germination A. Dormancy C. Amplification 5AF/ GerA FigP SpoVA complex complex complex Inner membrane AD Spore core K+, Na+, H+ B. Initiation of germination D. Dipicolinic acid (DPA) release L-alanine K+, Na+, H+ K+, Na+, H+ DPA Eichenberger (2024) Stages of spore germination DPA release Core hydration Loss of heat resistance Cortex hydrolysis Setlow Anthrax Liu et al 2014  Anthrax virulence factors Toxins expressed by the vegetative cell PA: protective antigen (carrier protein) LF: lethal factor PA+LF=LeTx lethal toxin EF: oedema factor PA+EF=EdTx oedema toxin Toxin genes are located on pXO1 plasmid Capsule surrounds the vegetative cell, composed of poly-D-glutamic acid (PDGA) capBCADE operon on pXO2 plasmid  Bioterrorism Read et al (2002) Science  Strains of B. anthracis Strains Plasmids Source Pathogenicity Ames ancestor pXO1+, pXO2+ isolated from dead cow (Sarita, TX) yes Ames Florida pXO1+, pXO2+ isolated from bioterror victim (Florida) yes Ames Porton Down pXO1-, pXO2- plasmids cured (Porton Down,UK) no Sterne 34F2 pXO1+, pXO2- vaccine strain no Rasko et al 2005 pXO1 plasmid (182 kb, 204 genes) LF PA EF PA: protective antigen LF: lethal factor EF: edema factor Okinaka et al. 1999  Anthrax toxins LF: lethal factor PA+LF=LeTx lethal toxin EF: edema factor PA+EF=EdTx edema toxin PA: protective antigen (carrier protein)  Endocytosis of anthrax toxins PA83 monomer is PA: protective antigen cleaved by protease LF: lethal factor (Furin) EF: edema factor PA20 is released ERK p38 JNK  LF=lethal factor Zn-metalloprotease that cleaves members of the MAPKK family Removes the docking site for MAPKs (mitogen activated protein kinases) on MAPKKs Pannifer et al. (2001)  EF=edema factor Adenylate cyclase, dependent on calmodulin (CaM) for activity Causes increase in cAMP levels Drum et al. (2002)  pXO2 plasmid (95 kb, 104 genes) Capsule surrounds the vegetative cell, composed of poly-D-glutamic acid (PDGA) capBCADE operon NCBI Capsule and S-layer C: capsule S: S-layer P: peptidoglycan m: membrane Mesnage et al. (1998) Model for capsule synthesis and assembly Richter et al 2008 C. difficile is a nosocomial agent Rupnik et al (2009) High risk of CDI after antibiotic treatment CD I= C. difficile infection Rupnik et al (2009) Microbiota provides resistance against infection Buffie & Pamer 2013 Germination of C. difficile is induced by bile salts Paredes-Sabja et al (2014) C. difficile toxins cause pseudomembranous colitis Shen (2012)  C. difficile toxins PaLoc (pathogenicity locus) located on the chromosome Toxins A (TcdA) and B (TcdB) Inactivation of Rho and Ras by glucosylation Rupnik et al (2009) Mechanism of action of C. difficile toxins Jank & Aktories 2008 Mechanism of action of C. difficile toxins Jank & Aktories 2008 Biofilms, fruiting bodies A biofilm is a matrix-encased microbial community Most bacteria are capable of forming biofilms Biofilms are found in close association with surfaces or at interfaces solid-air solid-liquid air-liquid 1 m A typical biofilm biofilm A biofilm often contains more than one microbial species Mixed-species biofilms predominate in most environments Single-species biofilms can exist in a variety of infections (e.g., Pseudomonas aeruginosa in cystic fibrosis)  The dental plaque is a mixed-species biofilm Letter to the Royal Society of London Antonie van Leeuwenhoek (1632-1723) Peters B M et al. Clin. Microbiol. Rev. 2012;25:193-213  Benefits of biofilm formation 1) Protection Resistance to physical forces (saliva, blood flow) Resistance to phagocytosis 2) Colonization of a favorable niche 3) Communal behavior Division of the metabolic burden Gene transfer  Biofilm formation Transition from planktonic (free-living cells) to sessile (surface-attached) cells Transition from loner to community-based existence 4) detachment Pillars and mushroom-like structures 1) attachment 2) colonization 3) growth O’Toole et al. (2000) and matrix formation Fluid-filled channels View from above Fluid-filled channels  Submerged biofilm (liquid-solid interface) Medium flow (e.g., saliva with dental plaque) Confocal scanning laser microscopy Vibrio cholerae Cells express GFP constitutively Branda et al. (2005)  Submerged biofilm (liquid-solid interface) Standing culture (no medium flow) Microtiter dish wells and crystal violet dye E. coli cells wild type sad mutant Facilitates high-throughput screens to identify genes involved in biofilm formation Isolation of sad mutants (surface attachment defective) Branda et al. (2005)  Biofilm at the air-liquid interface Pellicle formation Standing culture (no medium flow) Pellicle B. subtilis wild type mutant Branda et al. (2005)  Biofilm at the air-solid interface (=on plate) Colony morphology on LB agar plate Coccobacteria septica Rhodospirillum centenum Escherichia coli K-12 Pseudomonas aeruginosa Streptomyces Saccharomyces cerevisiae coelicolor Amycolatopsis Serratia marcescens wild type mutant Congo Red dye Bacillus subtilis Pseudomonas oryzihabitans Candida albicans Pseudomonas aeruginosa Mycobacterium smegmatis Vibrio cholerae Branda et al. (2005) Okegbe et al. (2014)  “Wrinkling” to maximize access to O2 Kempes et al. (2014) Okegbe et al. (2014)  Pseudomonas aeruginosa biofilms Opportunistic pathogen exploits breaks in the host defenses to initiate an infection (e.g., cystic fibrosis, AIDS, injury, burns) Planktonic cell Schaechter (flagellar motility) Twitching motility Mature biofilm (type IV pili) Aggregation requires twitching motility Pseudomonas aeruginosa Type IV pilus-defective mutant O’Toole et al. (2000)  Lactoferrin prevents biofilm formation Lactoferrin is a component of the innate immunity host response View from above Perturbs twitching motility of P. aeruginosa cells Side view Singh et al. Nature 417, 552-555 Quorum sensing signals control biofilm formation in P. aeruginosa las system (LasR) 2 AHL autoinducers (acyl-homoserine lactone) rhl system 1 PQS autoinducer (RhlR) (Pseudomonas quinolone signal) Lazdunski et al (2004)  Matrix production Pseudomonas aeruginosa Staphylococcus aureus Branda et al. (2005) Matrix is usually formed of EPS =extracellular polysaccharides (exopolysaccharides) Timescale of biofilm formation  Prokaryotic development cycles 1) Unicellular and cell-cycle independent Bacillus endospore formation 2) Unicellular and cell-cycle dependent Caulobacter swarmer cell differentiation 3) Multicellular via directed movement (cell-cycle independent) Myxobacteria fruiting body formation 4) Multicellular via directed growth (cell-cycle dependent) Streptomycetes sporulation  Myxobacteria fruiting body formation Fruiting body Myxospore Mounded aggregate not as resistant as an endospore, but more resistant than a vegetative cell Lysis of prey Swarm Goldman, B. S. et al. (2006) prey Proc. Natl. Acad. Sci. USA 103, 15200-15205  Species of Myxobacteria Myxobacteria are -proteobacteria Sporangioles (globular spore-containing structures) form at the tip of the fruiting body Myxococcus Myxococcus Myxococcus Stigmatella aurantiaca Chondromyces crocatus stipitatus fulvus xanthus Contains ~ 105 spores One sporangiole Several sporangioles  Motility of Myxobacteria Adventurous (A)-motility individual cell movement Social (S)-motility migration as a swarm important for predation, swarms kill and consume other microbial cells group action is necessary for killing and lysis  Adventurous motility Reversal of direction Slime trail Change direction ~every 5 min Sogaard-Andersen (2004)  A-motility system: gliding Gliding =motility on solid surfaces (no flagellum involved) Islam and Mignot (2015) Requires: focal adhesion complexes (Agl motor-Glt complex) cytoskeletal proteins (helical track) slime production  Focal adhesion complexes (FACs) and Glt apparatus Mignot et al. Science (2007) AglZ-YFP oscillates from pole to pole upon cellular reversals Islam and Mignot (2015) Focal adhesion complexes (FACs) and Glt apparatus Nan et al (2014) Slime production by Nls (Nozzle-Like-Structures) Nls secrete slime Wolgemuth et al (2002)  S-motility system: twitching Twitching motility: Cycles of extension, adhesion and retraction of type IV pili (Tfp) Fibrils on the cell surface interconnect neighboring cells to form an extracellular matrix Cells have to be within contact distance of each other Contact with extracellular matrix triggers retraction of pili Zhang et al (2012)  Reversal of direction Motility is a polarized process Unknown mechanism that involves Frz~P Zhang et al (2012) Aggregation is controlled by A- and C-signals A is a soluble signal A is a quorum-sensing signal inducing the first stages of aggregation (! here A does not stand for adventurous) C is a cell-contact signal C is morphogenetic and helps to shape the fruiting body A and C are produced in response to amino acid starvation  Mode of action of the A-signal Mixture of 6 amino acids (Trp, Pro, Phe, Tyr, Leu, Ile) and peptides containing those amino acids Stringent response signal Kaiser (2004) Stringent response (ppGpp synthesis) Response to amino acid starvation Uncharged tRNA Detects stalled ribosome (p)ppGpp synthesis Charged tRNA Braeken et al. (2006) Mode of action of the A-signal Mixture of 6 amino acids (Trp, Pro, Phe, Tyr, Leu, Ile) and peptides containing those amino acids Histidine kinase Two- component NtrC-like system response regulator Kaiser (2004) Expression of A-signal-dependent genes NtrC-like 54 activator proteins C. Wyman et al., Science 275, 1658 -1661 (1997) The C-signal regulates fruiting body formation At intermediate levels, C promotes aggregation At high levels, C induces sporulation  C is a cell-contact signal C is a 17kDa protein anchored in the outer membrane C is encoded by csgA CsgA is a precursor of C (activated by proteolysis) Kaiser (2004) C-signal concentration increases as a consequence of a positive feedback loop C induces phosphorylation of FruA Kaiser (2004) Note that fruA expression is dependent on A signal FruA~P acts on the Frz system to promote aggregation Stevens & Sogaard-Andersen (2005) C switches motility behavior from oscillatory to unidirectional (by decreasing the reversal frequency) Ensures that every cell in the swarm moves in the same direction FruA~P activates dev to induce sporulation Kaiser (2004) FruA~P is a DNA binding response regulator Genome sequence of Myxococcus xanthus 9.1 Mb, 69 % GC content One of the largest bacterial genomes Gene duplication and divergence 48% of the CDS have at least one paralog Goldman, B. S. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 15200-15205  Characteristics of the M. xanthus genome Selective amplification of genes for cell-cell signaling and transcription control 137 sensor and hybrid histidine kinases 97 STPK genes (serine threonine protein kinase) 53 EBP genes (enhancer binding proteins for 54 dependent genes-NtrC-like) Metabolic genes Genes for synthesis of branched chain a.a. are absent Leu, Ile, Val account for 1/5 of a.a. found in average proteins Consistent with extreme sensitivity to a.a. starvation  Analogy to Dictyostelium, a social ameobozoan Same habitat, solitary cells aggregate and differentiate upon starvation, but cellular morphology and genomic composition is completely different 600 µm Fruiting body Dictyostelium can ingest its preys, whereas myxobacteria need to lyse them first Migrating slug 200 µm Maeda (2005) Bdellovibrio bacteriovorus, another bacterial predator 3.8 Mb genome, 51 % GC content A fruiting body is a type of biofilm O’Toole et al. (2000) B. subtilis forms aerial, fruiting body-like structures Spores Reporter gene (lacZ) under the control of a sporulation promoter Gonzalez-Pastor (2001) “Wild” strains vs. ”domesticated” strains Laboratory strains of B. subtilis have lost the ability to form biofilms Spatial organization of B. subtilis biofilms Yellow: reporter for sporulation Blue: reporter for flagellum production Green: reporter for sporulation Red: reporter for matrix production Vlamakis et al. (2008) Antimicrobials Original culture of Penicillium  Definitions Disinfection = elimination of microbes from an object or inanimate surface Decontamination = treatment that renders an object or inanimate surface safe to handle Sterilization = elimination of all living organisms and viruses from a growth medium Sterilization by autoclaving Autoclave Produces steam under pressure =wet heat An oven would produce dry heat (=baking)  Sterilization by autoclaving Required time and temperature for sterilization: 10-15 minutes at 121°C Filter sterilization Size of the pores is the important parameter Historically, distinction between viruses and bacteria was made based on the size of the pores required for filtration  Filter types Depth filter Conventional membrane filter Random array of fibers Polymers with high tensile strength Air: HEPA filters Size of the holes depends on (high-efficiency particulate air) polymerization conditions (Liquid: used as prefilters to remove larger particles)  Nucleopore filters Polycarbonate films Pores made by nuclear radiation, enlarged by chemicals Used for sample concentration prior to SEM  Chemical agents for external use Sterilants = chemical agents that kill all forms of microbial life (including spores) Disinfectants = chemical agents that kill microorganisms from objects or inanimate surfaces (except spores) Antiseptics = chemical agents that kill microorganisms and are sufficiently nontoxic to be applied to living tissues  Sterilants and disinfectants Denaturing, oxidizing or alkylating agents (acting on proteins) or detergents (acting on membranes) Antiseptics Antimicrobial agents used in vivo Synthetic drugs are obtained by chemical synthesis Antibiotics are natural products  Effects of antimicrobials growth is inhibited, Bacteriostatic but resumes when Fungistatic toxic compound Viristatic is removed Bacteriocidal Fungicidal bacteria are killed Viricidal  Discovery of antimicrobials Thomson et al (2002) Brown and Wright (2016)  Discovery of antimicrobials Streptomycin (Nobel 1952) Penicillin (Nobel 1945) Prontosil (Nobel 1939)  The “magic bullet” concept =a drug that will kill the pathogen but not the other organisms Paul Ehrlich (1854-1915) Nobel prize winner 1908  Salvarsan (compound 606), the first antimicrobial Therapy against syphilis, discovered in 1909, clinical use in 1910 Contains arsenic Highly unstable in air Paul Ehrlich Sahachiro Hata (1873-1938) “The step from the laboratory to the patient's bedside (...) is extraordinarily arduous and fraught with danger."  Salvarsan (compound 606), the first antimicrobial Paul Ehrlich’s microscope Jewish Museum, Berlin Winau et al. (2004)  Sulfonamides, sulfa drugs Gerhard Domagk (1895-1964) Analog of p-aminobenzoic acid Treatment of Streptococcus infections Nobel prize winner 1939 (forced by the Nazi regime to turn it down) substrate of the dihydropteroate synthetase intermediate in the folic acid synthesis pathway  Folic acid Nucleic acid precursor vitamin  Trimethoprim Inhibits dihydrofolate reductase (tetrahydrofolate synthesis) Used in combination with sulfonamides Tetrahydrofolate biosynthesis pathway sulfonamide trimethoprim Targets of antimicrobials Spectrum of antimicrobials narrow spectrum broad spectrum (widespread use) very specific applications Annual worldwide use of antimicrobials -lactams (penicillins, cephalosporins and carbapenems) represent more than half  Penicillin structure Penicillin is a -lactam BBC Dorothy Crowfoot Hodgkin (1910-1994)  -lactams Penicillin Carbapenems Cephalosporins 5-member 5-member 6-member thiazolidine ring S in thiazolidine ring dihydrothiazine ring replaced by C Increase in use of antimicrobials (2000-2010) Van Boeckel et al (2014, The Lancet)  Inhibition of translation: macrocyclic lactones Erythromycin lactone = cyclic ester Inhibits protein synthesis (50S subunit) Macrolide ring Sterically blocks the progression of the growing peptide  Inhibition of transcription: macrocyclic lactones Rifampin/Rifampicin lactone = cyclic ester Inhibition of RNA polymerase (binds to -subunit) Resistance is acquired very easily (by mutation in -subunit)  Inhibition of translation: quinones Benzoquinone, or quinone is one of the two isomers of cyclohexadienedione Tetracycline Inhibits protein synthesis (30S subunit) Broad spectrum  Inhibition of DNA replication: quinolones Quinolones are synthetic antibacterial compounds (i.e. not antibiotics) Fluoroquinolones Inhibition of DNA gyrase Broad spectrum Nalidixic acid Ciprofloxacin  Mueller-Hinton medium (1941) Most widely used medium for Neisseria gonorrhea and N. meningitidis Used for antibiotic testing Jane Hinton John Howard Mueller (1919-2003) (1891-1954) Daughter of the first African-American Professor at Harvard University In 1949, one of the first two African-American women to become Doctors of Veterinary Medicine (Alfreda Johnson Webb is the other one) Antibiotic resistance  Antibiotic resistance 1945 2015 Nobel Prize for discovery of penicillin World Antibiotic Awareness Week 16-22 November 2015 Causes of antimicrobial resistance (AMR) Overprescribing of antibiotics  Rise in antibiotics use Annual consumption of antibiotics for human use: ~ 70 billion standard units Van Boeckel et al (2014, The Lancet) Example: retail sale of carbapenems Laxminarayan et al (2013, The Lancet)  Misuse of antibiotics Antibiotics kill bacteria, not viruses I think I need antibiotics for my IT’S A VIRUS! cold… Dr. Stacey Naito’s blog Patients not finishing their treatment MDR (= Multidrug-Resistant) Tuberculosis Zignol et al (2016), NEJM Overuse of antimicrobials in livestock Sub-therapeutic antibiotic treatment (STAT) = used in agriculture for growth promotion of livestock 63,151 ± 1560 tons/year for livestock Estimates of antimicrobial Largest five consumers consumption in OECD of antimicrobials in countries livestock 2010 2030 (projected) Van Boeckel et al (2015, PNAS) Deaths attributable to AMR every year by 2050 Deaths attributable to AMR every year by 2050 AMR impact Wernli et al (2017, PLoS Medicine)  Current burden of AMR in the USA CDC Report on Antibiotic Resistance Threats in the United States (2013) Top 18 drug-resistant threats CDC Hazard levels in the USA CDC Appearance of antibiotic resistance Candida albicans Acinetobacter spp. Gram-negative Enterococcus faecalis* Gram-positive Streptococcus pneumoniae Gram-positive/ acid-fast Mycobacterium tuberculosis* Fungus Salmonella typhi Haemophilus influenzae Neisseria gonorrhoeae Pseudomonas aeruginosa* Salmonella spp. Shigella spp. Staphylococcus aureus Year CDC Acquisition of antibiotic resistance Sommer et al (2017)  Resistance mechanisms 1) Modification of the antibiotic (e.g. hydrolysis) 2) Target site changes (e.g. mutation) 3) Reduced permeability/increased efflux 4) Alternative pathways Modifications of the antibiotic (e.g. -lactamase) (e.g. acetylation of chloramphenicol) Blair et al (2015)  Resistance to -lactams -lactamases Penicillin -Binding Protein Penicillin (PBP) -lactamase cuts here Target site modifications (e.g. mutation of rpoB) Blair et al (2015) Reduced membrane permeability/increased efflux Blair et al (2015) Resistance to Antibiotic B conferred by increased efflux Resistance to Antibiotic C conferred by reduced permeability Resistance mechanisms  Antibiotic resistance in Staphylococcus aureus Waves of resistance -lactamase Chambers and DeLeo (2009) Semisynthetic penicillins Antibiotic resistance in Staphylococcus aureus MRSA=Methicillin-resistant S. aureus Chambers and DeLeo (2009) MRSA: methicillin resistant Staphylococcus aureus Penicillin -Binding Protein* (PBP*) Penicillin mecA SCCmecI=Staphylococcal chromosome cassette mec I MRSA: methicillin resistant Staphylococcus aureus In the USA CDC (2013) Worldwide Grundmann et al. (2006, The Lancet) SCCmec: Staphylococcal Cassette Chromosome mec SCCmecI=Staphylococcal chromosome cassette mec I mecA: encodes a methicillin resistant variant of PBP2 ccr: cassette chromosome recombinase Grundmann et al. (2006) Chambers and DeLeo (2009)  Vancomycin inhibits cell wall synthesis Like penicillin, but mechanism is different Binds to the end of the pentapeptide chain (D-Ala-D-Ala) Vancomycin Basic unit NAG NAM peptide chain Antibiotic resistance in Staphylococcus aureus VRSA=Vancomycin-resistant S. aureus VISA=vancomycin-intermediate S. aureus Chambers and DeLeo (2009) Antibiotic resistance in Neisseria gonorrhoeae Cyanobacteria, Streptomyces, TB  Prokaryotic development cycles 1) Unicellular and cell-cycle independent Bacillus endospore formation Cyanobacteria heterocyst formation 2) Unicellular and cell-cycle dependent Caulobacter swarmer cell differentiation 3) Multicellular via directed movement (cell-cycle independent) Myxobacteria fruiting body formation 4) Multicellular via directed growth (cell-cycle dependent) Streptomycetes sporulation Bacterial phylogeny Battistuzzi et al. (2004) BMC Evolutionary Biology 2004, 4:44 Cyanobacteria  Cyanobacteria = Photosynthetic bacteria Fix carbon to produce carbohydrates and oxygen 6 CO2 + 12 H2O + light 6H12O6 + 6 O2 Sunlight as energy source, water as reductant Nitrogen for amino acid and nucleic acid biosynthesis? O2 interferes with N2 fixation process Nitrogenase required for conversion of N2 to NH4+ is inactivated by O2 N assimilation, conversion to Glu and Gln N represents ~15% of the dry weight of cells 4 1 Assimilation pathways converge 2 to glutamate and glutamine 3 (GS/GOGAT pathway) 1) Diffusion of ammonia 2) Processing of nitrogenous compounds (deaminases) 3) From nitrate (nitrate reductase) or nitrite (nitrite reductase) 4) N2 fixation N2-fixing cyanobacteria Issa et al. (2014)  Filamentous cyanobacteria and their genomes Can use ammonium, nitrate or atmospheric N2 as N source Nostoc sp. strain PCC 7120 7.2 Mb, 41 % GC content, 6 plasmids Anabaena variabilis 7.1 Mb, 41 % GC content, 3 plasmids Anabaena grown in the presence of nitrate Nitrate (NO3-) as nitrogen source Zhang et al. (2006) Chains of identical vegetative cells (all photosynthetic)  Anabaena grown in the absence of nitrate Two cell types Vegetative cells (photosynthesis) and heterocysts (N2 fixation) No nitrate heterocyst Zhang et al. (2006) Heterocyst = other cyst Heterocysts are regularly intercalated, every 10-20 cells  Cell specialization 20 m Heterocyst Larger cell Fluorescence due to chlorophyll (no chlorophyll, no photosynthesis) Golden and Yoon (2003)  Metabolic cooperation Heterocysts provide nitrogen source (as glutamine or glutamate) Vegetative cells provide carbon source (as carbohydrates)  Morphological characteristics of heterocysts HEP: polysaccharide layer HGL: glycolipid layer Protection from physical damage Hydrophobic barrier (to decrease rate of O2 entry) Muro-Pastor & Hess (2012) Metabolic characteristics of heterocysts Increased respiration rate To consume O2 that managed to cross the cell-envelope barriers Loss of photosystem II To prevent O2 production  Differentiation pathway Which cells will differentiate into heterocysts? =patterning Progression of the differentiation process? = temporal regulation Search for mutants Muro-Pastor & Hess (2012) Regulation of differentiation Muro-Pastor & Hess (2012) Sensing nitrogen starvation/GS-GOGAT pathway Accumulation of 2-OG (2-oxoglutarate, -ketoglutarate) Serves as C skeleton for NH4+ assimilation via the GS-GOGAT pathway GS = Glutamine synthase GOGAT = Glutamate synthase 2 molecules of Glutamate are produced or 1 Glutamate + 1 Glutamine Hodges, M. J. Exp. Bot. 2002  NtcA, the 2-OG sensor Accumulation of 2-OG triggers heterocyst development 2-OG binding stimulates NtcA DNA-binding activity Structure of NtcA monomer (model) CAP protein family Wisen et al. (2004)  HetR controls heterocyst differentiation hetR expression depends on ntcA Enhancement of ntcA expression requires hetR Positive regulatory loop HetR is specific for heterocyst differentiation, whereas NtcA has additional roles Gain-of-function allele of hetR results in near complete heterocyst formation (even in the presence of nitrate) The NtcA/HetR regulon Envelope formation Nitrogen fixation Inhibition of cell division Heterocysts are terminally differentiated (i.e. irreversible process)  Patterning, lateral inhibition pat (pattern formation) genes A differentiating cell inhibits differentiation of its neighbors Commitment Meeks and Elhai(2002)  Additional cell types, hormogonia, akinetes Hormogonium =Small motile filament dispersal Hornwort Flores & Herrero (2010) Akinetes are spore-like cells Formed in response to starvation Resistant to cold and desiccation (but sensitive to high temperature) Flores and Herrero et al. (2010) Larger size (15-20 m) Thicker cell wall Granulation (cyanophycin and glycogen)  Prokaryotic development cycles 1) Unicellular and cell-cycle independent Bacillus endospore formation Cyanobacteria heterocyst formation 2) Unicellular and cell-cycle dependent Caulobacter swarmer cell differentiation 3) Multicellular via directed movement (cell-cycle independent) Myxobacteria fruiting body formation 4) Multicellular via directed growth (cell-cycle dependent) Streptomycetes sporulation  Streptomyces belong to Actinobacteria Mycobacteria Mycobacterium tuberculosis 4.4 Mb, 66 % GC content Tuberculosis Mycobacterium leprae 3.3 Mb, 59 % GC content Leprosy Corynebacteria Corynebacterium diphtheriae 2.5 Mb, 54 % GC content Diphtheria Actinomycetes Mycelial, branching, spore forming  Actinomycetes Among the most numerous and ubiquitous soil bacteria Nocardia Streptomyces opportunistic pathogen antibiotic producer Important for C recycling Various types of spore-bearing structures Morphology of a Streptomyces coelicolor colony coelicolor =blue color Morphology of a Streptomyces coelicolor colony (exospores)  Genome of Streptomyces coelicolor Streptomyces coelicolor A3(2) 8.7 Mb, 72 % GC content, linear chromosome >20 clusters for known or predicted secondary metabolites High proportion of regulatory genes Linear chromosomal DNA is highly unstable Frequently undergoes large rearrangements at the extremities (deletions) often associated with the amplification of an adjacent sequence Streptomyces life cycle Angert (2005)  Substrate mycelium Red pigment =actinorhodin Willey et al. (2006) Filamentous Apical growth=grow by branching and extension of hyphal tips Limited number of septa (~ coenocytic fungi)  Hyphal tip extension Fluorescent vancomycin Reveals sites of nascent peptidoglycan 5 mm 5 mm DivIVA-GFP localizes at hyphal tips Flaerdh (2003)  Aerial mycelium Appear when nutrients are depleted Willey et al. (2006) Aerial hyphae grow up from the colony surface White, fuzzy appearance Turns grey (pigment in mature spores)  Model for aerial hyphae formation SapB (spore associated protein B) SapB is a surfactant reduces the surface tension at the air–water interface Chaplins Elliot and Talbot (2004) Chaplins are amyloid proteins Cross- structure (stacked -sheets) Gebbink et al (2005) Amyloid fibrils (Alzheimer disease) Chaplins assemble into rodlets Form a hydrophobic sheath on the cell surface Gebbink et al (2005)  bld mutants Mutations in the bld (bald) genes block the formation of the aerial mycelium Colonies with smooth, 'hairless' phenotype Signaling cascade Chater & Chandra (2006) Spore formation  whi mutants Chater &Chandra (2006) whi mutants white fuzzy aerial mycelium that fails to turn grey Cytokinesis and FtsZ-GFP localization Ladder of uniformly spaced Z rings Flaerdh (2003) Production of pigmented secondary metabolites Thomson et al (2002) Secondary metabolites Geosmin earthy smell of freshly plowed soil or after rainfall  Antibiotics produced by Streptomyces 70% of antibiotics are made by Actinomycetes (remaining 30% by filamentous fungi and nonactinomycetes bacteria) Incidence of active tuberculosis Pai et al (2016) 1.5 million deaths in 2014 (25% HIV positive) 2 billion people infected (5-10% could develop disease)  Tuberculosis White plague of the 17th - 19th centuries (consumption, phthisis) Health screenings at Ellis Island Tuberculosis Pavilion (North Brother Island)  Impact of tuberculosis on culture Movies Opera Books Fyodor Vampires Dostoevsky The Drunken Angel by Akira Kurosawa The Magic Mountain by Thomas Mann Mycobacterium tuberculosis Actinobacterium (high GC Gram positive) 4.4 Mb, 66% GC content Genome sequences and phylogeny Species Phyla Proteobacteria Battistuzzi et al. (2004) BMC Evolutionary Biology 2004, 4:44  Discovery of the tubercule bacillus, Mycobacterium tuberculosis (1882) Robert Koch (1843-1910) This represented a technical tour de force, Nobel Prize 1905 because the bacterium grows very slowly and is very difficult to stain! Kaufmann and Winau (2005), Nature Immunology  Acid fast staining Acid fast reveals mycolic acids Acid fast stain (carbolfuchsin)  Mycobacteria and mycolic acids Two membranes: inner membrane + mycomembrane Mycolic acids: waxy layer Abdallah et al 2007 Very impermeable, grow very slowly (tolerance to antibiotics, reduced rate of nutrient uptake?) Mycobacterial cell growth Mycobacteria divide asymmetrically Mycobacteria deposit new cell wall material asymmetrically at the tips of the bacilli. Dulberger et al 2019 Mycobacteria cell surface Dulberger et al 2019 Tuberculin (1890), Koch makes a mistake Tuberculin-a cure for tuberculosis? ‘The substance, with which the new treatment against tuberculosis is being performed, is a glycerol extract of pure culture of tubercle bacilli’ ‘… guinea pigs, which are well known to be highly susceptible to tuberculosis, no longer respond to infection with tubercle bacilli once they had been pretreated with such substances.’ Kaufmann and Schaible (2005), Trends Micro Tuberculin (1890), Koch makes a mistake Clinical assay: Tuberculin treatment on 769 patients 1% was cured 34% some improvement 55% no improvement 4% died Rudolf Virchow (1821-1902) showed that instead of killing the bacteria, tuberculin activated latent bacteria. Kaufmann and Schaible (2005), Trends Micro  Tuberculin skin test or Mantoux test Tuberculin is used for the diagnosis of tuberculosis Injecting 0.1 ml of tuberculin purified protein derivative (PPD) into the forearm Previous TB vaccination with BCG is a false positive CDC  Symptoms of TB Necrosis of the lungs X-rays reveal destroyed lung tissue High-resolution CT scan  The two stages of TB: stage I Stage I: Exposure, mild disease Public health campaign (1920s) Nunes-Alves et al. (2014)  Granuloma formation Mtb replicates in macrophages Macrophages produce cytokines Pai et al (2016) Granuloma More immune cells recruited to the site of infection Infection is contained, but bacteria are not killed Nunes-Alves et al. (2014)  The two stages of TB: stage II After a span of months or years (higher risk if immune system is weakened) Caseating Stage II: Pai et al (2016) granuloma Reactivation Non-contagious, static infection Nunes-Alves et al. (2014)  Discovery of streptomycin Streptomycin (Nobel 1952) Penicillin (Nobel 1945) Prontosil (Nobel 1939)  Streptomycin Isolated from Streptomyces griseus First antibiotic active against M. tuberculosis Inhibits protein synthesis (30S subunit) Use has decreased (resistance has developed, side effects are important)  Discovery of streptomycin Rutgers University Albert Schatz (1922-2005) Selman Waksman (1888-1973)  The controversy Nobel prize winner 1952 “for (…) ingenious, systematic and successful studies of soil microbes that led to the discovery of streptomycin.” Experiment eleven: Antagonistic Actinomycetes Schatz’s notebook (August 23rd, 1943) S. griseus isolated from leaf compost, straw compost and stable manure Special Collections and University Archives, Rutgers University Libraries  Waksman’s antibiotic team (1947) H. Boyd Woodruff David Hendlin (actinomycin and (fosfomycin) Albert Schatz streptothricin) (streptomycin) Ed Karow Elizabeth Horning (development of (clavacin and submerged fumigacin) fermentation) Christine Reilly (streptomycin development) Professor Selman Waksman Professor Robert Starkey Harry Katznelson D. Montgomery Reynolds (rhizosphere studies) (grisein) Woodruff H B Appl. Environ. Microbiol. 2014;80:2-8 The dimorphic life cycle of Caulobacter crescentus Article#9: Biofilms  Article#9: Biofilms Striations packed lengthwise across most of the biofilm  Article#9: Biofilms Mutants show alterations to the striation phenotype Article#9: Biofilms Article#9: Biofilms  Article#9: Biofilms wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries  Prokaryotic development cycles 1) Unicellular and cell-cycle independent Bacillus endospore formation Cyanobacteria heterocyst formation 2) Unicellular and cell-cycle dependent Caulobacter swarmer cell differentiation 3) Multicellular via directed movement (cell-cycle independent) Myxobacteria fruiting body formation 4) Multicellular via directed growth (cell-cycle dependent) Streptomycetes sporulation Asymmetric division in bacteria Reproduction by budding is not limited to yeast Life cycle of Hyphomicrobium, an asymmetric bacterium Asymmetric division in Caulobacter  Caulobacter is dimorphic Swarmer cell Stalked cell Stalk Flagellum Motile Holdfast Sessile (non motile) Dimorphic = 2 shapes Ecology of Caulobacter Govers and Jacobs-Wagner (2020)  Caulobacter is an -Proteobacterium Sinorhizobium meliloti 6.7 Mb Caulobacter crescentus 62.2 % GC 1 chromosome, 2 megaplasmids Root nodules (symbiosis with plants) Agrobacterium tumefaciens Mitochondria are derived from -proteobacteria 5.7 Mb Model organism 59 % GC content for cell cycle studies 2 chromosomes (1 linear, 1 circular) 4 Mb Ti-plasmid 67.2 % GC Crown gall disease (plant tumor) Genetic manipulation of plants Life cycle of Caulobacter Cell cycle takes 150 minutes at 30°C  Cell cycle of Caulobacter Stalked cell Cell division Swarmer cell G1 Loss of No M phase in bacteria flagellum G2 G1 G1/S S S/G2 Formation of stalk S phase DNA replication and formation Stalked cell of swarmer DNA replication (S phase), methylation and chromosome segregation DNA replication occurs only in the stalked cell Skerker and Laub (2004)  DnaA levels increase during S phase Stalked cell Cell division Swarmer cell G1 Loss of flagellum G2 G1 G1/S DnaA S S/G2 Formation of stalk Increase in DnaA levels S phase DNA replication and formation Stalked cell of swarmer Control of DNA replication by DnaA E. coli Promotes initiation of replication DnaA binding sites C. crescentus CtrA levels increase after S phase Stalked cell Cell division Swarmer cell Increase in CtrA levels G1 Loss of flagellum CtrA G2 G1 G1/S DnaA S S/G2 Formation of stalk S phase DNA replication and formation of swarmer Inhibition of DNA replication by CtrA DNA replication occurs once-and-only-once per cell cycle E. coli SeqA inhibits re-initiation DnaA binding sites C. crescentus CtrA binding sites CtrA~P inhibits DNA replication (in swarmer cells) and prevents re-initiation in S phase cells  CtrA is the master regulator of the cell cycle CtrA is conserved in most species of -proteobacteria ctrA is an essential gene CtrA is a response regulator that binds to DNA upon phosphorylation Asymmetric distribution of phosphorylated CtrA is crucial for cell cycle progression Regulation of CtrA expression and activity Transcription Phosphorylation Proteolysis Regulation of CtrA synthesis 2 promoters (P1 & P2)  Regulation of ctrA transcription CtrA~P binding sites Inhibition Activation Skerker and Laub (2004) Positive feedback loop results in rapid accumulation of CtrA  Role of methylation in ctrA transcription * CcrM DNA Methylation Methylation site If fully methylated, P1 is inactive P1 is activated just after replication of the ctrA locus (i.e., when the region becomes hemi-methylated) CtrA~P activates expression of ccrM, which encodes a DNA methyltransferase =negative feedback loop Activation of CtrA by phosphorylation Phosphorylation of CtrA is required for DNA binding Activation of CtrA by phosphorylation CtrA is a response regulator Phosphorelay? CtrA Which kinase?  Which kinase? Many candidates Cck cell-cycle kinase Ple pleiotropic phenotype (motility, phage sensitivity, stalk formation) Div cell division phenotype Phenotype of conditional cckA ts (temperature sensitive) mutant is similar to that of a ctrA ts mutant  CckA is a hybrid kinase Hybrid kinase=contains both a histidine kinase domain and a receiver domain ~P HK domain Receiver domain Subcellular localization of CckA-GFP Activation of CtrA by a phosphorelay hypothetical What is Hpt? Phosphotransfer profiling to identify Hpt ChpT is a histidine phosphotransferase that transfers phosphates from CckA to CtrA Biondi et al. (2006) CtrA phosphorylation (summary) Biondi et al (2006) Regulation of CtrA by proteolysis ClpXP is the protease that degrades CtrA (adaptor) ClpXP is a protease ClpXP specifically localizes to the stalked cell pole CtrA is specifically degraded in the stalked cell  Levels of CtrA and RcdA RcdA is an adaptor protein that directs CtrA to ClpXP Localization of ClpX-GFP Same results with ClpP-GFP  Structure of ClpP Cylindrical (14 subunits, 2 heptameric rings) Energy-dependent serine proteases Highly conserved throughout bacteria and eukaryotes Yu and Houry (2007)  Model for mechanism of action (ClpX or ClpA) In E. coli, ClpP forms complexes with AAA+ chaperones, ClpX and ClpA Other bacteria contain ClpA paralogs, such as ClpC, ClpE, and ClpL Yu and Houry (2007)  CtrA regulon by ChIP-chip analysis CtrA directly regulates about 100 genes Skerker and Laub (2004)  The CtrA regulon 3 main classes 1) Cell cycle processes (cell division, DNA methylation) 2) Regulatory genes 3) Polar morphogenesis (flagellum and pili)  Role of GcrA Stalked cell Cell division Swarmer cell G1 Loss of flagellum CtrA GcrA G2 G1 G1/S Increase in GcrA levels DnaA S S/G2 Formation of stalk S phase DNA replication and formation of swarmer  DnaA, CtrA and GcrA regulate each other DnaA activates gcrA expression GcrA activates ctrA expression CtrA represses gcrA expression GcrA accumulates as CtrA is proteolyzed J. Holtzendorff et al., Science 304, 983 -987 (2004) GcrA and CtrA have antagonistic functions J. Holtzendorff et al., Science 304, 983 -987 (2004) The GcrA regulon 3 main classes: 1) Cell cycle processes (DNA replication) 2) Regulatory genes 3) Polar morphogenesis J. Holtzendorff et al., Science 304, 983 -987 (2004)  Summary of cell cycle regulation Biondi et al (2006)  Asymmetric localization of flagellum and pili L. Shapiro et al., Science 298, 1942 -1946 (2002) Proteins involved in flagellar and pilus assembly localize to swarmer cell pole Mutants with impaired polarity ( tipN) wild type Flagellum is present but misplaced 15% of the cells have two stalks Cells are longer Huitema et al. (2006) TipN marks the new pole of the cell TipN (tip of new pole) Lam et al. (2006) Polarity in related -proteobacteria Jiang et al (2014) Polarity in related -proteobacteria  Components of the Caulobacter cytoskeleton Gitai (2005)  Control of FtsZ ring formation Caulobacter does not have a MinCD system FtsZ MipZ MipZ is an inhibitor of FtsZ polymerization Distantly related to ParA-like partitioning proteins and to MinD (18% identity) Thanbichler and Shapiro (2006)  MipZ interacts with ParB MipZ forms a complex with the partitioning protein ParB ParB binds to parS sites near the origin of replication Thanbichler and Shapiro (2006) MipZ localization follows segregation of oriC sites One of the duplicated oriC sites moves to the opposite pole Viollier et al. (2004) Thanbichler and Shapiro (2006)  Model for MipZ function Thanbichler and Shapiro (2006)  Control of cell shape Crescentin (encoded by creS gene) Determinant of the curved and helical shapes of C. crescentus (Vibrio-like shape) Sequence similarity to IF (intermediate filaments) Ausmees et al. (2003)  CreS-GFP forms a filamentous structure Colocalizes with the inner cell curvature Ausmees et al. (2003) The holdfast promotes attachment to surfaces Holdfast promotes attachment to surfaces Polysaccharide adhesin Measurement of holdfast size and thickness by AFM Tsang et al. (2006) Proc. Natl. Acad. Sci. USA 103, 5764-5768  Force measurement Force is on the order of 0.59 mNewtons (>68 N/mm2) (strongest ever measured for a biological adhesive) Tsang et al. (2006) Force is calculated from the amount of bending required to break the cell–pipette contact Infection Article#10: Streptomyces exploration  Article#10: Streptomyces exploration Ability to adopt a non-branching vegetative hyphal conformation and rapidly transverse solid surfaces Fungi trigger Streptomyces exploratory growth  Article#10: Streptomyces exploration Streptomyces explorer cells can communicate this exploratory behavior to other physically separated streptomycetes using an airborne volatile organic compound (VOC) Blue indicates VOC-induced alkalinity Explorer cells release a VOC that promotes exploratory growth in distantly located cells Exploratory growth can be communicated to unrelated streptomycetes Article#10: Streptomyces exploration VOCs inhibit the growth of other bacteria  Microbial pathogens =microbes that cause infectious diseases 1,407 currently recognized species of pathogens affecting humans 538 bacteria 317 fungi 287 helminths (parasitic worms) 208 viruses 57 protists Woolhouse (2006)  Deaths due to infectious diseases in the world Disease Deaths Pathogens Acute respiratory infections 4,259,000 Bacteria, viruses, fungi AIDS 2,040,000 Virus Diarrheal diseases 2,163,000 Bacteria, viruses, protists Tuberculosis 1,464,000 Bacterium Malaria 889,000 Protist Measles 424,000 Virus Other 2,561,000 Bacteria, viruses, protists, fungi WHO (2004)  Source of the pathogen Endogenous=opportunistic infection Other humans, e.g. sexually transmitted infections (STI) Animal vectors or reservoirs (e.g. insects or rodents) =zoonosis Food poisoning, e.g. lettuce (E. coli O157:H7) or cheese (Listeria), =foodborne illness Environmental sources, e.g. contaminated water (cholera)  Host range Wide variety of hosts (e.g. Pseudomonas aeruginosa, influenza) Specific to humans (e.g. Neisseria gonorrhoeae, HIV)  Infection 1) Entry Incubation period 2) Becoming established 3) Damage Disease Further exposure at local sites TOXICITY: COLONIZATION toxin effects are and local or systemic TISSUE EXPOSURE ADHERENCE INVASION GROWTH DAMAGE, to pathogens to skin or mucosa through epithelium Production of DISEASE virulence factors INVASIVENESS: further growth at original and distant sites Further exposure  Entry B. anthracis spores External tissues (cavities) that are contiguous with the exterior Internal tissues Schaechter (2006)  Entry Entry does not necessarily mean penetration into host cells (e.g. toxins) More than one anatomical site can be the site of entry of a single pathogen e.g. Cutaneous anthrax: skin (mild) Inhalation anthrax: lungs (serious) Schaechter (2006)  Becoming established 3 main factors: Size of the inoculum Invasiveness=invasive ability of the microbe State of the host’s defenses Further exposure at local sites TOXICITY: COLONIZATION toxin effects are and local or systemic TISSUE EXPOSURE ADHERENCE INVASION GROWTH DAMAGE, to pathogens to skin or mucosa through epithelium Production of DISEASE virulence factors INVASIVENESS: further growth at original and distant sites Further exposure  Size of the inoculum (LD50) LD50 (lethal dose 50) = dose of an agent that kills 50% of the animals in a test group  Disease = damage to host tissues 1) Cell death by lysis or apoptosis 2) Toxin action (e.g. tetanus, botulism, cholera) 3) Mechanical action (obstruction of vital passages) 4) Host response (e.g. septic shock) Toxins  Botulism and tetanus Paralysis induced by neurotoxins Agent of botulism Most potent and deadly neurotoxin (Botox) Clostridium botulinum Causes paralysis, asphyxia Found in improperly canned foods Agent of tetanus (lockjaw) Clostridium tetani binds to nerve terminals and retrograde transmission generalized muscular spasms, respiratory failure Botulinum toxin causes flaccid paralysis Tetanus toxin causes spastic paralysis  Host defenses Physical and chemical barriers Innate response (constitutive) Toll-like receptors (TLRs) Complement system Phagocytosis Adaptive response (induced) Antibodies Cell-mediated immunity  Barrier defenses Skin (salty and acidic) Body fluids (saliva, tears, mucus) Mucous membranes of the respiratory, urinary, and reproductive tracts Mucus traps microbes Low pH (stomach, skin)  Barrier defenses Mucosal Cutaneous barriers barrier Cutaneous barrier (skin and oral cavity) Keratinization Stratification, Keratin production, Loss of nuclei and organelles Anti-microbial peptides Cells involved in immune response Mucosal barrier (intestine) Seal Anti- microbial peptides  Inflammatory response Upon injury or infection Production of: Cytokines =signaling molecules of the immune response Histamine triggers dilatation and permeabilization of blood vessels Recruitment of phagocytic cells  Local response to infection Pathogen Macro- phage Movement Signaling of fluid molecules histamine Capi