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Bio305 Regulation of Bacterial
                    Virulence
               Professor Mark Pallen
Introductory Lectures
   1: Pathogen Biology
   2: Genetics of Bacterial Virulence
   3: Regulation of Bacterial Virulence

   Later lecture blocks from me on
       Bacterial Genomics
       Bacterial Protein Secretion
Learning Objectives
   At the end of this lecture, the student will be able to
       provide a definition of terms related to bacterial gene
        regulation
       describe the hierarchical regulation of bacterial gene
        expression
       outline the kinds of transcriptional regulators and
        regulatory mechanisms found in bacteria
       describe how gene expression can be analysed
        experimentally
Regulation of Virulence
A multi-layered hierarchy
   Changes in DNA sequence
       Gene amplification
       Genetic rearrangements e.g. flagellar phase variation
   Transcriptional Regulation
       Transcription Factors (TFs): proteins that bind DNA and alter
        transcription
       Simplest system: a TF that recognises a single signal and
        regulates expression of a single gene
   Translational Regulation
       Trpoperon
   Post-translational Regulation
       Stability of protein, controlled cleavage
       Covalent modifications
Pathogen gene expression
   Gene expression is regulated
       Inducible versus constitutive genes
       Wasteful if always constitutive
       Artificial constitutive constructs decrease fitness
   In response to changes in environment
       Signal sensing
       Signal transduction
Operons and Promoters
   Single genes rare: most genes are in operons
       multiple genes encoded in single polycistronic mRNA
       genes within an operon subject to common regulatory
        mechanisms
   Promoter
       DNA sequence that defines the binding site of RNA
        polymerase and transcription factors
       TFs function act as activators of transcription or
        repressors that prevent RNA polymerase binding to the
        promoter
   Operons often have more than one promoter and
    can be subject to a complex hierarchy of regulation
Operons and Promoters
Transcription factors
                          Dimerisation domain: may also be sensing domains




DNA binding domain fits
                                    Sequence typically contains
into major groove
                                    inverted repeats
Pathogen gene expression
   Transcriptional regulatory networks (TRNs)
       encompass TFs and their target genes
       Simple networks of single TF/single operon are rare
       Instead co-ordinate regulation of gene expression
   multiple genes/operons co-regulated
       by common regulator (regulon, e.g. DtxRregulon)
       by common stimulus (stimulon or response, e.g. iron-
        starvation response)
   TRNs overlap; signal transduction pathways are
    complex
       mutations in global regulators cause pleiotropic effects
       ~ 50 global TRNs in E. coli
Regulation of Pathogen Gene Expression
    A simple system: Diphtheria
   tox gene regulated by
    repressor DtxR
       an iron-activated TF
   Fe2+ binds DtxR which
    represses expression of tox
   Under iron limiting
    conditions, 2Fe-DtxR-tox
    operator dissociates and
    toxin gene is expressed
The DtxR regulon: not so simple!
Transcriptional Regulatory Networks
   Six basic network motifs      When combined can
    occur in TRNs                  produce complex
                                   unpredictable counter-
                                   intuitive effects,
                                   understandable only
                                   through sophisticated
                                   models
Global Regulation
                               Regulons combine in
                               ever-more complex TRNs
                               until they encompass all
                               gene expression in the
                               bacterial cell

                               Some regulators act
                               globally to co-ordinate
                               expression of 100s or
                               even 1000s of genes




Ma H et al. Nucl. Acids Res.
2004;32:6643-6649
Helix-Turn-Helix Regulators
   Many TFs contain helix-
    turn-helix motif          Stabilising helix
       recognition helix
       stabilizing helix
                                          turn
   AraC family
       ToxT in V. cholerae                   Recognition helix
       HilD, RamA in
        Salmonella
   LysR family
       QseA, QseD in EPEC
Signal transduction
   External signal not always
    transmitted directly to target to
    be regulated
       Can detected by a sensor and
        transmitted to regulatory
        machinery (signal transduction)
   Can be extensive multi-
    component signal transduction
    pathways with partner switching
       e.g. coupling protein secretion
        and gene regulation in type III
        secretion
Two-Component Regulatory Systems
   Common kind of signal transduction occurs in two-
    component regulatory systems
       Sensor kinase: (cytoplasmic or membrane) detects
        environmental signal and autophosphorylates
       Response regulator: (cytoplasm) DNA-binding protein
        that regulates transcription; phosphorylated by sensor
        kinase
   Some systems have multiple regulatory elements
   ~50 two-component systems in E. coli
   Potential for cross-talk
Two-Component Regulatory Systems
       Signal
                          Histidine sensor kinase



                    His
                P




                             Response regulator
                      P
       RNA      Asp
    polymeras
        e
Two-Component Regulatory Systems
   TCSs that regulate toxin gene expression
       BvgS/BvgA in Bordetellapertussis (pertussis toxin and
        adenylatecyclase toxin)
       VirS/VirR in Clostridumperfingens(alpha-toxin and others)
       AgrA/AgrC in S. aureus (numerous toxins)
       CovS/CovR in S. pyogenes(streptolysin S, streptokinase)
   TCSs that regulate other virulence factors
       OmpR/PhoP in enterics
       SsrA/SsrB in Salmonella Spi2
Quorum sensing and virulence
   mechanism by which bacteria
    assess their population density
       ensures sufficient number of
        cells present before initiating
        response that requires certain
        cell density to have effect
   Each species produces specific
    autoinducermolecule (blue)
       Diffuses freely across cell
        envelope
       Reaches high concentrations
        inside cell only if many cells are
        near
       Binds to specific activator and
        triggers transcription of specific
        genes (red)
   Several different classes of
    autoinducers
       Acylhomoserinelactone first to
        be identified
                                             http://upload.wikimedia.org/wikipedia/commons/c/cf/Qu
                                             orum_sensing_diagram.png
Bio305 Lecture on Gene Regulation in Bacterial Pathogens
Regulatory RNAs
Q: How can we study virulence gene
expression and its regulation?
Clues from DNA sequences
   Sequence Analysis allows you to identify
       Identify TFs by homology
       Promoter consensus sequences
       Binding sites for regulatory factors
           RpoN, HIS, Crp, Lrp, Fur, etc
       Operons
Pathogen gene expression
    DNA-protein interactions
   Gel retardation assays
       Run DNA alone
        alongside DNA and
        protein on gel
       DNA bound to protein
        retarded in gel
Pathogen gene expression
    DNA-protein interactions
   Footprintingassay
       Mix DNA with protein
       Perform limited                         Mix protein and labelled DNA

        digestion with DNAse I
                                 Protein
       Identify regions which   protects DNA
                                                           DNase
                                 from
        are protected from       nuclease
        digestion                                                protected




                                                        Footprint      A     C   G   T
Chromatin
Immunoprecipitation
   nucleoprotein in cells is
    cross-linked, extracted,
    sonicated to give
    sheared
   DNA fragments
   Anti-TF Ab used to
    enrich the TF-cross-
    linked DNA fragments.
   IP DNA and control DNA
    analysed using
    microarray (ChIP-chip)
    or high-throughput
    sequencing (ChIP-seq)
                           http://commons.wikimedia.org/wiki/File:ChIP-sequencing.svg
Measurement of pathogen gene expression
Expression must be                        Direct assay versus via
  measured under defined                   assay of reporter
  environmental conditions
                                              ease versus artefacts
 Stressful versus basal
       heat shock, acid stress,          Single gene versus
        starvation stress, etc             many
   In vitro versus in vivo               Opportunistic searches
       Broth or plate
       Inside cells, organs, animal
                                           versus global surveys
Reporter gene fusions
   Fuse reporter gene to test gene
   Exploit enzymatic activity of reporter gene product
   Easier to measure reporter gene product
       optimised universal assay
       maybe less toxic to cells
   Promoter traps to identify unknown genes
       Responding to stimulus
       Regulated by given regulator
lacZ fusions
promoter from test gene
                   rbs/ATG

                              promoterless lacZ

                  rbs/AUG
                                         mRNA



                                   beta-galactosidase


                             substrate          colour change
lacZ fusions

           promoterless lacZ                transposon




                                              Replica-plate onto X-gal
                                              plates




     High iron
                                                    Low iron


                 Select for further study
In Vivo Expression
Technology (IVET)
                                                        Esssential in
   A genetic approach positively                          host                   LacZ
    selects for bacterial genes
    specifically induced when bacteria
    infect their host, but not expressed
    under lab conditions
   IVET vectors contain random               Random DNA provides promoter
    promoter fragment and promoter-
    less gene that encodes selective
    marker required for survival in host
   Random integration of IVET vector
    into chromosome creates pool of
    recombinant pathogens
   Only bacteria that contain the
    selective marker fused to a gene
    that is transcriptionally active in the
    host are able to survive
   Post-selection screening for Lac-
    colonies finds promoters that are
    only active in vivo


                                                  http://commons.wikimedia.org/wiki/File:Mouse.svg
Measuring individual gene expression
   can be assayed by quantitative real-time reverse
    transcription polymerase chain reaction (RT-PCR)

              promoter                                   terminator
                                  transcript




                  1               2            3                4




                      1   2   3       4            1    2   3   4




                          PCR                          RT-PCR
Measuring global gene expression
   can be analysed using
       microarrays
       RNA-Seq
   Can be applied to
       in vitro conditions e.g. acid stress, heat shock
       in vivo conditions after isolation of bacterial RNA from
        infected cells and tissues
Microarrays
   Arrange large number
                               Control cells   Test cells
    of hybridisation targets
    in gridded array
   Variety of approaches
   Provides global
    genome-wide survey of
    1000s of genes
   Assay changes in
    expression of every
    gene after change in
    environment or in
    regulator mutant
RNA-Seq
Whole Transcriptome Shotgun Sequencing
   high-throughput sequencing of cDNA
   advantages over microarrays
       no probes or genome sequence needed
       unbiased view of transcriptome
       no interference from non-specific hybridisation
       discovery of novel features, e.g. small RNAs
       delineation of operons and untranslated regions
       improved sequence annotation
       precise high-resolution mapping of sequence data
       much greater dynamic range
       more discriminatory at high levels of gene expression
       more sensitive at very low levels of expression
   disadvantage: expense
RNA-Seq
   Starting material
    bacterial RNA
   Optional subtraction of
    tRNA and rRNA
   Generation of cDNA
    libraries
   High-throughput
    sequencing
   Bioinformatics
   Interpretation of cDNA
    sequencing read
    histograms
Summary
   Gene expression, operons, promoters
   Pathogen gene expression and its regulation
   Transcription factors: HTH, TCS, RNAs
   Methods to study virulence gene expression
   Bioinformatics, Gel retardation, Footprinting
   ChIP, Reporter gene fusions, IVET, RT-PCR
   Global gene expression: microarrays, RNA-Seq

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Bio305 Lecture on Gene Regulation in Bacterial Pathogens

  • 1. Bio305 Regulation of Bacterial Virulence Professor Mark Pallen
  • 2. Introductory Lectures  1: Pathogen Biology  2: Genetics of Bacterial Virulence  3: Regulation of Bacterial Virulence  Later lecture blocks from me on  Bacterial Genomics  Bacterial Protein Secretion
  • 3. Learning Objectives  At the end of this lecture, the student will be able to  provide a definition of terms related to bacterial gene regulation  describe the hierarchical regulation of bacterial gene expression  outline the kinds of transcriptional regulators and regulatory mechanisms found in bacteria  describe how gene expression can be analysed experimentally
  • 4. Regulation of Virulence A multi-layered hierarchy  Changes in DNA sequence  Gene amplification  Genetic rearrangements e.g. flagellar phase variation  Transcriptional Regulation  Transcription Factors (TFs): proteins that bind DNA and alter transcription  Simplest system: a TF that recognises a single signal and regulates expression of a single gene  Translational Regulation  Trpoperon  Post-translational Regulation  Stability of protein, controlled cleavage  Covalent modifications
  • 5. Pathogen gene expression  Gene expression is regulated  Inducible versus constitutive genes  Wasteful if always constitutive  Artificial constitutive constructs decrease fitness  In response to changes in environment  Signal sensing  Signal transduction
  • 6. Operons and Promoters  Single genes rare: most genes are in operons  multiple genes encoded in single polycistronic mRNA  genes within an operon subject to common regulatory mechanisms  Promoter  DNA sequence that defines the binding site of RNA polymerase and transcription factors  TFs function act as activators of transcription or repressors that prevent RNA polymerase binding to the promoter  Operons often have more than one promoter and can be subject to a complex hierarchy of regulation
  • 8. Transcription factors Dimerisation domain: may also be sensing domains DNA binding domain fits Sequence typically contains into major groove inverted repeats
  • 9. Pathogen gene expression  Transcriptional regulatory networks (TRNs)  encompass TFs and their target genes  Simple networks of single TF/single operon are rare  Instead co-ordinate regulation of gene expression  multiple genes/operons co-regulated  by common regulator (regulon, e.g. DtxRregulon)  by common stimulus (stimulon or response, e.g. iron- starvation response)  TRNs overlap; signal transduction pathways are complex  mutations in global regulators cause pleiotropic effects  ~ 50 global TRNs in E. coli
  • 10. Regulation of Pathogen Gene Expression A simple system: Diphtheria  tox gene regulated by repressor DtxR  an iron-activated TF  Fe2+ binds DtxR which represses expression of tox  Under iron limiting conditions, 2Fe-DtxR-tox operator dissociates and toxin gene is expressed
  • 11. The DtxR regulon: not so simple!
  • 12. Transcriptional Regulatory Networks  Six basic network motifs  When combined can occur in TRNs produce complex unpredictable counter- intuitive effects, understandable only through sophisticated models
  • 13. Global Regulation Regulons combine in ever-more complex TRNs until they encompass all gene expression in the bacterial cell Some regulators act globally to co-ordinate expression of 100s or even 1000s of genes Ma H et al. Nucl. Acids Res. 2004;32:6643-6649
  • 14. Helix-Turn-Helix Regulators  Many TFs contain helix- turn-helix motif Stabilising helix  recognition helix  stabilizing helix turn  AraC family  ToxT in V. cholerae Recognition helix  HilD, RamA in Salmonella  LysR family  QseA, QseD in EPEC
  • 15. Signal transduction  External signal not always transmitted directly to target to be regulated  Can detected by a sensor and transmitted to regulatory machinery (signal transduction)  Can be extensive multi- component signal transduction pathways with partner switching  e.g. coupling protein secretion and gene regulation in type III secretion
  • 16. Two-Component Regulatory Systems  Common kind of signal transduction occurs in two- component regulatory systems  Sensor kinase: (cytoplasmic or membrane) detects environmental signal and autophosphorylates  Response regulator: (cytoplasm) DNA-binding protein that regulates transcription; phosphorylated by sensor kinase  Some systems have multiple regulatory elements  ~50 two-component systems in E. coli  Potential for cross-talk
  • 17. Two-Component Regulatory Systems Signal Histidine sensor kinase His P Response regulator P RNA Asp polymeras e
  • 18. Two-Component Regulatory Systems  TCSs that regulate toxin gene expression  BvgS/BvgA in Bordetellapertussis (pertussis toxin and adenylatecyclase toxin)  VirS/VirR in Clostridumperfingens(alpha-toxin and others)  AgrA/AgrC in S. aureus (numerous toxins)  CovS/CovR in S. pyogenes(streptolysin S, streptokinase)  TCSs that regulate other virulence factors  OmpR/PhoP in enterics  SsrA/SsrB in Salmonella Spi2
  • 19. Quorum sensing and virulence  mechanism by which bacteria assess their population density  ensures sufficient number of cells present before initiating response that requires certain cell density to have effect  Each species produces specific autoinducermolecule (blue)  Diffuses freely across cell envelope  Reaches high concentrations inside cell only if many cells are near  Binds to specific activator and triggers transcription of specific genes (red)  Several different classes of autoinducers  Acylhomoserinelactone first to be identified http://upload.wikimedia.org/wikipedia/commons/c/cf/Qu orum_sensing_diagram.png
  • 22. Q: How can we study virulence gene expression and its regulation?
  • 23. Clues from DNA sequences  Sequence Analysis allows you to identify  Identify TFs by homology  Promoter consensus sequences  Binding sites for regulatory factors  RpoN, HIS, Crp, Lrp, Fur, etc  Operons
  • 24. Pathogen gene expression DNA-protein interactions  Gel retardation assays  Run DNA alone alongside DNA and protein on gel  DNA bound to protein retarded in gel
  • 25. Pathogen gene expression DNA-protein interactions  Footprintingassay  Mix DNA with protein  Perform limited Mix protein and labelled DNA digestion with DNAse I Protein  Identify regions which protects DNA DNase from are protected from nuclease digestion protected Footprint A C G T
  • 26. Chromatin Immunoprecipitation  nucleoprotein in cells is cross-linked, extracted, sonicated to give sheared  DNA fragments  Anti-TF Ab used to enrich the TF-cross- linked DNA fragments.  IP DNA and control DNA analysed using microarray (ChIP-chip) or high-throughput sequencing (ChIP-seq) http://commons.wikimedia.org/wiki/File:ChIP-sequencing.svg
  • 27. Measurement of pathogen gene expression Expression must be  Direct assay versus via measured under defined assay of reporter environmental conditions  ease versus artefacts  Stressful versus basal  heat shock, acid stress,  Single gene versus starvation stress, etc many  In vitro versus in vivo  Opportunistic searches  Broth or plate  Inside cells, organs, animal versus global surveys
  • 28. Reporter gene fusions  Fuse reporter gene to test gene  Exploit enzymatic activity of reporter gene product  Easier to measure reporter gene product  optimised universal assay  maybe less toxic to cells  Promoter traps to identify unknown genes  Responding to stimulus  Regulated by given regulator
  • 29. lacZ fusions promoter from test gene rbs/ATG promoterless lacZ rbs/AUG mRNA beta-galactosidase substrate colour change
  • 30. lacZ fusions promoterless lacZ transposon Replica-plate onto X-gal plates High iron Low iron Select for further study
  • 31. In Vivo Expression Technology (IVET) Esssential in  A genetic approach positively host LacZ selects for bacterial genes specifically induced when bacteria infect their host, but not expressed under lab conditions  IVET vectors contain random Random DNA provides promoter promoter fragment and promoter- less gene that encodes selective marker required for survival in host  Random integration of IVET vector into chromosome creates pool of recombinant pathogens  Only bacteria that contain the selective marker fused to a gene that is transcriptionally active in the host are able to survive  Post-selection screening for Lac- colonies finds promoters that are only active in vivo http://commons.wikimedia.org/wiki/File:Mouse.svg
  • 32. Measuring individual gene expression  can be assayed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) promoter terminator transcript 1 2 3 4 1 2 3 4 1 2 3 4 PCR RT-PCR
  • 33. Measuring global gene expression  can be analysed using  microarrays  RNA-Seq  Can be applied to  in vitro conditions e.g. acid stress, heat shock  in vivo conditions after isolation of bacterial RNA from infected cells and tissues
  • 34. Microarrays  Arrange large number Control cells Test cells of hybridisation targets in gridded array  Variety of approaches  Provides global genome-wide survey of 1000s of genes  Assay changes in expression of every gene after change in environment or in regulator mutant
  • 35. RNA-Seq Whole Transcriptome Shotgun Sequencing  high-throughput sequencing of cDNA  advantages over microarrays  no probes or genome sequence needed  unbiased view of transcriptome  no interference from non-specific hybridisation  discovery of novel features, e.g. small RNAs  delineation of operons and untranslated regions  improved sequence annotation  precise high-resolution mapping of sequence data  much greater dynamic range  more discriminatory at high levels of gene expression  more sensitive at very low levels of expression  disadvantage: expense
  • 36. RNA-Seq  Starting material bacterial RNA  Optional subtraction of tRNA and rRNA  Generation of cDNA libraries  High-throughput sequencing  Bioinformatics  Interpretation of cDNA sequencing read histograms
  • 37. Summary  Gene expression, operons, promoters  Pathogen gene expression and its regulation  Transcription factors: HTH, TCS, RNAs  Methods to study virulence gene expression  Bioinformatics, Gel retardation, Footprinting  ChIP, Reporter gene fusions, IVET, RT-PCR  Global gene expression: microarrays, RNA-Seq