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 VirulenceA 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

Trp operon 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

DNA binding domain fits into major groove

Dimerisation domain: may also be sensing domains

Sequence typically contains 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. DtxR regulon) 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 ExpressionA 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 occur in TRNs

When combined can 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 recognition helix stabilizing helix

AraC family ToxT in V. cholerae HilD, RamA in

Salmonella LysR family

QseA, QseD in EPEC

Stabilising helix

Recognition helix

turn

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

P

His

AspP

RNA polymera

se

Histidine sensor kinase

Response regulator

Signal

Two-Component Regulatory Systems TCSs that regulate toxin gene expression

BvgS/BvgA in Bordetella pertussis (pertussis toxin and adenylate cyclase toxin)

VirS/VirR in Clostridum perfingens (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 autoinducer molecule (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 Acyl homoserine lactone first to be

identified

http://upload.wikimedia.org/wikipedia/commons/c/cf/Quorum_sensing_diagram.png

Regulatory RNAs

Q: How can we study virulence gene expression and its regulation?

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

Clues from DNA sequences

Pathogen gene expressionDNA-protein interactions

Gel retardation assays Run DNA alone

alongside DNA and protein on gel

DNA bound to protein retarded in gel

Pathogen gene expressionDNA-protein interactions

Footprinting assay Mix DNA with protein Perform limited

digestion with DNAse I

Identify regions which are protected from digestion

A C G TFootprint

protected

Mix protein and labelled DNA

Protein protects DNA from nuclease

DNase

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 expressionExpression must be

measured under defined environmental conditions

Stressful versus basal heat shock, acid stress,

starvation stress, etc In vitro versus in vivo

Broth or plate Inside cells, organs,

animal

Direct assay versus via assay of reporter ease versus artefacts

Single gene versus many

Opportunistic searches 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

promoterless lacZ

beta-galactosidase

rbs/ATG

rbs/AUG

substrate colour change

mRNA

promoter from test gene

lacZ fusions

promoterless lacZ transposon

Replica-plate onto X-gal plates

High iron Low iron

Select for further study

In Vivo Expression Technology (IVET) A genetic approach positively

selects for bacterial genes specifically induced when bacteria infect their host, but not expressed under lab conditions

IVET vectors contain random 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

Esssential in host LacZ

Random DNA provides promoter

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

1 2 3 4

1 2 3 4 1 2 3 4

PCR RT-PCR

transcript

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

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

Control cells Test cells

RNA-SeqWhole 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