A study of Salmonella typhi lytic bacteriophages and their use in elucidating novel regulatory controls employed by

the bacterium to regulate Vi capsule expression

Derek Pickard, Sanger Institute,Microbiology and Evolution group,Hinxton, Cambridge CB10 1SA

• Unique human pathogen• Most prevalent in areas of the world with

poor sanitation and hygiene• Stagnant pools of water in slum areas are

particularly vulnerable to disease risk

Salmonella typhi – distinguishing features of risk

Salmonella typhi-distinguishing clinical manifestations

-Progressive fever (40 °C) - profuse sweating and gastroenteritis

-leukopenia- a decrease in number of circulating white blood cells-spleen and liver are enlarged (hepatosplenomegaly)

-Intestinal hemorrhage due to bleeding in congested Peyer's patches

-Intestinal perforation in the distal ileum: very serious complication

-Neuropsychiatric symptoms (described as "muttering delirium“ or "coma vigil"), with picking at bedclothes or imaginary objects.

-Encephalitis and general malaise

• Lipopolysaccharide (LPS): O9, O12• Vi antigen

- capsular exopolysaccharide- homopolymer of N-acetyl galacturonic acid- Modified by O acetylation, - consists of long linear

fibres, tightly bound via acetyl hydrogen bonds- masks the underlying O9 LPS - expression of the Vi capsule is tightly regulated

O9

Vi

The S.typhi bacterial cell surface..............

Overview: The ViaB locus and the known regulatory controls of expression

tviA tviB tviC tviD tviE vexA vexB vexC vexD vexE

cell surface anchoring

transcriptional regulator

UDP-GlcNAc 6-dehydrogenase

UDP-GlcNAcA/ UDP-GalNAc 4-epimerase

?

OmpR-EnvZ

RcsB-RcsC

The ViaB operon is encoded on a promiscuous conjugative transposon called SPI-7:Belong to diverse and highly related group of ICEs found within the Enterobacteriaceae

The classic range of S. typhi Vi

bacteriophage

-Originally these Vi targeting phage were isolated and identified in the 1930’s and 1940’s-Types I to IV- Craigie in 1936-Types V to VII- Desranleau in 1949-The latter used them extensively to treat Typhoid fever in actual clinical cases with great success-Probable side effects due to LPS levels in the phage preparations-Superceded by antibiotics and vaccines which led to a decline in phage therapy experiments and expertise

50nm

Type II

50nm

50nm

100nm

The ViI S. typhi phage- a myoviridae with a genome size of 160Kbp

ViI phage infects all known Vi expressing bacteria -S.typhi, S. Dublin, Citrobacter freundii and S. Typhimurium.

Other myoviridae phage related to S. typhi ViI and infecting other Enterobacteriaceae

1.PhaX1- infects E. Coli O157 plus possibly also ETECand Citrobacter2. CBA120- infects E. Coli O157 and some E. coli O783. SFP10- infects Salmonella typhimurium and E.coli O1574. SH19 - infects Salmonella typhimurium6. phiSbom-AG3: infects Shigella boydii and possibly alsoEnterobacter hormaeche7. LIMEstone1- infects Erwinia chyantheni

Even at the DNA level the synteny is remarkable........

TSP1 TSP2 TSP4

TSP3

TSP1 TSP2 TSP3

0-154aa0-245aa

TSP4

0-137aa 0-360aa424-1040aa170-594aa 246-927aa 161-846aa

Key:-Tail spike proteins

Bacterial receptor recognition modules of the phage TSPs-Potentially 4 diverse bacterial Species can be hosts for this phage

Generalised depiction of theTail Spike Proteins (TSPs) of the ViI-like family of bacteriophages.

• The group of ViI-like phages differs from any established phage genus by its characteristic morphology and genomic properties.

• The synteny and potential wide host range within the Enterobacteriaceae is a signature of this phage family

• DNA synteny means that genetic exchange and recombination between these phage is ongoing within ‘shared hosts’

• Exchange of tail spike proteins and thus hosts make this phage a useful research tool!

Making the S. typhi TraDIS mutant libraryWhat is TraDIS? Transposon Directed Insertion-site Sequencing

-It has similarity to a randomly generated Tn5 library but with a more dense coverage throughout the bacterial genome (and plasmids)-The transposon used lacks a transposase enzyme for stability reasons so needs to be supplemented into the reaction.- Each mutant in a library can be located using specific PCR primers to the transposable element

Illumina oligo specific transposon oligo

PCR fragments

STAGE 2. Using the STAGE 2. Using the S. typhiS. typhi transposon transposon library to investigate genes associated library to investigate genes associated

with Vi capsule biosynthesis and with Vi capsule biosynthesis and regulationregulationVi phage type II S. typhi BRD948 Vi+ve

transposon library

Mix at high MOI-Select for survivors on selective antibiotic platesMix at high MOI-Select for survivors on selective antibiotic plates -majority are Vi negative as expected-majority are Vi negative as expected

Vi –ve library Using bioinformatic analysis of DNA from surviving bacteria to pinpoint transposon insertion within genes so to identify

which mutants from the library survived the Vi phage treatment and then ask WHY.…!!

Genes identified in the TraDIS screen

Sys IDGene name

Log2 read ratio P-value Function

t4352 tviB -7.10 3x10-51Vi polysaccharide biosynthesis, UDP-glucose/GDP-mannose dehydrogenase

t4349 tviE -6.96 1.28E-49 Vi polysaccharide biosynthesis TviEt4353 tviA -6.66 2.79E-46 Vi polysaccharide biosynthesis regulatort4350 tviD -6.35 6.18E-43 Vi polysaccharide biosynthesist4011 yrfF -6.11 1.99E-40 putative membrane proteint4344 vexE -6.04 1.20E-39 Vi polysaccharide export proteint4351 tviC -5.80 2.68E-37 Vi polysaccharide biosynthesis protein, epimeraset4347 vexB -5.74 1.17E-36 Vi polysaccharide export inner-membrane proteint4345 vexD -5.59 3.05E-35 Vi polysaccharide export inner-membrane proteint4348 vexA -5.59 3.34E-35 Vi polysaccharide export proteint4346 vexC -5.08 1.54E-30 Vi polysaccharide export ATP-binding proteint1220 himA -3.84 1.51E-20 integration host factor alpha-subunitt4004 ompR -3.59 7.64E-19 two-component response regulator OmpRt3216 greA -3.16 5.74E-16 transcription elongation factort4005 envZ -2.44 7.98E-12 two-component sensor kinase EnvZt4386 efp -2.16 2.12E-10 elongation factor Pt2867 barA -2.15 2.50E-10 sensor proteint3205 nusA -2.02 1.03E-09 L factort0929 sirA -1.55 1.22E-07 invasion response-regulatort3500 oxyR -1.26 1.73E-06 hydrogen peroxide-inducible regulon activatort3474 rpoB -1.25 1.87E-06 DNA-directed RNA polymerase, beta-subunitt1627 topA -0.86 4.06E-05 DNA topoisomerase I, omega protein It2325 ppiB -0.75 8.91E-05 peptidyl-prolyl cis-trans isomerase Bt3095 parC -0.71 1.23E-04 topoisomerase IV subunit At1238 - -0.64 1.96E-04 conserved hypothetical protein

Prepare individual knockout of selected genes using the Wanner Red Recombinase system-

1.Preliminary phenotype analysis carried out2.RNA microarray analysis to investigate effect of knockout on gene expression

Vi Bacteriophage testing on mutant strain

Gene knocked out

  Slide agglutination profile

 

Type I

 

Type  II

 

Type  IV

 

Type  VI and VII

Vi ELISATitre 

yrlF O9 +++  Vi- - - - - <2rcsB O9 +++  Vi - - - - - <2oxyR O9 +++  Vi - - - - - <2sirA O9 +++  Vi +/- +/- - - +/- NDenvZ O9 +++  Vi + + + + + 128barA O9 +++  Vi +     + + + + 256greA O9 +++  Vi ++ ++ ++ ++ ++ 256efp O9 +++  Vi ++ ++ ++ ++ ++  512ihfA O9 +++  Vi +++ ++/+++ ++/+++ ++/+++ ++/   +++ 1024ppiB

O9 -       Vi +++ ++ ++ ++ ++ ND

ihfB O9 +/-    Vi +++ +++ +++ + +++ NDactP O9 -       Vi +++ ++++ ++++ ++++ ++++ NDphoN O9 -       Vi +++ ++++ ++++ ++++ ++++ NDBRD948 O9 -       Vi +++ ++++ ++++ ++++ ++++ >2048

1. Preliminary phenotype analysis carried out

Growth of phage on various mutant strains

BRD948 + ViII phage efp mutant + ViII phage barA mutant + ViII phage

BRD948 + ViVI phage efp mutant + ViVI phage barA mutant + ViVI phage

RNA microarray analysis to investigate effect of knockout on gene expression

1. igaA/yrfF - intracellular growth attenuator of the rcsB-rcsC two component regulator-sensor system. May work by interacting with RcsA protein as well

2. two component sensor-regulators systems control Vi expression . What is the relationship of OmpR-EnvZ and BarA-SirA in S. Typhi? Differential gene regulation of SPI-1/SPI-2 in relation toVi capsule

3. oxyR –Oxidative stress and LPS modification. Senses Oxidative stress but why does it knock out Vi capsule synthesis?

4. efp and greA- Involved with elongation and termination stages of transcription by direct modification of RNA Polymerases. Are they moderately affecting ability of phage to replicate or is it due to effects on cell metabolism and efficiency of RNA Polymerase transcription and elongation?

5. himA/himB- integration host factorbut most likely effects Vi gene expression due to effect on DNA topography

6. and finally.......rcsB and envZ are known to regulate Vi capsule synthesis and showed up in the TraDIS data.

What are the properties of the genes

identified in the TraDIS screen?

Conclusions 2

TraDIS has identified genes that switch off or repress Vi expression

Often this was not a all or nothing expression but varied from mutant to mutant......

Those mutants that could still be infected by Vi phage had log expression values of logFC values of -2.30E while those clones that Vi phage could not infect had logFC values of -9.00E

The identity of the gene or genes responsible for the acetyl-modification of the Vi capsule was not identified in the screen...

This is likely due to some level of redundancy with various genes able to carry out the acetylation of the Vi capsule

The barA-sirA two component regulatory system is known to be an important mechanism for differential gene regulation for a variety of genes required for virulence, and this now includes the Vi operon..

• Dave Goulding and Ana Luisa Toribio• Prof. Gordon Dougan• Keith Turner and Dam Turner• Sequencing teams for all the Vi phage work

and TraDIS• All the outside collaborators who sequenced

these other family of bacteriophage and to which we are all contributed to a paper about them

• Funding by the Wellcome Trust is gratefully acknowledged

Transposon mutagenesis of S. typhi BRD948 KanR

Transposomes

electroporation

Tn5-derivative

TranspositionKan

Commercially available recombinant

Transposase

Sys IDGene name

Input inserts

Output inserts

Input reads

Output reads

Log2 read ratio P-value Function

t4352 tviB 307 285 11914 1646207 -7.10 3x10-51 Vi polysaccharide biosynthesis, UDP-glucose/GDP-mannose dehydrogenaset4349 tviE 280 209 4659 592697 -6.96 1.28E-49 Vi polysaccharide biosynthesis TviEt4353 tviA 89 78 1721 184485 -6.66 2.79E-46 Vi polysaccharide biosynthesis regulatort4350 tviD 627 540 22768 1870584 -6.35 6.18E-43 Vi polysaccharide biosynthesist4011 yrfF 190 112 1263 94290 -6.11 1.99E-40 putative membrane proteint4344 vexE 141 81 3246 219685 -6.04 1.20E-39 Vi polysaccharide export proteint4351 tviC 224 164 5856 332427 -5.80 2.68E-37 Vi polysaccharide biosynthesis protein, epimeraset4347 vexB 168 131 5269 286393 -5.74 1.17E-36 Vi polysaccharide export inner-membrane proteint4345 vexD 218 159 6661 325843 -5.59 3.05E-35 Vi polysaccharide export inner-membrane proteint4348 vexA 206 164 9690 470499 -5.59 3.34E-35 Vi polysaccharide export proteint4179 actP 94 5 1216 49412 -5.23 6.59E-32 Sodium:solute symporter family proteint4346 vexC 177 142 18227 620241 -5.08 1.54E-30 Vi polysaccharide export ATP-binding proteint4225 phoN 208 3 5341 87540 -4.01 8.23E-22 nonspecific acid phosphatase precursort4209 dcuB 71 5 2371 39400 -4.00 9.93E-22 anaerobic C4-dicarboxylate transporter t1220 himA 8 2 60 2186 -3.84 1.51E-20 integration host factor alpha-subunitt4362 - 246 2 4107 55798 -3.73 8.40E-20 putative membrane proteint4004 ompR 47 16 272 4392 -3.59 7.64E-19 two-component response regulator OmpRt4356 - 271 7 5186 60540 -3.52 2.44E-18 hypothetical proteint4268 - 169 4 2629 30184 -3.47 5.12E-18 putative exported proteint3216 greA 32 16 527 5488 -3.16 5.74E-16 transcription elongation factort0012 dnaK 5 3 14 862 -3.08 1.77E-15 DnaK proteint4005 envZ 77 35 602 3708 -2.44 7.98E-12 two-component sensor kinase EnvZt4386 efp 51 17 300 1689 -2.16 2.12E-10 elongation factor Pt2867 barA 140 65 1780 8225 -2.15 2.50E-10 sensor proteint3205 nusA 18 5 108 743 -2.02 1.03E-09 L factort4313 - 27 1 220 968 -1.74 1.95E-08 putative membrane proteint0929 sirA 36 18 263 964 -1.55 1.22E-07 invasion response-regulatort3500 oxyR 66 30 717 1853 -1.26 1.73E-06 hydrogen peroxide-inducible regulon activatort3474 rpoB 5 1 9 159 -1.25 1.87E-06 DNA-directed RNA polymerase, beta-subunitt1627 topA 66 12 280 591 -0.86 4.06E-05 DNA topoisomerase I, omega protein It2325 ppiB 8 3 50 153 -0.75 8.91E-05 peptidyl-prolyl cis-trans isomerase Bt3095 parC 1 1 12 83 -0.71 1.23E-04 topoisomerase IV subunit At1238 - 78 2 788 1284 -0.64 1.96E-04 conserved hypothetical protein