Genetic Responses of Listeriamonocytogenes to High PressureProcessing (HPP)

ISOPOL XVI Savannah Georgia March 22 2007

JOHN BOWMAN

CLAUDIO BITTENCOURT

TOM ROSS

Australian Food Safety Centre of Excellence

Private Bag 52

University of Tasmania, Hobart,

Tasmania 7001

High-pressure processing of foods

Currently used as a postlethalitytreatment of many food types especially ready-to-eat meats, vegetable products, juices and various seafood products.

Shown to inactivate rapidly vegetative cells of pathogens(less effective against spores).

The Effect of HPP on Biological Systems

Based on the Le Chatelier principle HPP tends to:

- inhibit processes that result in a net volume increase

- benefit processes that result in a net volume decrease

- therefore biomolecular complexes are altered/damaged by HPP (e.g. ribosomes, DNA helices, septal rings) but small molecules (flavour compounds, vitamins etc.) are unaffected

- biological effect may be counteracted by solutes in food e.g. NaCl, sugars

L. monocytogenes and HPP: Some general findings

- The stationary growth phase state cross-protectsagainst HPP (membrane stabilisation, cell wall thickening etc.)

- Cross-protection effects have been shown to be enhanced inin foods due the presence of various solutes e.g. sea salts, sugars.Probably due to a counteractive effect in regards to macro-molecular stability.

- Inactivated cells may “resuscitate/recover” following storageeven after treatments that cause total inactivation (moredefined experiments needed here).

- Phenolic food additives may cause HPP-sensitisation(mechanism involves cell membrane destabilization?)

L. monocytogenes survival following HPP

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400 MPa – stationarygrowth phase

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Strain 2542, serotype 1/2a from smoked salmon

Project Aims:

•Determine the genetic responses occurring in Listeria monocytogenesfollowing HPP using a genomic microarray approach

•Determine functional changes induced by HPP based on gene expression data

•Identify relevant signalling and regulatoryelements

Custom Microarray for L. monocytogenes(based on strain EGD-e)

•2857 × 70mer oligomers derived from EDG-e genome (OperonTechnologies, Huntsville, AL, USA)•Arrayed by AGRF Ltd., Walter & Eliza Hall Institute of MedicalResearch, University of Melbourne

Caveats:•>98% identity (0-1 mismatches) with 847 genes of L. innocuaClip11262 genome ….however what is the degree of identity theoligos have with genes of other L. monocytogenes strains.

•A comparison was made between two 1/2a serotype strains (EGD-e and smoked salmon strain 2542)

•~97% genes were homologous between the strains as indicated by relative signal intensities

Strain 2542 was tested as it exhibited robust survival followingHPP treatments e.g. 1-3 log reduction at 400 MPa for 5 min Depending on growth phase

•Brain Heart Infusion (BHI) broth cultures log-phase

•Controls: BHI broth atmospheric pressure•Treatments: 400 MPa 5 min (temperature controlled)

L. monocytogenes hydrostatic pressure treatment microarray experiment

L. monocytogenes HPP treatment microarray experiment

HPP treatments cause wide ranging changes in the L. monocytogenes transcriptome:- 472 genes upregulated significantly (p<0.005, FDR)- 503 genes down regulated significantly (p<0.005, FDR)- 400 MPa and 600 MPa (5 min.) treatments correlate- 600 MPa treatments - weaker expression, more variable.

HPP/Gene expression L. monocytogenes

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tufB & rpoC expression under HPP (reverse transcriptase PCR)

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tufB (5 min)rpoC (5 min)tufB (15 min)rpoC (15 min)

R2 = 0.876

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Microarray gene expression log2 ratios

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agrB cspL

ftsE

flaA

ftsZ

uspAinlA

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pdhD

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gpmA

gadBhly

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Microarray/ Reverse Trancriptase-PCR comparison

HPP induced differential gene expression organised bygene functional category:

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E G D M H N L C P U I F O Q T K J

COG Classification

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HPP induced differential gene expression organised bygene functional category:

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COG Classification

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Increased expression

Reduced expressionCarbohydratetransport/metabolism

Energy production/conversion

HPP induced differential gene expression organised bygene functional category:

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COG Classification

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Cell division/chromosomalpartisioning

Cell envelopebiogenesis

Translation/ribosomes

•No obvious directly associated alternative RNAP sigma factor acting as a master regulator.•RNAP Delta Factor (rpoE) upregulated. In Bacillus subtilis shown to modulate RNAP activity, associated with increased transcription specificity (inhibits expression from weak promoters) and increases efficiency of RNA synthesis by enhancing RNAP recycling.

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iolmo0895 (sigB/sigma37)lmo0243 (sigH/sigma24)lmo0423 (sigma24)lmo1099 (TN916 sigma24)lmo1454 (rpoD/sigma70)lmo2461 (sigL/sigma54)lmo2560 (rpoE/delta factor)

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lmo0895(sigB/sigma37)

lmo0243(sigH/sigma24)

lmo0423 (sigma24)

lmo1099 (sigma24)

lmo1454(rpoD/sigma70)

lmo2461(sigL/sigma54)

lmo2560 (rpoE/delta factor

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400 MPa 600 MPa

HPP invokes a number of response regulators including a putativequorum sensory peptide system when exposed to HPP

agrBDCA homologs lmo0048-0051 in L. monocytogenes EGDe are homologousto the accessory gene regulator cluster of Staphylococcus aureus required for exoprotein/toxin

regulation and secretion in S. aureus

Appears important for protein secretion regulation in L. monocytogenes; agr mutants are much less virulent (Autret et al. 2003)

It is possible that agrDB may act as a regulator as it may code a regulatory RNA (as found in S. aureus).

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agrD agrB agrC agrA

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Listeria monocytogenes strongly upregulates certain coldshock protein (csp) genes when treated with HPP

- Shown previously by proteomic analysis (Wemekamp-Kamphuis et al. 2002, Wageningen University)- role as RNA chaperones preventing deleterious secondarystructure formation

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cspB(lmo2016)

cspL(lmo1364)

cspD(lmo1879) cspR *

(lmo0935)

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*spoU classrRNA methylase

Affects on transcription and translation:

HPP causes DNA damageDNA repair genes upregulatedrecU, recN, recQ, recR, dinG, dinP, putative DNA ligase etc.

HPP causes stalling of transcription by dissociation ofthe transcription complexPossibly overcome due to upregulation of antiterminators…. nusA, nusG and rho

HPP disrupts ribosomesIncreased ribosome and tRNA assembly, turnover and stabilisationtRNA – tgt, miaA, truB, rnpARibosomes- DEAD box helicases, a lot of ribosomal proteins

HPP stalls translationPost termination complex dissociation enhanced– fus (EF-G), infC* (IF3), frr

Fatty acid synthesis genes are upregulated- acyl-coenzyme A synthetase (acs) (2 fold increase)- biotin carboxyl carrier (accB) (2 fold increase)- ACP-S-malonyltransferase (fabD) (2-3 fold increase)- β-ketoacyl-acyl carrier protein synthases (fabH) (3-9 fold increase)- acpP (acyl carrier protein) 14-16 fold increase- appears to be linked to increased synthesis of fatty acid iso-15:0

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LOGPHASE

STATIONARYPHASE

Listeria monocytogenes upregulates flagella biosynthesis, chemotaxis, and protein secretion complex

1.Upregulation of flagella biosynthesis and chemotaxis

flaA - flagellin

flgBCGLK – flagella structural proteins

motAB – motor proteins

flhAF, fliDFIMNQ – type III secretion proteins, flagella

biosythesis

cheAMWY – chemotaxis proteins

2. Upregulation of the protein translocation complex

secEGY – protein conducting channel

secDFyajC-yidC homologs – membrane complex

secA (ATPase) - expression unchanged or reduced

1. Most septal ring (Z-ring) proteins are upregulatedftsAEIKLX, amiCftsZ has unchanged expression

2. Proteins involved in septal ring placement, maintenance of cell shape and chromosome segregation

mreB, minC, divIVA homologs (lmo1888, lmo2020); spoVG, homologs (lmo0196, lmo0197); several genes downstream from ftsK; xerCD homologs (lmo1277, lmo1955); gyrAB, typAhomolog – actin (lmo1067)

3. Teichoic acid and peptidoglycan synthesis and crosslinking

dapB, uppS, dacC, mltE, dltCD, glmSU, gtcA, mdoB, wcaA

Maintains/repairs the septal ring when exposed to HPP

Dividing cells

Septal Ring

Conclusions

- Listeria monocytogenes actively expresses many genesas a response to HPP

-At very high pressures and with time expression is diminishedsignificantly

-Overall responses suggest L. monocytogenes copes with HPPby maintenance and stabilisation of biomolecular complexesincluding ribosomes, septal ring, cell wall, protein secretion.

Core Partners

Core Members

Acknowledgements:

Michelle Bull (Food Science Australia)Cindy Stewart (Food Science Australia, now Univ. Chicago)AGRF Ltd.