Stress response in lactic acid bacteria and its implications in probiotic

STRESS RESPONSES IN LACTOBACILLI:

IMPLICATIONS IN PROBIOTICS

Diwas Pradhan

Dairy Microbiology Division

ICAR-National Dairy Research Institute

April, 2012

INTRODUCTION

• By definition,probiotics mustreach their targetsite alive toconfer a healthbenefit to thehost

• A plethora ofstresses needs tobe encountered

• LaCOG analyses employed to identify a set of LAB genes associated withvarious stress responses.

• HrcA involved in the control of the heat-shock protein expression

• Heat-shock proteins commonly under HrcA control and performingchaperonin-like functions (GroELS, DnaJK, GrpE) are universallyconserved.

• The involvement of CtsR in the regulation of class III stress proteins,including the Clp proteases and related functions.

• The corresponding Clp proteases appear to be universally present inthese LAB genomes.

• Majority of stress-tolerance genes are conserved among strains of aparticular species

• strain-specific survival capacities depend on their relative levels of expressionrather than their presence or absence.

Lactic Acid Bacteria Genomics

• The major HSPs:

o The classical chaperones DnaK, GroEL, and GroES (participate

in protein folding, protein translocation, and possibly higher-

order protein assembly)

o The Clp family of proteins (disposing heat damaged proteins)

play an indispensable role in protein quality

o Regulated by HrcA and CtsR regulon

• HtrA, FtsH, sHSPs, RecA, and Other Important Factors

Responses to Heat Stress

Varmanen, P. and Savijoki, K., 2011

• A number of CSPs are induced

• Function as transcriptional and translational regulators

• Also act as molecular chaperones

• Three csp genes (cspL, cspC, and cspP) identified inLactobacillus plantarum NC8. Overproduction causes distinctphenotypic effects

• CspL overproduction transiently alleviated the cold-shock impairmentof growth

• CspP overproduction enhanced the cryotolerance

• CspC overproduction improves growth adaptation at optimaltemperatures

Responses to Cold Stress

Capozzi, V., 2011

• Also induced heat-responsive genes sHSPsuggesting a cross-talk between the heat-and cold-stress responses

• An increase in C16:0 and C18:2 fatty acids inLactobacillus acidophilus grown at lowtemperature

Responses to Cold Stress

Capozzi, V., 2011

Cold-shock Response In Bacillus subtilis

• Biological modifications

related to temperature

perception and signal

transduction (red)

• Membrane adaptation

(yellow)

• Nucleoid structure and

transcription (blue)

• CSPs and translation

apparatus (green)

• Metabolism, protein

folding, and cell

differentiation (violet)

• Oxygen, Superoxide, Hydrogen Peroxide and theHydroxyl Radical(HO˙)

• SOD, thioredoxin reductase, peroxidase, glutathionereductase and RecA

• Incorporation of high concentrations of manganeseby Lb. plantarum

• SOD or manganese produce H2O2 from O2˙−.

Responses to Oxidative Stress

Cesselin, B., et al., 2011

Lb. plantarum, jonsohnii and casei

• Encodes cydABCD genes.

• Require heme and a menaquinone to activate respiration metabolism

A Respiratory Chain In LAB

• Activity and pH optima of the proton translocating

(H)-ATPase

• Acid tolerance response(ATR)

a. Decrease fluidity of CM- increase the concentration of saturated FAs or cyclopropane FA(C19:0)

b. Saturated to unsaturated FAs ratio- from 0.4 to 4.9

• Malate and Histidine contribute to acid adaptation

• His contributes to intracellular buffering

Acid stress

Broadbent, J. R., et. al., 2010

• Two-component regulatory systems (2CRS) andABC-type oligopeptide transport proteins

– (Opp) known to function as sensors for environmental change

• Differential expression of transposase genes

• Metabolism, Information storage, processing and Cellular processes genes were significantly altered

Acid stress

Model for Physiological Responses of GG to Bile Stress.

Koskenniemi, K. et al., 2011

• Self-Immunity: Immunity proteins on their cytoplasmicmembrane for protection against the producedbacteriocin.

• NisI and SpaI- lipoproteins anchored to the membrane surfacevia a lipid-modified N-terminal cysteine residue.

• ABC transporter(LanFEG)

• The two-component systems (TCSs) and extracellularsigma factors are involved in the intensive action towardcell wall active antibiotics.

Responses to Bacteriocins and Other Antimicrobials

Asaduzzaman, S. M. and Sonomoto, K., 2011

• Carbohydrate exhaustion ushers LAB into the NC state• Resuscitation has not been demonstrated yet.

• Switch to the catabolism of amino acids usingaminotransferases (ATases)

• Many of the regulatory or quorum-sensing molecules are notproduced.• The accumulation of compatible molecules enables cellular stability

• Loss of the ability to produce colonies• Accompanied by the production of Met and Ser into the medium

Responses to Starvation

Weimer, B. C., 2011

• Majority of analyses have generally focused on

one particular stress

• Need for network reconstruction based on

multiple stress-induced transcriptome profiles

• Not been reported for LAB to date

• Will help reveal the regulatory networks and

complete regulons involved.

Assessment of Multiple Stress Responses

and Regulatory Network Reconstruction

Bron, P. A., 2011

1. In Vitro approaches to identify RobustnessGenes in Lactic Acid Bacteria

• Comparative Genomic Hybridization

• DNA microarray technology

2. In Vivo Expression Technology

Functional Genomics Approaches to Unravel Lactic Acid Bacteria Stress

Responses

Bron, P. A. et. al., 2011

Zomer, A. and Sinderen, D. van., 2010

Stress Gene Regulatory Network Of B. breve UCC2003

• The CGH approach employsone-directional comparisonof gene-content profiles perstrain using genome widemicroarrays that aredesigned on the basis of thegenome of a single strain.

• Enables the construction ofhigh-resolution genomewide presence-absencepatterns for each of thestrains that is analyzed.

Comparative Genomic

Hybridization

genome sequence of a robust strain

DNA of a robust strain

DNA of the strain

Sequence present in both (grey)Sequence present in robust strain but normal strain (black)

19

Widely used technologyto identify several of the(conserved) geneticfactors regulated duringstress imposed on LAB

DNA microarray technology

Proteomics

• Powerful method thatallow the genomewide identification ofin vivo induced (ivi)promoters and theircorresponding genesutilizing a promotertrapping system.

In Vivo Expression

Technology(IVET)

Rediers, H. et al., 2005

Strategic Approach To Identify And Validate

Stress Tolerance Associated Genes And

Functions

Pregenomics Approaches

Additives

Pre-adaptation

Cross-Protection

Postgenomics Strategies

Overexpression of Stress Regulon Members by Genetic

Modification

Targeted Mutagenesis of Stress Regulators

Fermentation-Enhanced Probiotic Function

Improving Robustness

Bron, P. A., et. al., 2011

ADDITIVES

A straightforward way to improve stress response

Glucose, fructose, Tween-80, betaine, gum acacia etc.

PREADAPTATIONLAB have developed several conserved stress

responsesThe CtsR and HrcA regulators

Overlap in different stress regulons

Overexpression of Stress Regulon Membersby Genetic Modification

Alteration of expression levels ofgenes encoding stress regulon members or regulatorsprior to stress exposure

Targeted Mutagenesis of Stress Regulators

Manipulation of complete stress regulons by targeting the canonical stress regulator CtsR

• Exploits stress response systems of pathogenic bacteria.

• Heterologous expression of BetL from L. monocytogenes in

the Lactobacillus salivarius UCC118 resulted in a strain with

improved resistance to numerous stresses.

• A novel bile resistance mechanism- BilE

1. Operates by excluding bile from the cell

2. Not many homologues of the bilE operon have been identified in

any of the genomes of the probiotic organisms

Pathobiotechnology

Mills, S., et. al., 2011

Fermentation-Enhanced Probiotic

Function

Stress in the Context of Microbial Communities

Stress and the Cell Cycle

Stress and DNADamage Control

Stress-Induced Mutagenesis

Stress in the Context of the Single Cell

Sensing and Signaling Stress

Papadimitriou, K., and Kok, J., 2011

The ability of probiotic bacteria to survive the harshenvironments has been a major factor in their selectioncriteria.

Induction of the probiotic stress response through pre-adaptation strategies- not always successful.

Developing a molecular toolbox (throughpathobiotechnology, targeting indigenous defensestrategies), should ensure that the most functionallyactive strains can be confidently selected for probioticdevelopment.

CONCLUSIONS

Apprehensions on the use of Genetically Modifiedorganisms.

Studies which evaluate the safety of engineeredprobiotics are crucial if the technology is to gainacceptance.

In this way next-generation probiotic cultures will bebetter equipped to face technological andgastrointestinal challenges as well as meeting medicaldemands

Cont.