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Microbiological Research
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elative expression of bacterial and host specific genes associated with probioticurvival and viability in the mice gut fed with Lactobacillus plantarum Lp91
Molecular Biology Unit, Department of Dairy Microbiology, National Dairy Research Institute, Karnal, Haryana 132 001, IndiaDepartment of Food Engineering and Technology, Tezpur University, Napaam, Assam 784 028, India
a r t i c l e i n f o
rticle history:eceived 19 December 2012eceived in revised form 1 March 2013ccepted 13 April 2013vailable online 29 May 2013
a b s t r a c t
The present investigation was aimed at studying the relative expression of atpD (a key part of F1F0-ATPaseoperon), bsh (bile salt hydrolase), mub (mucus-binding protein) and MUC2 (mucin) genes in mouse modelfor establishing the in vivo functional efficacy of Lactobacillus plantarum Lp91 (MTCC5690) by reversetranscription-quantitative PCR (RT-qPCR). The atpD gene was significantly up-regulated to 2.0, 2.4 and3.2 folds in Lp91 after 15, 30 and 60 min transit in the stomach of mice. The maximal significant (P < 0.00)
eywords:actobacillus1F0-ATPaseile salt hydrolaseeverse transcription-quantitative PCRrobiotic
level of relative bsh gene expression was recorded in Lp91 with 41.6 fold in comparison to only 5.0 fold inreference strain Lp5276 after seven days of mice feeding. Simultaneously, mub gene expression increasedto 12.8 and 22.7 fold in both Lp91 and Lp5276, respectively. The expression level of MUC2 was at the levelof 1.6 and 2.1 fold in the host colon on administration with Lp91 and Lp5276 feeding, respectively. Hence,the expression of atpD, bsh, mub, MUC2 could be considered as prospective and potential biomarkers forscreening of novel probiotic lactobacillus strains for optimal functionality in the gut.
The human and animal gastrointestinal microbiota is a com-lex ecosystem composed of a diverse microbial population,hich is constantly exposed to endogenous microbes, exogenousathogenic and nonpathogenic microorganisms and varied food
ngredients. With recent advancement in metagenomic sequenc-ng, between 1000 and 1150 prevalent bacterial species inhabitinguman intestinal tract has been identified (Zoetendal et al. 2006;in et al. 2010). Some microorganisms can beneficially affect theost by improving the balance of commensal intestinal microflorand are referred to as probiotics (Chermesh and Eliakim 2006).he key members of probiotic group include species of the generaactobacillus, Bifidobacterium and Saccharomyces boulardii. Amonghe probiotic lactobacilli, Lactobacillus plantarum is a flexible andersatile organism that inhabits a wide array of environmentaliches including the human gastrointestinal (GI) tract (Sartor 2004;
e Vries et al. 2006) whose whole genome sequence has alreadyeen completed and is now available as public domain in the NCBIatabase (Kleerebezem et al. 2003). Administration of probiotics
can prevent or alleviate certain medical conditions including acutediarrhea, Irritable Bowel Syndrome, infection after surgery, can-cers, high serum cholesterol and diabetes, etc. Although, consumerinterest in probiotics has been growing enormously during the lastfew years (Arvanitoyannis and Van Houwelingen-Koukaliaroglou2005), the activities of probiotics in the gastrointestinal tractand the precise mechanisms by which they exert their health-modulating effects have not yet been clearly delineated (Marcoet al. 2006).
One of the important criteria for designating a microbial cul-ture as probiotic as per WHO/FAO guidelines is that these bacteriamust overcome the host’s physiological barrier including hostileacidic conditions (FAO/WHO 2002) and bile toxicity prevalent inthe stomach and duodenum respectively so that the selected straincould survive in adequate number to express their health promot-ing functions optimally in the gut. In this context, attempts weremade by some investigators to understand the role of atpD gene (inatp operon) in protecting the probiotic strains by maintaining theneutral pH (Azcarate-Peril et al. 2009; Duary et al. 2010; Walleniuset al. 2012) and bile salt hydrolase (bsh) by deconjugating the accu-mulated bile salts (Jones et al. 2008; Kim and Lee 2008; Duaryet al. 2012; Kumar et al. 2012) in probiotic Lactobacillus spp. under
different in vitro conditions. Cui et al. (2011) proposed two com-ponent systems (TCSs) consisting of histidine protein kinase (HPK)and a cytoplasmic response regulator (RR) for acid adaptation inLactobacillus delbrueckii subsp. bulgaricus. Besides, possessing acid
nd bile tolerance, probiotic bacteria also compete with intesti-al microorganisms for adhesion to mucus and epithelial cells byroduction of specific adhesion proteins (40–200 kDa). Adhesiono mucosa is also important for proliferation of probiotic cells withxtended transit especially in the lower part of the gut. Mucus bind-ng protein (mub, 353 kDa) and muscus adhesion promoting proteinmapA, 26 kDa) are the two key surface proteins expressed differen-ially among different species of lactobacilli, thus, promoting theirttachment to intestinal tissues (Buck et al. 2005; Ramiah et al.007; Kaushik et al. 2009; Duary et al. 2012). Adhesion is believedo be important for demonstrating the probiotic effects such asmmunomodulation (Valeur et al. 2004) and pathogen exclusionMack et al. 1999). In addition, the protective layer of mucus, com-rising of mucin (a complex mixture of large, highly glycosylatedroteins) (Dekker et al. 2002) and glycolipids, covers the gut epithe-
ial cells and offer attachment sites for bacterial colonization in theut. Among different epithelial mucin genes, MUC2 is the major gel-orming structural component of the mucus gel both in the smallnd large intestines (Porchet et al. 1999). Since, mucins consti-ute the major structural components of the mucus layer, changesn mucin quantity, secretion and structure could lead to dimin-shed protection of the colonic epithelium. The outcome of somereliminary studies using various in vitro and in vivo models sug-ests that administration of probiotics has the potential to induceUC2 gene expression in colonic epithelium and hence can offer
prospective strategy to prevent inflammatory diseases (Hoeblert al. 2006; Caballero-Franco et al. 2007). However, very little workas been done on these lines. Hence, this study was specificallyndertaken to look at the relative expression of atpD, bsh, Mub andapA genes of L. plantarum Lp91 (Microbial Type Culture Collec-
ion (MTCC) 5690) – a putative probiotic indigenous strain havingtrong immuno-modulatory, antioxidative and cholesterol lower-ng potentials (Achuthan et al. 2012; Duary et al. 2012a; Kumar et al.011) and MUC2 gene of the animal gut in mice model by reverseranscription-quantitative PCR (RT-qPCR).
. Materials and methods
.1. Bacterial strains and growth conditions
L. plantarum Lp91 (MTCC 5690; Microbial Type Culture Collec-ion and Gene Bank, Institute of Microbial Technology, Chandigarh,ndia), the subject of this study, was a laboratory isolate of humanrigin whose probiotic and colonization potential in Caco2 andT-29 cell lines were established in vitro (Duary et al. 2011) pre-iously in our lab as per FAO/WHO guidelines (FAO/WHO 2002).. plantarum Lp5276 (also designated as CSCC5276, NCDO82 orTTE-71034) (Kaushik et al. 2009; Duary et al. 2010, 2012) useds a reference strain in this study was procured from Dr. N.P. Shahrom Victoria University, Australia. The purity of the Lactobacillusultures was ascertained by Gram staining and microscopic exam-nation. The test and reference were propagated and maintained in
RS (de Man-Rogosa-Sharpe broth, HiMedia, India) broth at 37 ◦Cor 18 h. The active bacterial cultures were maintained in litmus
ilk (4 ◦C) and also as glycerol stocks (−70 ◦C).The identity of the Lactobacillus strains was ascertained by PCR
oth at genus and species level using genus specific LbLMA1/R-61 (Dubernet et al. 2002) and species specific Lpla2/Lpla3 primersSong et al. 2000) along with 16S rRNA sequencing.
.2. In vivo studies using mouse model
Swiss Albino Mice (4-6 week-old male) weighing 20.8 ± 0.1 g onverage were obtained from the Small Animal House at Nationalairy Research Institute, Karnal, India. They were housed with six
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animals per cage, in a room with 12/12-h light/dark cycle and anambient temperature of 20–25 ◦C. During the course of experiment,the animals were fed normal diet (bengal gram crushed: 58%, wheatstarch: 15%, groundnut cake: 10%, casein: 4%, refined oil: 4%, saltmixture: 4%, vitamin mixture: 0.2% and choline chloride: 0.2%) andwater ad libitum. The approval of Institute’s Animal Ethics Com-mittee was obtained before the conduct of the animal trial andthe mice were maintained in accordance with the National Insti-tute of Nutrition, India guidelines for the care and use of laboratoryanimals.
2.3. Preparation of bacterial cell suspension for oral feeding
Both the bacterial strains (Lp91 and Lp5276) were grown atexponential growth phase individually in MRS broth to an OD600of 2.0, harvested (3000 × g, 10 min, 4 ◦C), washed twice withphosphate-buffered saline (PBS, pH 7.4) and resuspended in PBS atthe appropriate cell concentration. Mice were divided into groupsof six animals each for studying the in vivo expression of acid andbile tolerance as well as expression of genes encoding MUC and sur-face layer proteins. For acid tolerance study, the mice were fed byoral intubation with 1.5 ml of the cell suspension (109cfu/ml) of theprobiotic strains and for control mice group sterile PBS alone wasfed (negative control). Animals were sacrificed after 0 min, 15 min,30 min and 1 h after oral intubation. The stomach was flushed withsterile PBS solution to collect the bacterial cells. For bile toleranceand surface proteins study, mice were fed orally for 7 days withlive cultures of lactobacilli resuspended in 200 �l of sterile PBS andPBS alone as a negative control. After a 7-day probiotic treatment,the animals were sacrificed, and the intestines were excised andflushed with sterile PBS to remove the adhered bacteria. Similarly,for investigating MUC2 gene expression, mice were fed orally for7 days with live cultures of lactobacilli resuspended in 200 �l ofsterile PBS. After a 7-day probiotic treatment, the animals weresacrificed, colons excised followed by removal of fecal matter andtransfer of the tissue to the liquid nitrogen container.
The intestinal tissues from colon of probiotic and control groupswere set apart for histopathological examination and stained withHE (Hematoxylin and Eosin, HiMedia, India) prior to microscopicexamination using standard protocols.
2.4. Extraction of RNA
Total RNA was recovered from 50 to 100 mg of the tissue sam-ple as well as bacteria by addition of 1 ml of TRI Reagent (Sigma,USA). In order to lyse the cells of lactobacilli, an aliquot of 10–15 �lof lysozyme (50 mg/ml of 10 mM Tris–HCl, pH 8.0) and 200 �l ofTris–HCl (10 mM, pH 8.0) were added and further processed forRNA extraction with TRI Reagent. RNA was suspended in 50 �l ofDEPC (diethylpyrocarbonate) treated/RNase free water and storedat −80 ◦C till further use. The purity of the total RNA extractedwas determined at 260/280 nm ratio and the integrity of RNA waschecked by electrophoresing on 1% agarose gel. Residual DNA wasremoved by treating RNA with RNase free DNaseI as per the instruc-tions furnished by the manufacturer (Promega, USA). Finally, thepurified RNA was stored at −80 ◦C until further use. DNase treatedRNA (1 �g) was transcribed into cDNA, using ImPromII reversetranscriptase kit (Promega, USA) and random primers following theprotocol as per the manufacturer’s instructions.
2.5. Primer design
The primers used in the investigation are listed inTable 1. Primers targeted against MUC2-Mus (Gene BankAcc. No. XM 001473263) and Gadph Mus (Gene Bank Acc.No. NC 000072) were designed by using Primer3plus software
A. Chandran et al. / Microbiological Research 168 (2013) 555– 562 557
Table 1Primer sequences of target and reference genes used for RT-qPCR.
Name Definition Forward primer (5′–3′) Reverse primer (5′–3′) Annealing temp. (◦C) Reference
atpD F1F0-ATPase �subunit
gcc aac ctg gtt cgt atg tg acc acg tcg tcg atc tta cc 54 Duary et al. (2010)
bsh Bile salt hydrolase atg ggc gga cta gga tta cc tgc cac tct ctg tct gca tc 54 Duary et al. (2012)mub Mucin binding
proteingta gtt act cag tga cga tca atg taa ttg taa agg tat aat cgg agg 54 Duary et al. (2012) and Ramiah
et al. (2007)mapA Mucus adhesion
promoting proteintgg att ctg ctt gag gta ag gac tag taa taa cgc gac cg 54 Duary et al. (2012) and Ramiah
et al. (2007)Gadph-bac Glyceraldehyde-3-
phosphatedehydrogenase
act gaa tta gtt gct atc tta gac gaa agt agt acc gat aac atc aga 54 Duary et al. (2012)
MUC2 Mus Mucin tgc cca gag agt ttg gag ag cct cac atg tgg tct ggt tg 54 This studyt ctg g
( gi).TTbrpgo
2
f(1a1toanMt5cgcrfirm
2
taqawsweod2adt
Gadph Mus Glyceraldehyde-3-phosphatedehydrogenase
acc cag aag act gtg gat gg tca gc
http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.che GC content of the primers was in the range of 40 and 60% andm from 55 to 60 ◦C. BLAST searches were performed against otheracterial genomes to determine the specificity of the primers. Toule out the possibility of primer dimer formation, the amplifiedroduct obtained with each primer pair was checked by agaroseel electrophoresis (2% agarose) followed by melt curve analysisf the RT-qPCR amplified products.
.6. LightCycler quantitative real-time PCR
Reverse transcription-quantitative PCR (RT-qPCR) was per-ormed for gene expression in a LightCycler 480 instrumentRoche), with Relative Quantification Software (version LCS480.5.0.39, Roche) with fluorescence signal detection (SYBR Green)fter each amplification cycle. Each reaction was performed in a0 �l reaction volume containing 5 �l of 2× SYBR Green PCR Mas-er Mix (Roche), 2.5 �l of properly diluted cDNA (typically 30 ng/�lf cDNA for all the genes used for RT-qPCR), 0.5 �l of each primert 10 �M and 1.5 �l of nuclease-free water. Negative controls (witho DNA template, only primer pair, water and 2× SYBR Green PCRaster Mix) for each primer set were included in each run. The
hermal cycle conditions included initial denaturation (95 ◦C for min), followed by amplification and quantification program of 40ycles (10 s at 95 ◦C, 15 s at annealing temperature for respectiveenes as shown in Table 1 and 15 s at 72 ◦C with a single fluores-ence measurement), melt curve program (60–95 ◦C with a heatingate of 0.11 ◦C s−1 and a continuous fluorescence measurement) andnally a cooling step at 40 ◦C. Measurement of gene expression withegard to each RNA extractions were obtained in triplicate, and theean of these values were used for further analysis.
.7. Data analysis of relative target gene expression
Generation of quantitative data by real-time PCR is based onhe number of cycles required for optimal amplification gener-ted fluorescence to reach a specific threshold of detection (theuantification cycle; Cq value) (Bustin et al. 2009). Real-time PCRmplification efficiencies were determined for each set of primersith the slope of a linear regression model (Pfaffl 2001). The cDNA
amples were diluted in a range of 100, 50, 25, 5, 1, and 0.25 ng andere used as RT-qPCR templates. The standard curves were gen-
rated by plotting the log cDNA values against Cq values obtainedver the range of dilutions. The slope of the curves was used toetermine the reaction efficiency (E) as E = 10[−1/slope] (Pfaffl et al.
002). The total expression ratio of the five genes of interest i.e.tpD, bsh, mub, mapA and MUC2 was tested for significance by a ran-omization test implemented in the relative expression softwareool (REST 2009) (www.gene-quantification.info) and the same was
ga tga cct tg 54 This study
also used in application for group wise comparison and statisticalanalysis of relative expression results in RT-qPCR (Pfaffl et al. 2002).Randomization test method, as a part of the REST software, wasused to assess statistical significance of up- or down-regulation ofthe target genes after normalization to the reference gene. For up-regulation, the factor of regulation is equal to the given value in theRandomization Data Output Box. While, for down-regulation, theregulation factor is illustrated as a reciprocal values (1/expressionratio) (Pfaffl et al. 2002). Randomization test randomly reallocatesthe observed values in different sample groups to the values in thecontrol group. Relative expression is then estimated for each of thenew groups and the effect of randomization in each group is eval-uated. If this effect is not significant, it provides sufficient evidencethat the observed treatment effect was not due to the random allo-cation. Statistical analysis was considered significant at P ≤ 0.05.Data that emerged from this study was presented in the form ofwhisker-box plots. The top and bottom of each box represent the25th- to 75th-percentile values while the 50th percentile (median)is the horizontal dotted line in the rectangle. The vertical bars rep-resent the minimum and maximum values that are not consideredoutliers.
2.8. Random amplification of polymorphic DNA (RAPD) profiles
Colonies were randomly picked into MRS broth from differentfecal count plates and grown for 16–18 h at 37 ◦C. The genomicDNA was then isolated by Pospiech and Neumann (1995) method.PCR with random primer OPBG-02 (5′ggaaagcca3′) was carried out.PCR reaction was performed in 25 �l reaction volume contain-ing 2 �l of genomic DNA, 2.5 �l PCR buffer, 2.0 �l of oligo primer(OPBG-02), 2.0 �l of 200 �M dNTP and 0.5 �l of 1.0 U Taq DNA poly-merase. The PCR products were electrophoresed on 1.5% agarose gelwith ethidium bromide (0.5 �g/ml) (Amersham Biosciences, USA)at 100 V using 1× TAE buffer. The PCR cycling parameters includedan initial denaturation of 95 ◦C/5 min, followed by 45 cycles each ofdenaturation (95 ◦C/30 s), annealing at (38 ◦C/30 s) and extension(72 ◦C/2 min) and final extension of 72 ◦C/10 min.
2.9. Statistical analysis
The data obtained from each animal experiment were expressedas mean ± standard deviation values and statistically analyzed withthe Statistical Package for the Social Science (SPSS for Windows,version 10.1, SPSS, Chicago).
3. Results
After ensuring the purity of the specific probiotic strains, theiridentity as L. plantarum was confirmed by using genus and species
Fig. 1. Genus and species specific PCR using primer LpLmlA1/161R1 andLpla3/Lpla2. (a) Genus specific PCR Lactobacillus cultures; (b) species specific PCRf
stoltlTpFt
ihLrgk(
3
tdpife(vtawcurfi1
Fo
or L. plantarum Lp91 and CSCC5276.
pecific primers. Genomic DNA from both the Lactobacillus cul-ures when subjected to genus PCR resulted in the amplificationf a 250 bp products on the agarose gel which was specific foractobacilli only (Fig. 1). After ascertaining the test cultures as Lac-obacillus, we directed our efforts to identify the culture at speciesevel by using Lpla3/Lpla2 set of primers specific for L. plantarum.he PCR assay conducted with the genomic DNA from the culturesroduced in an amplification of 248 bp PCR product as shown inig. 1, thereby, indicating that both the strains belonged to L. plan-arum.
In this study, the expression of atpD, bsh, mub and mapA genesn L. plantarum 91 under in vivo conditions and MUC2 gene of theost was investigated in mice vis a vis a reference probiotic strain. plantarum Lp5276 on their oral administration in the gut byeverse transcription-quantitative PCR (RT-qPCR). Two referenceenes Gadph bac and Gapdh Mus gene were the most stable house-eeping genes in the bacteria and host cell line model, respectivelydata not shown).
.1. Expression of atpD gene of Lp91 in mouse stomach
The survival of Lp91 in the mouse stomach administered withhe same strains after 15, 30 and 60 min was determined afteretermining the viable counts in the flushed samples on MRS agarlates (Fig. 2). The initial level of inoculum that was administered
ntragastrically in mouse was 9.5 log counts per ml for Lp91 and 9.9or Lp5276. The survival of Lp91 was slightly better than the refer-nce strain as can be reflected from their respective viable counts7.7 for Lp91 and 7.6 for Lp5276). However, after 60 min, the sur-ival of both the cultures registered less than one fold decline inhe gut as revealed by lower log counts i.e. 7.3 and 7.0 for Lp91nd Lp5276, respectively. Relative expression of the atpD alongith the reference gene (Gapdh bac) in the surviving bacterial
ells present in the stomach flushings was quantified by RT-qPCR
sing REST 2009 software. Specificity of primers for the target andeference genes used in RT-qPCR assay was confirmed by ampli-cation of a specific single product of the desired amplicon i.e.24 bp (atpD) and 147 bp (Gadph bac) and single product of specific
ig. 2. Survival of Lp91 and Lp5276 in the mouse stomach based on viable countsn MRS agar.
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melting temperatures of 82.01 and 79.5 ◦C for atpD and Gadph bac,respectively (Fig. 1S Supplementary information). The curves forrespective genes revealed high linearity (R2 > 0.98). The slopes ofatpD and Gadph bac curves were −3.3275 and −3.5005 respectivelythat indicated high RT-qPCR efficiencies (E) of 1.99 and 1.93, respec-tively (Fig. 2S). The target and reference genes were analyzed inthree different sets of trials each conducted in duplicate to accountfor any intra and inter assay variation on the same 96 well plate.From the data presented in Fig. 3, it is quite evident that atpD genewas significantly (P < 0.05) up regulated both in Lp91 and Lp5276in mouse stomach at 15, 30 and 60 min after oral administration.The fold increase in the expression of atpD gene in Lp91 2.0, 2.4 and3.2 after 15, 30 and 60 min transit, respectively. However, the cor-responding values for Lp5276 were slightly on higher side i.e. 2.4,4.1 and 3.4 clearly indicating a significant increase in the expres-sion of the targeted gene in both the test as well as the referencestrain.
3.2. Expression of bsh gene of LP91 in mouse gut
The expression of the ‘bsh’ gene in mouse gut was also investi-gated as was done for atpD described previously. The specificity ofthe primers for ‘bsh’ gene was ascertained by detecting the desiredamplicon (103 bp) and specific melting temperature (81.47 ◦C) (Fig.1S Supplementary information) along with RT-qPCR efficiency (E,1.96) (Fig. 2S). Amongst the two strains, there was 41.6 fold increasein the relative expression of bsh in Lp91 over the control (Fig. 4)indicating that the gene was up regulated significantly (P < 0.00)in comparison to that of Lp5276 wherein the expression level wasonly 5.0 fold.
3.3. Expression of mub and mapA genes of Lp91 in mouse gut
Both mub and mapA of the two probiotic strains subjected toRT-qPCR in the mouse gut resulted into amplification of the desiredPCR products of 150 and 156 bp with specific melting temperaturesof 83.63 and 85.27 ◦C, respectively (Fig. 1S Supplementary informa-tion) and RT-qPCR efficiencies (E) of 1.83 and 1.99, respectively (Fig.2S). Although, Lp91 was able to up-regulate the expression of mubgene significantly by 12.8 fold, it was lower than that recorded withthe reference strain wherein the level of expression was 22.7 foldsover the control (Fig. 4). The relative expression profile of mapAof both Lp91 and Lp5276 on the other hand was not significantlyaffected as the level of expression in both the strains were only1.0 and 1.3 fold respectively after probiotic administration in themouse gut.
3.4. Expression of MUC2 in mouse gut
The effect of Lp91 administration in the gut on the MUC2 geneexpression in mice model by Real Time PCR was also investigatedin this study and was compared with that of Lp5276. Amplificationof MUC2 and housekeeping Gapdh Mus gene resulted into specificsingle product of 123 and 124 bp with specific melting temper-ature of 83.77 and 87.72 ◦C, respectively (Fig. 1S Supplementaryinformation). The RT-qPCR efficiencies (E) of MUC2 and Gapdh Muswere 1.81 and 1.97, respectively (Fig. 2S). The relative expressionof MUC2 in mouse gut evoked with both the probiotic strains wassignificantly (P < 0.05) enhanced relative to control albeit at a muchlower level as can be reflected from their respective fold values of1.6 (Lp91) and 2.1 (Lp5276) only (Fig. 5).
3.5. Histopathological examination
It can be revealed from histological specimens of mouseintestinal tissue (Fig. 6) that there is increase thickness of
A. Chandran et al. / Microbiological Research 168 (2013) 555– 562 559
F mout bservar
ssmf
dPci
Fsp
ig. 3. Relative expression of atpD in L. plantarum Lp91 and L. plantarum Lp5276 inool (REST 2009)). Boxes represent the interquartile range, or the middle 50% of oepresent the minimum and maximum observations.
ubmucosal muscular tissue. None of the mice groups demon-trated any visible inflammation, also indicating improvedaintenance of the overall intestinal barrier function of probiotic
ed groups.The fecal samples were collected daily from the different groups,
iluted in dilution blank and plated on BCP-MRS (Bromo-Cresolurple-de Man-Rogosa-Sharpe broth, HiMedia, India) agar. Plateount suggested that a constant level of bacteria was maintainedn gut throughout the experimental period (Fig. 3S).
ig. 4. Relative expression of bsh, mub and mapA gene in L. plantarum Lp91 and L. plantarumoftware tool (REST 2009)). Boxes represent the interquartile range, or the middle 50% of
lots represent the minimum and maximum observations.
se stomach. Expression ratios were calculated (using relative expression softwaretions. The dotted line represents the median gene expression. Whisker-box plots
3.6. RAPD profiles of Lactobacillus isolates recovered from fecalsamples
RAPD DNA fingerprinting technique as described previouslywas used as a tool for identification of Lactobacillus isolates
recovered from fecal samples of experimental animals at strainlevel (Samarzija et al. 2002). The typical banding patterns of Lp91and Lp5276 using primer OPBG-02 have been projected in Fig. 7aand b. The analysis of RAPD profiles was carried out by plotting
Lp5276 in mouse gut. Expression ratios were calculated (using relative expressionobservations. The dotted line represents the median gene expression. Whisker-box
560 A. Chandran et al. / Microbiological R
Fig. 5. Relative expression of MUC2 gene in mouse gut on intervention with L.plantarum Lp91 and L. plantarum Lp5276 Expression ratios were calculated (usingrre
duSf9lroL
4
eaptittm
daoutfrw
elative expression software tool (REST 2009)). Boxes represent the interquartileange, or the middle 50% of observations. The dotted line represents the median genexpression. Whisker-box plots represent the minimum and maximum observations.
endrogram by unweighted pair group method analysis (UPGMA)sing Syngene software (Gene tool and Gene Directory; G-BoxYNGENE). The randomly selected Lactobacillus isolates recoveredrom fecal samples of mice groups administered with Lp91 showed1% similarity with the pure culture of Lp91. Similarly, the fecal iso-
ates from animals treated with Lp5276 showed a similarity witheference strain of Lp5276 culture, thereby, confirming the identityf the fecal isolates at strain level in conformity with Lp91 andp5276, respectively.
. Discussion
This study was undertaken primarily with the objective ofstablishing the in vivo functional efficacy of L. plantarum 91 –n indigenous putative probiotic strain which exhibited strongrobiotic attributes such as acid and bile tolerance and coloniza-ion potentials under in vitro conditions. The main purpose of thenvestigation was to look at the efficacy of this strain with regardo the expression of specific genes associated with acid and bileolerance under in vivo conditions to validate in vitro results in the
ouse model.Surviving the host’s physiological barrier i.e. high acidic con-
itions in stomach and bile salts toxicity due to excessiveccumulation in intestine are the two key pre-requisites along withther functional properties, to confirm probiotic status of a partic-lar organism (Zhang et al. 2011). The level of gene expression in
he probiotic cultures at low/acidic pH in the stomach may varyrom strain to strain. The pH changes in the environment have beeneported to influence expression of many genes in bacteria most ofhich are involved in maintaining the pH at neutral values around
Fig. 6. Microscopic (20×) imaging of intestinal histologic features of mic
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7.0 (Kullen and Klaenhammer 1999; Jacob et al. 2007). In manybacteria, the activity of F1F0ATPase increases as the pH of growthmedium decreases. However, the regulation of pH inducible pheno-types particularly under in vivo conditions prevalent in the gut hasnot been clearly elucidated as yet at molecular level. Ours is per-haps one of the fewer in vivo studies conducted in mouse model todemonstrate up regulation of atpD gene in lactobacilli in the mousestomach on oral administration. Our results indicate unequivocallythat atp operon is essential for the growth and survival of pro-biotic lactobacilli under acidic conditions which is in agreementwith the observation that activity of F1F0ATPase in some relatedbacteria was enhanced at low external pH as described previously(Kullen and Klaenhammer 1999; Koebmann et al. 2000). However,it was observed in our study that after an extended transit timeof 60 min in the mouse stomach, although the expression of atpDwas significantly upregulated in both in Lp91 and Lp5276, theircorresponding viable counts declined by less than one log cycletherein unlike those recorded after 15 and 30 min, where the countsremained more or less stable. This disparity in results could possi-bly be attributed to turning a small portion of probiotic populationinto dormant non-culturable forms after extended transient periodof 60 min due to acidic conditions prevalent in the stomach. How-ever, these variations in viable cell counts were minor and did notnegatively affect the expression of atpD. Besides this, the enhancedactivity of F1F0ATPase in the surviving viable Lp91 cells may alsoaccount for increased atpD expression induced as a result of highacidic pH in the stomach. The genes encoded on atp operon par-ticularly atpD had all the desirable features to serve as appropriatemolecular markers such as high genetic stability and wide distri-bution as reported in previous studies (Ludwig et al. 1993; Ludwigand Schleifer 1999). In one of our own recent studies carried outunder in vitro conditions, L. plantarum Lp91 exhibited high toler-ance to acidic stress conditions by significant increase in the relativeexpression of atpD gene by 4.7 fold at pH 2.5 after 90 min (Duaryet al. 2010).
Bile tolerance is another important key component used forscreening and selection of probiotic cultures for expressing theirhealth promoting and other important physiological functionsbesides their survival in the gut. The relative expression of bsh genewas up-regulated significantly in Lp91 under in vivo gut conditionswhen the strain was fed to mice Our results in this regard are inagreement with the findings of Bron et al. (2004) who also reportedsignificantly higher expression levels of bsh gene in L. plantarumstrains Lp0237 and Lp0075 (13 and 29 fold, respectively) duringpassage of these organisms through the mouse duodenum than inL. plantarum grown on MRS, thereby, substantiating the validity ofour findings. In a related study, Bron (2004) utilized both in vivoand in vitro studies as models for the human situation to describe
the identification and functional analysis of L. plantarum promotersand genes that play important role in the survival during intesti-nal passage of this organism. The expression levels of these geneswere dramatically increased in L. plantarum cells isolated from the
e intestine against L. plantarum Lp91 and Lp5276 fed mice groups.
A. Chandran et al. / Microbiological R
Fig. 7. RAPD profiles of lactobacillus isolates from fecal samples of mice using primerOPBG-02. (a) the typical banding patterns of Lp91; (b) the typical banding patternso
mrtmgorl3uactsrc
pptwuafooisoiuaeta
f Lp5276.
ouse duodenum relative to cells grown in MRS medium. Theseesults can eventually help in development of molecular modelshat can describe the behavior of LAB in GI tract which are funda-
ental for understanding the mechanism for survival in the hostileut environment especially in the duodenum due to accumulationf toxic levels of bile salts. Duary et al. (2012) in a recent study alsoeported up-regulation of the relative bsh gene expression at theevel of 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 folds after 1, 2 and
h respectively at 2% bile salt concentration in L. plantarum Lp91nder in vitro conditions. Hence, it is quite apparent from our resultss well as from those of other groups that the in vitro probioticharacteristics such as acid and bile tolerance can be replicated inhe in vivo situation as well in the mouse duodenum and stronglyuggest that presence of bile in the gut was the inducing envi-onmental factor in the expression of bsh gene under in vivoonditions.
Since mucin binding (mub) and mucus adhesion promotingroteins (mapA) constitute the two key surface layer proteins thatlay an important role in probiotic colonization and extendedransit in the gut, their level of expression in Lp91 in the mouse gutas also looked at in this study. Their expression was found to bepregulated significantly. Our results in this regard are in partialgreement with those of Ramiah et al. (2007) who also recorded 80old higher expression in mub gene in L. plantarum 423 in presencef 0.01% (w/v) mucin and 144 fold higher when grown in presencef 0.05% mucin under in vitro conditions. Our results however aren contradiction with those reported in a different study by theame group (Ramiah et al. 2009) who did not find any up regulationf mub, slp and EF-Tu by Lactobacillus acidophilus ATCC 4356 underncreasing concentrations of mucin although slp and EF-Tu werep-regulated significantly in the presence of bile and pancreatin
t normal conditions (0.3%, w/v). These results indicate that thexpression of these surface bound proteins particularly with regardo mub protein was highly dependent on the specific gut conditionsnd probably does not play an important role in adhesion of L.
esearch 168 (2013) 555– 562 561
acidophilus ATCC4356. The divergence in the outcome of thesestudies with regard to mub gene expression in different probioticlactobacilli could be attributed to some factors that can triggerthe up or down regulation of this gene under different conditions(in vitro/in vivo) and could be ascribed to differential behavior ofdifferent Lactobacillus species and strains as can be evidenced fromthe opposite response recorded with L. plantarum and L. acidophilus(Ramiah et al. 2007). Duary et al. (2012) have recently reportedthat mub gene was expressed at different levels in L. plantarum Lp9under in vitro conditions at different bile, pancreatin and mucinconcentrations at pH 6.5. However, the mapA gene expressionwas not significantly affected in our study under the aforesaidconditions which was at variance with those of Ramiah et al. (2007)and Duary et al. (2012) who also reported enhanced expression ofmapA gene in L. plantarum under different gut simulated in vitroconditions. This suggests that the expression of mub and mapAare characteristic of the species and may be controlled by variousfactors like presence of mucin, pH, origin of species, etc. whichpromote adherence of probiotic strains on gut epithelial cells.
In gut environmental condition, both mechanical and immuno-logical protection to body is provided by mucosal barriers fromexternal stimuli, which are mostly coated by mucus. It is now wellestablished that probiotics can influence mucosal cell interactionsand cellular stability by the enhancement of intestinal barrier func-tions through modulation of cytoskeletal and tight junction proteinphosphorylation. In this context, MUC2 recognized as the major gelforming mucin of the small and large intestine and constitutingthe main structural component of the mucus helps in the main-tenance of the tight barrier function to protect the gut againstinfections and its disruption as a result of metabolic inflamma-tory disorders. The results obtained on MUC2 expression inducedby orally administered Lp91 and Lp5276 in the mice gut are con-sistent with the findings of Caballero-Franco et al. (2007) who tooreported only a five fold increase in MUC2 expression in the mice gutafter seven days of administration of VSL#3 probiotic formula withconcomitant increase in mucin secretion relative to control. Ourresults are also partially in agreement with those of Gaudier et al.(2005) who hypothesized that VSL#3 probiotics could modulatemucin gene expression which would help in healing the inflamedmucosa. Once mucin is synthesized in the goblet cells and secretedto the intestinal surface, it forms an adherent layer that undergoescontinuous degradation and renewal. Increased MUC2 expressionshowed direct correlation with increased mucosal thickness, ascould be evidenced from the histopathological examination of themice gut after probiotic administration recorded in our study. Thus,enhancing the thickness of the mucosal layer through mediationof probiotic intervention could provide a strong protection to thegut against the infections along with minimization of the damageattributed to inflammatory disorders.
Hence, from the outcome of this study, it can be concluded thatour indigenous probiotic L. plantarum Lp91 is quite robust and hasthe potential to survive the hostile gut environment with extendedtransit in the gut with optimal functionality (acid and bile toler-ance). Because of these attributes, it can be explored as a candidateprobiotic in protecting the gut from infections and other intestinaldiseases through enhanced mucin production.
Acknowledgments
We gratefully acknowledge the Director, National DairyResearch Institute (NDRI, Karnal, India) for providing facilities to
carry out the study. The financial support received from IndianCouncil of Agricultural Research (ICAR, India) in terms of pro-viding fellowship to the first author of the paper is greatlyappreciated.
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ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.micres.2013.04.010.
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