# The X-prolyl dipeptidyl-peptidase PepX of Streptococcus thermophilus initially described as intracellular is also responsible for peptidase extracellular activity

Zeeshan Hafeez, Céline Cakir-Kiefer, Xavier Lecomte, Laurent Miclo, Annie Dary-Mourot

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Zeeshan Hafeez, Céline Cakir-Kiefer, Xavier Lecomte, Laurent Miclo, Annie Dary-Mourot. The X-prolyl dipeptidyl-peptidase PepX of Streptococcus thermophilus initially described as intracellular is also responsible for peptidase extracellular activity. Journal of Dairy Science, 2019, 102 (1), pp.113-123. 10.3168/jds.2018-14823. hal-02111674

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# The X-prolyl dipeptidyl-peptidase PepX of *Streptococcus thermophilus* initially described as intracellular is also responsible for peptidase extracellular activity

Zeeshan Hafeez,<fnref n="1" /> Céline Cakir-Kiefer,<fnref n="2" /> Xavier Lecomte,<fnref n="1" /> Laurent Miclo,<fnref n="1" />* and Annie Dary-Mourot<fnref n="1" />*†

## ABSTRACT

This study addresses the hypothesis that the extra-cellular cell-associated X-prolyl dipeptidyl-peptidase activity initially described in *Streptococcus thermophilus* could be attributable to the intracellular X-prolyl dipeptidyl-peptidase PepX. For this purpose, a PepX-negative mutant of *S. thermophilus* LMD-9 was constructed by interrupting the *pepX* gene and named LMD-9-Δ*pepX*. When cultivated, the *S. thermophilus* LMD-9 wild type strain grew more rapidly than its Δ*pepX* mutant counterpart. Thus, the growth rate of the LMD-9-Δ*pepX* strain was reduced by a factor of 1.5 and 1.6 in milk and LM17 medium (M17 medium supplemented with 2% lactose), respectively. The negative effect of the PepX inactivation on the hydrolysis of β-casomorphin-7 was also observed. Indeed, when incubated with this peptide, the LMD-9-Δ*pepX* mutant cells were unable to hydrolyze it, whereas this peptide was completely degraded by the *S. thermophilus* LMD-9 wild type cells. This hydrolysis was not due to leakage of intracellular PepX, as no peptide hydrolysis was highlighted in cell-free filtrate of wild type strain. Therefore, based on these results, it can be presumed that though lacking an export signal, the intracellular PepX might have accessed the β-casomorphin-7 externally, perhaps via its galactose-binding domain-like fold, this domain being known to help enzymes bind to several proteins and substrates. Therefore, the identification of novel distinctive features of the proteolytic system of *S. thermophilus* will further enhance its credibility as a starter in milk fermentation.

**Key words:** X-prolyl dipeptidyl peptidase, *Streptococcus thermophilus*, β-casomorphin-7

## INTRODUCTION

In developed countries, demand for healthy foods has escalated due to increased consumer perception about the diet-health relationship (Mollet and Rowland, 2002). Among these healthy foods, fermented dairy products remain of interest because they can be consumed daily without influencing the balance of diet. Because of the numerous auxotrophies of lactic acid bacteria (LAB), proteolysis of milk proteins is an essential phenomenon of milk fermentation that determines their growth in milk (Savijoki et al., 2006). Control of caseinolysis in dairy products is not only an economical challenge but also a health challenge. Besides the growth of LAB, casein breakdown generates peptides, which either contribute to the physicochemical properties or enhance the health benefits of the finished dairy products. In the latter case, they are designated as bioactive peptides, which are known to be potentially able to modulate different body functions. A convenient and natural way to give desired health attributes to dairy products is the generation of bioactive peptides via casein hydrolysis, and one of the major actors in caseinolysis is the proteolytic system of LAB (Hafeez et al., 2014).

The LAB *Streptococcus thermophilus* is the second most important species of industrial dairy starter after *Lactococcus lactis* and is widely used in the manufacture of various dairy products such as yogurt and hard cooked cheeses (Hols et al., 2005), mainly for its fast acidification (Dandoy et al., 2011) and organoleptic properties. Moreover, some strains of *S. thermophilus* have the capacity to generate a variety of bioactive peptides from milk caseins owing to its proteolytic system (Miclo et al., 2012; Chang et al., 2014).

The proteolytic system of *S. thermophilus* is generally organized in 4 main parts. The first and principal actor of this system is the proteinase PrtS, which is usually anchored to the cell wall and belongs to the subtilisin-like serine protease family (Fernandez-Espla et al., 2000). However, it must be underlined that PrtS

<footnote n="1">¹Université de Lorraine, Composés Alimentaires: Biofonctionnalités et Risques Neurotoxiques (CALBINOTOX), F-54000, Nancy, France</footnote>

<footnote n="2">²Université de Lorraine, INRA, Unité de Recherche Animal et Produits Animaux (URAFPA), F-54000, Nancy, France</footnote>

<footnote>Received March 26, 2018.</footnote>

<footnote>Accepted September 12, 2018.</footnote>

<footnote>*Co-senior authors.</footnote>

<footnote>†Corresponding author: annie.dary@univ-lorraine.fr</footnote>
is neither always present nor associated with the cell wall of *S. thermophilus* strains; for example, strain CNRZ1066 lacks the *prtS* gene, whereas *PrtS* is wall anchored in strain LMD-9 or can also exist as a free form in strain 4F44 (Delorme et al., 2010; Chang et al., 2012). After their liberation from caseins by *PrtS*, peptides can be internalized by oligo- and dipeptide transport system (Garault et al., 2002; Jameh et al., 2016) or further hydrolyzed by extracellular peptidases (Hafeez et al., 2013, 2015). Once inside the cell, the peptides are exposed for further breakdown to a pool of intracellular exo- and endopeptidases that make the final constituent of this system (Savijoki et al., 2006).

About 15 different intracellular peptidase activities have been described in several *S. thermophilus* strains (Rul and Monnet, 1997). Only some of these enzymes [aminopeptidase PepN (Rul et al., 1994), cysteine aminopeptidase PepC (Chapot-Chartier et al., 1994), acidic residue-specific aminopeptidase PepA (Rul et al., 1995), metallo-aminopeptidase PepS (Fernandez-Espla and Rul, 1999), dipeptidase (Rabier and Desmazeaud, 1973), endopeptidase PepO (Chavagnat et al., 2000), and Xaa-Pro dipeptidyl-peptidase PepX (Tsakalidou et al., 1998)] have been characterized biochemically or genetically after either purification or gene cloning. PepX (EC 3.4.14.11) is an enzyme that catalyzes the removal of N-terminal dipeptides (Xaa-Pro) by hydrolyzing the peptide bond implying the carboxylic function of a proline residue and belongs to the MEROPS peptidase family S15 (http://www.ebi.ac.uk/interpro/entry/IPR008252).

In a previous work we reported the presence of extracellular cell-associated aminopeptidase, carboxypeptidase, peptidyl-dipeptidase, and X-prolyl dipeptidyl-peptidase activities in *S. thermophilus* LMD-9 and CNRZ1066 (Hafeez et al., 2013, 2015). Such activities did not result from activity of the known extracellular protease *PrtS* and *HrtA*, as inactivation in the strain LMD-9 of each of them or of both did not preclude the extracellular peptidase activities observed (Hafeez et al., 2013, 2015). Unfortunately, genes encoding these extracellular cell-associated peptidase activities have not yet been identified among the numerous genome sequences of *S. thermophilus* strains that are now available, especially those of strains LMD-9 and CNRZ 1066. In addition, genome sequence analysis of these strains failed to detect any other X-prolyl dipeptidyl-peptidase gene except PepX. Despite the fact that PepX does not possess any sorting signal according to SignalP or PredTAT prediction tools (Bagos et al., 2010; Petersen et al., 2011), we tested the possibility that this enzyme, initially described as an intracellular peptidase, was

also responsible for the extracellular cell-associated X-prolyl dipeptidyl-peptidase activity we detected.

The strategy was to delete the *pepX* open reading frame in the *S. thermophilus* LMD-9 genome and replace it with the spectinomycin adenyl transferase gene, which provides resistance to the spectinomycin antibiotic. Growth of *S. thermophilus* LMD-9 and LMD-9-ΔpepX strains was characterized in 10% reconstituted skim milk and LM17 medium. Furthermore, the proteolysis experiments using the proline-rich peptide β-casomorphin-7, derived from bovine β-casein A1 protein variant (⁶⁰YPFPGPI⁶⁶; Brantl et al., 1979), were performed in the presence of intact cells of both strains in a nongrowth medium.

## MATERIALS AND METHODS

### Peptide Synthesis and Bacterial Growth Conditions

The peptide β-casomorphin-7 used in this study was synthesized by Genosphere Biotechnologies (Paris, France). The *S. thermophilus* LMD-9 strain ATCC BAA-491, whose genome sequence is known (Makarova et al., 2006), came from the American Type Culture Collection (Manassas, VA). The *S. thermophilus* LMD-9 and *S. thermophilus* LMD-9-ΔpepX (this study) strains were conserved at -80°C in 10% sterile reconstituted skim milk. Both strains were cultivated at 42°C in either 10% reconstituted skim milk or LM17 (M17 medium supplemented with 2% lactose; Terzaghi and Sandine, 1975). *Escherichia coli* DH5-α was preserved at -80°C in Luria-Bertani broth containing 11.4% glycerol and cultured aerobically at 37°C in Luria-Bertani broth (Sambrook and Russell, 2001). When needed, spectinomycin was added to the culture medium at a concentration of 300 µg/mL to select the LMD-9-ΔpepX mutant strain.

### DNA Extraction and PCR Amplifications

For DNA extraction, *E. coli* DH5-α and the 2 *S. thermophilus* strains used in this work were grown in Luria-Bertani broth and LM17 medium, respectively, as described by Hafeez et al. (2013). Plasmid pSET4s (Takamatsu et al., 2001) possessing the spectinomycin adenyltransferase gene *specR* was purified from *E. coli* DH5-α using a GeneJET Plasmid Miniprep Kit (Fermantas, Villebon-sur-Yvette, France) according to supplier recommendations, and genomic DNA of *S. thermophilus* was extracted according to Fischer et al. (1997). Plasmid pSET4s and genomic DNA of *S. thermophilus* LMD-9 were used as template DNA for amplification
of the *specR* gene and of upper (Up) and down (Dn)-flanked regions of the *pepX* gene, respectively (Figure 1). All PCR were conducted in a Mastercycler® pro thermal cycler (Eppendorf, Hamburg, Germany). Primers (Table 1) were designed using Primer3Plus software (Untergasser et al., 2007) and were purchased from Eurogentec (Serain, Belgium). To create overlap regions between flanked regions of the *pepX* and *specR* genes, a short nucleotide sequence homologous to the Up- and Dn-flanked regions of PepX was added to each of the primers used to amplify the specR fragment (Figure 1). The PCR amplifications and agarose gel electrophoresis were performed as described previously (Hafeez et al., 2013). Overlapping PCR reactions were performed as previously described (Lecomte et al., 2014). The PCR-amplified fragments were purified using a high pure PCR product purification kit (Roche Applied Science, Meylan, France), and sequence analyses of both DNA strands of PCR fragments were conducted by Beckman Coulter Genomics (Essex, UK).

## S. thermophilus LMD-9-ΔpepX Mutant Construction and Transformation

To construct the *S. thermophilus* LMD-9-ΔpepX mutant strain, the *pepX* gene [locus tag: STER_1633/STER_RS08015; National Center for Biotechnology Information (Bethesda, MD) reference sequence: NC_008532.1] of *S. thermophilus* LMD-9 was replaced by the *specR* gene using overlap extension PCR (Figure 1). First, an 862-bp upstream sequence (starting just before the start codon and comprising the promoter and ribosome binding site of the *pepX* gene) and an 825-bp downstream sequence (including 71 nucleotides before the stop codon and terminator of the *pepX* gene) were amplified from *S. thermophilus* LMD-9 genomic DNA using UpPepX_F/UpPepX_R and PepXDn_F/PepXDn_R primer couples, respectively (Table 1). The *specR* gene corresponding to a 1,200-bp sequence was amplified from pSET4s plasmid DNA using the UpPepX-Spec_F/Spec-PepXDn_R primer pair (Figure

<figure><img src="image_3.png" /></figure>

**Figure 1.** Schematic representation of different steps of overlap extension PCR to replace the *pepX* gene by the *specR* gene and resultant mutant locus. (a) Amplification of the PepX upper flanked region (UpPepX), resistance to antibiotic spectinomycin (specR), and PepX down flanked region (PepXDn) fragments using appropriate primer pairs. (b) The overlap extension PCR using the outermost primer pair and the 3 amplicons. (c) The resulting chromosomal locus carrying the *specR* gene obtained after transformation with the final overlap extension PCR amplicon. Dotted lines indicate the homologous/complementary regions. P, RBS, stop, and T correspond to promoter, ribosome binding site, stop codon, and transcription terminator of *pepX* gene, respectively.
1a). The amplicon specR contains extremities homologous to the UpPepX and PepXDn amplicons (Figure 1b). Then, the 3 amplicons (UpPepX, specR, and PepXDn) were mixed in equimolar concentrations and attached together using external primers UpPepX_F and PepXDn_R (Figure 1b). Finally, linear DNA fragment obtained by overlap extension PCR was introduced in *S. thermophilus* LMD-9 competent cells by natural transformation according to Hafeez et al. (2013). The validity of construction was checked by sequencing the different PCR fragments obtained using specific primers (Table 1).

## Growth of *S. thermophilus* LMD-9 and *S. thermophilus* LMD-9-ΔpepX

Growth of *S. thermophilus* LMD-9 wild type and ΔpepX mutant strains was monitored in 10% reconstituted skim milk and LM17 broth. Before growth in each medium, strains were inoculated at 1% in 10% reconstituted skim milk and grown overnight at 42°C to provide preculture. For studies in milk medium, optical density (OD) of the preculture, diluted 10× with EDTA solution 0.2%, pH 12 (a clarifying agent), was measured at 480 nm as previously described (Garault et al., 2000; Galia et al., 2016). Then, the preculture was diluted in 40 mL of 10% reconstituted skim milk to obtain an OD$_{480}$ nm of 0.01, and aliquots of 1.3 mL were incubated at 42°C. Afterward, OD$_{480}$ nm was measured after the same dilution using EDTA every 30 min until

the bacterial culture reached the stationary growth phase. Measurements were made in duplicate. In the case of the ΔpepX mutant strain, each medium and clarifying solution was supplemented with spectinomycin at 300 µg/mL.

To monitor growth in LM17 broth, preculture of each strain was diluted to an OD$_{600}$ nm of 0.01 in LM17 broth and cultured as in reconstituted skim milk. Then, the OD$_{600}$ nm was measured after 1 h and then every 30 min until the bacterial culture reached the stationary growth phase. Growth rates of both strains were calculated from the maximum slope of a logarithmic representation of growth curves assessed by OD measurements at 480 and 600 nm of bacterial cultures grown in reconstituted skim milk and LM17, respectively.

## Hydrolysis of Peptide β-Casomorphin-7

The ability of *S. thermophilus* LMD-9 and LMD-9-ΔpepX mutant strains to hydrolyze β-casomorphin-7 was evaluated in phosphate-acetate buffer (PAB; 12.5 mM, pH 6.5). For this purpose, both strains were grown in LM17 medium up to exponential phase (OD$_{600}$ nm of 1.00 ± 0.05), and cells were harvested by centrifugation at 3,100 × g for 10 min at 20°C. After twice washing with PAB, the cell pellet was resuspended in the same buffer to obtain an initial OD$_{600}$ nm of 1.00 ± 0.05. Then, the β-casomorphin-7 was added to the cell suspensions to a final concentration of 20 nmol/mL and incubated at 42°C under shaking at 200 rpm. In

<table><caption>**Table 1.** Primers used for the construction of the *Streptococcus thermophilus* LMD-9-ΔpepX mutant and the sequence verification of the construction obtained</caption><thead><tr><th>Primer</th><th>Sequence 5'-3'</th><th>Annealing temperature (°C)</th><th>Purpose</th></tr></thead><tbody><tr><td>UpPepX_F<sup>1</sup></td><td>GCAAGAGATGGTGAAGAAGAC</td><td rowspan="2">54.0</td><td rowspan="2">Amplify upper-flanked region of pepX gene</td></tr><tr><td>UpPepX_R</td><td>ATTCAAAAACCTCAAAAATAATACG</td></tr><tr><td>UpPepX-Spec_F</td><td>CGTATTATTTTTGAGGTTTTTGAATTGACTCCCGTCGTGTAGATAACTAG</td><td rowspan="2">58.0</td><td rowspan="2">Amplify specR gene</td></tr><tr><td>Spec-PepXDn_R</td><td>GCTCAAAGTCTGTGGTATAGAGAACCGCTACGATAACGCCTGTTT</td></tr><tr><td>PepXDn_F</td><td>GTTCTCTATACCACAGACTTTGAGC</td><td rowspan="2">54.0</td><td rowspan="2">Amplify down-flanked region of pepX gene</td></tr><tr><td>PepXDn_R<sup>1</sup></td><td>AACTGGTGTTTATCGTTTGC</td></tr><tr><td>PepXSpec1_F</td><td>AAAGGTAGAGAGCTTTAAACAAG</td><td rowspan="2">54.1</td><td rowspan="2">Sequencing</td></tr><tr><td>PepXSpec1_R</td><td>GGATACCTTATTGCACAACTTC</td></tr><tr><td>PepXSpec2_F</td><td>GCACGATTACGATTTTCTGG</td><td rowspan="2">57.9</td><td rowspan="2">Sequencing</td></tr><tr><td>PepXSpec2_R</td><td>AAATATCTCTTGCCAGTCACG</td></tr><tr><td>PepXSpec3_F</td><td>CTCCCCGTCGTGTAGATAAC</td><td rowspan="2">56.7</td><td rowspan="2">Sequencing</td></tr><tr><td>PepXSpec3_R</td><td>TTCCTTCTGAGGAATGTATCC</td></tr><tr><td>PepXSpec4_F</td><td>TGGTACCGTGGAATCATCCT</td><td rowspan="2">57.9</td><td rowspan="2">Sequencing</td></tr><tr><td>PepXSpec4_R</td><td>AGTGTGCTCAAAGTCTGTGG</td></tr><tr><td>PepXSpec5_F</td><td>TTTGGGAAATATTCATTCTAATTG</td><td rowspan="2">55.4</td><td rowspan="2">Sequencing</td></tr><tr><td>PepXSpec5_R</td><td>TGGTAACTTTGTAGGCGTTG</td></tr><tr><td>PepXSpec6_F</td><td>GACAAAACAATGGCCGTAAG</td><td rowspan="2">58.8</td><td rowspan="2">Sequencing</td></tr><tr><td>PepXSpec6_R</td><td>TTATGACAAGCGGACAAGTG</td></tr></tbody></table>

<footnote n="1">¹Primers were used for overlap extension PCR with annealing temperature of 50°C. UpPepX-Spec_F and Spec-PepXDn_R italic sequences are homologous to the reverse complement of UpPepX_R and PepXDn_F, respectively. F = forward; R = reverse.</footnote>
parallel, β-casomorphin-7 was incubated alone in PAB under the same conditions as a control of the peptide stability. Each sample (500 µL) aliquoted at 0, 1, 2, 4, 6, and 22 h of incubation was mixed with 15 µL of trifluoroacetic acid to stop enzyme activity, the cells were removed by filtration through a 0.45-µm filter (Phenomenex, Le Pecq, France), and the hydrolysis products were stored at -20°C until reversed-phase HPLC (RP-HPLC) analysis.

Stability of Extracellular Cell-Associated PepX Activity and Detection of Bacterial Cell Lysis

The stability of extracellular cell-associated X-prolyl dipeptidyl-peptidase activity in PAB (12.5 mM, pH 6.5) was explored as well as hydrolysis of β-casomorphin-7 by intracellular peptidases, particularly PepX, that may be released in the incubation medium as a result of spontaneous cell lysis. For this, S. thermophilus LMD-9 wild type and LMD-9-ΔpepX mutant strains were grown in LM17 medium to an OD<sub>600 nm</sub> of 1.00 ± 0.05 (see “Growth of S. thermophilus LMD-9 and S. thermophilus LMD-9-ΔpepX”). The cells recovered by centrifugation were washed twice with PAB and resuspended in it to obtain the initial OD. Both strains were then incubated at 42°C under shaking at 200 rpm. After 22 h of incubation, the cell pellet and supernatant were separated by centrifugation at 3,100 × g for 10 min at 20°C. The supernatant was filtered through a 0.22-µm filter (Merck Millipore Corp., Billerica, MA) and named cell-free filtrate (CFF). The cell pellet and CFF were incubated with β-casomorphin-7 (20 nmol/ mL), and samples drawn at 0 and 6 h of incubation were treated and analyzed as described above.

RP-HPLC Analysis of Hydrolysis Products

The RP-HPLC analysis of hydrolysis products from β-casomorphin-7 was conducted as described previously by Hafeez et al. (2013) with some modifications. Aliquots of hydrolysis products (70 µL) recovered at different time intervals were injected into a LiChrospher 100 RP-18 column (150 × 2 mm, 5-µm particle size, 10-nm porosity; Merck Millipore Corp.) connected to an Alliance 2690 HPLC unit (Waters, Milford, MA). Samples were eluted during 60 min by applying a gradient of 0.5% min⁻¹ acetonitrile containing 0.1% trifluoroacetic acid in water at a flow rate of 0.25 mL/min. The column temperature was maintained at 30°C. Detection of eluted samples was monitored using a photodiode array detector 996 (Waters) between 200 and 300 nm. The percentage of residual peptide quantity was calculated by comparing the peak area at 215 nm obtained at the

chosen time of hydrolysis with that of the initial area of the peptide at 0 min of hydrolysis.

RESULTS

Characterization of S. thermophilus LMD-9 and LMD-9-ΔpepX Mutant Growth in Milk and LM17

The growth behavior of S. thermophilus LMD-9 wild type and S. thermophilus LMD-9-ΔpepX strains was evaluated in reconstituted skim milk (medium poor in free AA and peptides but rich in caseins) and LM17 (medium rich in AA and peptides). After overnight preculture in 10% reconstituted skim milk, both strains were inoculated in each medium and OD was measured either at 480 nm for reconstituted milk or at 600 nm for LM17 broth.

The S. thermophilus LMD-9 wild type strain grew faster in milk than the ΔpepX mutant and reached the exponential growth phase after 3 h of incubation, whereas the lag phase was prolonged for the ΔpepX mutant strain, and it took almost 4 h to attain exponential growth phase. After 6 h of incubation, the wild type strain was already in the stationary phase, whereas the ΔpepX mutant strain was still in the exponential growth phase. The difference in growth rates (determined from the maximum slope of a logarithmic representation of growth curves assessed by OD<sub>480nm</sub> measurements) of both strains was obvious in the exponential growth phase (Figure 2). During this phase, growth rate of the wild type strain was higher by a factor of 1.5 compared with the ΔpepX mutant counterpart. Growth rate of the ΔpepX mutant strain was not influenced by the presence or absence of spectinomycin in the growth medium (Figure 2; Table 2).

After 4 h of incubation in LM17 medium, both S. thermophilus LMD-9 and LMD-9-ΔpepX mutant strains were in their exponential growth phase (Figure 2) as determined by the OD measurements at 600 nm. However, a difference in growth rate was also observed in the exponential growth phase: the growth rate of the wild type strain was 0.95, which corresponded to about 1.6-fold higher growth rate compared with the ΔpepX mutant strain (Table 2). As before, the presence of spectinomycin had no significant effect on the mutant growth because the mutant showed more or less similar growth pattern in the absence of this antibiotic. Thus, the results obtained for both media suggest an important role of the peptidase PepX during growth because the growth rate of the wild type strain in the 2 media—milk (a medium poor in AA and peptides) and LM17 (a medium rich in AA and peptides) is higher than that of the mutant.
<table><caption>**Table 2.** Growth rates (GR; h⁻¹) and generation times (GT; h) of *Streptococcus thermophilus* LMD-9 wild type and LMD-9-ΔpepX mutant strains during their growth in milk and LM17 media</caption><thead><tr><th rowspan="2">Strain</th><th colspan="2">Milk</th><th colspan="2">LM17</th></tr><tr><th>GR</th><th>GT</th><th>GR</th><th>GT</th></tr></thead><tbody><tr><td>LMD-9</td><td>0.42</td><td>0.71</td><td>0.95</td><td>0.33</td></tr><tr><td>LMD-9-ΔpepX + spectinomycin</td><td>0.27</td><td>1.1</td><td>0.56</td><td>0.51</td></tr><tr><td>LMD-9-ΔpepX</td><td>0.27</td><td>1.1</td><td>0.58</td><td>0.54</td></tr></tbody></table>

Hydrolysis of β-Casomorhin-7 by PepX

The *S. thermophilus* LMD-9 wild type strain has the capacity to hydrolyze proline residue-rich bioactive peptide β-casomorphin-7 through its cell-associated X-prolyl dipeptidyl-peptidase activity. Indeed, we previously demonstrated by liquid chromatography/ electrospray ionization MS that when incubated with cells of *S. thermophilus* LMD-9, β-casomorphin-7 was hydrolyzed into the shorter peptides FPGA, GPI, FP, and YP (Hafeez et al., 2013). Therefore, to investigate whether the deficiency of intracellular PepX has an influence on this type of activity, the β-casomorphin-7 was incubated at 42°C in PAB (25 mM, pH 6.5) for 22 h with the nonproliferating cells of *S. thermophilus* LMD9 wild type and LMD-9-ΔpepX mutant strain, which were beforehand grown to the exponential growth phase in LM17 medium. The breakdown pattern of the

peptide was then compared between the 2 strains. The RP-HPLC chromatograms of β-casomorphin-7 showed that no hydrolysis of the peptide occurred after 4 h of contact with cells of the *S. thermophilus* LMD-9-ΔpepX mutant strain (Figure 3b), and the peptide degradation was almost insignificant even after 22 h of incubation as determined by calculating the residual peptide quantity (Table 3). On the other hand, a significant portion of the peptide was already hydrolyzed (84% loss of the initial peptide quantity) after 4 h of exposure to the cells of *S. thermophilus* LMD-9 wild type strain (Figure 3c). Moreover, the residual peptide quantity calculation exposed that the peptide was fully degraded by the wild type strain after 22 h of contact because no peptide traces were observed at the end of the incubation period (Table 3). Total hydrolysis of β-casomorphin-7 by *S. thermophilus* LMD-9 was an expected result, as the wild type strain was shown to possess an extracellular X-prolyl dipeptidyl-peptidase activity. Moreover, this hydrolysis cannot be correlated with the instability of the peptide because it remained intact during the 22-h incubation period under the same conditions without bacterial cells (data not shown). Conversely, the inactivation of the *pepX* gene led to an absence of hydrolysis of β-casomorphin-7 even after 22 h of incubation. This shows that the peptidase PepX, initially described as intracellular, was responsible for the extracellular X-prolyl dipeptidyl-peptidase activity previously described in the wild type LMD-9 strain.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Growth curves of *Streptococcus thermophilus* LMD-9 wild type (filled circles) and *S. thermophilus* LMD-9-ΔpepX in the presence (filled triangles) and absence (empty squares) of spectinomycin in (a) milk and (b) LM17 medium. Values correspond to the mean of 2 assays.</figcaption></figure>
<table><caption>**Table 3.** Quantity (% ± SEM) of intact β-casomorphin-7 recovered at different time intervals after incubation with nonproliferating cells of *Streptococcus thermophilus* LMD-9 wild type and LMD-9-ΔpepX mutant</caption><thead><tr><th rowspan="2">Incubation time (h)</th><th colspan="2">β-Casomorphin-7 remaining</th></tr><tr><th><i>S. thermophilus</i> LMD-9</th><th><i>S. thermophilus</i> LMD-9-ΔpepX</th></tr></thead><tbody><tr><td>1</td><td>83 ± 5</td><td>100</td></tr><tr><td>2</td><td>50 ± 2</td><td>100</td></tr><tr><td>4</td><td>16 ± 1</td><td>100</td></tr><tr><td>6</td><td>3.0 ± 0.1</td><td>97 ± 2</td></tr><tr><td>22</td><td>0</td><td>90.5 ± 2</td></tr></tbody></table>

## Stability of Cell-Associated PepX Activity and Control of Integrity of Bacterial Cells

The *S. thermophilus* strains possess a variety of intra-cellular peptidases that could be released in incubation medium as a result of spontaneous cell lysis. To check the integrity of bacterial cells as well as the stability of cell-associated PepX activity, *S. thermophilus* LMD-9 wild type and LMD-9-ΔpepX mutant strain were grown in LM17 medium up to an OD<sub>600 nm</sub> of 1.00 ± 0.05 and then incubated for 22 h in PAB. The potential hydrolysis of β-casomorphin-7 was investigated in the obtained cell pellet and CFF. No significant peptide hydrolysis in CFF of both strains was observed even after 6 h of incubation, whereas the peptide was significantly hydrolyzed by the cell pellet of the wild type strain, contrary to the mutant strain (Figure 4). This thus confirmed an undetectable cell lysis and release of intracellular peptidases in the incubation medium. Similarly, hydrolysis of the peptide by the cell pellet of the wild type strain confirmed the stability of the cell-associated PepX activity.

## DISCUSSION

Besides the cell wall protease PrtS and peptide transport system, the proteolytic system of *S. thermophilus* comprises a variety of intracellular and extracellular cell wall-associated peptidases (Savijoki et al., 2006; Hafeez et al., 2014). Peptidases play an essential role in *S. thermophilus* growth in milk because they release AA from casein-derived peptides, previously generated by the hydrolysis of caseins by PrtS. The Xaa-Pro dipeptidyl-peptidase PepX of *S. thermophilus* is postulated to be important for the hydrolysis of proline-rich peptides because bovine milk caseins are a group of proline-rich proteins, particularly β-casein and κ-casein containing 16.7 and 11.8% proline residues, respectively (Casey and Meyer, 1985; Smid et al., 1991; El-Agamy, 2017). Four proline-specific peptidases were reported in *S. thermophilus*: PepX, aminopeptidase P, a prolinase, and a prolidase. PepX displayed the highest activity of all the aminopeptidases found in this bacterium species

(Rul and Monnet, 1997). The hydrolysis of different substrates and peptides containing proline residue(s) by the intracellular peptidase PepX has been widely described in different *S. thermophilus* strains (Meyer

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Reversed-phase HPLC chromatograms of β-casomorphin-7 (a, control) after 4 h of incubation with cells of (b) *Streptococcus thermophilus* LMD-9-ΔpepX mutant and (c) *S. thermophilus* LMD-9 wild type strain in phosphate-acetate buffer (12.5 mM, pH 6.5). Peak corresponding to β-casomorphin-7 is represented by filled circle.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Comparison of reversed-phase HPLC chromatograms obtained after 6 h of hydrolysis of the β-casomorphin-7 by cell pellet (solid line) and cell-free filtrate (CFF; dashed line) of (a) *Streptococcus thermophilus* LMD-9 wild type and (b) *S. thermophilus* LMD-9-Δ*pepX* mutant strain. For clarity, the superimposed chromatogram of β-casomorphin-7 obtained from CFF of LMD-9-Δ*pepX* mutant strain was shifted to the right. After 22 h of incubation of both strains in phosphate-acetate buffer (12.5 mM, pH 6.5), cell suspensions were centrifuged to obtain the cell pellet and the CFF. Both the cell pellet and the CFF were further incubated with the β-casomorphin-7 for a period of 6 h.</figcaption></figure>

and Jordi, 1987; Tsakalidou et al., 1998), but to our knowledge the implication of intracellular PepX in extracellular cell-associated X-prolyl dipeptidyl-peptidase activity has never been reported.

In this work, we showed that the inactivation of the *pepX* gene had a negative effect on the growth of the *S. thermophilus* LMD-9-Δ*pepX* mutant in the 2 media tested (i.e., milk and LM17). The overall growth of the Δ*pepX* mutant appeared to be more markedly decreased in milk than in the LM17 medium (Figure 2). Nevertheless, growth rates of the Δ*pepX* mutant strain were decreased almost equally in both media. This decrease cannot be attributed to the presence of the *specR* gene used in the construction of the mutant (Figure 1) because the presence or absence of spec tinomycin had not influenced the growth of the Δ*pepX* mutant regardless of the culture medium. As reported in *Lactobacillus helveticus* (Vesanto et al., 1995), the transcription of the *pepX* gene is a typical feature of the exponential phase. Therefore, if it is true, it is not surprising that the inactivation of PepX leads to a decreased growth of the corresponding mutant because during the exponential phase protein synthesis is active, and so the bacterium needs AA. However, several conflicting results about the implication of PepX or other peptidases in the growth of LAB in milk and other complex growth media have to be cited here. Some of them support our findings, whereas others op-

pose them. It has been stated that the PepX-negative mutant of *Lactobacillus helveticus* CNRZ32 grew in milk at a decreased growth rate but not in de Man, Rogosa and Sharpe medium (Yüksel and Steele, 1996). Likewise, Mierau et al. (1996) reported a decrease in growth rate for a single or multiple peptidase-deficient mutants of *L. lactis* compared with the wild type strain when grown in milk, but no difference in growth rates of wild type and mutants was observed in GM17. In another study conducted on the PepX-deficient mutant of *L. lactis*, the authors on one hand observed that PepX is not essential for growth in milk because the PepX mutant grows normally in milk but on the other hand proposed that alteration in expression of the *pepX* gene may lead to dramatic changes in the peptide composition of finished fermented milk products (Mayo et al., 1993). Cow milk is a medium poor in free AA and peptides and rich in caseins, which are proteins with a high content of proline residues (about 17% for β-casein; 1 of the 2 major caseins of bovine milk according to UniProtKB P02666). As it was shown that PepX displayed the highest activity of all the aminopeptidases of *S. thermophilus* (Rul and Monnet, 1997), its inactivation was expected to affect the growth of the Δ*pepX* mutant in milk. Conversely, LM17 is a medium rich in AA and peptides that are provided by meat, soy, yeast extracts, and casein hydrolysates. The effect of inactivation of PepX on the growth of the mutant that we observed
in this medium could result from the incapacity of the
ΔpepX mutant to hydrolyze peptides rich in proline
residues brought by casein hydrolysates.

In this work, we brought genetic proof of the im-
plication of PepX in the extracellular degradation of
β-casomorphin-7 that did not result from cellular lysis.
Indeed, we showed that the *S. thermophilus* LMD-9-
ΔpepX mutant strain was unable to hydrolyze the
β-casomorphin-7 in a nonproliferating growth medium.
In contrast, β-casomorphin-7 was fully hydrolyzed in
the same conditions by the cells of the wild type strain,
and this observation is consistent with the previous
studies in which hydrolysis of proline-rich peptides by
the extracellular cell-associated X-prolyl dipeptidyl-
peptidase of *S. thermophilus* strains has been estab-
lished (Hafeez et al., 2013, 2015). The spontaneous lysis
of cells and eventual release of intracellular peptidases
in the incubation medium leading to degradation of the
peptide can be ruled out because no peptide hydrolysis
was observed after 6 h of incubation in CFF of the wild
type strain. Kiefer-Partsch et al. (1989) also observed
hydrolysis of β-casomorphin-7 by the PepX of *L. lac*
*tis*. Further, in a recent work, extracellular peptidase
activity was postulated in *L. lactis*. Indeed, analysis of
the extracellular peptidome of this bacterium showed
peptides mainly originating from surface proteins and
having been generated by PrtP, HtrA, or an unknown
extracellular peptidase (Guillot et al., 2016).

How the intracellular PepX reaches the cell mem-
brane or the wall to become extracellular remains un-
known, especially as shaving experiments conducted on
the strain LMD-9 did not allow its detection (Lecomte
et al., 2014). In bacteria, most of the exported proteins
display a Sec-signal peptide that target them to the
Sec secretion pathway. Other Sec-independent secretion
pathways have been described, such as the twin-arginine
translocation secretion pathway that was distinguished
by the presence of a twin-arginine consensus motif in
the signal peptide (Berks, 1996; Berks et al., 2000).
Nevertheless, some bacterial proteins have been found
to be secreted without any apparent signal peptide due
to a nonclassical secretion (Bendtsen et al., 2005). Con-
cerning *S. thermophilus*, if the protease PrtS displays a
Sec-signal peptide, the sequence analysis of PepX did
not reveal any N-terminal signal sequence that could
account for translocation from the cytoplasm but high-
lighted the presence of a galactose-binding domain-like
fold (IPR008979). Several studies revealed that the
galactose-binding domain-like fold confers the ability
to proteins and enzymes to bind to specific ligands
such as cell-surface-attached carbohydrate substrates
(Gaskell et al., 1995; Boraston et al., 2002; Newstead et
al., 2005), phosphatidylserine-rich procoagulant mem-
branes (Macedo-Ribeiro et al., 1999), single-stranded

DNA (Marintchev et al., 1999), and several proteins
(Kitami et al., 2011). Further, it has been proposed
that the C-terminal moiety of PepX of *L. lactis* could
act as a tropic factor toward the cell membrane (Tan et
al., 1992; Rigolet et al., 2002). Finally, the secretion of
proteins by a nonclassical manner could be controlled
by the AA composition, the secondary structure, and
the presence of disordered regions (Bendtsen et al.,
2004, 2005). Concerning PepX, SecretomeP software
(http://www.cbs.dtu.dk/services/SecretomeP) gives a
probability of nonclassical secretion of 0.449, very close
to the threshold set at 0.5. Thus, this type of secretion
cannot be excluded in the case of PepX. Therefore, for
all these reasons, we propose that the intracellular PepX
might (1) be located in the plasmic membrane with its
catalytic domain orientated toward peptidoglycan or
(2) cross the cell membrane due to its galactose-binding
domain-like fold or by a nonclassical manner and then
is buried inside the peptidoglycan. In both hypotheses,
β-casomorphin-7 or small peptides would be hydrolyzed
after their penetration into the peptidoglycan.

To conclude, this study provides the first evidence of
the potential implication of intracellular PepX in the
hydrolysis of proline-rich peptides extracellularly and
that a single PepX might be responsible for the concur-
rent intra- and extracellular enzyme activity. Identifica-
tion of novel distinctive features of a proteolytic system
of *S. thermophilus* will further enhance its credibility as
a starter in milk fermentation.

ACKNOWLEDGMENTS

The authors thank Lucas Leguaye and David Bel-
haddad [Composés Alimentaires: Biofonctionnalités
et Risques Neurotoxiques (CALBINOTOX), Nancy,
France] for their technical help.

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