In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii.

Lactobacillus delbrueckii, the type species of the genus Lactobacillus, is widely recognized as the primary starter culture in the dairy industry due to its proteolytic activity, which enables it to growth in milk. In this study, a comprehensive genomic analysis of the proteolytic system was conducted on L. delbrueckii strains. The analysis included 27 genomes of L. delbrueckii, with a specific focus on the key enzyme involved in this system, the cell envelope-associated proteinase (CEP). The amino acid sequences, as well as the protein-structure prediction of the CEPs, were compared. Additionally, syntenic analysis of the genomic locus related to the CEPs revealed high conservation in L. delbrueckii subsp. bulgaricus strains, while L. delbrueckii subsp. lactis strains exhibited greater variability, including the presence of insertion sequences, deletions, and rearrangements. Finally, the CEP promoter region and putative regulatory elements responsible for controlling the expression of the proteolytic system in lactobacilli were investigated. Our genomic analysis and in silico characterization of the CEPs contribute to our understanding of proteolytic activity and the potential applications of these lactic acid bacteria in the dairy industry. Further research in this area will expand our knowledge and potential practical uses of these findings.

Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infect Human Intestinal Epithelial Cells and Induce Moderate Inflammation.

Klebsiella pneumoniae is an opportunistic pathogen that can produce moderate and severe infections in immunosuppressed hosts. In recent years, an increase in the isolation of hypermucoviscous carbapenem-resistant K. pneumoniae with sequence type 25 (ST25) in hospitals in Norwest Argentina was observed. This work aimed to study the virulence and inflammatory potential of two K. pneumoniae ST25 strains (LABACER01 and LABACER27) in the intestinal mucosa. The human intestinal Caco-2 cells were infected with the K. pneumoniae ST25 strains, and their adhesion and invasion rates and changes in the expression of tight junction and inflammatory factors genes were evaluated. ST25 strains were able to adhere and invade Caco-2 cells, reducing their viability. Furthermore, both strains reduced the expression of tight junction proteins (occludin, ZO-1, and claudin-5), altered permeability, and increased the expression of TGF-ß and TLL1 and the inflammatory factors (COX-2, iNOS, MCP-1, IL-6, IL-8, and TNF-α) in Caco-2 cells. The inflammatory response induced by LABACER01 and LABACER27 was significantly lower than the one produced by LPS or other intestinal pathogens, including K. pneumoniae NTUH-K2044. No differences in virulence and inflammatory potential were found between LABACER01 and LABACER27. In line with these findings, no major differences between the strains were found when the comparative genomic analysis of virulence factors associated with intestinal infection/colonization was performed. This work is the first to demonstrate that hypermucoviscous carbapenem-resistant K. pneumoniae ST25 infects human intestinal epithelial cells and induces moderate inflammation.

Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-ß, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

The Mucus Binding Factor Is Not Necessary for Lacticaseibacillus rhamnosus CRL1505 to Exert Its Immunomodulatory Activities in Local and Distal Mucosal Sites.

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-ß and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505.

The Mucus-Binding Factor Mediates Lacticaseibacillus rhamnosus CRL1505 Adhesion but Not Immunomodulation in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 possesses immunomodulatory activities in the gastrointestinal and respiratory tracts when administered orally. Its adhesion to the intestinal mucosa does not condition its beneficial effects. The intranasal administration of L. rhamnosus CRL1505 is more effective than the oral route at modulating immunity in the respiratory tract. Nonetheless, it has not yet been established whether the adherence of the CRL1505 strain to the respiratory mucosa is needed to provide the immune benefits to the host. In this study, we evaluated the role of adhesion to the respiratory mucosa of the mucus-binding factor (mbf) knock-out L. rhamnosus CRL1505 mutant (Δmbf CRL1505) in the context of a Toll-like receptor 3 (TLR3)-triggered innate immunity response. In vitro adhesion studies in porcine bronchial epitheliocytes (PBE cells) indicated that L. rhamnosus Δmbf CRL1505 adhered weakly compared to the wild-type strain. However, in vivo studies in mice demonstrated that the Δmbf CRL1505 also reduced lung damage and modulated cytokine production in the respiratory tract after the activation of TLR3 to a similar extent as the wild-type strain. In addition, the mutant and the wild-type strains modulated the production of cytokines and antiviral factors by alveolar macrophages in the same way. These results suggest that the Mbf protein is partially involved in the ability of L. rhamnosus CRL1505 to adhere to the respiratory epithelium, but the protein is not necessary for the CRL1505 strain to exert its immunomodulatory beneficial effects. These findings are a step forward in the understanding of molecular interactions that mediate the beneficial effects of nasally administered probiotics.

Bacterium-like Particles from Corynebacterium pseudodiphtheriticum as Mucosal Adjuvant for the Development of Pneumococcal Vaccines.

Previously, it was shown that intranasally (i.n.) administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or CP-derived bacterium-like particles (BLPs) improve the immunogenicity of the pneumococcal conjugate vaccine (PCV). This work aimed to deepen the characterization of the adjuvant properties of Cp and CP-derived BLPs for their use in the development of pneumococcal vaccines. The ability of Cp and CP-derived BLPs to improve both the humoral and cellular specific immune responses induced by i.n. administered polysaccharide-based commercial pneumococcal vaccine (Pneumovax 23®) and the chimeric recombinant PSPF (PsaA-Spr1875-PspA-FliC) protein was evaluated, as well as the protection against Streptococcus pneumoniae infection in infant mice. Additionally, whether the immunization protocols, including Cp and CP-derived BLPs, together with the pneumococcal vaccines can enhance the resistance to secondary pneumococcal pneumonia induced after inflammatory lung damage mediated by the activation of Toll-like receptor 3 (TLR3) was assessed. The results showed that both Cp and CP-derived BLPs increased the immunogenicity and protection induced by two pneumococcal vaccines administered through the nasal route. Of note, the nasal priming with the PSPF T-dependent antigen co-administered with Cp or CP-derived BLPs efficiently stimulated humoral and cellular immunity and increased the resistance to primary and secondary pneumococcal infections. The CP-derived BLPs presented a stronger effect than live bacteria. Given safety concerns associated with live bacterium administration, especially in high-risk populations, such as infants, the elderly, and immunocompromised patients, BLPs emerge as an attractive mucosal adjuvant to improve the host response to pneumococcal infections and to enhance the vaccines already in the market or in development.

Oral Administration of Lacticaseibacillus rhamnosus CRL1505 Modulates Lung Innate Immune Response against Klebsiella pneumoniae ST25.

Orally administered Lacticaseibacillus rhamnosus CRL1505 enhances respiratory immunity, providing protection against respiratory viruses and Streptococcus pneumoniae. However, the capacity of the CRL1505 strain to improve respiratory immunity against Gram-negative bacterial infections has not been evaluated before. The aim of this work was to evaluate whether the Lcb. rhamnosus CRL1505 was able to beneficially regulate the respiratory innate immune response and enhance the resistance to hypermucoviscous KPC-2-producing Klebsiella pneumoniae of the sequence type 25 (ST25). BALB/c mice were treated with the CRL1505 strain via the oral route and then nasally challenged with K. pneumoniae ST25 strains LABACER 01 or LABACER 27. Bacterial cell counts, lung injuries and the respiratory and systemic innate immune responses were evaluated after the bacterial infection. The results showed that K. pneumoniae ST25 strains increased the levels of TNF-α, IL-1ß, IL-6, IFN-γ, IL-17, KC and MPC-1 in the respiratory tract and blood, as well as the numbers of BAL neutrophils and macrophages. Mice treated with Lcb. rhamnosus CRL1505 had significantly lower K. pneumoniae counts in their lungs, as well as reduced levels of inflammatory cells, cytokines and chemokines in the respiratory tract and blood when compared to infected controls. Furthermore, higher levels of the regulatory cytokines IL-10 and IL-27 were found in the respiratory tract and blood of CRL1505-treated mice than controls. These results suggest that the ability of Lcb. rhamnosus CRL1505 to help with the control of detrimental inflammation in lungs during K. pneumoniae infection would be a key feature to improve the resistance to this pathogen. Although further mechanistic studies are necessary, Lcb. rhamnosus CRL1505 can be proposed as a candidate to improve patients' protection against hypermucoviscous KPC-2-producing strains belonging to the ST25, which is endemic in the hospitals of our region.

The Respiratory Commensal Bacterium Corynebacterium pseudodiphtheriticum as a Mucosal Adjuvant for Nasal Vaccines.

Previously, we demonstrated that nasally administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or its bacterium-like particles (BLPs) increase the resistance of mice against bacterial and viral respiratory pathogens by modulating the innate immunity. In this work, we evaluated the ability of Cp and BLPs to stimulate alveolar macrophages, and to enhance the humoral immune response induced by a commercial vaccine against Streptococcus pneumoniae. In the first set of experiments, Cp or the BLPs were incubated with primary cultures of murine alveolar macrophages and the phagocytic activity, and the production of cytokines was evaluated. The results revealed that Cp and BLPs were efficiently phagocyted by respiratory macrophages and that both treatments triggered the production of TNF-α, IFN-γ, IL-6, and IL-1ß. In the second set of experiments, 3-week-old Swiss mice were intranasally immunized at days 0, 14, and 28 with the pneumococcal vaccine Prevenar®13 (PCV), Cp + PCV, or BLPs + PCV. On day 33, samples of bronco-alveolar lavages (BAL) and serum were collected for the study of specific antibodies. In addition, immunized mice were challenged with S. pneumoniae serotypes 6B or 19F on day 33 and sacrificed on day 35 (day 2 post-infection) to evaluate the resistance to the infection. Both Cp + PCV and BLPs + PCV groups had higher specific serum IgG and BAL IgA antibodies than the PCV control mice. In addition, the mice that were immunized with Cp + PCV or BLPs + PCV had lower lung and blood pneumococcal cell counts as well as lower levels of BAL albumin and LDH, indicating a reduced lung damage compared to the control mice. Improved levels of anti-pneumococcal antibodies were also detected in the serum and BAL samples after the challenges with the pathogens. The results demonstrated that C. pseudodiphtheriticum 090104 and its bacterium-like particles are capable of stimulating the respiratory innate immune system serving as adjuvants to potentiate the adaptive humoral immune response. Our study is a step forward in the positioning of this respiratory commensal bacterium as a promising mucosal adjuvant for vaccine formulations aimed at combating respiratory infectious diseases.

Lactiplantibacillus plantarum Strains Modulate Intestinal Innate Immune Response and Increase Resistance to Enterotoxigenic Escherichia coli Infection.

Currently, probiotic bacteria with not transferable antibiotic resistance represent a sustainable strategy for the treatment and prevention of enterotoxigenic Escherichia coli (ETEC) in farm animals. Lactiplantibacillus plantarum is among the most versatile species used in the food industry, either as starter cultures or probiotics. In the present work, the immunobiotic potential of L. plantarum CRL681 and CRL1506 was studied to evaluate their capability to improve the resistance to ETEC infection. In vitro studies using porcine intestinal epithelial (PIE) cells and in vivo experiments in mice were undertaken. Expression analysis indicated that both strains were able to trigger IL-6 and IL-8 expression in PIE cells in steady-state conditions. Furthermore, mice orally treated with these strains had significantly improved levels of IFN-γ and TNF-α in the intestine as well as enhanced activity of peritoneal macrophages. The ability of CRL681 and CRL1506 to beneficially modulate intestinal immunity was further evidenced in ETEC-challenge experiments. In vitro, the CRL1506 and CRL681 strains modulated the expression of inflammatory cytokines (IL-6) and chemokines (IL-8, CCL2, CXCL5 and CXCL9) in ETEC-stimulated PIE cells. In vivo experiments demonstrated the ability of both strains to beneficially regulate the immune response against this pathogen. Moreover, the oral treatment of mice with lactic acid bacteria (LAB) strains significantly reduced ETEC counts in jejunum and ileum and prevented the spread of the pathogen to the spleen and liver. Additionally, LAB treated-mice had improved levels of intestinal IL-10 both at steady state and after the challenge with ETEC. The protective effect against ETEC infection was not observed for the non-immunomodulatory TL2677 strain. Furthermore, the study showed that L. plantarum CRL1506 was more efficient than the CRL681 strain to modulate mucosal immunity highlighting the strain specific character of this probiotic activity. Our results suggest that the improved intestinal epithelial defenses and innate immunity induced by L. plantarum CRL1506 and CRL681 would increase the clearance of ETEC and at the same time, protect the host against detrimental inflammation. These constitute valuable features for future probiotic products able to improve the resistance to ETEC infection.

Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal Epithelial Cells and Macrophages.

Lactobacillus delbrueckii subsp. lactis CRL 581 beneficially modulates the intestinal antiviral innate immune response triggered by the Toll-like receptor 3 (TLR3) agonist poly(I:C) in vivo. This study aimed to characterize further the immunomodulatory properties of the technologically relevant starter culture L. delbrueckii subsp. lactis CRL 581 by evaluating its interaction with intestinal epithelial cells and macrophages in the context of innate immune responses triggered by TLR3. Our results showed that the CRL 581 strain was able to adhere to porcine intestinal epithelial (PIE) cells and mucins. The CRL 581 strain also augmented the expression of antiviral factors (IFN-α, IFN-ß, Mx1, OAS1, and OAS2) and reduced inflammatory cytokines in PIE cells triggered by TLR3 stimulation. In addition, the influence of L. delbrueckii subsp. lactis CRL 581 on the response of murine RAW macrophages to the activation of TLR3 was evaluated. The CRL 581 strain was capable of enhancing the expression of IFN-α, IFN-ß, IFN-γ, Mx1, OAS1, TNF-α, and IL-1ß. Of note, the CRL 581 strain also augmented the expression of IL-10 in macrophages. The results of this study show that the high proteolytic strain L. delbrueckii spp. lactis CRL 581 was able to beneficially modulate the intestinal innate antiviral immune response by regulating the response of both epithelial cells and macrophages relative to TLR3 activation.

Characterization of Weissella viridescens UCO-SMC3 as a Potential Probiotic for the Skin: Its Beneficial Role in the Pathogenesis of Acne Vulgaris.

Previously, we isolated lactic acid bacteria from the slime of the garden snail Helix aspersa Müller and selected Weissella viridescens UCO-SMC3 because of its ability to inhibit in vitro the growth of the skin-associated pathogen Cutibacterium acnes. The present study aimed to characterize the antimicrobial and immunomodulatory properties of W. viridescens UCO-SMC3 and to demonstrate its beneficial effect in the treatment of acne vulgaris. Our in vitro studies showed that the UCO-SMC3 strain resists adverse gastrointestinal conditions, inhibits the growth of clinical isolates of C. acnes, and reduces the adhesion of the pathogen to keratinocytes. Furthermore, in vivo studies in a mice model of C. acnes infection demonstrated that W. viridescens UCO-SMC3 beneficially modulates the immune response against the skin pathogen. Both the oral and topical administration of the UCO-SCM3 strain was capable of reducing the replication of C. acnes in skin lesions and beneficially modulating the inflammatory response. Of note, orally administered W. viridescens UCO-SMC3 induced more remarkable changes in the immune response to C. acnes than the topical treatment. However, the topical administration of W. viridescens UCO-SMC3 was more efficient than the oral treatment to reduce pathogen bacterial loads in the skin, and effects probably related to its ability to inhibit and antagonize the adhesion of C. acnes. Furthermore, a pilot study in acne volunteers demonstrated the capacity of a facial cream containing the UCO-SMC3 strain to reduce acne lesions. The results presented here encourage further mechanistic and clinical investigations to characterize W. viridescens UCO-SMC3 as a probiotic for acne vulgaris treatment.

Immunobiotic Feed Developed with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and the Soymilk By-Product Okara Improves Health and Growth Performance in Pigs.

Lactobacillus delbrueckii subsp. delbrueckii TUA4408L is able to differentially modulate the innate immune response of porcine intestinal epithelial cells triggered by TLR4 activation. This strain also has a remarkable ability to grow on plant substrates. These two immunological and biotechnological characteristics prompted us to evaluate whether the soymilk by-product okara fermented with the TUA4408L strain can serve as an immunobiotic feed with the ability to beneficially modulate the intestinal immunity of piglets after weaning to improve their productivity. Our in vivo studies demonstrated that the administration of immunobiotic TUA4408L-fermented okara feed significantly increased piglet growth performance and meat quality. These positive effects were associated with the ability of the TUA4408L-fermented okara feed to beneficially modulate both intestinal microbiota and immunity in pigs. The immunobiotic feed improved the abundance of the beneficial bacteria Lactobacillus and Lactococcus in the gut of pigs, reduced blood markers of inflammation, and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These findings indicate that the immunobiotic TUA4408L-fermented okara feed could be an economical and environmentally friendly option to improve the growth performance and immune health of pigs.

Metabolic shift in the production of corrinoid compounds by Lactobacillus coryniformis in the absence of purines.

Lactobacillus coryniformis CRL 1001 and L. reuteri CRL 1098 have the complete genes necessary to synthesize pseudo-cobalamin as final product in a vitamin B12 free commercial medium. Unlike vitaminB12 (the most biologically active form), the pseudo-cobalamin contains adenine instead of 5,6-dimethlbenzimidazole (DMB) in the Coα-ligand. Considering the vitamin B12-gene clusters of these bacteria, the aim of this work was to analyze the production of corrinoids with DMB (vitamin B12) instead of adenine (pseudo-B12) as lower ligand base in a vitamin B12 free chemically defined medium (CDM) without purines. Genome-wide screening of genes related to purine metabolism showed that both strains possess all pur genes necessary for the synthesis of inositol monophosphate, the main precursor for purine biosynthesis. Accordingly, both strains were able to grow in B12 free CDM without purines, with the supplementation of different synthetic intermediaries. Isolated compounds with positive vitamin B12 activity were quantified and characterized by LC/MS-MS. Total corrinoids values were higher for both strains in comparison to those obtained in vitaminB12 free commercial medium. Interestingly, CRL 1001 strain synthesized cobalamin, suggesting that this strain is able to activate DMB as nitrogenous base instead adenine when it is in excess in a purine-free medium. The present paper represents the first demonstration of a partial metabolic shift to produce vitamin B12 in a Lactobacillus strain.

Immunomodulatory Properties of a γ-Aminobutyric Acid-Enriched Strawberry Juice Produced by Levilactobacillus brevis CRL 2013.

Gamma-aminobutyric acid (GABA) plays a key role in mammals as the major inhibitory neurotransmitter of the central nervous system. Although GABA may not be able to cross the human blood-brain barrier, it was approved as a food ingredient because of its benefits to the host after oral administration including anti-hypertensive, anti-depressant and anti-inflammatory activities. Considering the current trend toward the development of new functional and natural products and that microbial fermentation is one of the most promising methods to produce this non-protein amino acid, the in situ production of GABA through fermentation of strawberry and blueberry juices by the efficient GABA producer strain, Levilactobacillus brevis (formerly known as Lactobacillus brevis) CRL 2013, was evaluated. A high GABA production (262 mM GABA) was obtained after fermenting strawberry juice supplemented with yeast extract for 168 h, being GABA yield significantly higher in strawberry juices than in the blueberry ones. Thus, GABA-enriched fermented strawberry juice (FSJ) was selected to carry out in vivo and in vitro studies. The in vitro functional analysis of the GABA-enriched FSJ demonstrated its ability to significantly decrease the expression of cox-2 gene in LPS stimulated RAW 264.7 macrophages. In addition, in vivo studies in mice demonstrated that both, L. brevis CRL 2013 and the GABA-enriched FSJ were capable of reducing the levels of peritoneal, intestinal and serum TNF-α, IL-6, and CXCL1, and increasing IL-10 and IFN-γ in mice exposed to an intraperitoneal challenge of LPS. Of note, the GABA-enriched FSJ was more efficient than the CRL 2013 strain to reduce the pro-inflammatory factors and enhance IL-10 production. These results indicated that the CRL 2013 strain exerts anti-inflammatory effects in the context of LPS stimulation and that this effect is potentiated by fermentation. Our results support the potential use of L. brevis CRL 2013 as an immunomodulatory starter culture and strawberry juice as a remarkable vegetable matrix for the manufacture of GABA-enriched fermented functional foods capable of differentially modulating the inflammatory response triggered by TLR4 activation.

YebC, a putative transcriptional factor involved in the regulation of the proteolytic system of Lactobacillus.

The proteolytic system of Lactobacillus plays an essential role in bacterial growth, contributes to the flavor development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. In this work, a genomic analysis of all genes involved in the proteolytic system of L. delbrueckii subsp. lactis CRL 581 was performed. Genes encoding the cell envelope-associated proteinase, two peptide transport systems, and sixteen peptidases were identified. The influence of the peptide supply on the transcription of 23 genes involved in the proteolytic system of L. delbrueckii subsp. lactis was examined after cell growth in a chemically defined medium (CDM) and CDM supplemented with Casitone. prtL, oppA 1, optS, optA genes as well as oppDFBC and optBCDF operons were the most highly expressed genes in CDM; their expression being repressed 6- to 115-fold by the addition of peptides. The transcriptional analysis was confirmed by proteomics; the up-regulation of the PrtL, PepG, OppD and OptF proteins in the absence of peptides was observed while the DNA-binding protein YebC was up-regulated by peptides. Binding of YebC to the promoter region of prtL, oppA 1, and optS, demonstrated by electrophoretic mobility shift assays, showed that YebC acts as a transcriptional repressor of key proteolytic genes.