A Commensal Bifidobacterium longum Strain Prevents Gluten-Related Immunopathology in Mice through Expression of a Serine Protease Inhibitor.

Microbiota-modulating strategies, including probiotic administration, have been tested for the treatment of chronic gastrointestinal diseases despite limited information regarding their mechanisms of action. We previously demonstrated that patients with active celiac disease have decreased duodenal expression of elafin, a human serine protease inhibitor, and supplementation of elafin by a recombinant Lactococcus lactis strain prevents gliadin-induced immunopathology in the NOD/DQ8 mouse model of gluten sensitivity. The commensal probiotic strain Bifidobacterium longum NCC2705 produces a serine protease inhibitor (Srp) that exhibits immune-modulating properties. Here, we demonstrate that B. longum NCC2705, but not a srp knockout mutant, attenuates gliadin-induced immunopathology and impacts intestinal microbial composition in NOD/DQ8 mice. Our results highlight the beneficial effects of a serine protease inhibitor produced by commensal B. longum strains.IMPORTANCE Probiotic therapies have been widely used to treat gastrointestinal disorders with variable success and poor mechanistic insight. Delivery of specific anti-inflammatory molecules has been limited to the use of genetically modified organisms, which has raised some public and regulatory concerns. By examining a specific microbial product naturally expressed by a commensal bacterial strain, we provide insight into a mechanistic basis for the use of B. longum NCC2705 to help treat gluten-related disorders.

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease.

BACKGROUND: Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. METHODS: Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. RESULTS: The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. CONCLUSIONS: A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications.

Lactic acid bacteria (LAB) are promising vectors of choice to deliver active molecules to mucosal tissues. They are recognized as safe by the World Health Organization and some strains have probiotic properties. The wide range of potential applications of LAB-driven mucosal delivery includes control of inflammatory bowel disease, vaccine delivery, and management of auto-immune diseases. Because of this potential, strategies for the display of proteins at the surface of LAB are gaining interest. To display a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall, by a covalent link using sortase mediated anchoring via the LPXTG motif, or by non-covalent liaisons employing binding domains such as LysM or WxL. Both the stability and functionality of the displayed proteins will be affected by the kind of anchor used. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli. Although it is not necessarily so that surface-display is the preferred localization in all cases, it has been shown that for certain applications, such as delivery of the human papillomavirus E7 antigen, surface-display elicits better biological responses, compared to cytosolic expression or secretion. Recent developments include the display of peptides and proteins targeting host cell receptors, for the purpose of enhancing the interactions between LAB and host. Surface-display technologies have other potential applications, such as degradation of biomass, which is of importance for some potential industrial applications of LAB.

Dissecting the respective roles of microbiota and host genetics in the susceptibility of Card9-/- mice to colitis.

BACKGROUND: The etiology of inflammatory bowel disease (IBD) is unclear but involves both genetics and environmental factors, including the gut microbiota. Indeed, exacerbated activation of the gastrointestinal immune system toward the gut microbiota occurs in genetically susceptible hosts and under the influence of the environment. For instance, a majority of IBD susceptibility loci lie within genes involved in immune responses, such as caspase recruitment domain member 9 (Card9). However, the relative impacts of genotype versus microbiota on colitis susceptibility in the context of CARD9 deficiency remain unknown. RESULTS: Card9 gene directly contributes to recovery from dextran sodium sulfate (DSS)-induced colitis by inducing the colonic expression of the cytokine IL-22 and the antimicrobial peptides Reg3ß and Reg3γ independently of the microbiota. On the other hand, Card9 is required for regulating the microbiota capacity to produce AhR ligands, which leads to the production of IL-22 in the colon, promoting recovery after colitis. In addition, cross-fostering experiments showed that 5 weeks after weaning, the microbiota transmitted from the nursing mother before weaning had a stronger impact on the tryptophan metabolism of the pups than the pups' own genotype. CONCLUSIONS: These results show the role of CARD9 and its effector IL-22 in mediating recovery from DSS-induced colitis in both microbiota-independent and microbiota-dependent manners. Card9 genotype modulates the microbiota metabolic capacity to produce AhR ligands, but this effect can be overridden by the implantation of a WT or "healthy" microbiota before weaning. It highlights the importance of the weaning reaction occurring between the immune system and microbiota for host metabolism and immune functions throughout life. A better understanding of the impact of genetics on microbiota metabolism is key to developing efficient therapeutic strategies for patients suffering from complex inflammatory disorders. Video Abstract.

Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using in vitro and in vivo approaches.

Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable in vitro experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the Lactobacillus and Bifidobacterium genera, were screened using three different sets of in vitro experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: Lactobacillus gasseri PI41, Lacticaseibacillus rhamnosus PI48 and Bifidobacterium animalis subsp. lactis PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, L. gasseri PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of in vitro experiments combined with in vivo assessments allowed us to identify a promising probiotic candidate strain, L. gasseri PI41, in the context of IBS.

Immunotherapy of allergic diseases using probiotics or recombinant probiotics.

Allergic diseases affect up to 30% of the western population, and their prevalence is increasing. Probiotics are able to modulate the mucosal immune response, and clinical trials demonstrated that specific strains, especially lactic acid bacteria (LAB) ones, reduce allergic symptoms. Moreover, the use of recombinant probiotics has been evaluated as possible strategies for the immunotherapy of allergic diseases. The production and delivery of allergens by recombinant LAB in concert with their ability to induce a Th1-type immune response have been shown to be a promising mucosal vaccination strategy in mouse model. The aim of this article is to review the applications of probiotics in allergy immunotherapy with a special focus on recombinant LAB delivering proteins or DNA.

Assessment of the safety of Levilactobacillus brevis CNCM I-5321, a probiotic candidate strain isolated from pulque with anti-proliferative activities.

Gut dysbiosis has been strongly correlated with colorectal cancer (CRC) development and the use of probiotics to modulate this imbalance represents a potential and promising therapy to prevent and treat CRC. For this reason, the identification of novel probiotic strains from diverse origins has widely increased in recent years, including traditional fermented foods. In this work we describe a new strain previously isolated from pulque (a traditional Mexican beverage), Levilactobacillus brevis CNCM I-5321, which may represent an interesting probiotic candidate to prevent and treat cancer. Indeed, our results show that CNCM I-5321 displays significant and specific antiproliferative capacities in human intestinal cancer cell lines (HT-29, HTC-116 and Caco-2 cells), but not in normal cells (FH cells). In addition, CNCM I-5321 is able to induce: (1) a pro-inflammatory immune response through stimulation of interleukin (IL)-2, IL-6, IL-12 and IL-17 cytokines and (2) apoptosis via activation of caspase 8. On the other hand, a minimum inhibitory concentration (MIC) assay revealed phenotypic resistance of this strain to ampicillin and chloramphenicol. However, no known transferable determinants were found in the genome of CNCM I-5321, thus this probiotic candidate presents no risk of horizontal transfer to the intestinal bacterial population. Finally, the safety status of CNCM I-5321 was evaluated using an innovative model of chicken embryo chorioallantoic membrane (CAM) to assess undesirable and/or toxic effects. Overall, our results support that CNCM I-5321 strain is non-pathogenic and safe for potential use as an anti-cancer candidate in human and animal medicine.

Gut barrier-microbiota imbalances in early life lead to higher sensitivity to inflammation in a murine model of C-section delivery.

BACKGROUND: Most interactions between the host and its microbiota occur at the gut barrier, and primary colonizers are essential in the gut barrier maturation in the early life. The mother-offspring transmission of microorganisms is the most important factor influencing microbial colonization in mammals, and C-section delivery (CSD) is an important disruptive factor of this transfer. Recently, the deregulation of symbiotic host-microbe interactions in early life has been shown to alter the maturation of the immune system, predisposing the host to gut barrier dysfunction and inflammation. The main goal of this study is to decipher the role of the early-life gut microbiota-barrier alterations and its links with later-life risks of intestinal inflammation in a murine model of CSD. RESULTS: The higher sensitivity to chemically induced inflammation in CSD mice is related to excessive exposure to a too diverse microbiota too early in life. This early microbial stimulus has short-term consequences on the host homeostasis. It switches the pup's immune response to an inflammatory context and alters the epithelium structure and the mucus-producing cells, disrupting gut homeostasis. This presence of a too diverse microbiota in the very early life involves a disproportionate short-chain fatty acids ratio and an excessive antigen exposure across the vulnerable gut barrier in the first days of life, before the gut closure. Besides, as shown by microbiota transfer experiments, the microbiota is causal in the high sensitivity of CSD mice to chemical-induced colitis and in most of the phenotypical parameters found altered in early life. Finally, supplementation with lactobacilli, the main bacterial group impacted by CSD in mice, reverts the higher sensitivity to inflammation in ex-germ-free mice colonized by CSD pups' microbiota. CONCLUSIONS: Early-life gut microbiota-host crosstalk alterations related to CSD could be the linchpin behind the phenotypic effects that lead to increased susceptibility to an induced inflammation later in life in mice. Video Abstract.

Over-production of exopolysaccharide by Lacticaseibacillus rhamnosus CNCM I-3690 strain cutbacks its beneficial effect on the host.

Most lactobacilli produce extracellular polysaccharides that are considered to contribute to the probiotic effect of many strains. Lacticaseibacillus rhamnosus CNCM I-3690 is an anti-inflammatory strain able to counterbalance gut barrier dysfunction. In this study ten spontaneous variants of CNCM I-3690 with different EPS-production were generated and characterized by their ropy phenotype, the quantification of the secreted EPS and genetic analysis. Amongst them, two were further analysed in vitro and in vivo: an EPS over-producer (7292) and a low-producer derivative of 7292 (7358, with similar EPS levels than the wild type (WT) strain). Our results showed that 7292 does not have anti-inflammatory profile in vitro, and lost the capacity to adhere to the colonic epithelial cells as well as the protective effect on the permeability. Finally, 7292 lost the protective effects of the WT strain in a murine model of gut dysfunction. Notably, strain 7292 was unable to stimulate goblet cell mucus production and colonic IL-10 production, all key features for the beneficial effect of the WT strain. Furthermore, transcriptome analysis of colonic samples from 7292-treated mice showed a down-regulation of anti-inflammatory genes. Altogether, our results point out that the increase of EPS production in CNCM I-3690 impairs its protective effects and highlight the importance of the correct EPS synthesis for the beneficial effects of this strain.

Inactivation of the ybdD gene in Lactococcus lactis increases the amounts of exported proteins.

Random insertional mutagenesis performed on a Lactococcus lactis reporter strain led us to identify L. lactis ybdD as a protein-overproducing mutant. In different expression contexts, the ybdD mutant shows increased levels of exported proteins and therefore constitutes a new and attractive heterologous protein production host. This study also highlights the importance of unknown regulatory processes that play a role during protein secretion.

Maternal dietary omega-3 deficiency worsens the deleterious effects of prenatal inflammation on the gut-brain axis in the offspring across lifetime.

Maternal immune activation (MIA) and poor maternal nutritional habits are risk factors for the occurrence of neurodevelopmental disorders (NDD). Human studies show the deleterious impact of prenatal inflammation and low n-3 polyunsaturated fatty acid (PUFA) intake on neurodevelopment with long-lasting consequences on behavior. However, the mechanisms linking maternal nutritional status to MIA are still unclear, despite their relevance to the etiology of NDD. We demonstrate here that low maternal n-3 PUFA intake worsens MIA-induced early gut dysfunction, including modification of gut microbiota composition and higher local inflammatory reactivity. These deficits correlate with alterations of microglia-neuron crosstalk pathways and have long-lasting effects, both at transcriptional and behavioral levels. This work highlights the perinatal period as a critical time window, especially regarding the role of the gut-brain axis in neurodevelopment, elucidating the link between MIA, poor nutritional habits, and NDD.

An intranasal administration of Lactococcus lactis strains expressing recombinant interleukin-10 modulates acute allergic airway inflammation in a murine model.

BACKGROUND: Around 300 million people world-wide suffer from asthma, and the prevalence of allergic diseases has increased. Much effort has been used in the study of mechanisms involved in the immune response observed in asthma to intervene for the treatment of this condition. During inflammation in asthma, Th2 cytokines and eosinophils are essential components of the host immune system. Furthermore, for therapeutic interventions against this disease, IL-10 is an important cytokine because it has a central role in the regulation of inflammatory cascades. OBJECTIVE: To evaluate the immunomodulatory effect of Lactococcus lactis strains expressing recombinant IL-10 in a mouse model of ovalbumin (OVA)-induced acute airway inflammation. METHODS: L. lactis expressing recombinant IL-10 in a cytoplasmic (LL-CYT) or secreted form (LL-SEC) and wild-type (LL-WT) were used. IL-10 production by the recombinant strains was evaluated by ELISA. After an intranasal administration of L. lactis producing recombinant IL-10 and the induction of acute allergic airway inflammation in mice, blood samples were collected to detect IgE anti-OVA, and bronchoalveolar lavage (BAL) was harvested for eosinophil count. Additionally, the lungs were collected for the detection of the eosinophil peroxidase (EPO) activity, measurement of cytokines and chemokines and evaluation of pathology. RESULTS: Mice that received LL-CYT and LL-SEC strains showed a significant decrease in eosinophils numbers, EPO activity, anti-OVA IgE and IgG1 levels, IL-4 and CCL3 production and pulmonary inflammation and mucus hypersecretion, compared with the asthmatic group. Only the LL-CYT/OVA group showed reduced levels of IL-5, CCL2, CCL5 and CCL11. CONCLUSION: Treatment with L. lactis producing recombinant IL-10 used in this study (LL-CYT and LL-SEC) modulated experimental airway inflammation in the mouse model independently of Treg cells. Additionally, the LL-CYT strain was more efficient in the suppression of lung inflammation.

Toll-like receptor 2 is critical for induction of Reg3 beta expression and intestinal clearance of Yersinia pseudotuberculosis.

OBJECTIVE: Yersinia pseudotuberculosis causes ileitis and mesenteric lymphadenitis by mainly invading the Peyer's patches that are positioned in the terminal ileum. Whereas toll-like-receptor 2 (TLR2) controls mucosal inflammation by detecting certain microbiota-derived signals, its exact role in protecting Peyer's patches against bacterial invasion has not been defined. DESIGN: Wild-type, Tlr2-, Nod2- and MyD88-deficient animals were challenged by Y pseudotuberculosis via the oral or systemic route. The role of microbiota in conditioning Peyer's patches against Yersinia through TLR2 was assessed by delivering, ad libitum, exogenous TLR2 agonists in drinking water to germ-free and streptomycin-treated animals. Bacterial eradication from Peyer's patches was measured by using a colony-forming unit assay. Expression of cryptdins and the c-type lectin Reg3 beta was quantified by quantitative reverse transcriptase polymerase chain reaction analysis. RESULTS: Our data demonstrated that Tlr2-deficient mice failed to limit Yersinia dissemination from the Peyer's patches and succumbed to sepsis independently of nucleotide-binding and oligomerisation domain 2 (NOD2). Recognition of both microbiota-derived and myeloid differentiation factor 88 (MyD88)-mediated elicitors was found to be critically involved in gut protection against Yersinia-induced lethality, while TLR2 was dispensable to systemic Yersinia infection. Gene expression analyses revealed that optimal epithelial transcript level of the anti-infective Reg3 beta requires TLR2 activation. Consistently, Yersinia infection triggered TLR2-dependent Reg3 beta expression in Peyer's patches. Importantly, oral treatment with exogenous TLR2 agonists in germ-free animals was able to further enhance Yersinia-induced expression of Reg3 beta and to restore intestinal resistance to Yersinia. Lastly, genetic ablation of Reg3 beta resulted in impaired clearance of the bacterial load in Peyer's patches. CONCLUSIONS: TLR2/REG3 beta is thus an essential component in conditioning epithelial defence signalling pathways against bacterial invasion.

Behavior and viability of spontaneous oxidative stress-resistant Lactococcus lactis mutants in experimental fermented milk processing.

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild-type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress-resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial cells.

We recently demonstrated that noninvasive food-grade Lactococcus lactis (L. lactis) can deliver eukaryotic expression plasmid in mammalian cells in vitro. Here, we evaluated, in vivo, whether a eukaryotic expression plasmid carried by lactococci can translocate to the epithelial cells of the intestinal membrane. The strain LL(pLIG:BLG1) carrying one plasmid containing a eukaryotic expression cassette encoding beta-lactoglobulin (BLG), a major allergen of cow's milk, was orally administered by gavage to mice. BLG cDNA was detected in the epithelial membrane of the small intestine of 40% of the mice and BLG was produced in 53% of the mice. Oral administration of LL(pLIG:BLG1) induced a low and transitory Th1-type immune response counteracting a Th2 response in case of further sensitization. We demonstrated for the first time the transfer of a functional plasmid to the epithelial membrane of the small intestine in mice by noninvasive food-grade lactococci.

Opposing effect of Lactobacillus on in vitro Klebsiella pneumoniae in biofilm and in an in vivo intestinal colonisation model.

Beneficial bacteria represent potential sources of therapy, particularly in the battle against antibiotic-resistant pathogens. The Gram-negative bacillus Klebsiella pneumoniae is not only a paradigm of multi-resistant opportunistic pathogen, but it is also able to colonise the human intestine and displays a high capacity to form biofilm. In this study, the anti-biofilm activity of 140 neutralised Lactobacillus supernatants was assessed against K. pneumoniae. Among the 13 strains whose supernatant significantly impaired biofilm formation, Lactobacillus plantarum CIRM653 was selected because it was also able to impair K. pneumoniae preformed biofilm, independently of a bactericidal effect. Mixed K. pneumoniae/L. plantarum CIRM653 biofilms had reduced tridimensional structures associated with a significant decrease in K. pneumoniae biomass. Further investigation showed that L. plantarum CIRM653 supernatant induced transcriptional modifications of K. pneumoniae biofilm-related genes, including down-regulation of the quorum sensing-related lsr operons and over-expression of type 3 pili structure genes. Increased production of type 3 pili was validated by Western-blot, hemagglutination and adhesion assays. L. plantarum CIRM653 activity against K. pneumoniae was also assessed in a murine intestinal colonisation model: a constant faecal pathogen burden was observed, as against a gradual decrease in the control group. These results reveal that an in vitro a priori attracting anti-biofilm activity of Lactobacillus might be counterbalanced by an in vivo behaviour in a complex microbiota environment with potential deleterious dispersal of highly adherent K. pneumoniae cells, raising the question of the accuracy of in vitro assays in screening of beneficial microbes.

Anti-nociceptive effect of Faecalibacterium prausnitzii in non-inflammatory IBS-like models.

Visceral pain and intestinal dysbiosis are associated with Irritable Bowel Syndrome (IBS), a common functional gastrointestinal disorder without available efficient therapies. In this study, a decrease of Faecalibacterium prausnitzii presence has been observed in an IBS-like rodent model induced by a neonatal maternal separation (NMS) stress. Moreover, it was investigated whether F. prausnitzii may have an impact on colonic sensitivity. The A2-165 reference strain, but not its supernatant, significantly decreased colonic hypersensitivity induced by either NMS in mice or partial restraint stress in rats. This effect was associated with a reinforcement of intestinal epithelial barrier. Thus, F. prausnitzii exhibits anti-nociceptive properties, indicating its potential to treat abdominal pain in IBS patients.

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis.

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.