A first-in-human, open-label Phase 1b and a randomised, double-blind Phase 2a clinical trial in recent-onset type 1 diabetes with AG019 as monotherapy and in combination with teplizumab.

AIMS/HYPOTHESIS: We hypothesised that islet beta cell antigen presentation in the gut along with a tolerising cytokine would lead to antigen-specific tolerance in type 1 diabetes. We evaluated this in a parallel open-label Phase 1b study using oral AG019, food-grade Lactococcus lactis bacteria genetically modified to express human proinsulin and human IL-10, as a monotherapy and in a parallel, randomised, double-blind Phase 2a study using AG019 in combination with teplizumab. METHODS: Adults (18-42 years) and adolescents (12-17 years) with type 1 diabetes diagnosed within 150 days were enrolled, with documented evidence of at least one autoantibody and a stimulated peak C-peptide level >0.2 nmol/l. Participants were allocated to interventions using interactive response technology. We treated 42 people aged 12-42 years with recent-onset type 1 diabetes, 24 with Phase 1b monotherapy (open-label) and 18 with Phase 2a combination therapy. In the Phase 2a study, after treatment of the first two open-label participants, all people involved were blinded to group assignment, except for the Data Safety Monitoring Board members and the unblinded statistician. The primary endpoint was safety and tolerability based on the incidence of treatment-emergent adverse events, collected up to 6 months post treatment initiation. The secondary endpoints were pharmacokinetics, based on AG019 detection in blood and faeces, and pharmacodynamic activity. Metabolic and immune endpoints included stimulated C-peptide levels during a mixed meal tolerance test, HbA1c levels, insulin use, and antigen-specific CD4+ and CD8+ T cell responses using an activation-induced marker assay and pooled tetramers, respectively. RESULTS: Data from 24 Phase 1b participants and 18 Phase 2a participants were analysed. No serious adverse events were reported and none of the participants discontinued AG019 due to treatment-emergent adverse events. No systemic exposure to AG019 bacteria, proinsulin or human IL-10 was demonstrated. In AG019 monotherapy-treated adults, metabolic variables were stabilised up to 6 months (C-peptide, insulin use) or 12 months (HbA1c) post treatment initiation. In participants treated with AG019/teplizumab combination therapy, all measured metabolic variables stabilised or improved up to 12 months and CD8+ T cells with a partially exhausted phenotype were significantly increased at 6 months. Circulating preproinsulin-specific CD4+ and CD8+ T cells were detected before and after treatment, with a reduction in the frequency of preproinsulin-specific CD8+ T cells after treatment with monotherapy or combination therapy. CONCLUSIONS/INTERPRETATION: Oral delivery of AG019 was well tolerated and safe as monotherapy and in combination with teplizumab. AG019 was not shown to interfere with the safety profile of teplizumab and may have additional biological effects, including changes in preproinsulin-specific T cells. These preliminary data support continuing studies with this agent alone and in combination with teplizumab or other systemic immunotherapies in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03751007, EudraCT 2017-002871-24 FUNDING: This study was funded by Precigen ActoBio.

Intestinal Delivery of Proinsulin and IL-10 via Lactococcus lactis Combined With Low-Dose Anti-CD3 Restores Tolerance Outside the Window of Acute Type 1 Diabetes Diagnosis.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12-20 or InsB13-2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

Interleukin-27 Is a Potential Rescue Therapy for Acute Severe Colitis Through Interleukin-10-Dependent, T-Cell-Independent Attenuation of Colonic Mucosal Innate Immune Responses.

BACKGROUND: If treatment with intravenous steroids fail, inflammatory bowel disease patients with acute severe colitis face systemic anti-tumor necrosis factor biologic rescue therapy or colectomy. Interleukin (IL)-27 is a cytokine with an immunosuppressive role in adaptive immune responses. However, the IL-27 receptor complex is also expressed on innate immune cells, and there is evidence that IL-27 can impact the function of innate cell subsets, although this particular functionality in vivo is not understood. Our aim was to define the efficacy of IL-27 in acute severe colitis and characterize novel IL-27-driven mechanisms of immunosuppression in the colonic mucosa. METHODS: We assessed oral delivery of Lactococcus lactis expressing an IL-27 hyperkine on the innate immune response in vivo in a genetically intact, noninfective, acute murine colitis model induced by intrarectal instillation of 2,4,6-trinitrobenzenesulfonic acid in SJL/J mice. RESULTS: IL-27 attenuates acute severe colitis through the reduction of colonic mucosal neutrophil infiltrate associated with a decreased CXC chemokine gradient. This suppression was T cell independent and IL-10 dependent, initially featuring enhanced mucosal IL-10. IL-27 was associated with a reduction in colonic proinflammatory cytokines and induced a multifocal, strong, positive nuclear expression of phosphorylated STAT-1 in mucosal epithelial cells. CONCLUSION: We have defined novel mechanisms of IL-27 immunosuppression toward colonic innate immune responses in vivo. Mucosal delivery of IL-27 has translational potential as a novel therapeutic for inflammatory bowel disease, and it is a future mucosal directed rescue therapy in acute severe inflammatory bowel disease.

Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti-CD3 Depends on the Induction of Foxp3-Positive T Cells.

The introduction of ß-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4+Foxp3+ T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-ß partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3+ Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4+Foxp3+ T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.

Recombinant Lactococcus lactis can make the difference in antigen-specific immune tolerance induction, the Type 1 Diabetes case.

Especially in western civilizations, immune diseases that are driven by innocuous (auto- or allo-) antigens are gradually evolving to become pandemic threats. A particularly poignant example is type 1 diabetes, where young children are confronted with the perspective and consequences of total pancreatic ß-cell destruction. Along these disquieting observations we find ourselves equipped with impressively accumulating molecular immunological knowledge on the ins and outs of these pathologies. Often, however, it is difficult to translate this wealth into efficacious medicines. The molecular understanding, the concept of oral tolerance induction, the benefit of using recombinant Lactococcus lactis therein and recent openings towards their clinical use may well enable turning all colors to their appropriate fields on this Rubik's cube.

Oral delivery of glutamic acid decarboxylase (GAD)-65 and IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice.

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370-575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional ß-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4(+)Foxp3(+)CD25(+) regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.

Engineering lactococci and lactobacilli for human health.

Food-grade lactic acid bacteria (LAB) are good candidates for the development of oral vectors, and are attractive alternatives to attenuated pathogens, for mucosal delivery strategies. In this review, we summarize recent results on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of this work has been based on the model LAB, Lactococcus lactis, which is suitable for the heterologous expression of therapeutic proteins. Recombinant lactococci and lactobacilli strains expressing antiproteases and antioxidant enzymes have been tested successfully for their prophylactic and therapeutic effects in murine models of colitis. Recombinant lactococci secreting autoantigens have been found to be effective for the treatment of type 1 diabetes. Also, recombinant lactococci delivering DNA were able to prevent a bovine ß-lactoglobulin (BLG)-allergic reaction in mice. We believe that these various coherent findings demonstrate the potential value of using LAB, particularly lactococci and lactobacilli strains, to develop novel vectors for the therapeutic delivery of proteins to mucosal surfaces. Further tests and in particular human clinical trials are now important next steps to conclude on the benefit of these approaches for human health.

Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice.

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.

Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines.

The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L. casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, T cell infiltration and inflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD.

AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis.

Non-clinical studies, focusing on the pharmacodynamics (PD), pharmacokinetics (PK) and safety pharmacology of genetically modified Lactococcus lactis (L. lactis) bacteria, engineered to secrete human Trefoil Factor 1 (hTFF1), were performed to provide proof-of-concept for the treatment of oral mucositis (OM) patients. L. lactis strain sAGX0085 was constructed by stably inserting an htff1 expression cassette into the bacterial genome, and clinically formulated as a mouth rinse (coded AG013). PD studies, using different oral dosing regimens, were performed in a clinically relevant hamster model for radiation-induced OM. The PK profile was assessed in healthy hamsters and in hamsters with radiation-induced OM. In addition, in vitro and in vivo safety pharmacology studies were conducted, in pooled, complement-preserved human serum, and in neutropenic hamsters and rats respectively. Topical administration of L. lactis sAGX0085/AG013 to the oral mucosa significantly reduced the severity and course of radiation-induced OM. PK studies demonstrated that both living L. lactis bacteria, as well as the hTFF1 secreted, could be recovered from the administration site for maximum 24h post-dosing, without systemic exposure. The in vitro and in vivo safety pharmacology studies confirmed that L. lactis sAGX0085 could not survive in systemic circulation, not even under neutropenic conditions. The results from the PD, PK and safety pharmacology studies reported here indicate that in situ secretion of hTFF1 by topically administered L. lactis bacteria provides a safe and efficacious therapeutic tool for the prevention and treatment of OM.

Modulation of gut-associated lymphoid tissue functions with genetically modified Lactococcus lactis.

Lactic acid bacteria are a group of taxonomically diverse, Gram-positive food-grade bacteria that have been safely consumed throughout history. The lactic acid bacterium Lactococcus lactis, well-known for its use in the manufacture of cheese, can be genetically engineered and orally formulated to deliver therapeutic proteins in the gastrointestinal tract. This review focuses on the genetic engineering of Lactococcus lactis to secrete high-quality, correctly processed bioactive molecules derived from a eukaryotic background. The therapeutic applications of these genetically modified strains are discussed, with special regards to immunomodulation.

Immunomodulation by genetically engineered lactic acid bacteria.

The taxonomically diverse lactic acid bacteria (LAB) are unified by their capability to produce lactic acid from carbohydrates by fermentation. The LAB Lactococcus (L.) lactis has been characterized into great detail and is increasingly used as a production host for heterologous proteins. L. lactis is a non-pathogenic and non-colonizing LAB species and can be efficiently engineered to produce proteins of viral, bacterial or eukaryotic origin, both intra- or extracellularly. Importantly, orally formulated L. lactis strains (ActoBiotics), engineered to synthesize and secrete therapeutic peptides and proteins in the gastrointestinal tract, are already in advanced stages of preclinical and clinical development. This review focuses on the genetic engineering of LAB in general and L. lactis in specific to secrete high-quality, correctly processed, bioactive molecules derived from a eukaryotic background. The therapeutic applications of these genetically modified strains are discussed, as well as the need for a sound environmental containment strategy, and a detailed review is presented on Lactococcus strains engineered to produce specific antigens, antibodies, cytokines and trefoil factors, with special regards to immunomodulation.

Actobiotics as a novel method for cytokine delivery.

Interleukin-10 (IL-10) is central in immune downregulation, but so far its use in inflammatory diseases remains cumbersome. For treatment of inflammatory bowel disease, adequate amounts of IL-10 must reach the intestinal lining. Systemic injection of a pharmacologically active doses of recombinant human (rh) IL-10 results in very low mucosal levels of protein and severe toxicity and side effects. In animal models, topical and active delivery of IL-10 by ingestion of recombinant Lactococcus lactis (L. lactis) was shown to be a valuable alternative. Starting thereof we have developed a novel pharmaceutical platform. Our expertise and TopAct (topical and active) delivery technology allows use of recombinant L. lactis- ActoBiotics- in clinical practice. Here we discuss the development of recombinant L. lactis for intestinal delivery of rhIL-10 in humans.

Characterization of ApuB, an extracellular type II amylopullulanase from Bifidobacterium breve UCC2003.

The apuB gene of Bifidobacterium breve UCC2003 was shown to encode an extracellular amylopullulanase. ApuB is composed of a distinct N-terminally located alpha-amylase-containing domain which hydrolyzes alpha-1,4-glucosidic linkages in starch and related polysaccharides and a C-terminally located pullulanase-containing domain which hydrolyzes alpha-1,6 linkages in pullulan, allowing the classification of this enzyme as a bifunctional class II pullulanase. A knockout mutation of the apuB gene in B. breve UCC2003 rendered the resulting mutant incapable of growth in medium containing starch, amylopectin, glycogen, or pullulan as the sole carbon and energy source, confirming the crucial physiological role of this gene in starch metabolism.

Paracellular entry of interleukin-10 producing Lactococcus lactis in inflamed intestinal mucosa in mice.

BACKGROUND: Genetically modified Lactococcus lactis secreting interleukin-10 (IL-10) has been demonstrated to provide localized delivery of a therapeutic agent through active in situ synthesis in murine colitis. At present, many aspects of the exact mechanism by which the beneficial effect of the IL-10-producing L. lactis on the mucosa is mediated remain to be clarified. METHODS: Our aim was to determine the interaction of L. lactis with the intestinal mucosa. Therefore, we administered IL-10-producing L. lactis to healthy mice and in 2 mouse models of chronic colitis. Paraffin sections of ileum and colon samples were examined with confocal and transmission electron microscopy. Ileum and colon homogenates were prepared after flushing and after removal of mucus layer and epithelium. These homogenates and homogenates of mesenteric lymph nodes and spleen were plated on agar and immunoblotting for L. lactis and IL-10 was performed. RESULTS: Both confocal and electron microscopy showed the presence of lactococci in inflamed intestinal mucosa of mice with colitis. We recovered viable bacteria that could still produce IL-10 from homogenates of inflamed ileum and colon of which mucous and epithelial layers were removed. We did not find lactococci in mesenteric lymph nodes or in the spleen of mice with colitis. CONCLUSIONS: This study demonstrates uptake of IL-10-secreting L. lactis by the paracellular route in inflamed mucosal tissue. We suggest that IL-10 production by L. lactis residing inside the mucosa in the vicinity of responsive cells can improve the local action of interleukin-10 in inflamed tissue and the efficiency of the treatment.

Oral administration of an IL-10-secreting Lactococcus lactis strain prevents food-induced IgE sensitization.

BACKGROUND: Because tolerance to food is potentially modulated by IL-10, strategies to prevent food allergy should favor an increased delivery of IL-10 to the gut. OBJECTIVES: We hypothesized that administration of a Lactococcus lactis transfected to secrete murine IL-10 could prevent sensitization in a mouse model of food allergy. METHODS: Before each oral sensitization with beta-lactoglobulin in the presence of cholera toxin, young mice were administered the transfected Lactococcus lactis. Antigen-induced anaphylaxis after oral challenge assessed clinical protection achieved by the pretreatment. Serum and feces antigen-specific antibody concentrations were sequentially measured. Antibody titers were correlated with antibody and IL-10-secreting cell numbers in the spleen and in Peyer patches. RESULTS: Pretreatment with transfected Lactococcus lactis contributed to diminish anaphylaxis significantly, and inhibit antigen-specific serum IgE and IgG(1) production strongly. In addition, transfected Lactococcus lactis increased the production of antigen-specific IgA in the gut. Variations of antibody levels in the serum and the gut correlated with the numbers of antibody-producing cells. In addition, the presence of exogenous IL-10 in the gut by transfected Lactococcus lactis induced IL-10 secretion by Peyer patches cells. Increased IL-10 titers were also measured in the plasma. CONCLUSION: These results suggest that a microorganism bioengineered to deliver IL-10 in the gut can decrease food-induced anaphylaxis and provide an option to prevent IgE-type sensitization to common food allergens. CLINICAL IMPLICATIONS: Nonpathogenic IL-10-producing microorganisms in the gut could have a potential to prevent systemic food-induced anaphylaxis.

Therapeutic drug delivery by genetically modified Lactococcus lactis.

Food-grade bacteria have been consumed throughout history without associated pathologies and are, therefore, absolutely safe to ingest. Unexpectedly, Lactococcus lactis (L. lactis), known from cheese production, can be genetically engineered to constantly secrete satisfactory amounts of bioactive cytokines. Both of these features enabled the development of a new kind of topical delivery system: topical and active delivery of therapeutic proteins by genetically modified micro-organisms. The host organism's record inspired the development of applications that target intestinal diseases. In a variety of mouse models, chronic colon inflammation can be successfully treated with (interleukin) IL-10-secreting L. lactis. Trefoil factor (TFF) producer strains have also been shown to be very effective in the treatment of acute colitis. Such novel therapeutic strains are textbook examples of genetically modified (GM) organisms. There are legitimate concerns with regard to the deliberate release of GM micro-organisms. On development of these applications, therefore, we have engineered these bacteria in such a way that biological containment is guaranteed. The essential gene thyA, encoding thymidylate synthase, has been exchanged for IL-10. This makes the GM strain critically dependent on thymidine. Lack of thymidine, for example, resulting from thymidine consumption by thyA-deficient strains-will irreversibly lead to induced "thymidine-less death." This accomplishment has created the possibility of using this strategy for application in human medicine.

Intracellular accumulation of trehalose protects Lactococcus lactis from freeze-drying damage and bile toxicity and increases gastric acid resistance.

Interleukin-10 (IL-10) is a promising candidate for the treatment of inflammatory bowel disease. Intragastric administration of Lactococcus lactis genetically modified to secrete IL-10 in situ in the intestine was shown to be effective in healing and preventing chronic colitis in mice. However, its use in humans is hindered by the sensitivity of L. lactis to freeze-drying and its poor survival in the gastrointestinal tract. We expressed the trehalose synthesizing genes from Escherichia coli under control of the nisin-inducible promoter in L. lactis. Induced cells accumulated intracellular trehalose and retained nearly 100% viability after freeze-drying, together with a markedly prolonged shelf life. Remarkably, cells producing trehalose were resistant to bile, and their viability in human gastric juice was enhanced. None of these effects were seen with exogenously added trehalose. Trehalose accumulation did not interfere with IL-10 secretion or with therapeutic efficacy in murine colitis. The newly acquired properties should enable a larger proportion of the administered bacteria to reach the gastrointestinal tract in a bioactive form, providing a means for more effective mucosal delivery of therapeutics.

Comparative and functional analysis of sortase-dependent proteins in the predicted secretome of Lactobacillus salivarius UCC118.

Surface proteins are important factors in the interaction of probiotic and pathogenic bacteria with their environment or host. We performed a comparative bioinformatic analysis of four publicly available Lactobacillus genomes and the genome of Lactobacillus salivarius subsp. salivarius strain UCC118 to identify secreted proteins and those linked to the cell wall. Proteins were identified which were predicted to be anchored by WXL-binding domains, N- or C-terminal anchors, GW repeats, lipoprotein anchors, or LysM-binding domains. We identified 10 sortase-dependent surface proteins in L. salivarius UCC118, including three which are homologous to mucus-binding proteins (LSL_0152, LSL_0311, and LSL_1335), a collagen-binding protein homologue (LSL_2020b), two hypothetical proteins (LSL_1838 and LSL_1902b), an enterococcal surface protein homologue (LSL_1085), a salivary agglutinin-binding homologue (LSL_1832b), an epithelial binding protein homologue (LSL_1319), and a proteinase homologue (LSL_1774b). However, two of the genes are gene fragments and four are pseudogenes, suggesting a lack of selection for their function. Two of the 10 genes were not transcribed in vitro, and 1 gene showed a 10-fold increase in transcript level in stationary phase compared to logarithmic phase. The sortase gene was deleted, and three genes encoding sortase-dependent proteins were disrupted. The sortase mutant and one sortase-dependent protein (mucus-binding homologue) mutant showed a significant reduction in adherence to human epithelial cell lines. The genome-wide investigation of surface proteins can thus help our understanding of their roles in host interaction.