Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci.

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

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.

The lactococcal ICE-ome encodes a repertoire of exchangeable traits with potential industrial relevance.

Dairy industries apply selected lactococcal strains and mixed cultures to produce diverse fermented products with distinctive flavor and texture properties. Innovation of the starter culture functionality in cheese applications embraces natural biodiversity of the Lactococcus species to identify novel strains with alternative flavor or texture forming capacities and/or increased processing robustness and phage resistance. Mobile genetic elements (MGE), like integrative conjugative elements (ICEs) play an important role in shaping the biodiversity of bacteria. Besides the genes involved in the conjugation of ICEs from donor to recipient strains, these elements also harbor cargo genes that encode a wide range of functions. The definition of such cargo genes can only be achieved by accurate identification of the ICE boundaries (delimiting). Here, we delimited 25 ICEs in lactococcal genome sequences with low contig numbers using insertion-sites flanking single-copy core-genome genes as markers for each of the distinct ICE-integrases we identified previously within the conserved ICE-core genes. For ICEs in strains for which genome information with large numbers of contigs is available, we exemplify that CRISPR-Cas9 driven ICE-curing, followed by resequencing, allows accurate delimitation and cargo definition of ICEs. Finally, we compare and contrast the cargo gene repertoire of the 26 delimited lactococcal ICEs, identifying high plasticity among the cargo of lactococccal ICEs and a range of encoded functions that is of apparent industrial interest, including restriction modification, abortive infection, and stress adaptation genes.

Detection and classification of the integrative conjugative elements of Lactococcus lactis.

Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.

Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Functional and practical insights into three lactococcal antiphage systems.

The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various Skunavirus members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against Skunavirus members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.IMPORTANCEIn the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.

Raffinocyclicin is a novel plasmid-encoded circular bacteriocin produced by Lactococcus raffinolactis with broad-spectrum activity against many gram-positive food pathogens.

This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.

The effect of seed germination and Bacillus spp. on the ripening of plant cheese analogs.

Consumer demand for plant cheeses is increasing, but challenges of improving both flavor and quality remain. This study investigated the microbiological and physicochemical impact of seed germination and fermentation with Bacillus velezensis and Bacillus amyloliquefaciens on the ripening of plant cheese analogs. Chlorine treatment or addition of Lactiplantibacillus plantarum and Lactococcus lactis controlled microbial growth during seed germination. Lp. plantarum and Lc. lactis also served as starter cultures for the acidification of soy and lupine milk and were subsequently present in the unripened plant cheese as dominant microbes. Acidification also inhibited the growth and metabolic activity of bacilli but Bacillus spores remained viable throughout ripening. During plant cheese ripening, Lc. lactis was inactivated before Lp. plantarum and the presence of bacilli during seed germination delayed Lc. lactis inactivation. Metagenomic sequencing of full-length 16S rRNA gene amplicons confirmed that the relative abundance of the inoculated strains in each ripened cheese sample exceeded 99%. Oligosaccharides including raffinose, stachyose, and verbascose were rapidly depleted in the initial stage of ripening. Both germination and the presence of bacilli during seed germination had impact on polysaccharide hydrolysis during ripening. Bacilli but not seed germination enhanced proteolysis of plant cheese during ripening. In conclusion, the use of germination with lactic acid bacteria in combination with Bacillus spp. exhibited the potential to improve the quality of ripened plant cheeses with a positive effect on the reduction of hygienic risks. IMPORTANCE: The development of novel plant-based fermented food products for which no traditional templates exist requires the development of starter cultures. Although the principles of microbial flavor formation in plant-based analogs partially overlap with dairy fermentations, the composition of the raw materials and thus likely the selective pressure on the activity of starter cultures differs. Experiments that are described in this study explored the use of seed germination, the use of lactic acid bacteria, and the use of bacilli to reduce hygienic risks, to acidify plant milk, and to generate taste-active compounds through proteolysis and fermentative conversion of carbohydrates. The characterization of fermentation microbiota by culture-dependent and culture-independent methods also confirmed that the starter cultures used were able to control microbial communities throughout 90 d of ripening. Taken together, the results provide novel tools for the development of plant-based analogs of fermented dairy products.

Scrutinizing a Lactococcus lactis mutant with enhanced capacity for extracellular electron transfer reveals a unique role for a novel type-II NADH dehydrogenase.

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis, however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis. NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms.

The tal gene of lactococcal bacteriophage TP901-1 is involved in DNA release following host adsorption.

Temperate P335 phage TP901-1 represents one of the best-characterized Gram-positive phages regarding its structure and host interactions. Following its reversible adsorption to the polysaccharidic side-chain of the cell wall polysaccharide of its host Lactococcus cremoris 3107, TP901-1 requires a glucosylated cell envelope moiety to trigger its genome delivery into the host cytoplasm. Here, we demonstrate that three distinct single amino acid substitutions in the Tal protein of TP901-1 baseplate are sufficient to overcome the TP901-1 resistance of three L. cremoris 3107 derivatives, whose resistance is due to impaired DNA release of the phage. All of these Tal alterations are located in the N-terminally located gp27-like domain of the protein, conserved in many tailed phages. AlphaFold2 predictions of the Tal mutant proteins suggest that these mutations favor conformational changes necessary to reposition the Tal fiber and thus facilitate release of the tape measure protein from the tail tube and subsequent DNA ejection in the absence of the trigger otherwise required for phage genome release. IMPORTANCE: Understanding the molecular mechanisms involved in phage-host interactions is essential to develop phage-based applications in the food and probiotic industries, yet also to reduce the risk of phage infections in fermentations. Lactococcus, extensively used in dairy fermentations, has been widely employed to unravel such interactions. Phage infection commences with the recognition of a suitable host followed by the release of its DNA into the bacterial cytoplasm. Details on this latter, irreversible step are still very scarce in lactococci and other Gram-positive bacteria. We demonstrate that a component of the baseplate of the lactococcal phage TP901-1, the tail-associated lysin (Tal), is involved in the DNA delivery into its host, L. cremoris 3107. Specifically, we have found that three amino acid changes in Tal appear to facilitate structural rearrangements in the baseplate necessary for the DNA release process, even in the absence of an otherwise required host trigger.

Potential role of salivary lactic acid bacteria in pathogenesis of oral lichen planus.

BACKGROUND: Emerging evidence emphasized the role of oral microbiome in oral lichen planus (OLP). To date, no dominant pathogenic bacteria have been identified consistently. It is noteworthy that a decreased abundance of Streptococcus, a member of lactic acid bacteria (LAB) in OLP patients has been commonly reported, indicating its possible effect on OLP. This study aims to investigate the composition of LAB genera in OLP patients by high-throughput sequencing, and to explore the possible relationship between them. METHODS: We collected saliva samples from patients with OLP (n = 21) and healthy controls (n = 22) and performed 16 S rRNA gene high-throughput sequencing. In addition, the abundance of LAB genera was comprehensively analyzed and compared between OLP and HC group. To verify the expression of Lactococcus lactis, real time PCR was conducted in buccal mucosa swab from another 14 patients with OLP and 10 HC. Furthermore, the correlation was conducted between clinical severity of OLP and LAB. RESULTS: OLP and HC groups showed similar community richness and diversity. The members of LAB, Lactococcus and Lactococcus lactis significantly decreased in saliva of OLP cases and negatively associated with OLP severity. In addition, Lactococcus and Lactococcus lactis showed negative relationship with Fusobacterium and Aggregatibacter, which were considered as potential pathogens of OLP. Similarly, compared with healthy controls, the amount of Lactococcus lactis in mucosa lesion of OLP patients was significantly decreased. CONCLUSIONS: A lower amount of Lactococcus at genus level, Lactococcus lactis at species level was observed in OLP cases and associated with disease severity. Further studies to verify the relationship between LAB and OLP, as well as to explore the precise mechanism is needed.

Unearthing Lactococcus lactis and Scheffersomyeces symbionts from edible wood-boring beetle larvae as a bio-resource for industrial applications.

BACKGROUND: Gut microbiota have several advantages in influencing the host nutrition, metabolism, immunity and growth. However, the understanding of the gut microbiota in key edible wood-boring beetle larvae remain largely undefined. In the present study, the characteristics of the gut microbiota of two edible wood-boring species (Titocerus jaspideus and Passalus punctiger) from two indigenous forested areas were investigated. RESULTS: Over 50% of Amplicon Sequence Variants (ASVs) constituted of Firmicutes in T. jaspideus. The dominant phyla in both beetle species were Bacteroidota (4.20-19.79%) and Proteobacteria (15.10-23.90%). Lactococcus lactis was the most abundant and core prokaryote in the guts of T. jaspideus. The fungi identified in the gut of both insects belong to the phylum Obazoa (66%) and Ascomycota (> 15%). Scheffersomyeces sp. was the core eukaryote recorded. The diversity of gut microbiota in both insect species did not vary significantly. Most of the prokaryotic genes expressed were predominantly associated with biosynthesis and metabolism. CONCLUSION: Our findings demonstrated that Lactococcus lactis and Scheffersomyeces are core gut microbes of wood boring beetle larvae with desirable probiotic properties and promising use in food product fermentation for improved growth performance, gut barrier health, intestinal flora balance and immune protection for human and animals. Further studies to highlight the latest medical-based applications of L. lactis as live-delivery vector for the administration of therapeutics against both communicable and non-communicable diseases are warranted.

Lactococcus lactis CNCM I-5388 versus NCDO2118 by its GABA hyperproduction ability, counteracts faster stress-induced intestinal hypersensitivity in rats.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and in vitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, in vivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients.

Analysis of the cell wall binding domain in bacteriocin-like lysin LysL from Lactococcus lactis LAC460.

Wild-type Lactococcus lactis strain LAC460 secretes prophage-encoded bacteriocin-like lysin LysL, which kills some Lactococcus strains, but has no lytic effect on the producer. LysL carries two N-terminal enzymatic active domains (EAD), and an unknown C-terminus without homology to known domains. This study aimed to determine whether the C-terminus of LysL carries a cell wall binding domain (CBD) for target specificity of LysL. The C-terminal putative CBD region of LysL was fused with His-tagged green fluorescent protein (HGFPuv). The HGFPuv_CBDlysL gene fusion was ligated into the pASG-IBA4 vector, and introduced into Escherichia coli. The fusion protein was produced and purified with affinity chromatography. To analyse the binding of HGFPuv_CBDLysL to Lactococcus cells, the protein was mixed with LysL-sensitive and LysL-resistant strains, including the LysL-producer LAC460, and the fluorescence of the cells was analysed. As seen in fluorescence microscope, HGFPuv_CBDLysL decorated the cell surface of LysL-sensitive L. cremoris MG1614 with green fluorescence, whereas the resistant L. lactis strains LM0230 and LAC460 remained unfluorescent. The fluorescence plate reader confirmed the microscopy results detecting fluorescence only from four tested LysL-sensitive strains but not from 11 tested LysL-resistant strains. Specific binding of HGFPuv_CBDLysL onto the LysL-sensitive cells but not onto the LysL-resistant strains indicates that the C-terminus of LysL contains specific CBD. In conclusion, this report presents experimental evidence of the presence of a CBD in a lactococcal phage lysin. Moreover, the inability of HGFPuv_CBDLysL to bind to the LysL producer LAC460 may partly explain the host's resistance to its own prophage lysin.

Isolation and characterization of lactic acid bacteria producing a potent anti-listerial bacteriocin-like inhibitory substance (BLIS) from chhurpi, a fermented milk product.

Nowadays, the bacteriocin industries have seen significant growth, supplanting chemical preservatives in its ability to improve the shelf-life and safety of food. The increasing customer desire to use natural preservatives has fueled advancing bacteriocin research. The objective of this study was to identify lactic acid bacteria (LAB) that produce bacteriocin-like inhibitory substance (BLIS) and have strong anti-listerial activity. We have identified and analyzed a LAB obtained from chhurpi samples, a popular milk-derived product in the Himalayan regions of India and Nepal. The strain was studied and identified based on its morphological, biochemical, and physiological characteristics. Furthermore, the molecular 16s-rDNA analysis suggests that the strain was Lactococcus sp. RGUAM1 (98.2% similar to Lactococcus lactis subsp. hordniae NBRC 100931T). The isolated strain can produce a potent BLIS, which has shown efficacy against three gram-positive bacteria responsible for food spoilage, such as Listeria monocytogenes (MTCC 657), Staphylococcus aureus subsp. aureus (MTCC 87), Lactobacillus plantarum (MTCC 1407), Lactobacillus paraplantarum (MTCC 12904). The scanning electron microscope (SEM) image illustrates that the crude cell-free supernatant (CFS) disrupts the cell envelope, leading to the release of cellular contents and the clustering of cells. In addition, this BLIS can easily withstand a wide range of pH (2-12), temperature (up to 100 °C for 15 min), bile salt (0.3% W/V), salinity (4% W/V), and enzyme activity of 1600 AU/ml against Listeria monocytogenes. Our research offers a robust framework and valuable insights into bio-preservation and its potential applications in diverse food products.

The expression of HIV-1 tat in Lactococcus lactis.

Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis.

Construction of recombinant Lactococcus expressing thymosin and interferon fusion protein and its application as an immune adjuvant.

BACKGROUND: In recent years, biosafety and green food safety standards have increased the demand for immune enhancers and adjuvants. In the present study, recombinant food-grade Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing thymosin Tα1 and chicken interferon fusion protein was constructed. RESULTS: The in vitro interactions with macrophages revealed a mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate both macrophage J774-Dual™ NF-κB and interferon regulator (IRF) signaling pathways. In vitro interactions with chicken peripheral blood mononuclear cells (PBMCs) demonstrated that a mixture of recombinant r-L. lactis-Tα1-IFN significantly enhanced the expression levels of interferon (IFN)-γ, interleukin (IL)-10, CD80, and CD86 proteins in chicken PBMCs. Animal experiments displayed that injecting a lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate the proliferation of T cells and antigen-presenting cells in chicken PBMCs. Moreover, 16S analysis of intestinal microbiota demonstrated that injection of the lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly improve the structure and composition of chicken intestinal microbiota, with a significant increase in probiotic genera, such as Lactobacillus spp. Results of animal experiments using the lysis mixture of recombinant r-L. lactis-Tα1-IFN as an immune adjuvant for inactivated chicken Newcastle disease vaccine showed that the serum antibody titers of the experimental group were significantly higher than those of the vaccine control group, and the expression levels of cytokines IFN-γ and IL-2 were significantly higher than those of the vaccine control group. CONCLUSION: These results indicate that food-safe recombinant r-L. lactis-Tα1-IFN has potential as a vaccine immune booster and immune adjuvant. This study lays the foundation for the development of natural green novel animal immune booster or immune adjuvant.

Immunogenicity and protective efficacy of a recombinant lactococcus lactis vaccine against HSV-1 infection.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) is a major cause of viral encephalitis, genital mucosal infections, and neonatal infections. Lactococcus lactis (L. lactis) has been proven to be an effective vehicle for delivering protein antigens and stimulating both mucosal and systemic immune responses. In this study, we constructed a recombinant L. lactis system expressing the protective antigen glycoprotein D (gD) of HSV-1. RESULTS: To improve the stability and persistence of antigen stimulation of the local mucosa, we inserted the immunologic adjuvant interleukin (IL)-2 and the Fc fragment of IgG into the expression system, and a recombinant L. lactis named NZ3900-gD-IL-2-Fc was constructed. By utilizing this recombinant L. lactis strain to elicit an immune response and evaluate the protective effect in mice, the recombinant L. lactis vaccine induced a significant increase in specific neutralizing antibodies, IgG, IgA, interferon-γ, and IL-4 levels in the serum of mice. Furthermore, in comparison to the mice in the control group, the vaccine also enhanced the proliferation levels of lymphocytes in response to gD. Moreover, recombinant L. lactis expressing gD significantly boosted nonspecific immune reactions in mice through the activation of immune-related genes. Furthermore, following the HSV-1 challenge of the murine lung mucosa, mice inoculated with the experimental vaccine exhibited less lung damage than control mice. CONCLUSION: Our study presents a novel method for constructing a recombinant vaccine using the food-grade, non-pathogenic, and non-commercial bacterium L. lactis. The findings indicate that this recombinant vaccine shows promise in preventing HSV-1 infection in mice.

The strain-dependent cytostatic activity of Lactococcus lactis on CRC cell lines is mediated through the release of arginine deiminase.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. RESULTS: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. CONCLUSION: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability.

Oral administration of recombinant Lactococcus lactis expressing largemouth bass (Micropterus salmoides) IFNa3 protein enhances immune response against largemouth bass virus (LMBV) infection.

Largemouth bass virus (LMBV) is a highly pathogenic pathogen that often causes high mortality of affected largemouth bass and significant financial losses. Type I interferon as an effective and broad spectrum tool has been successfully used for therapeutic or prophylactic treatment some viral infections. However, the implementation of immunotherapies based on interferon administration to combat LMBV infections has not been reported. And Lactic Acid Bacteria (LAB) are a powerful vehicle for expressing cytokines or immunostimulant peptides at the gastrointestinal level after oral administration. In this study, Lactococcus lactis (L. lactis) expression system with lactose as a screening marker was utilized to express the Micropterus salmoides interferon a3 (IFNa3) protein and orally administered to largemouth bass. The genetically engineered strain pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 was successfully constructed, and its potential to elicit immune protection response by oral administration was evaluated. After orally administration, the recombinant L. lactis was detected in guts of experimental fish and remained detectable for 72 h. Additionally, IFNa3 was able to enhance the test fish's immune response, as determined by the relatively increased mRNA relative expression of immune-related genes in the liver, spleen, and kidney tissues, including IFN-γ, TNF-α, IL-1ß, IL-8, IgM and IgT. Following LMBV challenge, the experiment group of pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 exhibited a 70 % survival rate, while survival rate were 15 % in the PBS control group, 45 % in the pNZ8149/L. lactis NZ3900 group. Furthermore, the viral load in the surviving fish was significantly lower than that of the control groups. These findings suggest that oral administration of recombinant L. lactis producing IFNa3 induces largemouth bass immune responses at a systemic level to effective prevent and combat of LMBV infection.