Probiotic cultivated meat: bacterial-based scaffolds and products to improve cultivated meat.

Cultivated meat is emerging to replace traditional livestock industries, which have ecological costs, including land and water overuse and considerable carbon emissions. During cultivated meat production, mammalian cells can increase their numbers dramatically through self-renewal/proliferation and transform into mature cells, such as muscle or fat cells, through maturation/differentiation. Here, we address opportunities for introducing probiotic bacteria into the cultivated meat industry, including using them to produce renewable antimicrobials and scaffolding materials. We also offer solutions to challenges, including the growth of bacteria and mammalian cells, the effect of probiotic bacteria on production costs, and the effect of bacteria and their products on texture and taste. Our summary provides a promising framework for applying microbial composites in the cultivated meat industry.

Imaging Flow Cytometry to Study Microbial Autoaggregation.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet. The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Digestive exophagy of biofilms by intestinal amoeba and its impact on stress tolerance and cytotoxicity.

The human protozoan parasite Entamoeba histolytica is responsible for amebiasis, a disease endemic to developing countries. E. histolytica trophozoites colonize the large intestine, primarily feeding on bacteria. However, in the gastrointestinal tract, bacterial cells form aggregates or structured communities called biofilms too large for phagocytosis. Remarkably, trophozoites are still able to invade and degrade established biofilms, utilizing a mechanism that mimics digestive exophagy. Digestive exophagy refers to the secretion of digestive enzymes that promote the digestion of objects too large for direct phagocytosis by phagocytes. E. histolytica cysteine proteinases (CPs) play a crucial role in the degradation process of Bacillus subtilis biofilm. These proteinases target TasA, a major component of the B. subtilis biofilm matrix, also contributing to the adhesion of the parasite to the biofilm. In addition, they are also involved in the degradation of biofilms formed by Gram-negative and Gram-positive enteric pathogens. Furthermore, biofilms also play an important role in protecting trophozoites against oxidative stress. This specific mechanism suggests that the amoeba has adapted to prey on biofilms, potentially serving as an untapped reservoir for novel therapeutic approaches to treat biofilms. Consistently, products derived from the amoeba have been shown to restore antibiotic sensitivity to biofilm cells. In addition, our findings reveal that probiotic biofilms can act as a protective shield for mammalian cells, hindering the progression of the parasite towards them.

Metabolic inputs in the probiotic bacterium Lacticaseibacillus rhamnosus contribute to cell-wall remodeling and increased fitness.

Lacticaseibacillus rhamnosus GG (LGG) is a Gram-positive beneficial bacterium that resides in the human intestinal tract and belongs to the family of lactic acid bacteria (LAB). This bacterium is a widely used probiotic and was suggested to provide numerous benefits for human health. However, as in most LAB strains, the molecular mechanisms that mediate the competitiveness of probiotics under different diets remain unknown. Fermentation is a fundamental process in LAB, allowing the oxidation of simple carbohydrates (e.g., glucose, mannose) for energy production under oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture of LGG. Furthermore, fermentation alters cell envelope remodeling and peptidoglycan biosynthesis, which leads to altered cell wall thickness, aggregation properties, and cell wall composition. In addition, fermentable sugars induced the secretion of known and novel metabolites and proteins targeting the enteric pathogens Enterococcus faecalis and Salmonella enterica Serovar Typhimurium. Overall, our results link simple carbohydrates with cell wall remodeling, aggregation to host tissues, and biofilm formation in probiotic strains and connect them with the production of broad-spectrum antimicrobial effectors.

The roles of calcium signaling and calcium deposition in microbial multicellularity.

Calcium signaling is an essential mediator of signal-controlling gene expression in most developmental systems. In addition, calcium has established extracellular functions as a structural component of biogenic minerals found in complex tissues. In bacteria, the formation of calcium carbonate structures is associated with complex colony morphology. Genes promoting the formation of biogenic minerals are essential for proper biofilm development and protection against antimicrobial solutes and toxins. Here we review recent findings on the role of calcium and calcium signaling as emerging regulators of biofilm formation in beneficial bacteria, as well as essential mediators of biofilm formation and virulence in human pathogens. The presented analysis concludes that the new understanding of calcium signaling may help to improve the performance of beneficial strains for sustainable agriculture, microbiome manipulation, and sustainable construction. Unraveling the roles of calcium may also promote the development of novel therapies against biofilm infections that target calcium uptake, calcium sensors, and calcium carbonate deposition.

A Pivotal Role for Mycobactin/mbtE in Growth and Adaptation of Mycobacterium abscessus.

Mycobacterium abscessus is an emerging pathogen that critically depends on iron for growth and pathogenesis. The acquisition of iron in Mycobacterium tuberculosis is governed by siderophores called mycobactins, synthesized by the mbt gene cluster, but the role of this gene cluster in the adaption of M. abscessus to iron limitation is not characterized. We identified an M. abscessus Tn_mutant with interruption of the mbtE gene (MAB_2248c), a central component of mycobactin biosynthesis. We tested this isolate growth characteristic, dependency on supplements, and transcriptomic response, comparing it to the response of wild-type (WT) bacteria in iron-limiting conditions. We also compare the structure of the mbt gene cluster across several mycobacteria. The Tn_mbtE mutant had a substantial, but not absolute, growth defect, which was more substantial in iron-limited media. Supplementation with mycobactin-J, hemin, blood, and surprisingly, albumin, salvaged the poor growth. Similarly, secreted mature (carboxy)-mycobactins from WT bacteria rescued the Tn_mbtE mutant during iron deprivation. The transcriptomic response of the Tn_mbtE mutant involved the upregulation of genes known to be implicated in iron homeostasis and was comparable to that of WT bacteria grown in iron-limiting conditions. Interestingly, the response was not identical to the response of M. tuberculosis to iron limitation. The mbt gene cluster and mycobactins play important roles in the physiology of M. abscessus. (Carboxy)-mycobactin is secreted from WT bacteria and can serve as "public good." The role of several iron-homeostasis related genes (like ideR) may differ between M. abscessus and Mtb. IMPORTANCE Mycobacterium abscessus is an emerging human pathogen belonging to the nontuberculous mycobacteria (NTM) family, causing severe pulmonary disease in compromised individuals. How this bacterium acquires iron is poorly understood. Here, we provide the first characterization of the role(s) the mbtE gene required for the biosynthesis of siderophore mycobactin in M. abscessus. We show that the gene mbtE is required for growth during iron deprivation and can be compensated by several supplements, including, surprisingly, albumin. We also show the transcriptomic response of the mbtE-mutant is comparable to the response of the parental strain to iron starvation and seems different from the response of M. tuberculosis. These results indicate the importance of studying mycobactin in M. abscessus and NTM strains. Understanding this pathway is central to understanding the acquisition of iron within hosts and its role in pathogenesis, which in turn may facilitate the development of antimycobacterial therapeutics.

Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.

Lactobacillaceae are Gram-positive rods, facultative anaerobes, and belong to the lactic acid bacteria (LAB) that frequently serve as probiotics. We systematically compared five LAB strains for the effects of different carbohydrates on their free-living and biofilm lifestyles. We found that fermentable sugars triggered an altered carrying capacity with strain specificity during planktonic growth. In addition, heterogeneous response to fermentable sugar was manifested in microbial aggregation (measured by imaging flow cytometry), colony development, and attachment to mucin. The acid production capacities of the strains were compatible and could not account for heterogeneity in their differential carrying capacity in liquid and on a solid medium. Among tested LAB strains, L. paracasei, and L. rhamnosus GG survived self-imposed acid stress while L. acidophilus was extremely sensitive to its own glucose utilization acidic products. The addition of a buffering system during growth on a solid medium significantly improved the survival of most tested probiotic strains during fermentation, but the formation of biofilms and aggregation capacity were responsive to the carbohydrate provided rather than to the acidity. We suggest that the optimal performance of the beneficial microbiota members belonging to Lactobacillaceae varies as a function of the growth model and the dependency on a buffering system.

Host-Biofilm Interactions.

In most natural, clinical and industrial settings, microorganisms preferentially exist in biofilms, structured communities that associate with biotic and abiotic surfaces [...].

Targeting Persistent Biofilm Infections: Reconsidering the Topography of the Infection Site during Model Selection.

The physiology of an organism in the environment reflects its interactions with the diverse physical, chemical, and biological properties of the surface. These principles come into consideration during model selection to study biofilm-host interactions. Biofilms are communities formed by beneficial and pathogenic bacteria, where cells are held together by a structured extracellular matrix. When biofilms are associated with a host, chemical gradients and their origins become highly relevant. Conventional biofilm laboratory models such as multiwall biofilm models and agar plate models poorly mimic these gradients. In contrast, ex vivo models possess the partial capacity to mimic the conditions of tissue-associated biofilm and a biofilm associated with a mineralized surface enriched in inorganic components, such as the human dentin. This review will highlight the progress achieved using these settings for two models of persistent infections: the infection of the lung tissue by Pseudomonas aeruginosa and the infection of the root canal by Enterococcus faecalis. For both models, we conclude that the limitations of the conventional in vitro systems necessitate a complimentary experimentation with clinically relevant ex vivo models during therapeutics development.

The roles of intracellular and extracellular calcium in Bacillus subtilis biofilms.

In nature, bacteria reside in biofilms- multicellular differentiated communities held together by an extracellular matrix. This work identified a novel subpopulation-mineral-forming cells-that is essential for biofilm formation in Bacillus subtilis biofilms. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. Consistently, biofilm development is prevented by the inhibition of calcium uptake. Our results provide a clear demonstration of the orchestrated production of calcite exoskeleton, critical to morphogenesis in simple prokaryotes.

Calcium carbonate mineralization is essential for biofilm formation and lung colonization.

Biofilms are differentiated microbial communities held together by an extracellular matrix. µCT X-ray revealed structured mineralized areas within biofilms of lung pathogens belonging to two distant phyla - the proteobacteria Pseudomonas aeruginosa and the actinobacteria Mycobacterium abscessus. Furthermore, calcium chelation inhibited the assembly of complex bacterial structures for both organisms with little to no effect on cell growth. The molecular mechanisms promoting calcite scaffold formation were surprisingly conserved between the two pathogens as biofilm development was similarly impaired by genetic and biochemical inhibition of calcium uptake and carbonate accumulation. Moreover, chemical inhibition and mutations targeting mineralization significantly reduced the attachment of P. aeruginosa to the lung, as well as the subsequent damage inflicted by biofilms to lung tissues, and restored their sensitivity to antibiotics. This work offers underexplored druggable targets for antibiotics to combat otherwise untreatable biofilm infections.

The Products of Probiotic Bacteria Effectively Treat Persistent Enterococcus faecalis Biofilms.

Objectives: Enterococcus faecalis is a Gram-positive commensal bacterium that possesses various survival and virulence factors, including the ability to compete with other microorganisms, invade dentinal tubules, and resist nutritional deprivation. E. faecalis is associated with persistent endodontic infections where biofilms formed by this bacterium in the root canal frequently resist dental therapies. Aseptic techniques, such as the inclusion of sodium hypochlorite, are the most commonly used methods to treat E. faecalis infections within the root canal system. In this work, we assess the effectiveness of probiotic strains to prevent the regrowth of E. faecalis biofilm cells treated by sodium hypochlorite irrigation. Methods: First, methods are presented that evaluate the effects of short-term exposure to sodium-hypochlorite on established E. faecalis. Next, we evaluate the effects of the secreted products of probiotic strains on biofilm cells and planktonic cells. Results: Sodium hypochlorite, the treatment conventionally used to decontaminate infected root canal systems, was extremely toxic to planktonic bacteria but did not fully eradicate biofilm cells. Furthermore, low concentrations of sodium hypochlorite induced eDNA dependent biofilms. Strikingly, conditioned medium from the probiotic bacteria Lactobacillus plantarum and Lactobacillus casei was sufficient to fully prevent the regrowth of treated biofilms while showing reduced potency towards planktonic cells. Conclusion: Sodium hypochlorite irrigations may contribute to the persistence of biofilm cells if used at concentrations lower than 3%. Probiotic strains and their products represent a new reservoir of biofilm therapies for E. faecalis infections formed in the root canal system.

Resolving the conflict between antibiotic production and rapid growth by recognition of peptidoglycan of susceptible competitors.

Microbial communities employ a variety of complex strategies to compete successfully against competitors sharing their niche, with antibiotic production being a common strategy of aggression. Here, by systematic evaluation of four non-ribosomal peptides/polyketide (NRPs/PKS) antibiotics produced by Bacillus subtilis clade, we revealed that they acted synergistically to effectively eliminate phylogenetically distinct competitors. The production of these antibiotics came with a fitness cost manifested in growth inhibition, rendering their synthesis uneconomical when growing in proximity to a phylogenetically close species, carrying resistance against the same antibiotics. To resolve this conflict and ease the fitness cost, antibiotic production was only induced by the presence of a peptidoglycan cue from a sensitive competitor, a response mediated by the global regulator of cellular competence, ComA. These results experimentally demonstrate a general ecological concept - closely related communities are favoured during competition, due to compatibility in attack and defence mechanisms.

Imaging flow cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members.

In nature, bacteria frequently reside in differentiated communities or biofilms. These multicellular communities are held together by self-produced polymers that allow the community members to adhere to the surface as well as to neighbor bacteria. Here, we report that exopolysaccharides prevent Bacillus subtilis from co-aggregating with a distantly related bacterium Bacillus mycoides, while maintaining their role in promoting self-adhesion and co-adhesion with phylogenetically related bacterium, Bacillus atrophaeus. The defensive role of the exopolysaccharides is due to the specific regulation of bacillaene. Single cell analysis of biofilm and free-living bacterial cells using imaging flow cytometry confirmed a specific role for the exopolysaccharides in microbial competition repelling B. mycoides. Unlike exopolysaccharides, the matrix protein TasA induced bacillaene but inhibited the expression of the biosynthetic clusters for surfactin, and therefore its overall effect on microbial competition during floating biofilm formation was neutral. Thus, the exopolysaccharides provide a dual fitness advantage for biofilm-forming cells, as it acts to promote co-aggregation of related species, as well as, a secreted cue for chemical interference with non-compatible partners. These results experimentally demonstrate a general assembly principle of complex communities and provides an appealing explanation for how closely related species are favored during community assembly. Furthermore, the differential regulation of surfactin and bacillaene by the extracellular matrix may explain the spatio-temporal gradients of antibiotic production within biofilms.

Molecular genetics for probiotic engineering: dissecting lactic acid bacteria.

The composition of the gut microbiome is greatly influenced by nutrition and dietary alterations which can also induce large temporary microbial shifts. However, the molecular mechanisms that promote these changes remain to be determined. Species of the family Lactobacillaceae and Bacillus species are genetically manipulatable bacteria that are naturally found in the human gastrointestinal (GI) tract and are often considered models of beneficial microbiota. Here, we identify specific conserved molecular pathways that play a key role in host colonization by beneficial members of the microbiota. In particular, we highlight three pathways important to the success of lactic acid bacteria (LAB) in the GI tract: glycolysis and fermentation, microbial communication via membrane vesicles, and condition-dependent antibiotic production. We elaborate on how the understanding of these circuits can lead to the development of novel therapeutic approaches to combat GI tract infections.

Bacillus subtilis Colonization of Arabidopsis thaliana Roots Induces Multiple Biosynthetic Clusters for Antibiotic Production.

Beneficial and probiotic bacteria play an important role in conferring immunity of their hosts to a wide range of bacterial, viral, and fungal diseases. Bacillus subtilis is a Gram-positive bacterium that protects the plant from various pathogens due to its capacity to produce an extensive repertoire of antibiotics. At the same time, the plant microbiome is a highly competitive niche, with multiple microbial species competing for space and resources, a competition that can be determined by the antagonistic potential of each microbiome member. Therefore, regulating antibiotic production in the rhizosphere is of great importance for the elimination of pathogens and establishing beneficial host-associated communities. In this work, we used B. subtilis as a model to investigate the role of plant colonization in antibiotic production. Flow cytometry and imaging flow cytometry (IFC) analysis supported the notion that Arabidopsis thaliana specifically induced the transcription of the biosynthetic clusters for the non-ribosomal peptides surfactin, bacilysin, plipastatin, and the polyketide bacillaene. IFC was more robust in quantifying the inducing effects of A. thaliana, considering the overall heterogeneity of the population. Our results highlight IFC as a useful tool to study the effect of association with a plant host on bacterial gene expression. Furthermore, the common regulation of multiple biosynthetic clusters for antibiotic production by the plant can be translated to improve the performance and competitiveness of beneficial members of the plant microbiome.

The NDR kinase-MOB complex FgCot1-Mob2 regulates polarity and lipid metabolism in Fusarium graminearum.

Members of the NDR (nuclear Dbf2-related) protein-kinase family are essential for cell differentiation and polarized morphogenesis. However, their functions in plant pathogenic fungi are not well understood. Here, we characterized the NDR kinase FgCot1 and its activator FgMob2 in Fusarium graminearum, a major pathogen causing Fusarium head blight (FHB) in wheat. FgCot1 and FgMob2 formed a NDR kinase-MOB protein complex. Localization assays using FgCot1-GFP or FgMob2-RFP constructs showed diverse subcellular localizations, including cytoplasm, septum, nucleus and hyphal tip. ΔFgcot1 and ΔFgmob2 exhibited serious defects in hyphal growth, polarity, fungal development and cell wall integrity as well as reduced virulence in planta. In contrast, lipid droplet accumulation was significantly increased in these two mutants. Phosphorylation of FgCot1 at two highly conserved residues (S462 and T630) as well as five new sites synergistically contributed its role in various cellular processes. In addition, non-synonymous mutations in two MAPK (mitogen-activated protein kinase) proteins, FgSte11 and FgGpmk1, partially rescued the growth defect of ΔFgmob2, indicating a functional link between the FgCot1-Mob2 complex and the FgGpmk1 signalling pathway in regulating filamentous fungal growth. These results indicated that the FgCot1-Mob2 complex is critical for polarity, fungal development, cell wall organization, lipid metabolism and virulence in F. graminearum.