Bacterial Cell Surface Research Articles

A conserved bacterial genetic basis for commensal-host specificity.

Animals selectively acquire specific symbiotic gut bacteria from their environments that aid host fitness. To colonize, a symbiont must locate its niche and sustain growth within the gut. Adhesins are bacterial cell surface proteins that facilitate attachment to host tissues and are often virulence factors for opportunistic pathogens. However, the attachments are often transient and nonspecific, and additional mechanisms are required to sustain infection. In this work, we use live imaging of individual symbiotic bacterial cells colonizing the gut of living Drosophila melanogaster to show that Lactiplantibacillus plantarum specifically recognizes the fruit fly foregut as a distinct physical niche. L. plantarum establishes stably within its niche through host-specific adhesins encoded by genes carried on a colonization island. The adhesin binding domains are conserved throughout the Lactobacillales, and the island also encodes a secretion system widely conserved among commensal and pathogenic bacteria.

Synthesis and characterization of new quaternized galactomannan derivatives with antibacterial activity.

The continuous increase in bacterial resistance requires the study and development of new biocompatible antibacterial materials. Galactomannan gum was extracted from endosperms of Dimorphandra gardneriana seeds, modified through quaternization reactions (with NaOH/CHPTAC), and evaluated for antimicrobial activity. Four quaternized derivatives were obtained (QG1-QG4). The derivatives were characterized by FTIR, NMR, and rheology, which confirmed the cationization. The galactomannan structure was theoretically studied, and Zeta potential exhibited positive increase in the charge of quaternized derivatives, changing from -3.2mV (unmodified galactomannan) to +59.35mV (QG4). Elemental analysis revealed an increase in degree of substitution (DS) between the derivatives, of which QG4 exhibited the highest DS (0.51). QG4 exhibited a bactericidal effect in the antimicrobial test for determination of minimal inhibitory and bactericidal concentrations, against Gram-positive bacteria. Atomic force microscopy showed an increase in bacterial cell surface roughness and decrease in cell height after treatment with QG4. QG3 and QG4 demonstrated antibiofilm potential, showing 50% and 40% of inhibition, respectively. After MTT assay, cell viability was maintained approximately 100% for all derivatives at concentrations of 20mg/mL on RAW 264.7 macrophages and 86% with 250μg/mL on L929 cells. Galactomannan was successfully modified, demonstrating strong antibacterial activity, concomitant to the increase in DS, Zeta potential and reduction of molar mass, with good biocompatibility. Therefore, this study presents a new material with potential for development of formulations for biomedical applications, from industrial waste, strengthening of the D. gardneriana production chain.

Cooperation between Coagulase and von Willebrand factor binding protein in Staphylococcus aureus fibrin pseudocapsule formation

The major human pathogen Staphylococcus aureus forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms.Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen.We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of S. aureus biofilm infections as antibiotics are more effective against actively growing cells.Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.

Exploring biosorption potential of cow dung derived novel strain Cellulosimicrobium cellulans KHP3 in the remediation of Chromium (Cr), Copper (Cu) and Zinc (Zn)

The present work reports the bioremediation ability of cow dung isolated bacterial strain Cellulosomicrobium cellulans KHP3 identified by applying PCR amplification and rRNA gene sequencing. The potential of C. cellulans KHP3 to tolerate different concentrations of heavy metals was investigated by growing the novel strain on nutrient agar medium supplemented with increasing concentrations (in ppm) of heavy metals i.e., Chromium (50–1500) Copper (50–200), and Zinc (50–200). The strain KHP3 displayed maximum tolerable concentration (MTC) of 1500 ppm against Chromium while 200 ppm was observed for Copper and Zinc. The strain KHP3 was found to be very effective in the removal of Chromium as indicated by 59.8% removal percentage followed by Zinc (36.6%) and Copper (20.4%) after 72 h of incubation at 37 °C. The higher biosorption capacity of our strain was achieved against Chromium (349.6 mg/g) followed by Zinc (198.8 mg/g) and Copper (58.6 mg/g) which reflects the potential of bacterial strain to absorb a greater amount of heavy metal ions. Following bioremediation assay, the strain C. cellulans KHP3 was subjected to FE-SEM coupled with EDS and FTIR to confirm surface morphological and physiochemical changes resulting from heavy metal adsorption. FE-SEM observation provided substantial information on the immobilization ofChromium, Copperand Zincions on the bacterial cell surface while EDS peaks revealed surface bio-sorption of these heavy metals. The binding of heavy metals on the surface of C. cellulans KHP3 was proved through FTIR study which revealed the involvement of various functional groups acting as surface ligand such as hydroxyl, carbonyl, and amine present on bacterial surface. Moreover, studies based on Freundlich, Langmuir, and Temkin isotherm models were also done to further validate the adsorption mechanism of our strain. The significant R 2 values of 0.9983 and 0.9852 observed in case of Chromium and Zinc respectively depicted monolayer adsorption. On the other hand, R 2 value 0.7959 in case of Copper significantly revealed multilayer adsorption mechanism. The findings highlight the biosorption ability of Cellulosomicrobium cellulans KHP3 for heavy metals that may be explored in the remediation of environmental pollutants.

Iron-Tannin Coating Reduces Clearance and Increases Tumor Colonization of Systemically Delivered Bacteria.

Engineered bacteria offer a novel approach to targeted cancer therapy, but challenges remain in delivering enough bacteria safely for effective treatment. Previous efforts have used either a native or synthetic coating to achieve better control over the half-life of bacteria in the body but have limitations in delivery or versatility. In this work, we optimized and evaluated a synthetic coating for probiotic Escherichia coli Nissle 1917 to increase its half-life in blood and thereby increase the bioavailability of intravenous doses of bacteria to colonize and treat tumors. Using a simple one-pot chemical process, we coated bacteria with iron and tannic acid (FeTA) to form a temporary adhesive protective coating surrounding the bacterial cell surface. The iron to tannic acid ratio of the coating was optimized for intravenous use, and FeTA-coated bacteria of several different genetic backgrounds showed 15-fold higher survival in blood survival assays for up to 4 hours. We found that the FeTA coating reduced both complement-mediated bacterial killing and phagocyte-mediated bacterial killing in vitro. As a result, systemic delivery of attenuated bacteria had up to 60% colonization efficiency of FeTA-coated bacteria in an orthotopic breast cancer mouse model compared to 10% for the non-coated control, all the while maintaining a two-fold decrease in weight loss of attenuated bacteria compared to wild-type. Altogether, we show that an optimized FeTA coating significantly extends the half-life and colonization efficiency of engineered bacteria, overcoming a key limitation of their application in cancer therapy.

Architectural dissection of adhesive bacterial cell surface appendages from a "molecular machines" viewpoint.

The ability of bacteria to interact with and respond to their environment is crucial to their lifestyle and survival. Bacterial cells routinely need to engage with extracellular target molecules, in locations spatially separated from their cell surface. Engagement with distant targets allows bacteria to adhere to abiotic surfaces and host cells, sense harmful or friendly molecules in their vicinity, as well as establish symbiotic interactions with neighboring cells in multicellular communities such as biofilms. Binding to extracellular molecules also facilitates transmission of information back to the originating cell, allowing the cell to respond appropriately to external stimuli, which is critical throughout the bacterial life cycle. This requirement of bacteria to bind to spatially separated targets is fulfilled by a myriad of specialized cell surface molecules, which often have an extended, filamentous arrangement. In this review, we compare and contrast such molecules from diverse bacteria, which fulfil a range of binding functions critical for the cell. Our comparison shows that even though these extended molecules have vastly different sequence, biochemical and functional characteristics, they share common architectural principles that underpin bacterial adhesion in a variety of contexts. In this light, we can consider different bacterial adhesins under one umbrella, specifically from the point of view of a modular molecular machine, with each part fulfilling a distinct architectural role. Such a treatise provides an opportunity to discover fundamental molecular principles governing surface sensing, bacterial adhesion, and biofilm formation.

Combinatorial control of type IVa pili formation by the four polarized regulators MglA, SgmX, FrzS, and SopA.

Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in translocation across surfaces, surface adhesion, biofilm formation, and virulence. T4aP-dependent translocation crucially depends on the number of pili. To address how the number of T4aP is regulated, we focused on M. xanthus, which assembles T4aP at the leading cell pole and is a model organism for T4aP biology. Our results support a model whereby the four proteins MglA, SgmX, FrzS, and the newly identified SopA protein establish a highly intricate interaction network for orchestrating T4aP formation at the leading cell pole. This network allows for combinatorial regulation of the number of T4aP resulting in discrete levels of T4aP-dependent motility.

Active-bromide and surfactant synergy for enhanced microfouling control.

Biofilms are structured microbial communities encased in a matrix of self-produced extracellular polymeric substance (EPS) and pose significant challenges in various industrial cooling systems. A nuclear power plant uses a biocide active-bromide for control of biological growth in its condenser cooling system. This study is aimed at evaluating the anti-bacterial and anti-biofilm efficacy of active-bromide against planktonic and biofilm-forming bacteria that are commonly encountered in seawater cooling systems. The results demonstrated that active-bromide at the concentration used at the power plant (1 ppm) exhibited minimal killing activity against Pseudomonas aeruginosa planktonic cells. The bacterial cell surface hydrophobicity assay using Staphylococcus aureus and P. aeruginosa indicated that Triton-X 100 significantly decreased the hydrophobicity of planktonic cells, enhancing the susceptibility of the cells to active-bromide. Biofilm inhibition assays revealed limited efficacy of active-bromide at 1 ppm concentration, but significant inhibition at 5 ppm and 10 ppm. However, the addition of a surfactant, Triton-X 100, in combination with 1 ppm active-bromide displayed a synergistic effect, leading to significant biofilm dispersal of pre-formed P. aeruginosa biofilms. This observation was substantiated by epifluorescence microscopy using a live/dead bacterial assay that showed the combination treatment resulted in extensive cell death within the biofilm, as indicated by a marked increase in red fluorescence, compared to treatments with either agent alone. These findings suggest that active bromide alone may be insufficient for microfouling control in the seawater-based condenser cooling system of the power plant. Including a biocompatible surfactant that disrupts established biofilms (microfouling) can significantly improve the efficacy of active bromide treatment.

Recovery of platinum nanoparticles by the anaerobic mixed consortium: Platinum (IV) ion reduction, microbial community dynamics and functional genes identification

Removal and recovery of platinum (Pt) from platinum-contaminated wastestreams is beneficial to tackle the increasing supply risk and prevent adverse environmental impacts. Reduction soluble Pt (IV) to Pt nanoparticles using biological method is a plausible approach. However, current studies mainly focus on pure bacterial cultures to reduce Pt (IV). This study evaluates Pt (IV) removal via bioreduction by anaerobic granular sludge with the addition of exogenous electron donors and elucidates underlying mechanisms. Results show that formate and ethanol exhibited significant effects of Pt (IV) reduction, while acetate, lactate and pyruvate showed limited effects. The reduced products Pt nanoparticles were deposited on bacterial cell surface and periplasm. Moreover, Bacteroidetes, Patescibacteria and Proteobacteria were dominant bacteria during the Pt (IV) reduction process. Pt (IV) reduction was mainly mediated by enzymatic processes. Overall, these findings deepen understanding of Pt (IV) bioreduction by anaerobic mixed consortium.

Peptide-Driven Assembly of Magnetic Beads for Potentiometric Sensing of Bacterial Enzyme at a Subcellular Level.

Bacterial enzymes with different subcellular localizations play a critical ecological role in biogeochemical processing. However, precisely quantifying enzymes localized at certain subcellular levels, such as extracellular enzymes, has not yet been fully realized due to the complexity and dynamism of the bacterial outer membrane. Here we present a magneto-controlled potentiometric sensing platform for the specific detection of extracellular enzymatic activity. Alkaline phosphatase (ALP), which is one of the crucial hydrolytic enzymes in the ocean, was selected as the target enzyme. Magnetic beads functionalized with an ALP-responsive self-assembled peptide (GGGGGFFFpYpYEEE, MBs-peptides) prevent negatively charged peptides from entering the bacterial outer membrane, thereby enabling direct potentiometric sensing of extracellular ALP both attached to the bacterial cell surface and released into the surrounding environment. The dephosphorylation-triggered assembly of peptide-coupled magnetic beads can be directly and sensitively measured by using a magneto-controlled sensor. In this study, extracellular ALP activity of Pseudomonas aeruginosa at concentrations ranging from 10 to 1.0 × 105 CFU mL-1 was specifically and sensitively monitored. Moreover, this magneto-controlled potentiometric method enabled a simple and accurate assay of ALP activity across different subcellular localizations.

A New Bacterial Chassis for Enhanced Surface Display of Recombinant Proteins.

Bacterial surface display is a valuable biotechnology technique for presenting proteins and molecules on the outer surface of bacterial cells. However, it has limitations, including potential toxicity to host bacteria and variability in display efficiency. To address these issues, we investigated the removal of abundant non-essential outer membrane proteins (OMPs) in E. coli as a new strategy to improve the surface display of recombinant proteins. We targeted OmpA, a highly prevalent OMP in E. coli, using the lambda red method. We successfully knocked out ompA in two E. coli strains, K-12 MG1655 and E. coli BL-21, which have broad research and therapeutic applications. We then combined ompA knockout strains and two OMPs with three therapeutic proteins including an anti-toxin enzyme (ClbS), interleukin 18 (IL-18) for activating cytotoxic T cells and an anti- CTLA4 nanobody (αCTLA4) for immune checkpoint blockade. A total of six different display constructs were tested for their display levels by flow cytometry, showing that the ompA knockout strains increased the percentage as well as the levels of display in bacteria compared to those of isogenic wild-type strains. By removing non-essential, highly abundant surface proteins, we develop an efficient platform for displaying enzymes and antibodies, with potential industrial and therapeutic applications. Additionally, the enhanced therapeutic efficacy opens possibilities for live bacteria-based therapeutics, expanding the technology's relevance in the field. The online version contains supplementary material available at 10.1007/s12195-024-00819-w.

Synthesis of colicin Ia neoglycoproteins: tools towards glyco-engineering of bacterial cell surfaces.

Colicins are antimicrobial proteins produced by certain strains of Escherichia coli that function as offensive weapons against closely-related competitor strains. Their bactericidal properties and narrow bacterial targeting range has made them of therapeutic interest. Furthermore, the applications of engineered non-bactericidal colicins are of interest as a cell surface-directed protein anchor for decorating E. coli with biomolecules. We previously demonstrated that an engineered non-bacteriocidal colicin E9 could be used to label bacterial cells with multiple biomolecules including glycans. Herein we extend our approach to colicin Ia, constructing mannose-presenting colicin la neoglycoproteins, through N-terminal organocatalyst-mediated protein aldol ligation (OPAL), or maleimide ligation targeting an internal cysteine. This work further highlights the potential utility of engineered colicins for non-genetic glyco-engineering of the E. coli cell surface.

Bacterial cell surface characterization by phage display coupled to high-throughput sequencing

The remarkable capacity of bacteria to adapt in response to selective pressures drives antimicrobial resistance. Pseudomonas aeruginosa illustrates this point, establishing chronic infections during which it evolves to survive antimicrobials and evade host defenses. Many adaptive changes occur on the P. aeruginosa cell surface but methods to identify these are limited. Here we combine phage display with high-throughput DNA sequencing to create a high throughput, multiplexed technology for surveying bacterial cell surfaces, Phage-seq. By applying phage display panning to hundreds of bacterial genotypes and analyzing the dynamics of the phage display selection process, we capture important biological information about cell surfaces. This approach also yields camelid single-domain antibodies that recognize key P. aeruginosa virulence factors on live cells. These antibodies have numerous potential applications in diagnostics and therapeutics. We propose that Phage-seq establishes a powerful paradigm for studying the bacterial cell surface by identifying and profiling many surface features in parallel.

Effect of O-Polysaccharide Modifications on Successful Plant Colonization by Bacteria

O-polysaccharides of gram-negative bacteria are a highly variable component of the lipopolysaccharide molecules located at the cell wall surface and involved in microbial interaction with plant and animal cells. Activity of prophage genes often results in various non-stoichiometric modifications (methylation, acetylation, etc.) of glycans at bacterial cell surface. The share of modified O-polysaccharides increases during the stationary growth phase and results in increased hydrophobicity of microbial surface. Bacterial cells with different hydrophobicity showed difference in attachment to plant roots. Increased cell hydrophobicity index was found to result in a significant increase in the number of adsorbed microorganisms per unit root length. Thus, acetyl transferase and methyl transferase genes of viral origin may be indirectly involved in successful colonization of plant roots by rhizosphere bacteria.

Comparative kinetic study of Pb(II) and Cd(II) metal ions removal from aqueous solutions by Lactobacillus helveticus, Limosilactobacillus fermentum, and Lactiplantibacillus pentosus

ABSTRACT In this study, selected lactic acid bacteria, including Lactobacillus helveticus, Limosilactobacillus fermentum, and Lactiplantibacillus pentosus, were evaluated for their potential to remove Pb(II) and Cd(II) ions from aqueous solutions. The highest removal efficiency was achieved at a pH value of 4. Kinetic modeling using the pseudo-first-order, pseudo-second-order, and intraparticle diffusion models demonstrated that the intraparticle diffusion model provided the best fit for describing the adsorption process. The results of the study indicated that among the three bacteria tested, L. fermentum exhibited the highest maximum adsorption capacity for Pb(II) at 4.95 mg g−1, followed by L. helveticus at 4.91 mg g−1 and L. pentosus at 4.08 mg g−1. In contrast, for Cd(II) adsorption, L. helveticus showed the highest maximum adsorption capacity at 4.38 mg g−1, followed by L. pentosus at 3.45 mg g−1 and L. fermentum at 2.86 mg g−1. The mechanism of Pb(II) and Cd(II) ions removal by LAB strains involves adsorption onto the bacterial cell surfaces, where interactions such as ion exchange, electrostatic attraction, and complexation play crucial roles. Overall, L. helveticus, L. fermentum, and L. pentosus hold promising potential for various applications in wastewater treatment, particularly in the removal of heavy metal ions.

Biogenesis and Functionality of Sortase-Assembled Pili in Gram-Positive Bacteria.

A unique class of multimeric proteins made of covalently linked subunits known as pili, or fimbriae, are assembled and displayed on the gram-positive bacterial cell surface by a conserved transpeptidase enzyme named pilus-specific sortase. Sortase-assembled pili are produced by a wide range of gram-positive commensal and pathogenic bacteria inhabiting diverse niches such as the human oral cavity, gut, urogenital tract, and skin. These surface appendages serve many functions, such as molecular adhesins, immunomodulators, and virulence determinants, that significantly contribute to both the commensal and pathogenic attributes of producer microbes. Intensive genetic, biochemical, physiological, and structural studies have been devoted to unveiling the assembly mechanism and functions, as well as the utility of these proteins in vaccine development and other biotechnological applications. We provide a comprehensive review of these topics and discuss the current status and future prospects of the field.

Structure and replication of Pseudomonas aeruginosa phage JBD30.

Bacteriophages are the most abundant biological entities on Earth, but our understanding of many aspects of their lifecycles is still incomplete. Here, we have structurally analysed the infection cycle of the siphophage Casadabanvirus JBD30. Using its baseplate, JBD30 attaches to Pseudomonas aeruginosa via the bacterial type IV pilus, whose subsequent retraction brings the phage to the bacterial cell surface. Cryo-electron microscopy structures of the baseplate-pilus complex show that the tripod of baseplate receptor-binding proteins attaches to the outer bacterial membrane. The tripod and baseplate then open to release three copies of the tape-measure protein, an event that is followed by DNA ejection. JBD30 major capsid proteins assemble into procapsids, which expand by 7% in diameter upon filling with phage dsDNA. The DNA-filled heads are finally joined with 180-nm-long tails, which bend easily because flexible loops mediate contacts between the successive discs of major tail proteins. It is likely that the structural features and replication mechanisms described here are conserved among siphophages that utilize the type IV pili for initial cell attachment.

Alginate Cryogels for Rapid Hemostasis and Toluidine Blue-Mediated Photodynamic Inactivation of Bacteria.

The development of new wound dressings with fast hemostatic and bactericidal properties for prehospital care is critical. Antibacterial photodynamic therapy (aPDT) has attracted attention due to its broad-spectrum antibacterial activity and minimal bacterial resistance. However, photosensitizers used in aPDT often face issues such as poor water solubility, short-lived singlet oxygen (1O2), and limited 1O2 diffusion range. In this study, sodium alginate was covalently modified with the photosensitizer toluidine blue O (TBO) and phenylboronic acid (PBA). The modified alginate was then cross-linked with Ca(II) ions and lyophilized to form a cryogel, named SA@Ca(II)@TBO@PBA (SCTP). This cryogel functions as an antibacterial photodynamic wound dressing. The chemical immobilization of TBO and PBA enhanced the cryogel's targeting ability. PBA formed reversible covalent bonds with diol groups on bacterial cell surfaces, allowing the cryogel to capture bacteria effectively and enhance aPDT. The bactericidal efficiency of the cryogel was tested through in vitro antibacterial assays, and its hemostatic properties were confirmed in vivo. The results indicate that this cryogel has excellent hemostatic and antibacterial capabilities, showing great promise as a wound dressing for clinical applications.

Cryo-electron microscopy in the study of virus entry and infection.

Viruses have been responsible for many epidemics and pandemics that have impacted human life globally. The COVID-19 pandemic highlighted both our vulnerability to viral outbreaks, as well as the mobilization of the scientific community to come together to combat the unprecedented threat to humanity. Cryo-electron microscopy (cryo-EM) played a central role in our understanding of SARS-CoV-2 during the pandemic and continues to inform about this evolving pathogen. Cryo-EM with its two popular imaging modalities, single particle analysis (SPA) and cryo-electron tomography (cryo-ET), has contributed immensely to understanding the structure of viruses and interactions that define their life cycles and pathogenicity. Here, we review how cryo-EM has informed our understanding of three distinct viruses, of which two - HIV-1 and SARS-CoV-2 infect humans, and the third, bacteriophages, infect bacteria. For HIV-1 and SARS-CoV-2 our focus is on the surface glycoproteins that are responsible for mediating host receptor binding, and host and cell membrane fusion, while for bacteriophages, we review their structure, capsid maturation, attachment to the bacterial cell surface and infection initiation mechanism.

Predicting the immunomodulatory activity of probiotic lactic acid bacteria using supervised machine learning in a Cornu aspersum snail model

In the process of screening for probiotic strains, there are no clearly established bacterial phenotypic markers which could be used for the prediction of their in vivo mechanism of action. In this work, we demonstrate for the first time that Machine Learning (ML) methods can be used for accurately predicting the in vivo immunomodulatory activity of probiotic strains based on their cell surface phenotypic features using a snail host-microbe interaction model. A broad range of snail gut presumptive probiotics, including 240 new lactic acid bacterial strains (Lactobacillus, Leuconostoc, Lactococcus, and Enterococcus), were isolated and characterized based on their capacity to withstand snails' gastrointestinal defense barriers, such as the pedal mucus, gastric mucus, gastric juices, and acidic pH, in association with their cell surface hydrophobicity, autoaggregation, and biofilm formation ability. The implemented ML pipeline predicted with high accuracy (88 %) strains with a strong capacity to enhance chemotaxis and phagocytic activity of snails’ hemolymph cells, while also revealed bacterial autoaggregation and cell surface hydrophobicity as the most important parameters that significantly affect host immune responses. The results show that ML approaches may be useful to derive a predictive understanding of host-probiotic interactions, while also highlighted the use of snails as an efficient animal model for screening presumptive probiotic strains in the light of their interaction with cellular innate immune responses.