Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

Stuknytė, Milda; Brockmann, Eeva-Christine; Huovinen, Tuomas; Guglielmetti, Simone; Mora, Diego; Taverniti, Valentina; Arioli, Stefania; Noni, Ivano De; Lamminmäki, Urpo

doi:10.1128/aem.03057-13

Cited by 8 publications

(5 citation statements)

References 40 publications

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“…In addition, evidence that both phagocytosis and constitutive macropinocytosis contributed to the uptake of strain NCFM was provided (18). Lack of stimulation of plasma membrane Toll-like receptors (TLRs) prior to endocytosis was also shown to be a prerequisite for strong IFN-␤/IL-12p70 induction (18) by L. acidophilus NCFM, whose S-layer protein shares a high level of similarity with that of L. helveticus MIMLh5 (73% identity and 83% positivity) (19).…”

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confidence: 99%

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

Taverniti

Marengo

Fuglsang

et al. 2019

Appl Environ Microbiol

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Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production. IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

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confidence: 99%

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

Taverniti

Marengo

Fuglsang

et al. 2019

Appl Environ Microbiol

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“…Monoclonal antibodies (mAbs) are more stable and reproducible and would be an attractive alternative for QC applications. A few studies reported the mAbs applied to LAB and probiotics, such as Stuknyte and colleagues who developed a mAb targeting L. helveticus SLP (from a different strain) for its detection in cheese by western blot (Stuknyte et al 2014). Another study also reported the development of anti-bifidobacteria mAbs used for immuno-culture methods (Amrouche et al 2006).…”

Section: Antibody Productionmentioning

confidence: 99%

Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products

2018

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Aims: Classical microbiology techniques are the gold standard for probiotic enumeration. However, these techniques are limited by parameters of time, specificity and incapacity to detect viable but nonculturable (VBNC) microorganisms and nonviable cells. The aim of the study was to evaluate flow cytometry as a novel method for the specific quantification of viable and nonviable probiotics in multistrain products. Methods and Results: Custom polyclonal antibodies were produced against five probiotic strains from different species (Bifidobacterium bifidum R0071, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium longum ssp. longum R0175, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011). Evaluation of specificity confirmed that all antibodies were specific at least at the subspecies level. A flow cytometry method combining specific antibodies and viability assessment with SYTO â 24 and propidium iodide was applied to quantify these strains in three commercial products. Analyses were conducted on two flow cytometry instruments by two operators and compared with classical microbiology using selective media. Results indicated that flow cytometry provides higher cell counts than classical microbiology (P < 0Á05) in 73% of cases highlighting the possible presence of VBNC. Equivalent performances (repeatability and reproducibility) were obtained for both methods. Conclusions: This study showed that flow cytometry methods can be applied to probiotic enumeration and viability assessment. Combination with polyclonal antibodies can achieve sufficient specificity to differentiate closely related strains. Significance and Impact of the Study: Flow cytometry provides absolute and specific quantification of viable and nonviable probiotic strains in a very short time (<2 h) compared with classical techniques (>48 h), bringing efficient tools for research and development and quality control.

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“…The S-layer is built from one or more (glyco)-protein subunits, Slayer proteins (SLPs), that self-assemble into a highly exible and dynamic lattice through an entropy-driven process, allowing for structural adaptation in response to changing environmental conditions 4,[6][7][8][9][10] . So far, S-layers have been shown to serve various functions, including cell stability, adhesion, molecular sieve, and aiding in osmotic stress adaptation 6,[11][12][13] . Still, each S-layer's exact functionality and assembly properties remain unclear.…”

Section: Introductionmentioning

confidence: 99%

SymProFold - Structural prediction of symmetrical biological assemblies

Buhlheller,

Sagmeister,

Grininger

et al. 2024

Preprint

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Symmetry in nature often arises from self-assembly processes and serves a function. Our automated pipeline SymProFold leverages the high accuracy of the AlphaFold-Multimer predictions to derive symmetrical assemblies like two-dimensional S-layer arrays or spherical viral capsids from their protein sequence, verified with available experimental data on the cellular level. S-layers, found on many bacterial and archaeal cells, are vital for facilitating cell adhesion, evading the immune system, and providing protection against stress. However, their experimental structural characterization remains challenging because of their self-assembly property and high sequence variability. SymProFold now offers new avenues for exploring functionalities and designing targeted applications in diverse fields like nanotechnology, biotechnology, medicine, and material and environmental sciences.

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Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

Cited by 8 publications

References 40 publications

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products

SymProFold - Structural prediction of symmetrical biological assemblies

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