Systematic Exploration of the Glycoproteome of the Beneficial Gut Isolate <b><i>Lactobacillus rhamnosus </i></b>GG

Abstract

Glycoproteins form an interesting class of macromolecules involved in bacterial-host interactions, but they are not yet widely explored in Gram-positive and beneficial species. Here, an integrated and widely applicable approach was followed to identify putative bacterial glycoproteins, combining proteome fractionation with 2D protein and glycostained gels and lectin blots. This approach was validated for the microbiota isolate Lactobacillus rhamnosus GG. The approach resulted in a list of putative glycosylated proteins receiving a ‘glycosylation score'. Ultimately, we could identify 41 unique glycosylated proteins in L. rhamnosus GG (6 top-confidence, 10 high-confidence and 25 putative hits; classification based on glycosylation score). Most glycoproteins are associated with the cell wall and membrane. Identified glycoproteins include proteins involved in transport, translation, and sugar metabolism processes. A robust screening resulted in a comprehensive mapping of glycoproteins in L. rhamnosus GG. Our results reflect the glycosylation of sugar metabolism enzymes, transporters, and other proteins crucial for cell physiology. We hypothesize that protein glycosylation can confer an extra level of regulation, for example by affecting enzyme functions. This is the first systematic study of the glycoproteome of a probiotic and beneficial gut isolate.

Surface (S) Layer Proteins of Lactobacillus acidophilus Block Virus Infection via DC-SIGN Interaction

“…Another explanation is that the DC-SIGN-S-layer interaction is triggering signaling pathways that require time to achieve their antiviral effect. In fact, there is ample evidence in the literature that bacterially derived surface proteins, including S-layer, are capable of triggering a variety of signaling pathways (Tytgat et al, 2016). S-layer can induce production of the anti-inflammatory cytokine IL-10 in immature DCs (Konstantinov et al, 2008), while in macrophage cells, it induces production of TNF-alpha, a pro-inflammatory cytokine (Taverniti et al, 2013).…”

Specific properties of probiotic strains: relevance and benefits for the host

“…Interaction between pili and whey proteins depends on pH; at acidic pH, pili are immobilized in the collapsed EPS layer; and at neutral pH, pili are easily accessible for interaction with whey proteins [70]. Assessment of probiotic bacteria wall using imaging and molecular modalities is important to evaluate or predict role in adhesion properties and immunomodulatory potential of the strain [39,[71][72][73]. Growing knowledge on nanomechanics of pili can help to design nanomaterials (potential prebiotics) capable of promote adhesive properties for benefits of bacterial-host interactions [74].…”

“…These studied basic bacterial characteristics can contribute to many secondary properties of probiotic species. Thus, relatively neutral to trigger biological effects properties like elasticity of bacterial wall can be measured using subcellular imaging [39,[71][72][73]; in newly discovered strains to predict their probiotic potential and justify stratification strains on potential for immunomodulatory activity [39].…”

Growth Mode and Carbon Source Impact the Surfaceome Dynamics of Lactobacillus rhamnosus GG

“…Washed cells at the same density were treated with 10 μM 4,6′-diamidino-2-phenylindole (DAPI; Molecular Probes, Thermo Scientific, Wilmington, DE, United States) to stain all bacterial populations and with antiserum raised against His 6 – SpaC to detect SpaC expression. Antiserum against purified His 6 – SpaC of L. rhamnosus GG was raised in rabbits using routine immunization procedures as described previously (Tytgat et al, 2016). The staining was carried out by using indirect immunofluorescence as described previously (Kainulainen et al, 2012) with minor modifications.…”