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Recrystallized S-layer proteins from a probiotic bacterium A model to probe structural and nanomechanical changes of bacterial surface upon temperature or pH changes

International audienceParacrystalline bacterial surface protein layers (S layers) are common constituents of the bacterial cell wall. These layers originate from the assembly of strain-variable single protein species, the S layer proteins (SLPs), into paracrystalline lattices that eventually cover the entire surface of the bacteria [1]. S layers are thought to play a role in maintaining integrity of the bacteria or in mediating cell-host crosstalk through surface recognition [2]. Thus, S layer-carrying bacteria can show interesting probiotic potential [3]. Such functions may depend on the actual structure of both the SLP and their paracristalline assembly when delivered into the host’s intestine, i.e., after food preparation and ingestion. When isolated, the SLPs spontaneously self-assemble into mono or bilayer paracrystalline arrays on a wide range of substrates and can be used as models for the detailed investigation of bacterial S layers. In the probiotic Propionibacterium freudenreichii, SLPs were shown to play a strain-dependent central role in bacterium/host interactions, including immunomodulation [4]. In the present study, SLP A, isolated from P. freudenreichii strain CIRM BIA 118 was recrystallized at 25°C and pH 6.7 in HEPES buffer then submitted to either increasing temperatures up to 45°C or decreasing pH values down to pH 3.0, relevant to foodmaking and digestion. In the initial conditions, SLP A assembled in hexagonal paracrystalline bilayer as evidenced by atomic force microscopy (AFM). Solid-state Nuclear Magnetic Resonance (NMR) indicated that the internal structure of this protein, (in the S layer array or in solution) exhibited many (~70%) disordered regions, also containing significant amount of bound water. When submitted to heating at 35 or 45°C, the structure of the paracrystalline array was maintained but exhibited decreasing elasticity as probed by atomic force spectroscopy (AFS). When submitted to acidification, the structure and elasticity of the paracrystalline array was maintained at pH 5.0 but varied at pH 3.0. The results were interpreted in terms of changes in the exposed disordered regions of the protein when assembled in the S layer. Such changes may determine the nature and extent of bacterium/host interactions.The authors aknowledge Valérie Briard for mass spectrometry identification of SLP A

Comparison of 2002 AECG and 2016 ACR/EULAR classification criteria and added value of salivary gland ultrasonography in a patient cohort with suspected primary Sjögren’s syndrome

Abstract Background The objective was to evaluate concordance between 2002 American-European Consensus Group (AECG) and 2016 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for primary Sjögren’s syndrome (pSS) and to assess how salivary gland ultrasonography (SGUS) might improve the classification of patients. Methods Patients with suspected pSS underwent a standardised evaluation, including SGUS, at inclusion into the single-centre Brittany DIApSS cohort. Agreement between the two criteria sets was assessed using Cohen’s κ coefficient. Characteristics of discordantly categorised patients were detailed. Results We prospectively included 290 patients between 2006 and 2016, among whom 125 (43%) met ACR/EULAR criteria and 114 (39%) also met AECG criteria; thus, 11 (4%) patients fulfilled only ACR/EULAR, no patients AECG only, and 165 (57%) patients neither criteria set. Concordance was excellent (κ = 0.92). Compared to patients fulfilling both criteria sets, the 11 patients fulfilling only ACR/EULAR criteria had similar age and symptom duration but lower frequencies of xerophthalmia and xerostomia (p < 0.01 for each) and salivary gland dysfunction (p < 0.01); most had systemic involvement (91%), including three (27%) with no sicca symptoms; 91% had abnormal salivary gland biopsy and 46% anti-Sjögren's-syndrome-related antigen A (anti-SSA); 64% were diagnosed with pSS by the physician. SGUS was abnormal in 12% of the 165 patients fulfilling no criteria set. Including SGUS among the ACR/EULAR criteria increased sensitivity from 87.4% to 91.1% when physician diagnosis was the reference standard. Conclusions Agreement between AECG and ACR/EULAR criteria sets is excellent. ACR/EULAR criteria are slightly more sensitive and classified some patients without sicca symptoms as having pSS. Including SGUS in the ACR/EULAR criteria may further improve their sensitivity

Recrystallized S‑Layer Protein of a Probiotic Propionibacterium: Structural and Nanomechanical Changes upon Temperature or pH Shifts Probed by Solid-State NMR and AFM

Surface protein layers (S layers) are common constituents of the bacterial cell wall and originate from the assembly of strain-dependent surface layer proteins (Slps). These proteins are thought to play important roles in the bacteria’s biology and to have very promising technological applications as biomaterials or as part of cell-host cross-talk in probiotic mechanism. The SlpA from Propionibacterium freudenreichii PFCIRM 118 strain was isolated and recrystallized to investigate organization and assembly of the protein using atomic force microscopy and solid-state ¹H and ¹³C-nuclear magnetic resonance. SlpA was found to form hexagonal <i>p</i>1 monolayer lattices where the protein exhibited high proportions of disordered regions and of bound water. The lattice structure was maintained, but softened, upon mild heating or acidification, probably in relation with the increasing mobilities of the disordered protein regions. These results gave structural insights on the mobile protein regions exposed by S layer films, upon physiologically relevant changes of their environmental conditions