Synthetic LPETG-containing peptide incorporation in the Staphylococcus aureus cell-wall in a sortase a- and growth phase-dependent manner

The majority of Staphylococcus aureus virulence- and colonization- associated surface proteins contain a pentapeptide recognition motif (LPXTG). This motif can be recognized and cleaved by sortase A (SrtA) which is a membrane-bound transpeptidase. After cleavage these proteins are covalently incorporated into the peptidoglycan. Therefore, SrtA plays a key role in S. aureus virulence. We aimed to generate a substrate mimicking this SrtA recognition motif for several purposes: to incorporate this substrate into the S. aureus cell-wall in a SrtA-dependent manner, to characterize this incorporation and to determine the effect of substrate incorporation on the incorporation of native SrtA-dependent cell-surface-associated proteins. We synthesized substrate containing the specific LPXTG motif, LPETG. As a negative control we used a scrambled version of this substrate, EGTLP and a S. aureus srtA knockout strain. Both substrates contained a fluorescence label for detection by FACScan and fluorescence microscope. A spreading assay and a competitive Luminex assay were used to determine the effect of substrate treatment on native LPXTG containing proteins deposition in the bacterial cell-wall. We demonstrate a SrtA-dependent covalent incorporation of the LPETG-containing substrate in wild type S. aureus strains and several other Gram-positive bacterial species. LPETG-containing substrate incorporation in S. aureus was growth phase-dependent and peaked at the stationary phase. This incorporation negatively correlated with srtA mRNA expression. Exogenous addition of the artificial substrate did not result in a decreased expression of native SrtA substrates (e.g. clumping factor A/B and protein A) nor induced a srtA knockout phenotype

Salivary Total Protease Activity Based on a Broad-Spectrum Fluorescence Resonance Energy Transfer Approach to Monitor Induction and Resolution of Gingival Inflammation

OBJECTIVE: Salivary total protease and chitinase activities were measured by a broad-spectrum fluorescence resonance energy transfer approach as predictors of induction and resolution of gingival inflammation in healthy individuals by applying an experimental human gingivitis model. METHODS: Dental biofilm accumulated (21 days, Induction Phase) by omitting oral hygiene practices followed by a 2-week Resolution Phase to restore gingival health in an experimental gingivitis study. Plaque accumulation, as assessed by the Turesky Modification of the Quigley-Hein Plaque Index (TQHPI), and gingival inflammation, assessed using the Modified Gingival Index (MGI), scores were recorded and unstimulated saliva was collected weekly. Saliva was analysed for total protein, albumin, total protease activity and chitinase activity (n = 18). RESULTS: The TQHPI and MGI scores, as well as total protease activity, increased until day 21. After re-establishment of oral hygiene, gingival inflammation levels returned to values similar to baseline (day 0). Levels of protease activity decreased significantly, but not to baseline values. Furthermore, 'fast' responders, who responded immediately to plaque, exhibited significantly higher proteolytic activity throughout the experimental course than 'slow' responders, who showed a lagged inflammatory response. CONCLUSION: The results indicate that differential inflammatory responses encompass inherent variations in total salivary proteolytic activities, which could be further utilised in contemporary diagnostic, prognostic and treatment modalities for periodontal diseases

Biotribological properties of xerostomia patient saliva and its enhancement

The study aimed to quantify the lubricating properties of chewing stimulated whole saliva from healthy controls (n=22), from patients suffering from primary Sjögren’s syndrome (n=37) and from patients undergoing head-and-neck radiotherapy (n=34). Materials and Methods All participants had to complete the Xerostomia Inventory questionnaire to score dry mouth sensation. Lubrication was measured using an ex vivo tongue-enamel friction system in terms of Relief and Relief period. MUC5b and total protein concentrations of the saliva samples were measured by an enzyme-linked immunosorbent assay and a bicinchoninic acid assay, respectively. Results Relief of Sjögren’s patients saliva and post-irradiation patients saliva was similar compared with healthy controls, but saliva from post-irradiation patients lubricated significantly better than saliva from Sjögren’s patients. The Relief period was similar between the three groups. The Relief and Relief period were higher for saliva samples post-irradiation compared to pre-irradiation. MUC5b and total protein concentrations were comparable in all groups. MUC5b and total protein output were significantly lower in patients subjected to radiotherapy compared to saliva from healthy controls and pre-irradiation patients. MUC5b concentrations positively correlated with lubricating properties of post-irradiation patient saliva. Conclusions The lubricating properties of patient saliva were not any worse than healthy controls. Lower flow rate leads to lower availability of saliva in the oral cavity and decreases the overall output of protein and MUC5b, which might result in an insufficient replenishing of the mucosal salivary film. Clinical Relevance An insufficient replenishing might underlie the sensation of a dry mouth and loss of oral function. In the talk I will explain biomaterials related strategies, yet ex vivo, to enhance salivary lubrication despite of low flowrates

Lubricating properties of chewing stimulated whole saliva from patients suffering from xerostomia

OBJECTIVES: The study aimed to quantify the lubricating properties of chewing stimulated whole saliva from healthy controls (n = 22), from patients suffering from primary Sjögren's syndrome (n = 37) and from patients undergoing head-and-neck radiotherapy (n = 34). MATERIALS AND METHODS: All participants had to complete the Xerostomia Inventory questionnaire to score dry mouth sensation. Lubrication was measured using an ex vivo tongue-enamel friction system in terms of Relief and Relief period. MUC5b and total protein concentrations of the saliva samples were measured by an enzyme-linked immunosorbent assay and a bicinchoninic acid assay, respectively. RESULTS: Relief of Sjögren's patients' saliva and post-irradiation patients' saliva was similar compared with healthy controls, but saliva from post-irradiation patients lubricated significantly better than saliva from Sjögren's patients. The Relief period was similar between the three groups. The Relief and Relief period were higher for saliva samples post-irradiation compared to pre-irradiation. MUC5b and total protein concentrations were comparable in all groups. MUC5b and total protein output were significantly lower in patients subjected to radiotherapy compared to saliva from healthy controls and pre-irradiation patients. MUC5b concentrations positively correlated with lubricating properties of post-irradiation patient saliva. CONCLUSIONS: The lubricating properties of patient saliva were not any worse than healthy controls. Lower flow rate leads to lower availability of saliva in the oral cavity and decreases the overall output of protein and MUC5b, which might result in an insufficient replenishing of the mucosal salivary film. CLINICAL RELEVANCE: An insufficient replenishing might underlie the sensation of a dry mouth and loss of oral function

Enhanced leishmanicidal activity of cryptopeptide chimeras from the active N1 domain of bovine lactoferrin

wo antimicrobial cryptopeptides from the N1 domain of bovine lactoferrin, lactoferricin (LFcin17-30) and lactoferrampin (LFampin265-284), together with a hybrid version (LFchimera), were tested against the protozoan parasite Leishmania. All peptides were leishmanicidal against Leishmania donovani promastigotes, and LFchimera showed a significantly higher activity over its two composing moieties. Besides, it was the only peptide active on Leishmania pifanoi axenic amastigotes, already showing activity below 10 μM. To investigate their leishmanicidal mechanism, promastigote membrane permeabilization was assessed by decrease of free ATP levels in living parasites, entrance of the vital dye SYTOX Green (MW = 600 Da) and confocal and transmission electron microscopy. The peptides induced plasma membrane permeabilization and bioenergetic collapse of the parasites. To further clarify the structural traits underlying the increased leishmanicidal activity of LFchimera, the activity of several analogues was assessed. Results revealed that the high activity of these hybrid peptides seems to be related to the order and sequence orientation of the two cryptopeptide moieties, rather than to their particular linkage through an additional lysine, as in the initial LFchimera. The incorporation of both antimicrobial cryptopeptide motifs into a single linear sequence facilitates chemical synthesis and should help in the potential clinical application of these optimized analogues