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Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degradation. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compound screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can determine the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind concanavalin A (ConA), the assay concept can be adapted to glycans that bind other lectins.

Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degradation. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compound screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can determine the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind concanavalin A (ConA), the assay concept can be adapted to glycans that bind other lectins.

Pseudomonasinfections are common among hospitalized, immunocompromised, and chronic lung disease patients. These infections are recalcitrant to common antibacterial therapies due to inherent antibiotic resistance. To meet the need of new anti-Pseudomonasdrugs, a sensitive, homogenous, and robust assay was developed with the aim of identifying inhibitors of acyl–coenzyme A synthetases (ACSs) from Pseudomonas. Given the importance of fatty acids for in vivo nutrition of Pseudomonas, such inhibitors might have the potential to reduce the bacterial fitness during infection. The assay, based on a coupled reaction between the Pseudomonasspp. ACS and the firefly luciferase, allowed the identification of three classes of inhibitors by screening of a diverse compound collection. These compounds were confirmed to reversibly bind ACS with potencies in the micromolar range. Two classes were found to compete with acyl–coenzyme A, while the third one was competitive with fatty acid binding. Although these compounds inhibit the bacterial ACS in cell-free assays, they show modest or no effect on Pseudomonasgrowth in vitro.

Pseudomonasinfections are common among hospitalized, immunocompromised, and chronic lung disease patients. These infections are recalcitrant to common antibacterial therapies due to inherent antibiotic resistance. To meet the need of new anti-Pseudomonasdrugs, a sensitive, homogenous, and robust assay was developed with the aim of identifying inhibitors of acyl–coenzyme A synthetases (ACSs) from Pseudomonas. Given the importance of fatty acids for in vivo nutrition of Pseudomonas, such inhibitors might have the potential to reduce the bacterial fitness during infection. The assay, based on a coupled reaction between the Pseudomonasspp. ACS and the firefly luciferase, allowed the identification of three classes of inhibitors by screening of a diverse compound collection. These compounds were confirmed to reversibly bind ACS with potencies in the micromolar range. Two classes were found to compete with acyl–coenzyme A, while the third one was competitive with fatty acid binding. Although these compounds inhibit the bacterial ACS in cell-free assays, they show modest or no effect on Pseudomonasgrowth in vitro.

ABSTRACTStaphylococcus lugdunensisis a coagulase-negative staphylococcus that is a commensal of humans and an opportunistic pathogen. It can cause a spectrum of infections, including those that are associated with the ability to form biofilm, such as occurs with endocarditis or indwelling medical devices. The genome sequences of two strains revealed the presence of orthologues of the icagenes that are responsible for synthesis of poly-N-acetylglucosamine (PNAG) that is commonly associated with biofilm in other staphylococci. However, we discovered that biofilm formed by a panel of S. lugdunensisisolates growing in iron-restricted medium was susceptible to degradation by proteases and not by metaperiodate, suggesting that the biofilm matrix comprised proteins and not PNAG. When the iron concentration was raised to 1 mM biofilm formation by all strains tested was greatly reduced. A mutant of strain N920143 lacking the entire locus that encodes iron-regulated surface determinant (Isd) proteins was defective in biofilm formation under iron-limited conditions. An IsdC-null mutant was defective, whereas IsdK, IsdJ, and IsdB mutants formed biofilm to the same level as the parental strain. Expression of IsdC was required both for the primary attachment to unconditioned polystyrene and for the accumulation phase of biofilm involving cell-cell interactions. Purified recombinant IsdC protein formed dimers in solution and Lactococcus lactiscells expressing only IsdC adhered to immobilized recombinant IsdC but not to IsdJ, IsdK, or IsdB. This is consistent with a specific homophilic interaction between IsdC molecules on neighboring cells contributing to accumulation of S. lugdunensisbiofilm in vivo.

Toll-like receptors (TLRs) are the most important class of innate pattern recognition receptors (PRRs) by which host immune and non-immune cells are able to recognize pathogen-associated molecular patterns (PAMPs). Most mammalian species have 10 to 15 types of TLRs. TLRs are believed to function as homo- or hetero-dimers. TLR2, which plays a crucial role in recognizing PAMPs fromStaphylococcus aureus, forms heterodimers with TLR1 or TLR6 and each dimer has a different ligand specificity. Staphylococcal lipoproteins, Panton-Valentine toxin and Phenol Soluble Modulins have been identified as potent TLR2 ligands. Conversely, the ligand function attributed to peptidoglycan and LTA remains controversial. TLR2 uses a MyD88-dependent signaling pathway that results in NF-κB translocation into the nucleus and activation of the expression of pro-inflammatory cytokine genes. Recognition rouses both an inflammatory response, culminating in the phagocytosis of bacteria, and an adaptive immune response, with the presentation of resulting bacterial compounds to T cells. Here, recent advances on the recognition ofS. aureus by TLRs are presented and discussed, as well as the new therapeutic opportunities deriving from this new knowledge.