Gingipains from Porphyromonas gingivalis drive Alzheimer’s pathology and can be blocked with small-molecule inhibitors.

Objective. To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA).Methods. The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and ␣-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry.Results. Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or ␣-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry.Conclusion. Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalismediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.Rheumatoid arthritis (RA) is characterized by disease-specific autoimmunity to citrullinated proteins. Citrullination is a posttranslational modification of arginine residues that is mediated by the family of peptidylarginine deiminases (PADs). Citrullinated fibrin(ogen) and ␣-enolase are 2 of the physiologic proteins that are targeted by anti-citrullinated protein antibodies in RA (1-5). Fibrinogen is the precursor of fibrin, and autoanMs. Wegner and Drs.

Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time-and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63. IMPORTANCE ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far.Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.