The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function

Protein secretion systems are vital for prokaryotic life, as they enable bacteria to acquire nutrients, communicate with other species, defend against biological and chemical agents, and facilitate disease through the delivery of virulence factors. In this review, we will focus on the recently discovered type IX secretion system (T9SS), a complex translocon found only in some species of the Bacteroidetes phylum. T9SS plays two roles, depending on the lifestyle of the bacteria. It provides either a means of movement (called gliding motility) for peace-loving environmental bacteria or a weapon for pathogens. The best-studied members of these two groups are Flavobacterium johnsoniae, a commensal microorganism often found in water and soil, and Porphyromonas gingivalis, a human oral pathogen that is a major causative agent of periodontitis. In P. gingivalis and some other periodontopathogens, T9SS translocates proteins, especially virulence factors, across the outer membrane (OM). Proteins destined for secretion bear a conserved C-terminal domain (CTD) that directs the cargo to the OM translocon. At least 18 proteins are involved in this still enigmatic process, with some engaged in the post-translational modification of T9SS cargo proteins. Upon translocation across the OM, the CTD is removed by a protease with sortase-like activity and an anionic LPS is attached to the newly formed C-terminus. As a result, a cargo protein could be secreted into the extracellular milieu or covalently attached to the bacterial surface. T9SS is regulated by a two-component system; however, the precise environmental signal that triggers it has not been identified. Exploring unknown systems contributing to bacterial virulence is exciting, as it may eventually lead to new therapeutic strategies. During the past decade, the major components of T9SS were identified, as well as hints suggesting the possible mechanism of action. In addition, the list of characterized cargo proteins is constantly growing. The actual structure of the translocon, situated in the OM of bacteria, remains the least explored area; however, new technical approaches and increasing scientific attention have resulted in a growing body of data. Therefore, we present a compact up-to-date review of this topic

Structure-based mechanism of cysteine-switch latency and of catalysis by pappalysin-family metallopeptidases

Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54–Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Å revealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1′. Moreover, residue Cys23 within a conserved cysteine–glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Å structure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1′ to S6′. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms.This study was supported in part by grants from Spanish, Catalan, US American (NIH/NIDR) and Polish (NCN) public agencies (BFU2015-64487R; MDM-2014-0435; Fundacio´ ‘La Marato´ de TV3’ 201815 and 2017SGR3, 2015/17/B/NZ1/ 00666, 2016/21/B/NZ1/00292, and R21DE026280). MK was recipient of a scholarship from the Polish Ministry of Science and Higher Education (1306/MOB/IV/2015/0, ‘Mobilnoc´ Plus’). The Structural Biology Unit of IBMB was a ‘Marı´a de Maeztu’ Unit of Excellence of the Spanish Ministry of Science, Innovation and Universities (2015–2019)

Development of a novel, high-affinity ssDNA trypsin inhibitor

Inhibitors of serine proteases are not only extremely useful in the basic research but are also applied extensively in clinical settings. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach we developed a family of novel, single-stranded DNA aptamers capable of specific trypsin inhibition. Our most potent candidate (T24) and its short version (T59) were thoroughly characterised in terms of efficacy. T24 and T59 efficiently inhibited bovine trypsin with Ki of 176 nM and 475 nM, respectively. Interestingly, in contrast to the majority of known trypsin inhibitors, the selected aptamers have superior specificity and did not interact with porcine trypsin or any human proteases tested. These included plasmin and thrombin characterised by trypsin-like substrate specificity. Our results demonstrate that SELEX may be successfully employed in the development of potent and specific DNA based protease inhibitors

Proteolytic Inactivation of LL-37 by Karilysin, a Novel Virulence Mechanism of Tannerella forsythia

Tannerella forsythia is a gram-negative bacterium strongly associated with the development and/or progression of periodontal disease. Here, we have shown that a newly characterized matrix metalloprotease-like enzyme, referred to as karilysin, efficiently cleaved the antimicrobial peptide LL-37, significantly reducing its bactericidal activity. This may contribute to the resistance of T. forsythia to the antibacterial activity of LL-37, since their vitality was found not to be affected by LL-37 at concentrations up to 2.2 μM. Furthermore, proteolysis of LL-37 by karilysin not only abolished its ability to bind lipopolysaccharide (LPS) to quench endotoxin-induced proinflammatory activity, but LL-37 cleavage also caused the release of active endotoxin from the LPS/LL-37 complex. Proteolytic inactivation of LL-37 bactericidal activity by karilysin may protect LL-37-sensitive species in the subgingival plaque and maintain the local inflammatory reaction driven by LPS from gram-negative bacteria. Consequently, the karilysin protease may directly contribute to periodontal tissue damage and the development and/or progression of chronic periodontitis

The tetrapeptide displays a Ki value similar to that of the peptide 15 lead structure.

<p>Shown are chemdraw structure models of the peptide versions 15-0, 15-5, 15-6 and 15-7. All truncated versions of peptide15 and intact peptide15 had low micromolar Ki values.</p

Phage ELISA test of nineteen selected clones after four panning rounds, peptide sequences and estimated apparent afffinity.

<p>A) Optical density responses detected after the binding of peptide-phage to immobilized Kly18 are shown. Background binding to BSA has been subtracted for each clone. Clones with signals above 0.5 were deemed positive and further sequenced. B) Peptide-phage clones were tested in inhibition ELISA for estimation of affinity. All clones (except clone 13) shared the WXP motif.</p

Peptide15 inhibits the proteolytic activity of Kly18.

<p>Peptide15 was pre-incubated with Kly18 in varying concentrations followed by addition of the substrate FITC-casein. The peptide15 inhibitory effect was seen as a decrease in Relative Fluorescence Units (RFU) in a dose-response manner. Error bars represent standard deviations between three experiments.</p

Peptide15 displays the characteristics of a competitive inhibitor.

<p>Shown are 1/V-1/S plot of peptide15 inhibition of Kly18. Kly18 was pre-incubated with fixed concentrations of peptide15 (10 µM and 30 µM) for 30 minutes, followed by incubation with varying concentrations of FITC-casein (5–50 µg/ml). As seen from the Lineweaver-Burke plot the Y-intercept is approximately the same for un-inhibited Kly18 as Kly18 inhibited with 10 µM and 30 µM, respectively. This suggested that peptide15 was a competitive inhibitor of Kly18. Linear regression lines were made using SigmaPlot 11.0. Data represent mean values from three experiments per substrate concentration.</p