KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure

Abstract

Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.

First Cultivation of Health-Associated Tannerella sp. HOT-286 (BU063)

“…HOT-286 by flow cytometry and used multiple displacement amplification to generate a collection of single cell-amplified genomes with predicted sizes from 3.44 to 4.07 Mb. Putative virulence genes of T. forsythia were detected by comparative analysis with the HOT-286 genomes and included genes encoding the PrtH, BspA, NanH, and KLIKK proteases (Beall et al 2014;Ksiazek et al 2015). Beall et al (2014) reported a surprisingly high level of strain polymorphism and substantial nucleotide divergence among the various genomes of Tannerella sp.…”

Type IX secretion system : characterization of an effector protein and an insight into the role of c-terminal domain dimeration in outer membrane translocation.

“…During translocation to the inner membrane, the SP is cleaved off and promirolase folds in the periplasm. The conserved CTD serves as a signal for transport by the T9SS [29], which moves mirolysin across the outer membrane to the extracellular milieu. During the journey through the periplasm, mirolysin is maintained as an inactive zymogen by the N-terminal profragment (NTP).…”

“…In case of T. denticola the virulence factors identified include cell surface proteins, lipooligosacharide, cytotoxic metabolites, leucine-rich repeat proteins, trypsin-like protease activity, dentilisin etc., which accentuate the periodontal disease by invasion and destruction of host tissues [25][26][27][28]. So far very few virulence factors were identified with respect to T. forsythia, among which a cysteine protease named PrtH and KLIKK proteases are the only proteolytic enzymes that are well characterized [29,30].…”

“…Among them metalloprotease karilysin, which belongs to M10A family and serine protease mirolase that belongs to S8A family, has been well characterized. Also, karilysin along with mirolysin exert a synergistic ability to evade the complement system of the host and thus contributing the survival of T. forsythia within the hostile environment of the inflamed periodontal pocket [29,[31][32][33][34][35][36][44][45][46][47]. The T9SS cargo protein (with the intact SP and CTD) from the cytoplasm is guided by the SP towards the inner membrane and is transported into the periplasm with the SP cleaved off at the IM.…”

“…Our knowledge has been increased immensely with the discovery of a novel family of six secretory proteases in T. forsythia that includes three serine proteases (mirolase, miropsin-1, and miropsin-2) and three metalloproteases (karilysin, mirolysin, and forsilysin). These proteases [29]. The domain at the C-terminus shares sequence similarity with the conserved C-terminal domain (CTD), which serves as a signal for the translocation of P. gingivalis and T. forsythia proteins across the outer membrane via a type IX secretion system (T9SS) [51,63,64] forsythi7 [45,46,65,66].…”