Secretion of hemolysin A by E. coli, described four decades ago, was the first protein secretion system discovered in Gram-negative bacteria (diderm bacteria; Goebel and Hedgpeth, 1982). Since then, eight other protein secretion pathways have been characterized in these prokaryotes, which have a cell envelope consisting of the inner membrane (IM) and the outer membrane (OM) separated by the periplasm. They are now referred to as type x secretion systems (T1SS–T9SS; reviewed in Abdallah et al., 2007; Gerlach and Hensel, 2007; Remaut et al., 2008; Desvaux et al., 2009; Goyal et al., 2014; Costa et al., 2015; Abby et al., 2016). Secretion systems in diderm bacteria are considered gateways through the OM that transport cargo with the help of either dedicated IM and periplasmic proteins or the Sec, Tat, and holins systems that first transport cargo to the periplasm. In fact, the Sec, Tat, and holins pathways, which transport proteins across the cytoplasmic membrane, are universal among bacteria, eukaryotes, and even archaea (Hutcheon and Bolhuis, 2003; Denks et al., 2014; Berks, 2015; Saier and Reddy, 2015). Therefore, secretion may be either a single-step process in which substrates (proteins or DNA) are translocated through a designated cell envelope-spanning structure (T1SS, T3SS, T4SS, and T6SS) or a two-step process in which the substrates first cross the IM into the periplasm using the Sec/Tat/holins systems, then are directed to the OM translocon. The final destinations of secreted cargos are diverse: they may stay attached to the surface of the OM, be released into the extracellular milieu, or be injected into the cytoplasm of a target cell (Costa et al., 2015; Abby et al., 2016).

Secretion systems perform numerous physiological functions essential for cell propagation and fitness within a specific ecological niche. They facilitate nutrient acquisition, communication with the environment, attachment to various surfaces, defense against host antimicrobial systems, and delivery of virulence factors at a precise location such as a eukaryotic cell (Letoffe et al., 1994; Henke and Bassler, 2004; Gerlach and Hensel, 2007; Rondelet and Condemine, 2013; Gaytan et al., 2016; Hachani et al., 2016; Majerczyk et al., 2016). However, none of the above adaptations can be assigned solely to one type of secretion.

The presence of protein secretion systems varies among phylogenetic lineages of diderm bacteria. Proteobacteria encode the broadest range of described secretion types, whereas other clades have a strong preference for only one or two types (e.g., Fusobacteria possess only T5SS; Chlamydiae, T3SS and T5SS). The most widespread systems are T1SS and T5SS; conversely, T2SS is rarely detected outside Proteobacteria (Abby et al., 2016).

In this review, we will cover the current knowledge regarding the recently discovered type IX secretion system (T9SS), also known as the Por secretion system (PorSS) or PerioGate. T9SS is exclusively present in the Bacteroidetes phylum, in a majority of its species (62% out of 97 genomes available; Sato et al., 2010; McBride and Zhu, 2013; Abby et al., 2016).

Uncovering and characterizing this unique secretion system was a gradual process over the last two decades and originated from studies of the Gram-negative, non-motile, anaerobic bacterium Porphyromonas gingivalis. P. gingivalis is a human oral pathogen that is a major causative agent of periodontitis, and, along with two other bacteria, Tannerella forsythia and Treponema denticola, forms the so-called red complex (Hajishengallis, 2015). Besides being a key pathogen in periodontitis, P. gingivalis is implicated in many systemic illnesses such as atherosclerosis (Kebschull et al., 2010), aspiration pneumonia (Benedyk et al., 2016), rheumatoid arthritis (RA; Laugisch et al., 2016), and even cancer (Whitmore and Lamont, 2014; Gao et al., 2016).

An important initial finding was that P. gingivalis produces potent proteolytic enzymes called gingipains (Kgp, RgpA, and RgpB; discussed in more detail later in this review; Pike et al., 1994; Pavloff et al., 1995; Curtis et al., 1999). Gingipains are essential virulence factors responsible for corrupting host innate defense mechanisms (Potempa et al., 2003; Hajishengallis, 2015). They are secreted in large amounts and are mainly attached to the surface of the OM, but are also partially released in a soluble form into the extracellular milieu (Pike et al., 1994; Rangarajan et al., 1997). Because none of the genes associated with known protein secretion systems could be found in the P. gingivalis genome, it was suspected that this bacterium had developed a unique OM translocon.

The search for this novel secretion system was greatly facilitated by the observation that colonies of P. gingivalis deficient in gingipain activity lack black pigmentation while growing on blood agar plates (Figure 1; Okamoto et al., 1998; Shi et al., 1999). Colony pigmentation results from the accumulation of heme on the surface of P. gingivalis cells, a process dependent on the proteolytic activity and hemagglutinin- and heme/hemoglobin-binding activity of gingipains (Smalley et al., 1998; Sroka et al., 2001). Spontaneous white/beige mutants were occasionally observed, and this phenotype was associated with, among other things, decreased cell surface-associated proteolytic activity (McKee et al., 1988; Shah et al., 1989). The discovery of the essential role of secreted, cell-bound gingipains in heme acquisition meant that pigmentation could be used as an easy screening tool for mutations blocking gingipain secretion. Of note, as potent virulence factors, gingipains were of particular interest for elucidating the role of P. gingivalis in the development of periodontitis.

Several high-throughput transposon mutagenesis studies were performed, resulting in the characterization of various pigment-less clones. Early studies associated this phenotype with the impaired activity of trypsin-like proteases and diminished hemagglutination and heme acquisition by mutants (Hoover and Yoshimura, 1994; Genco et al., 1995). Later investigations found aberrations in polysaccharide synthesis and disruption of kgp (one of the gingipains; Simpson et al., 1999; Chen et al., 2000; Abaibou et al., 2001; Shoji et al., 2002). Finally, Sato et al. (2005) identified in their transposon study porT (PG0751/PGN_0778), the first gene encoding a protein involved in the secretion of gingipains. Their mutated, non-pigmented strain had impaired gingipain activity. Moreover, gingipains accumulated in the periplasm as enzymatically inactive proenzymes instead of being exported outside the cell. A database search (BLASTP) found that PorT is present only in some species of the Bacteroidetes phylum, such as Porphyromonas gingivalis, Cytophaga hutchinsonii, and Prevotella intermedia, and absent from many other phylum proteomes like Bacteroides thetaiotaomicron and Bacteroides fragilis (Sato et al., 2005). Two years later, another gene, sov (PG0809/PGN_0832), was implicated in the secretion of gingipains, showing a mutation phenotype identical to the one observed for the porT mutation (Saiki and Konishi, 2007).

Finally, the 2010 comparison of the porT-positive proteomes/genomes of C. hutchinsonii and P. gingivalis with the porT-negative species B. thetaiotaomicron resulted in a list of 55 genes (in addition to porT) potentially involved in the secretion mechanism. Subsequent isogenic mutagenesis of all selected genes resulted in the identification of 11 genes (including porT and sov) associated with gingipain transport across the OM and gingipain activation. Because these proteins do not have sequence similarity to components of any other known secretion system, it was assumed to be a novel secretion system and was originally called the Por secretion system (PorSS; Sato et al., 2010; Nakayama, 2015). To be consistent with the existing nomenclature of secretion systems in diderm bacteria, the system was later designated the type IX secretion system or T9SS.

The comparative analysis of genomes carried out in a search for porT homologs revealed that T9SS is exclusively present in the Bacteroidetes phylum (Sato et al., 2005). Numerous studies on P. gingivalis show that T9SS is involved in virulence factor secretion, which damages human tissues and dysregulates immune responses (Potempa et al., 2003; Yoshimura et al., 2008; Sato et al., 2013; Bielecka et al., 2014; Taguchi et al., 2015). In addition, T. forsythia and Prevotella intermedia (another oral pathogenic bacteria) use this secretion pathway to disseminate their effector proteins (Nguyen et al., 2007; Veith et al., 2013; Narita et al., 2014; Tomek et al., 2014; Ksiazek et al., 2015b). Consequently, it is plausible that more pathogens from the Bacteroidetes phylum carrying porT homologs are utilizing this mechanism for virulence factor secretion. Although no experimental data are available to support this, it is likely that T9SS is a molecular weapon aimed at various host cells, similar to many other secretion systems (especially T3SS and T6SS).

Among Bacteroidetes' porT-positive species, there are many non-pathogenic environmental microorganisms such as C. hutchinsonii and F. johnsoniae. Both bacteria are aerobes ubiquitously distributed in soil and are capable of digesting macromolecules such as cellulose and chitin, respectively (Stanier, 1942, 1947). They are motile microorganisms that use a movement mechanism called gliding motility (Jarrell and McBride, 2008; Nakane et al., 2013). Surprisingly, the core T9SS genes are a subset of those necessary for gliding (gldK: ortholog of P. gingivalis porK, gldL/porL, gldM/porM, gldN/porN, sprA/sov, sprE/porW, and sprT/porT; Sato et al., 2010; McBride and Zhu, 2013; Shrivastava et al., 2013; McBride and Nakane, 2015). Moreover, secretion of chitinase and cellulase requires T9SS, meaning the system functions as a non-invasive tool used for movement and food acquisition in these bacteria (Kharade and McBride, 2014; Zhu and McBride, 2014; Yang et al., 2016).

The detailed mechanisms and regulation of T9SS in gliding motility and food scavenging are still under investigation and may reveal additional functions (even in non-gliding species).

Presently, 18 genes from a total of 29 candidates have been proven essential for proper T9SS function in P. gingivalis by deletion mutagenesis studies (Heath et al., 2016). Deletion of any of these genes results in the white pigmentation phenotype and accumulation of cargos (e.g., gingipains) in the periplasm. Some of these proteins build the core structures in the IM and OM, some play regulatory or accessory roles, and others are involved in post-translationally modifying cargo proteins (Table 1). Many aspects of their functions have yet to be discovered.

Genes encoding T9SS components are scattered around the P. gingivalis genome. The exception is a group of five genes, porP-porK-porL-porM-porN, that are co-transcribed (Vincent et al., 2016). In many other Bacteroidetes species, the operon structure of these genes is conserved [databases: STRING (Snel et al., 2000), DOOR (Dam et al., 2007; Mao et al., 2009), ProOpDB (Taboada et al., 2012), OperonDB (Pertea et al., 2009)]. Orthologs of the porP gene (sprP in some gliding motility bacteria) show the most variation, as the gene can be located in different genomic loci (e.g., F. johnsoniae Fjoh_3477 vs. gldK/Fjoh_1853), and, even if they precede porK, they remain as separate transcriptional units (e.g., C. hutchinsonii sprP/CHU_0170 and gldK/CHU_0171; Zhu and McBride, 2014). The rest of the P. gingivalis T9SS genes are either single units or predicted to be in 2–5 gene operons (Figure 2) with genes unrelated to T9SS structure and function. In addition, none of the adjacent genes encode T9SS cargo proteins.

Protein secretion using T9SS is a two-step process. First, the cargo proteins are guided by a classical signal peptide to the Sec machinery in the IM. During translocation, the signal peptide is cleaved off by type I signal peptidase, and the cargo is released into the periplasm. Although, the Sec pathway has not been experimentally analyzed in P. gingivalis, the screening of Bacteroidetes genomes confirmed that the system is mostly conserved (McBride and Zhu, 2013). In the periplasm, transported proteins fold into a stable conformation, as indicated from the accumulation of their soluble forms in the periplasm of T9SS secretory mutants. Whether the cargo proteins require a chaperone(s) to assist in folding and/or guiding them to the OM translocon is still unknown.

A common feature of all T9SS cargo proteins is the conserved CTD that targets T9SS cargo proteins to the OM translocon. The function of the CTD was first recognized while studying the secretion and processing of the RgpB (PG0506/PGN_1466) gingipain. The protein without the C-terminal Ig-like domain of 72 amino acid residues was not secreted, but accumulated in the periplasm of the mutated P. gingivalis strain in its truncated form (Seers et al., 2006). A parallel study confirmed this observation, showing that the integrity of the CTD is essential for RgpB secretion, as even truncating the C-terminal by two residues hinders transport across the OM. The same effect is caused by mutating the highly conserved residues at the C-terminus of the CTD (Nguyen et al., 2007). The elegant follow-up investigations with CTDs from different P. gingivalis T9SS cargo proteins (HBP35/PG0616/PGN_0659, CPG70/PG0232/PGN_0335, P27/PG1795/no PGN, and RgpB) genetically fused to GFP found that GFP was secreted and post-translationally modified by P. gingivalis in the same way as the native T9SS cargos. The secretion/modification signal was narrowed down to the last 22 residues of the CTD domain (Shoji et al., 2011), and proteomic analysis revealed cleaved CTDs in the culture medium (Veith et al., 2013).

Taken together, these findings suggested the existence of a C-terminal-sorting peptidase responsible for the proteolytic removal of the CTD during the cargos' translocation across the OM. The postulated sortase was identified in P. gingivalis as PorU, a surface-located cysteine peptidase that shares significant sequence similarity with gingipains (see previous section; Glew et al., 2012). Analysis of the cleavage sites of T9SS cargos in P. gingivalis revealed a PorU preference toward polar or acidic amino acid residues (Ser, Thr, Asn, Asp) at the carbonyl site (P1′ position) and small amino acid residues (such as Gly, Ser, Ala) at the amide site (P1 position; Glew et al., 2012; Veith et al., 2013). This low specificity of PorU was confirmed when the amino acids surrounding the cleavage site (P1–P1′) in RgpB were mutated. Of note, this did not affect the secretion of the gingipain (Zhou et al., 2013).

Essential T9SS genes, including porT, porV, sov, porP, porK, porL, porM, and porN, are regulated at the transcriptional level by a signaling pathway composed of the PorXY two-component system (TCS) and an extracytoplasmic function (ECF) sigma factor (SigP/PG0162/PGN_0274; Kadowaki et al., 2016). In contrast to the majority of TCSs, in which the components are encoded within the same operon, the porX and porY genes occur at separate loci within the P. gingivalis chromosome. Despite this unusual genomic organization, the activation of the PorXY TCS is canonical. PorY has a modular architecture typical for a histidine kinase (HK) and undergoes autophosphorylation at His193, as shown by radiolabeled [³²P-γ]ATP. The phosphate group is then transferred to the conserved Asp58 residue in the receiver domain of PorX, which functions as the response regulator (RR). To compensate for the lack of a DNA-binding domain in the RR, PorX interacts with SigP, which directly binds the promotor regions of T9SS genes. The SigP protein level is very low in the porX-deletion mutant, suggesting a stabilizing function for PorX on SigP (Kadowaki et al., 2016). Disruption of the PorXY TCS results in the dysfunction of T9SS, which manifests as the decrease of Rgp and Kgp activity, as well as the impaired processing of gingipains (Sato et al., 2010).

PorX can also modulate the T9SS architecture directly by interacting with the cytoplasmic domain of PorL (Vincent et al., 2016). The N-terminal domain of PorX is similar to RRs belonging to the CheY family, which are involved in chemotaxis. After phosphorylation, the CheY protein binds to the C-ring of flagella, which changes the direction of flagellar movement (Roman et al., 1992; Sagi et al., 2003). Due to the fact that T9SS was proposed to be a rotary apparatus enabling the rotary movement of SprB adhesin in gliding bacteria (Shrivastava et al., 2015), it has been speculated that the PorX mechanism might be similar to that of CheY (Vincent et al., 2016). However, its role in P. gingivalis cells will likely be different as this bacterium is non-motile.

There are other studies reporting the changes in a T9SS protein's expression profile under specific circumstances. In a PorZ-deletion strain, some of the T9SS genes (including porT, porV, and porN), together with genes encoding CTD-cargo peptidases (RgpB, Kgp, and CPG70), are upregulated, whereas the expression of other T9SS genes (such as porQ, porW, sov, and porU) is not changed (Lasica et al., 2016). Additionally, the gliding motility protein GldN (orthologous of P. gingivalis PorN) of Flavobacterium psychrophilum is significantly upregulated under iron-limited growth conditions and in vivo (LaFrentz et al., 2009). The expression of T9SS proteins must be strictly regulated to fine-tune the energy-absorbing secretion of proteins into the environment. However, a precise environmental signal has not been identified and our knowledge about T9SS regulation is still limited.

Only a few secretion systems are dedicated to carrying a single cargo protein; examples are HlyA in E. coli and HasA in S. marcescens for T1SS (Kanonenberg et al., 2013), and PulA in K. oxytoca and LT toxin in E. coli for T2SS (Rondelet and Condemine, 2013). The majority of secretion systems translocate many proteins of similar or diverse functions [e.g., T3SS; Gaytan et al., 2016]. In many respects, T9SS is one of the most robust secretion systems, which, in P. gingivalis alone, facilitates secretion of up to 35 cargos bearing the CTD (see Table 2), many of which are implicated in bacterial pathogenicity. In fact, experiments conducted to characterize the important virulence factors (the gingipains RgpA, RgpB, and Kgp) contributed to the discovery of T9SS. Below, we briefly describe only the most important cargos from the point of view of P. gingivalis virulence. References to other cargo proteins can be found in Table 2.

The mechanism of protein secretion by T9SS was mostly studied in P. gingivalis and gliding bacteria. Apart from a different subset of secreted proteins reflecting bacterial habitats, the mechanism of action is the same. Briefly, T9SS cargo proteins are directed to the T9SS machinery by the CTD, which is removed during secretion. Then, secreted proteins may be modified and attached to the surface by A-LPS (P. gingivalis), stay associated with the cell through polysaccharides, or be released (gliding bacteria; McBride and Nakane, 2015; Nakayama, 2015). However, analysis of T9SS in another member of the red complex, T. forsythia, revealed some interesting differences.

T. forsythia is covered with a two-dimensional crystalline surface (S-) layer that is thought to function as a protective coat, working as an external sieve and ion trap (Sleytr and Beveridge, 1999; Messner et al., 2010). It also mediates adhesion and subsequent invasion into human gingival epithelial cells (Sakakibara et al., 2007) and delays recognition of the bacterium by the host innate immune system (Sekot et al., 2012). The S-layer is composed of the glycosylated proteins TfsA (Tanf_03370) and TfsB (Tanf_03375). Deleting porU (Tanf_02580), porT (Tanf_10520), sov (Tanf_04410), or porK (Tanf_02360) results in the lack of an S-layer, which can be observed by transmission electron microscope (Narita et al., 2014; Tomek et al., 2014). In those mutants, both components of the S-layer are trapped within the periplasm, but, unlike in P. gingivalis, they are modified by O-glycosylation through the addition of multiple copies of a complex oligosaccharide using a general glycosylation pathway operating in Bacteroidetes (Coyne et al., 2013; Posch et al., 2013; Tomek et al., 2014). Nevertheless, TfsA and TfsB trapped in the periplasm are much smaller than both proteins in the wild-type cells, indicating that, upon secretion, both proteins are modified by a second glycan attachment in a manner different than O-glycosylation. It is speculated that, as in P. gingivalis, it could be a variant of LPS (Tomek et al., 2014).

T9SS cargo proteins in T. forsythia have two different types of CTD. The “classical” CTD associated with proteins from other Bacteroidetes species is found in TfsA, TfsB, and leucine rich protein BspA (Veith et al., 2009; Tomek et al., 2014). By contrast, a family of six proteases, three metalloproteases (karilysin, mirolysin, and forsilysin) and three serine proteases (mirolase, miropsin-1, and miropsin-2), bear a nearly identical CTD that shares very limited sequence similarity with the classical CTD. Because these six CTDs end with a KLIKK sequential motif, the enzymes are referred to as KLIKK proteases (Ksiazek et al., 2015b). The KLIKK proteases possess a unique structure and undergo extensive autoproteolytic processing (Cerda-Costa et al., 2011; Lopez-Pelegrin et al., 2015). Their activities, such as degrading complement proteins and LL-37 (the crucial antimicrobial peptide in the human oral cavity), may contribute to T. forsythia virulence through evading innate immunity (Jusko et al., 2015; Koneru et al., 2017).

In stark contrast to the other CTD proteins of T. forsythia, KLIKK proteases seem to be secreted directly into the extracellular medium, as shown for miropsin-2 (Tanf_06530), karilysin (Tanf_06550), and forsilysin (Tanf_06225) (Narita et al., 2014). Supporting this, proteomic analysis of the T. forsythia OM identified 13 of 26 proteins bearing the classical CTD, including TfsA, TfsB, and BspA (Tanf_04820), but none of the KLIKK proteases (Veith et al., 2009). Conversely, four KLIKK proteases, forsilysin, miropsin-2 (Friedrich et al., 2015), mirolase (Tanf_00440), and karilysin (Veith et al., 2015), were found in outer membrane vesicles (OMVs), although with a low Mascot score. This discrepancy could be explained by the transient presence of these proteases in the periplasm before they enter the OM translocon of T9SS. Interestingly, all three of the KLIKK proteases characterized thus far (karilysin, mirolase, and mirolysin) can remove the CTD during autoprocessing (Karim et al., 2010; Ksiazek et al., 2015a; Koneru et al., 2017). Collectively, the available data suggest that the KLIKK proteases are secreted into the extracellular milieu without removal of the CTD. This finding is similar to the secretion of PorU and PorZ from P. gingivalis, where the CTD is also not removed during secretion, although proteins stay associated with the cell surface (Lasica et al., 2016).

In this review, we summarized the biochemical and structural data concerning the recently discovered T9SS identified in a majority of the bacterial species belonging to the Bacteroidetes phylum (Sato et al., 2010; McBride and Zhu, 2013). The system has been investigated predominantly in human oral pathogens, such as P. gingivalis and T. forsythia, and environmental saprophytes, such as F. johnsoniae and C. hutchinsonii. It seems to be a major mechanism of protein secretion in these bacteria however, some families from Bacteroidetes were reported to possess other secretion systems e.g., T1SS or T6SS (Russell et al., 2014; Wilson et al., 2015; Abby et al., 2016; Chatzidaki-Livanis et al., 2016; Wexler et al., 2016; Ibrahim et al., 2017). Notably, both systems allow for direct substrate translocation from bacterial cytoplasm to the cell exterior, while T9SS cargos do not omit the periplasmic space during their secretion.

The role of T9SS is to ensure cell survival and fitness in response to the microorganisms' habitat by providing transportation of proteins necessary for, among other things, virulence, nutrition, and movement (gliding motility). Hence, the variety of secreted proteins even within a single species is large and comprises numerous adhesins and hydrolytic enzymes used for attachment and degradation of large organic compounds such as proteins, cellulose, and chitin (Guo et al., 2010; McBride and Nakane, 2015).

The cargo proteins of this system (Table 2) are equipped with the classical signal peptide for Sec-dependent translocation to the IM and the conserved CTD that directs them further to the secretion machinery in the OM. The recognition signal is mostly embedded within the IgSF-like tertiary structure of the CTD (de Diego et al., 2016; Lasica et al., 2016) and likely located within the 22 amino acid residues composing the sequential motifs of PxGxYVV and KxxxK in the two most C-terminal β-strands (Shoji et al., 2011; Veith et al., 2013).

Currently, for P. gingivalis cells, there are 16 proteins recognized as the structural and/or functional components of the translocon and two additional elements involved in T9SS regulation (Table 1). None of these proteins are fully characterized, so their structure, mode of reciprocal interactions, and precise roles in secretion are still obscure. Nevertheless, a contemporary general concept of T9SS structure and function based on available data is presented in Figure 3. Verification of this model requires extensive structural and functional investigations to elucidate the mechanism of CTD recognition and cleavage, passage of cargos though the OM translocon, attachment of a glucan moiety, and anchoring of cargos onto the cell surface, their release into the environment, or their assembly into gliding motility machinery.

AL analyzed literature, wrote the paper (excluding MK and MM sections), prepared the figures and tables. MK wrote the Mechanism of secretion and T9SS T. forsythia sections. MM wrote the Regulation section. JP edited the manuscript. All authors read and approved the full manuscript.