Propiedades importantes de anillos de Galois

Secretin monomers are translocated to the periplasm via the SecBA-SecYEG system. However, the subsequent folding and targeting pathway is not well understood and varies between secretins. Some secretins are localised to the OM with the help of an OM lipoprotein, or pilotin, which interacts with a dedicated pilotin-binding domain at the C-terminus of the secretin. Representative secretin-pilotin pairs from T2SS and T3SS, whose targeting has been studied in some detail, are PulD-PulS and InvG-InvH (Crago & Koronakis, 1998, Guilvout et al., 2006). Some secretins, like HxcQ (T2SS), BfpB and TcpQ of the type 4 pilus assembly system, contain a lipoprotein-like domain and they are targeted to the OM by fatty acylation – therefore they contain an “intramolecular” pilotin. However, secretins BfpB and TcpQ require additional partner proteins for OM localisation (Viarre et al., 2009). Removal of the pilotin, pilotin-binding-domain, or lipid-attachment motifs, all result in the mislocalisation

of secretins to the IM. In contrast to other pilotin-dependent secretins, PulD is degraded in the absence of its cognate pilotin, PulS. Altogether, published work on several secretins from different secretion systems points to a basic common domain architecture and varied requirements for additional folding, multimerisation, membrane insertion and OM targeting domains and accessory factors.

Pilotin-dependent secretins are probably shuttled to the OM via the Lol lipoprotein-sorting pathway (Collin et al., 2011, Okon et al., 2008). Given that multimerisation of InvG and HxcQ is not affected by the absence of the pilotin or fatty-acylation, these localisation factors are likely to have no role in the formation of the multimer. However, it has been suggested for N. meningitidis

PilQ secretin that the Bam complex and additional cognate lipoproteins (besides pilotins) encoded by the respective secretion system gene clusters, may have a function in multimerisation or stabilisation of the multimer. Yet, this is the only known case of a secretin requiring the Bam complex (Carbonnelle et al., 2005, Collin et al., 2007, Volokhina et al., 2009, Voulhoux et al., 2003). In contrast to the secretins discussed above, that require accessory factors or domains for OM targeting and/or multimerisation, the pIV secretin of the FFSS has no known localisation factors and membrane fractionation shows the localisation of pIV to both the IM and OM (Russel & Kazmierczak, 1993). Similarly, V. cholerae T2SS secretin, EpsD, appeared not to require additional localisation proteins. However, a chromosomally-encoded pilotin, AspS, has recently been identified (Dunstan et al., 2013).

Even though pilotins efficiently target their cognate secretins to the OM, depending on the secretin under investigation, there is always a variable IM- associated pool of secretin multimers (Crago & Koronakis, 1998, Daefler et al., 1997a), suggestive that pilotins sequester secretins, decreasing their IM association, rather than acting as chaperones. The exception is PulS, which protects the PulD secretin from proteolysis in the periplasm. In the absence of cognate pilotins, secretins that are dependent upon them are mislocalised entirely to the IM (Crago & Koronakis, 1998, Daefler & Russel, 1998, Guilvout et al., 2006). Fusing the pilotin-binding domain of PulD (PulDPBD) to a self-targeted

secretin, pIV, and concurrently expressing the pilotin, PulS, together with pIV−PulDPBD allows efficient targeting to the OM. Furthermore, proteolysis is

mediated by the PulDPBD domain, and interaction of PulS with pIV-PulDPBD

prevents degradation, independent of PulS lipidation and of OM targeting, as long as the interaction is maintained (Carbonnelle et al., 2005, Daefler et al., 1997a, Koo et al., 2008). Together, these findings suggest a model where secretin monomers are sequestered by their lipopeptide pilotin, or localisation factor, before they insert into the IM. Proteolysis of unsequestered secretins secures the OM targeting pathway and at the same time protects the host from the formation of potentially toxic secretin channels in the IM.

Whether secretins are transported to the OM as monomers or, multimers, ready for insertion remains to be revealed. The sheer size of secretin multimers (up to 1500 kDa) would make it exceedingly difficult to pass through the murein layer, even if a cognate transport protein exists. Substitution of pIV residue P375

(Russel, 1994) prevents efficient multimerisation of pIV. However, these substitution mutants were still found associated with both the IM and OM fractions, suggesting that secretins cross the periplasm as monomers and association of monomers with membranes could precede multimerisation and insertion (Russel, 1994). This observation poses the question of causality between multimerisation and membrane insertion of self-targeting secretins.

From these observations a model can be proposed, whereby OM biogenesis of secretins occurs by co-targeting the secretin monomers associated with a lipoprotein to the OM, followed by multimerisation and insertion into the membrane. Alternatively, lipoproteins may not act as pilots; rather they only prevent premature multimerisation/insertion by titrating monomers away from the IM. OM targeting could be a consequence of the OM lipoprotein itself being targeted to the OM by the Lol pathway. The involvement of the Lol lipoprotein- sorting pathway in targeting the secretin to the OM has yet to be confirmed.