Pseudomonas aeruginosa Major Pseudopilin XcpT is Incorporated into The Type IV Pilus Under Native Conditions

Abstract

<p>Retractable surface appendages Type IV pili (T4P) are one of the major virulence determinants in the opportunistic pathogen <em>Pseudomonas aeruginosa </em>(Pa), that is the leading cause of mortality in CF patients. T4P are heteropolymers composed of the major-pilin subunit PilA and the less-abundant minor pilins (MPs), FimU/PilV/W/X/E. Pilins share high sequence and structural similarity with pseudopilins (XcpT/U/V/W/X), that are proposed to form a periplasmic-structure in the evolutionarily related Type II secretion system (T2SS). Similar to T4P system, the T2SS is a multi-subunit complex that spans the inner (IM) and the outer (OM) membranes. It involves a two-step process facilitating the secretion of toxins into the extracellular milieu from the periplasm.</p> <p>Using immunogold TEM analysis and Western blot we identified, under native conditions, the major pseudopilin of T2SS XcpT, is incorporated into the T4P appendage, thus appearing on the surface. This is in contrast to previous studies reporting, the otherwise periplasmic structure, the pseudopilus appears on the surface only upon over-expression of XcpT. Further, we identified this incorporation is strictly dependent on PilA expression, such that levels of surface-XcpT co-varied with the levels of surface-PilA. However, XcpT incorporation into the T4P fiber did not affect T4P-mediated twitching motility or T2SS-mediated elastase secretion. Based on these observations we proposed two explanations. Firstly, given the similarity between XcpT and type IV pilins, it is possible the pseudopilin is recognized by the T4P machinery and therefore is incorporated into the pilus. Secondly, since XcpT incorporation does not affect T4P-mediated motility, it may affect other properties of T4P, such adherence during biofilm formation, previously associated with surface-exposed pseudopilus. In addition, we also identified enhanced expression of <em>fimU</em> and <em>pilX</em> MPs drastically increased elastase secretion, through a yet to be discovered mechanism. Regardless, our results present an alternative role of both minor pilins and XcpT in their non-native systems suggesting there is more overlap between the T4P and T2S systems than previously appreciated. Further exploration of this overlap will aid in the study of the two systems in Pa, as well as in other pathogens.</p>