XcpX Controls Biogenesis of the Pseudomonas aeruginosa XcpT-containing Pseudopilus

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that is involved in severe nosocomial infections and is also a key agent in the early death of patients suffering from cystic fibrosis (1Govan J.R. Deretic V. Microbiol. Rev. 1996; 60: 539-574Crossref PubMed Google Scholar). Remarkably, P. aeruginosa possesses several secretion systems that achieve the extracellular release of a wide variety of secreted toxins and enzymes, which are essential virulence factors for the bacterial pathogenesis. Among them, the most prolific pathway is the type II secretion system (2Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (223) Google Scholar), which is used by proteins as diverse as elastase, lipase, phospholipases, or exotoxin A. This pathway follows a two-step process, in which the type II-dependent exoproteins are the the or the A. A. PubMed Scopus Google Scholar). The protein is the by a called the secreton (2Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (223) Google Scholar). P. aeruginosa two different type II systems as the Xcp used extracellular to required for the secretion of exotoxin or (2Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (223) Google and the involved in the secretion of the molecular A. A. Microbiol. PubMed Scopus Google Scholar). systems are Gram-negative P. the type II are of and a called in P. aeruginosa (2Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (223) Google Scholar), in systems as as in type IV pili This is a which is for the of and and Microbiol. 1996; PubMed Scopus Google 1996; PubMed Scopus Google or XcpT-X and A. A. Microbiol. PubMed Scopus Google A. A. Microbiol. PubMed Scopus Google PubMed Google A. A. Microbiol. PubMed Scopus Google Scholar). The results in the of a the the pilin is assembled in called the type IV pili A. PubMed Scopus Google Scholar). components of the type II secretion share with proteins involved in the type IV are the and Microbiol. PubMed Scopus Google Scholar), the and A. PubMed Scopus Google Scholar), and the proteins and The when several that the of the type II secretion systems could be assembled in multifibrillar called the type II pseudopilus P. PubMed Scopus Google P. PubMed Scopus Google A. A. A. PubMed Scopus Google Scholar). Most interestingly, the major type IV pilin have which not to be part of the structure and for which was The Xcp pseudopilins can be into one major and A. A. Microbiol. PubMed Scopus Google A. PubMed Scopus Google Scholar). Among the XcpX is A. A. Microbiol. PubMed Scopus Google Scholar). Indeed, is than and the in pseudopilins and This to a for the assembly of type IV into the structure PubMed Scopus Google Scholar). major to be in the of the type II pseudopilus, most is the of its in the secretion this we several pseudopilus We that a of the Xcp components is to the assembly of the pseudopilus. We that the to have the characteristics that the assembly in a we that the XcpX is a key in the pseudopilus We that this control can be a interaction between XcpX and XcpT. and and used in this are in and in or The and used for into P. aeruginosa using the of Pseudomonas with the the for and and for P. and The of P. aeruginosa was as previously A. A. A. PubMed Scopus Google used in this and to and or of and in A. A. A. PubMed Scopus Google of the in of the in of the in of the in of the in of the in of the in of the in of the in of the in of the in of the in of the and from to part of the in of the in of the in of the in of the in 60: PubMed Google V. PubMed Scopus Google and to and in a used in this and to and or A. PubMed Scopus Google P. PubMed Scopus Google and in Google A. PubMed Scopus Google PubMed Scopus Google the with the into the of the and the the with the into the of the and the the into the with the the into the with the the into the with the for the of XcpT with a to the into the for the of XcpX with a to the into the the into the the A. A. A. PubMed Scopus Google the into the the into the the into the the into the the into the the into the the into the the into the the into the the into the the into the from the the into the the from the the into the the into the into the into the into the into the into the into the into the into the into the into the into the into the and to and in a of the used for the of the are as the of the using as and the the of the using as and the the of the using as and the the of the using the as and the the of the using as and the the of the using as and the The the of XcpT was with each of the the of the pseudopilins and The by using and the and The into the and with and into the or for of the XcpT and XcpX in was using as and the and The a XcpT. The was into the and was into the or and The and using and as and the and that we have the of the to the The into the and into the with the a with the the and the are the control of the of also a the and using or as and the and a a using the and a and with the shown that the of the in The into the and into the with the and the the the and the are the control of the of P. aeruginosa with in the and was used as to pseudopili from type IV pili and The was in this by to of XcpT. The was with of and of bacterial and in the by and the was The pseudopili by The pseudopili was in a of directly by or and and as previously A. A. A. PubMed Scopus Google Scholar). of the pseudopili was a The was in by the into as by P. Rev. Biophys. PubMed Scopus Google Scholar). The was into a with and into the using an The was equipped with a The was The the was with the PubMed Scopus Google Scholar). is for PubMed Scopus Google Scholar). and using was The into and for in a as the one previously A. A. A. PubMed Scopus Google for from the with a and into of this was a 5 for by of directly the bacterial with the with in for The with and with a of the XcpT or the in for the with a of the or for the by a of and with a a of to pseudopili was previously A. A. A. PubMed Scopus Google Scholar). used is from P. A. PubMed Scopus Google in 5 of with in a a a of the was and The bacterial by using an with a and of the or in with or or or the bacterial an of of the proteins was by for a of the The bacterial and in of and the The by a to 5 the by for from the by into a of the and was for with was by for The proteins and with proteins used for this of in and and for of was and the for the The was by for in and for and of the of the Xcp the and from and to the by used for and for and for The a an with into the and and into the The an of the with the the this the protein could be in the The called and was from the of the in The proteins called and and the to the in the of the protein from the the for XcpT or for the and in by and of the the proteins from and in of the a with by an system of the with and with the to the several with with proteins with with Xcp and and proteins using with a of 5 The for and for as by and and was by and in and used for in 5 of each protein was and in of and of of was to the was for which the was by of of each was directly a The was directly with or with the the of the The by in the and by from the or not for in in a and the of in a and the proteins with the and by using the of using the of the have shown previously P. A. PubMed Scopus Google that the P. aeruginosa type II protein secretion system is able to its major XcpT into a multifibrillar structure that we called the pseudopilus. using a P. aeruginosa for type IV pili and in to between extracellular and the The was in this to the control of the and pseudopili isolated as and and in and The bacterial from the by The pseudopili in a of the and directly used for The of the pseudopili using and P. A. PubMed Scopus Google and the of multifibrillar in is that used in can the of the and PubMed Scopus Google PubMed Google A. Google Scholar). to could be the with we used the for with by pseudopili that as with a of the showed when the fibers are are to the previously and the pseudopili to be and to have we that the pseudopili assembled by the Xcp secreton of P. aeruginosa are and that be of the used for pseudopili and We used the as to and to a for the pseudopilus By using of and into of as a was with and using a a structure could be This is the assembly of the pseudopilus that could be from A. A. A. PubMed Scopus Google and by other P. PubMed Scopus Google A. P. A. Microbiol. 2004; PubMed Scopus Google Scholar). The into the to four other pseudopilins are the Xcp which share the characteristics as XcpT and the pilin PilA. We pseudopilins XcpT or able to in a The the pseudopilins and XcpX in the control of the and The into P. aeruginosa and the to the that of XcpT of or XcpX proteins to be not XcpT is the most in the PubMed Google Scholar). We that the of the is involved in the of XcpT. We between the of the and the of the four other The of the to the to the was with the of each of the four other the The proteins the of to the by the of one of the pseudopilins and XcpX which a was The proteins and for and for the in a to in P. The different in the type and of the pseudopilins was by using the The of proteins is and are to with and We in or pseudopilins could be The different by and as The in show that in to none of the four other pseudopilins or by or assembled in a pseudopilus We that in the we none of the pseudopilins XcpT are assembled into a pseudopilus. Moreover, when the of XcpT was assembled into a pseudopilus in a that also one of the or none of proteins in the pseudopilus using and the not that pseudopilins such as are not part of an extracellular pseudopilus be that the the or of the protein secretion in the for the that proteins are of pseudopilins to form a pseudopilus. of the was a was with an of proteins from or with an the assembly of pseudopilins into a pseudopilus. of the results by and of pseudopilus assembly by the The of by and from to of the Xcp pseudopilins the as is in the The the pseudopilins are able or not to into a Xcp for II Xcp components are required for assembly of the type II pseudopilus. we have shown previously A. A. A. PubMed Scopus Google Scholar), in P. aeruginosa that of XcpT the the type II Xcp and and the type IV pili assembly system to the pseudopilus assembly the Xcp we a in which the and this the Xcp machinery is the system to XcpT We that this was in secretion and type IV a type of secretion not of the 12 was in the The 12 for the secretion by a that the as by the a not We the in 12 The in that pseudopilus and XcpT extracellular was by and pseudopilus assembly was by The results are in and in each of the Xcp components is required for not to be the for pseudopilus Indeed, the or able to be that the pseudopili II and not the the or or XcpT the assembled a pseudopilus. We that the 12 Xcp proteins required for the secretion process, a number of secreton components are essential for pseudopilus The absence of for in pseudopilus was that the for pseudopilus assembly as for secretion A. P. Microbiol. PubMed Scopus Google Scholar). Moreover, in the P. aeruginosa P. A. PubMed Scopus Google Scholar), the is in with We that the of this homology with the protein We that the could pseudopilus components essential for pseudopilus is in the as II pseudopilus indicate the of the to or not a pseudopilus is the that the of the is by the of the XcpT of pseudopilus assembly by the that pseudopilus assembly between pseudopili and The that the of could be by the assembly of the pseudopilus the for is in the as indicate the of the to or not a pseudopilus is the that the of the is by the of the XcpT of pseudopilus assembly by the that pseudopilus assembly between pseudopili and The that the of could be by the assembly of the pseudopilus the for in a to be that is between components to one of the systems and we a in which the and the have This was called this we of the or for pseudopilus assembly and as the in the Indeed, pseudopilus assembly was for the and not for the and not that and are essential components for the pseudopilus biogenesis, whereas and are the for XcpT into the involved in pseudopilus the pseudopilins could be involved in a control of its biogenesis. of the of in pseudopilus biogenesis, we that the four the is essential for pseudopilus assembly Indeed, the of the XcpT not the of pseudopili by not Moreover, the also not release of XcpT in the Conversely, results that and XcpX are not essential for pseudopilus assembly and the that XcpT could be in the and of the We that is the required for the assembly of XcpT into pseudopilus. XcpX the the of XcpT in the is with an and which a in the XcpT and an of the pseudopilus. using that the of XcpT to the assembly of to and to pseudopili as with the the of XcpX an of XcpT into we that of XcpX could with pseudopilus The was into a and in a that the We pseudopilus by and is shown in the that the release of XcpT into the extracellular was XcpX results by which showed that the extracellular of XcpT was with a in the number of assembled pseudopili not We that the of XcpX could to the control of XcpT into of with have previously that pseudopilus assembly the and of P. aeruginosa A. A. A. PubMed Scopus Google Scholar). We the number of pseudopili with the was with to for and a to form bacterial whereas the was as and a could not be with an XcpT that the is to the of the pseudopilus. interestingly, when XcpT was in an the to be in with isolated as with was with a and by such as and we could that the of XcpX results in an in pseudopilus The of XcpX for XcpX The XcpX is of a and the of the the PubMed Scopus Google that this the interaction between pilin subunits type IV We of a the of this we used an in the the was for a A. A. Microbiol. PubMed Scopus Google Scholar). for the other the the of XcpX was with the of the whereas the of a was to a protein called The was into the and in the of XcpT with in a of XcpT. Indeed, pseudopili a of in the of XcpT was as with the that XcpT and This in the of XcpT is in this as with was with the type and Moreover, with a that and we could not pseudopilus using not We that the of a in XcpX the of this and the control by XcpX XcpT and pseudopilus XcpX of that XcpT and XcpX a severe in pseudopilus assembly that XcpX with XcpT Most interestingly, we by with that the of XcpT is when XcpT and XcpX are in the XcpT could be in the of whereas is when XcpT is and Moreover, this for a of XcpT is when the form and the of XcpX was not by the of XcpT and this is when XcpX or the and that XcpX could have a XcpT. to this we the of XcpT in different in proteins from the and with the The used in a shown in XcpT is by in a is of with a of of the of XcpT is when XcpX is and of the XcpT protein is whereas is of XcpT was in the of the the of XcpT was and We that the of XcpX and XcpT in the could that to a of XcpT. XcpX with a key of XcpX in the control of pseudopilus assembly XcpT We this destabilization results from a interaction between XcpX and XcpT. this we used an in with The the and the of XcpT and XcpX by that an was and The into the and in as proteins called and and used for in using and The results in the show that whereas and molecular in the and to the as a of of each one is with with an molecular to a between and we this with control or and to a with an the we the of each The to the the molecular and the two and This was by not we a interaction between and The results here the assembly by the type II secreton of P. aeruginosa of the major XcpT into a structure that we called type II pseudopilus A. A. A. PubMed Scopus Google Scholar). We have several such as which are the required for the assembly and are the by the We that one of XcpX, controls the length and number of assembled and we that to the molecular mechanism underlying this Our results can be directly with in with the secreton of P. PubMed Scopus Google P. PubMed Scopus Google A. P. A. Microbiol. 2004; PubMed Scopus Google Scholar), also The of the P. aeruginosa have shown previously A. A. A. PubMed Scopus Google that the pseudopilus assembled by the Xcp secreton in P. aeruginosa as a multifibrillar structure that form of The secreton of also the into of P. PubMed Scopus Google P. PubMed Scopus Google Scholar). for of the pseudopili by using we that most of the pseudopili as than previously The between and be to the used for the for Indeed, is that induces that could the of such as pili A. Google Scholar). A. A. Google the by and the of to for that the the of in We that the pseudopili of P. aeruginosa are which This with the of the type II secreton (2Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (223) Google an be by one in the assembly of a extracellular pseudopilus the complex, secretion of type II is as we previously A. A. A. PubMed Scopus Google Scholar). Moreover, PubMed Scopus Google have shown that the XcpT directly with the suggesting that the pseudopilus and the are in the secretion using the secreton in of the assembly secretion P. PubMed Scopus Google P. PubMed Scopus Google Scholar). could be one that in the are to the assembly of the whereas a number of are of pseudopilus and for secretion of the in this the of secreton components was have the number of secretion secretion and to be the By in we the major XcpT in the P. in the of the XcpT in P. 12 Xcp components are essential for the secretion this we that Xcp proteins are not required for pseudopilus assembly could be as a of the components than the secretion process, and for pseudopilus assembly a P. PubMed Scopus Google have that the pseudopilins and from to and also for the assembly of the XcpT into a This that could a pseudopilus or that absence not in a or with to pseudopilus to the the secreton P. PubMed Scopus Google Scholar), we that the is not essential for the assembly of XcpT into pseudopilus is a protein A. A. 1996; PubMed Google Scholar), which with A. A. PubMed Scopus Google V. A. PubMed Scopus Google Scholar). an interaction results in a between and Moreover, that of the required the interaction with the of V. PubMed Google Scholar). the absence of we a of the and a of is for pseudopilus whereas and are not this that a of and is to pseudopilus assembly not protein secretion. is essential for protein is that the of this is not to also be to for The in the absence of a of pseudopili could be the bacterial was II and Indeed, with type IV pili from was shown that the the the by PubMed Scopus Google Scholar). and are of the Microbiol. PubMed Scopus Google Scholar), we that pseudopilus the A. P. Microbiol. PubMed Scopus Google have previously that in P. aeruginosa the can in the secretion process, and we that also for pseudopilus of the in that none of the is able to form a pseudopilus such as XcpT Moreover, that proteins are not part of the extracellular pseudopilus P. PubMed Scopus Google have shown that the not of the four other We that is the required for pseudopilus an of the to be essential for the assembly of the P. PubMed Scopus Google Scholar). could that an early such as the and is the of the pseudopilus, in with the components of the Xcp pseudopilin, XcpX, to a crucial not in pseudopilus Indeed, results showed in the absence of XcpX, pseudopilus assembly the number and length of pseudopili II and of XcpX with or pseudopili We that XcpX could control pseudopilus by the of and we to the molecular mechanism involved in this we that XcpX can be with XcpT we showed that of XcpX with XcpT the of XcpT that XcpT was to in an in when with XcpX that XcpX with XcpT and that interaction induces that XcpT and its to Remarkably, the with the type XcpX are when using the protein and We that with XcpT than XcpX, the and the destabilization of this in pili by the a assembly is the major that the extracellular and the whereas and are subunits with the PubMed Scopus Google Scholar). is to be by the of the to the in the of the and in an of multiple of the major and of of to the PubMed Scopus Google also that the interaction of the with the Indeed, the assembled pili than of the type Moreover, of in the the type pili The that control length was not Furthermore, the the in length as the in this the was by of that the was for the control of PubMed Scopus Google a in which the is to in the of with and of the This mechanism for the pili and type pili by PubMed Scopus Google Biochim. Biophys. Acta. 2004; 1694: PubMed Scopus Google Scholar). the the of XcpX or its to an of the pseudopilus assembly and or to a that XcpX have a in the of pseudopilus and also indicate that XcpX we a in which XcpX could a in the assembly of XcpT into the of P. aeruginosa or type IV pili, which are and two are required for is involved in the assembly of the major pilin into pili, and the is required for of the PubMed Scopus Google PubMed Scopus Google Scholar). The extracellular pseudopilus is XcpT The of the pseudopilus in the type II secreton not the of the 5 XcpT A. A. A. PubMed Scopus Google that the pseudopilus to protein secretion (2Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (223) Google A. A. A. PubMed Scopus Google Scholar). is to that the to and the pseudopilus not be as as for type IV of this type II secretion systems are to one the pseudopilus assembly and or be required for whereas is XcpX is a and as with XcpT. We have shown that XcpX and XcpT interact and that the to be crucial for pseudopilus the of between XcpX and XcpT is and be to pseudopilus The XcpX interaction with an XcpT in the pseudopilus its This is the type IV pili by PubMed Scopus Google Scholar). a for the in and pseudopilins to the a is between the the of a pilin assembled into the and the the of an This could interaction and The of the in XcpX of XcpT by XcpX we have the which with the pseudopilus, and with its Furthermore, by an in XcpX, we have a of pseudopilus assembly that could be by the of the the of the pseudopilus. an could be by the to an of the Moreover, we that XcpX could pseudopilus as as Indeed, we that XcpX can interact with XcpT and that this XcpT. we that XcpX could of the pseudopilus by in XcpT that its and its an and of the pseudopili subunits be an to the of type IV pseudopilus not a as is the for type IV pili PubMed Scopus Google Scholar), could the of subunits and a a in this be We for and for and for of the