Xcp Proteins Research Articles

In silico prediction of putative antimicrobial targets in Xanthomonas citri pv. punicae using genome subtraction approach.

Xanthomonas citri pv. punicae (Xcp) is the causative agent of bacterial blight disease in pomegranate and severely affects its production. The current control strategies for this disease provide inadequate protection. Identifying novel bactericide target proteins in pathogenic bacteria and formulating selective chemicals against those proteins is an effective approach to containing the disease. In this study, we used the genome subtraction approach and identified 595 Xcp proteins that are non-homologous to the pomegranate proteome, of which 69 are found to be essential proteins. These 69 proteins are considered possible drug target proteins in Xcp. Further, these proteins were subjected to subcellular localization, KEGG pathway, and virulent prediction analysis. Our systematic bioinformatics analysis deciphered 33 virulent proteins, of which two are iron complex outer membrane receptors, and the third is a T4SS PilQ protein localized in the outer membrane. These outer membrane-localized proteins are potential candidate targets for antibacterial agents, and the two iron complex outer membrane receptor proteins show homology with the Drug bank listed drug target sequences. From this study, we inferred that PilQ could be considered a novel antimicrobial target of Xcp, and therefore we deciphered the PilQ protein-protein interacting partners and phylogenetic relatedness. We have also predicted the physiochemical properties, secondary, and tertiary structure of PilQ protein which will be helpful in the design of antimicrobials. The identification of Xcp specific targets is the first step towards the development of a chemical control agent that is more selective with minimum environmental impact.

Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa

In Gram-negative bacteria, most of the sec-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of 12 Xcp proteins (XcpA and XcpP to XcpZ) organized as a multiproteic complex within the envelope. In this study, by a co-purification approach using a His-tagged XcpZ as a bait, XcpY and XcpZ were found associated together to constitute the most stable functional unit so far isolated from the P. aeruginosa secreton. This subcomplex was also found to interact with XcpR and XcpS to form a XcpRSYZ complex which was isolated under native conditions. Another component, XcpP was not found to be associated to the complex but the results suggest that it can transiently interact with the XcpYZ subcomplex in vivo.

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.

Role of XcpP in the functionality of the Pseudomonas aeruginosa secreton

In Gram-negative bacteria, most signal-peptide-dependent exoproteins are secreted via the type II secretion system (T2SS or secreton). In Pseudomonas aeruginosa, T2SS consists of twelve Xcp proteins (XcpA and XcpP to XcpZ) thought to be organized as a multiproteic complex within the envelope. Although well conserved, T2SS are known to be species-specific, namely for distant organisms, and this characteristic was thought to involve XcpP. To check which domain of XcpP could be involved in the species specificity, hybrid proteins were generated using protein domain swapping between P. aeruginosa XcpP and homolog proteins of either Erwinia chrysanthemi or Pseudomonas alcaligenes. The results obtained with hybrid proteins constructed by exchanging the C-terminal domains of P. aeruginosa and E. chrysanthemi suggested that XcpP interacts with XcpQ, probably via its C-terminal domain. More interestingly, the data obtained with a hybrid protein containing the C-terminal part of the P. alcaligenes XcpP homolog, showed that the wild-type C-terminal end plays a very important role in the function of the protein and is required both for a correct interaction with XcpQ and for modulating the opening of the secreton channel.

Identification of XcpZ domains required for assembly of the secreton of Pseudomonas aeruginosa.

Most of the exoproteins secreted by Pseudomonas aeruginosa are transported via the type II secretion system. This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton. We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton. In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis. We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton. Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.

Protein secretion mechanisms in Gram-negative bacteria

Gram-negative bacteria have developed a variety of secretion pathways to secrete toxins and enzymes into the extracellular medium. These pathways are very different with respect to their functional mechanism and complexity, and each system has its own advantages and limitations, regarding the number, size, folding state and fate of their substrates. Pseudomonas aeruginosa secretes many different proteins into the extracellular medium, using at least four secretion pathways. Most of the exoproteins are secreted via the type II system, composed of the 12 Xcp proteins. The only outer membrane protein of the system, XcpQ, belongs to a large family of proteins, designated secretins, which participate in a variety of different transport processes. Other Xcp proteins, XcpT-X, show homology to the subunits of the retractile type IV pili. Further analogies between the type II system and the assembly of retractile pili suggest a mechanism for type II secretion, in which a pilus-like structure, composed of XcpT-X, facilitates the transport of exoproteins through the channel formed by the secretin XcpQ.

Structure-function analysis of XcpP, a component involved in general secretory pathway-dependent protein secretion in Pseudomonas aeruginosa.

The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P. aeruginosa secretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.

Assembly of XcpR in the cytoplasmic membrane is required for extracellular protein secretion in Pseudomonas aeruginosa.

A broad range of extracellular proteins secreted by Pseudomonas aeruginosa use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12 xcp gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type P. aeruginosa strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an xcpY mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.

Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa.

Protein secretion in gram-negative bacteria is often dependent on the general secretory pathway (GSP). In Pseudomonas aeruginosa, this system requires at least 12 Xcp (Gsp) proteins, which are proposed to constitute a multiprotein complex localized in the bacterial envelope. Hitherto, little was known about the mutual interactions between Xcp proteins. In this study, mutants affected in the xcpZ gene encoding a bitopic inner-membrane protein were analysed to investigate the role of this protein in the architecture of the secretory machinery. The absence of XcpZ resulted in a decreased amount of XcpY. Reciprocally, XcpZ was not detectable in a xcpY mutant, demonstrating a mutual stabilization of these two proteins. These results strongly suggest that XcpZ and XcpY interact within the functional secretory apparatus.

The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family)

The xcp gene products in Pseudomonas aeruginosa are required for the secretion of proteins across the outer membrane. Four of the Xcp proteins, XcpT, U, V and W, present sequence homology to the subunits of type IV pili at their N-termini, and they were therefore designated pseudopilins. In this study, we characterized the xcpX gene product, a bitopic cytoplasmic membrane protein. Remarkably, amino acid sequence comparisons also suggested that the XcpX protein resembles the pilins and pseudopilins at the N-terminus. We show that XcpX could be processed by the prepilin peptidase, PilD/XcpA, and that the highly conserved glycine residue preceding the hydrophobic segment could not be mutated without loss of the XcpX function. We, therefore, classified XcpX (GspK) as the fifth pseudopilin of the system.

Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion.

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional crosslinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PIlC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus or PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.

Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa.

Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa. Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features. Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane. To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization. We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.

Protein secretion in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to habe disseminate widely among Gram-negative bacteria.

Protein secretion inPseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to habe disseminate widely among Gram-negative bacteria.

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase.

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periplasm via a signal sequence-dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram-negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to -X, are presented. The N-termini of four of the encoded Xcp proteins display similarity to the N-termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide-binding site, ATP hydrolysis may provide energy for both systems.