Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium.

Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

Protein secretion systems in Pseudomonas aeruginosa: A wealth of pathogenic weapons.

Pathogenic microorganisms have to face hostile environments while colonizing and infecting their hosts. Unfortunately, they can cope with it and have evolved a number of complex secretion systems, which direct virulence factors either at the bacterial cell surface into the environmental extracellular milieu or into the host cell cytosol. Six different classes of secretion systems have been described so far, currently identified as type I secretion system (T1SS) up to type VI secretion system (T6SS). The Gram-negative opportunistic human pathogen Pseudomonas aeruginosa possesses a broad panel of secretion systems. Five of the six secretion machines characterized in Gram-negative bacteria are at P. aeruginosa disposal, sometimes in several copies. All these machines are dedicated to the specific secretion of exoproteins, which display various activities useful for bacterial adaptation to the environment or for bacterial pathogenicity. This review will summarize the functional organization of these different secretion systems, which could constitute potential targets for therapeutic treatment of patients infected by one of the most potent nosocomial pathogens identified nowadays.

HxcQ liposecretin is self-piloted to the outer membrane by its N-terminal lipid anchor.

Secretins are an unusual and important class of bacterial outer membrane (OM) proteins. They are involved in the transport of single proteins or macromolecular structures such as pili, needle complexes, and bacteriophages across the OM. Secretins are multimeric ring-shaped structures that form large pores in the OM. The targeting of such macromolecular structures to the OM often requires special assistance, conferred by specific pilotins or pilot proteins. Here, we investigated HxcQ, the OM component of the second Pseudomonas aeruginosa type II secretion system. We found that HxcQ forms high molecular mass structures resistant to heat and SDS, revealing its secretin nature. Interestingly, we showed that HxcQ is a lipoprotein. Construction of a recombinant nonlipidated HxcQ (HxcQnl) revealed that lipidation is essential for HxcQ function. Further phenotypic analysis indicated that HxcQnl accumulates as multimers in the inner membrane of P. aeruginosa, a typical phenotype observed for secretins in the absence of their cognate pilotin. Our observations led us to the conclusion that the lipid anchor of HxcQ plays a pilotin role. The self-piloting of HxcQ to the OM was further confirmed by its correct multimeric OM localization when expressed in the heterologous host Escherichia coli. Altogether, our results reveal an original and unprecedented pathway for secretin transport to the OM.

Transcriptome and secretome analyses of the adaptive response of Pseudomonas aeruginosa to suboptimal growth temperature.

Pseudomonas aeruginosa is an opportunistic pathogen involved in several diseases, including cystic fibrosis and nosocomial infections. Although the behavior of this bacterium at 37 degrees C has been intensively studied, little is known about its capacity to adapt and survive at suboptimal temperatures, such as those encountered in hospitals. In this work, transcriptomic and proteomic analyses were used to identify factors that allow P. aeruginosa to become established at room temperature (close to 25 degrees C) and thus facilitate host infections. Since the virulence of this pathogen is multifactorial and dependent on the extracellular release of toxins and degradative enzymes targeted to the host by several secretory systems, the study focused on genes activated at 25 degrees C, namely, those encoding either components of the secretory machinery or secreted proteins. These observations were enhanced by 2D-PAGE analyses, which showed that the production of effectors from type I and type II secretion systems (respectively, proteases AprA and PrpL) and of a hemolysin co-regulated protein (Hcp) related to the type VI secretion system was specifically stimulated when the growth temperature was lowered from 37 to 25 degrees C. The results provide a fundamental basis for investigating the processes that allow P. aeruginosa to adapt to suboptimal growth temperatures and which thereby promote nosocomial infection.

XphA/XqhA, a novel GspCD subunit for type II secretion in Pseudomonas aeruginosa.

The opportunistic human pathogen bacterium Pseudomonas aeruginosa secretes various exoproteins in its surrounding environment. Protein secretion involves different secretory systems, including the type II secretion system, or T2SS, that is one of the most efficient secretory pathways of P. aeruginosa. There are two T2SS in this bacterium, the quorum-sensing-regulated Xcp system and the Hxc system, which is only present under phosphate-limiting conditions. Like T2SS of other bacteria, the Xcp T2SS is species specific, and this specificity mainly involves two proteins, XcpP (GspC family) and the secretin XcpQ (GspD family), which are the gatekeepers of the system. Interestingly, an orphan secretin, XqhA, was previously reported as being able to functionally replace the XcpQ secretin. In this study, we identified another gene, which we named xphA (xcpP homologue A), which is located next to xqhA. We showed that deletion of the xphA gene in an xcpP mutant caused the disappearance of the residual secretion observed in this mutant strain, indicating that the protein XphA plays a role in the secretion process. Our results also revealed that complementation of an xcpP/xcpQ mutant can be obtained with the gene couple xphA/xqhA. The XphA and XqhA proteins (the P(A)Q(A) subunit) could thus form, together with XcpR-Z, a functional hybrid T2SS. A two-dimensional polyacrylamide gel electrophoresis analysis showed that except for the aminopeptidase PaAP, for which secretion is not restored by the P(A)Q(A) subunit in the xcpP/xcpQ deletion mutant, each major Xcp-dependent exoprotein is secreted by the new hybrid machinery. Our work supports the idea that components of the GspC/GspD families, such as XphA/XqhA or XcpP/XcpQ, are assembled as a specific tandem within the T2SS. Each of these pairs may thus confer a different level of secretion specificity, as is the case with respect to PaAP. Finally, using a chromosomal xphA-lacZ fusion, we showed that the xphA-xqhA genes are transcribed from an early stage of bacterial growth. We thus suggest that the P(A)Q(A) subunit might be involved in the secretion process at a different growth stage than XcpP/XcpQ.

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.

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.

Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening.

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.

Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus.

Gram-negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two-step process. The first step is the translocation of signal peptide-bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12-15 proteins (Gsp) depending on the organism. Even though the systems are conserved, heterologous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a model organism to determine which domains of XcpP (GspC member) are involved in specificity. By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled-coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.

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.