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.     

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.     

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.     

Deciphering the Xcp Pseudomonas aeruginosa type II secretion machinery through multiple interactions with substrates

The type II secretion system enables gram-negative bacteria to secrete exoproteins into the extracellular milieu. We performed biophysical and biochemical experiments to identify systematic interactions between Pseudomonas aeruginosa Xcp type II secretion system components and their substrates. We observed that three Xcp components, XcpP(C), the secretin XcpQ(D), and the pseudopilus tip, directly and specifically interact with secreted exoproteins. We established that XcpP(C), in addition to its interaction with the substrate, likely shields the entire periplasmic portion of the secreton. It can therefore be considered as the recruiter of the machinery. Moreover, the direct interaction observed between the substrate and the pseudopilus tip validates the piston model hypothesis, in which the pseudopilus pushes the substrate through the secretin pore during the secretion process. All together, our results allowed us to propose a model of the different consecutive steps followed by the substrate during the type II secretion process.     

嗜麦芽寡养单胞菌D2株2套Ⅱ型分泌系统

嗜麦芽寡养单胞菌D2株经前期研究发现有2套Ⅱ型分泌系统（T2SS）,根据嗜麦芽寡养单胞菌K279a、R551—3、JV3、D457的T2SS基因簇序列,以及D2株的部分测序结果设计引物,采用基因移步法逐一扩增2套T2SS基因序列,产物连接至T载体,经酶切鉴定后测序、拼接。发现有22个完整的开放多码框架（ORF）,比对分析后发现T2SSl的基因簇与同种属细菌相应基因簇序列同源性均在80％以上,对应氨基酸序列同源性均能达到97％以上,而T2SS2基因簇与K279a、R551—3对应基因序列和氨基酸序列的同源性均不及T2SS1高。2套T2SS的GspE、F、I基因同源性在50％以上,其他对应基因同源性在35％～68％之间不等,氨基酸序列同源性则为15％～61％。2套他ss与铜绿假单胞菌PA01的同源性高于不同科的小肠结肠炎耶尔森菌。T2SS的序列测定及同源性分析为进一步研究细菌蛋白分泌机制奠定基础。     

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.     

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).     

On the path to uncover the bacterial type II secretion system

Gram-negative bacteria have evolved several secretory pathways to release enzymes or toxins into the surrounding environment or into the target cells. The type II secretion system (T2SS) is conserved in Gram-negative bacteria and involves a set of 12 to 16 different proteins. Components of the T2SS are located in both the inner and outer membranes where they assemble into a supramolecular complex spanning the bacterial envelope, also called the secreton. The T2SS substrates transiently go through the periplasm before they are translocated across the outer membrane and exposed to the extracellular milieu. The T2SS is unique in its ability to promote secretion of large and sometimes multimeric proteins that are folded in the periplasm. The present review describes recently identified protein-protein interactions together with structural and functional advances in the field that have contributed to improve our understanding on how the type II secretion apparatus assembles and on the role played by individual proteins of this highly sophisticated system.     

铜绿假单胞菌中Ⅲ型分泌系统调控机制及针对性治疗的研究进展

铜绿假单胞菌是临床上重要的条件致病菌,具有多种毒力因子且极易产生耐药性。Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS)是铜绿假单胞菌中重要的毒性因子分泌系统,该菌通过Ⅲ型分泌系统将多种毒力因子注入到真核宿主细胞内并逃逸宿主细胞免疫系统的清除,引起宿主细胞相应的病理变化。对Ⅲ型分泌系统的研究,不仅有助于明确铜绿假单胞菌的致病机理,更可为临床治疗和药物研发提供理论基础。本文主要对铜绿假单胞菌中Ⅲ型分泌系统的结构、功能、调控机制以及针对性治疗策略等方面的研究进行了综述。     

The accessory protein TagV is required for full Type VI secretion system activity in Serratia marcescens

The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter- and intra-species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well-characterised core proteins necessary for effector delivery (TssA-M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS-dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ-independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins.     

副溶血弧菌的Ⅲ型分泌系统

摘要：副溶血弧菌是一种嗜盐性革兰氏阴性短杆菌,主要引起食物中毒性肠胃炎,还可引起水生动物疾病。除了耐热直接溶血毒素和耐热直接溶血相关毒素外,近年发现的副溶血弧菌两套Ⅲ型分泌系统也与该菌的致病性密切相关。Ⅲ型分泌系统1位于染色体1上,主要贡献对宿主细胞的细胞毒性,介导宿主细胞的自体吞噬作用,最后导致细胞死亡。Ⅲ型分泌系统2位于位于染色体2的毒力岛上,具有肠毒性。本文扼要介绍副溶血弧菌Ⅲ型分泌系统的组成、功能及相关转录调控机制。     

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.     

The crystal structure of a binary complex of two pseudopilins: EpsI and EpsJ from the type 2 secretion system of Vibrio vulnificus

Type II secretion systems (T2SS) translocate virulence factors from the periplasmic space of many pathogenic bacteria into the extracellular environment. The T2SS of Vibrio cholerae and related species is called the extracellular protein secretion (Eps) system that consists of a core of multiple copies of 11 different proteins. The pseudopilins, EpsG, EpsH, EpsI, EpsJ and EpsK, are five T2SS proteins that are thought to assemble into a pseudopilus, which is assumed to interact with the outer membrane pore, and may actively participate in the export of proteins. We report here biochemical evidence that the minor pseudopilins EpsI and EpsJ from Vibrio species interact directly with one another. Moreover, the 2.3 Å resolution crystal structure of a complex of EspI and EpsJ from Vibrio vulnificus represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS. Both EpsI and EpsJ appear to be structural extremes within the family of type 4a pilin structures solved to date, with EpsI having the smallest, and EpsJ the largest, “variable pilin segment” seen thus far. A high degree of sequence conservation in the EpsI:EpsJ interface indicates that this heterodimer occurs in the T2SS of a large number of bacteria. The arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helical character of proteins assembled into a pseudopilus.     

Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system

Type IV secretion systems (T4SS) are utilized by a wide range of Gram negative bacteria to deliver protein and DNA substrates to recipient cells. The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/Icm systems found in the pathogens Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Here we report the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. Based on biochemical and genetic analysis, we discerned the existence of a critical five-protein subassembly that spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a remarkable conservation of the core subassembly in these evolutionarily distant type IV secretion machines.     

绿脓杆菌特有的Tsi2三维结构——一种全新卷曲螺旋构象的类抗毒素蛋白

绿脓杆菌(Pseudomonas aeruginosa)利用六型分泌系统(T6SS)向其他竞争性细菌分泌毒素效应分子Tse2,这是一种新发现的绿脓杆菌获得生存优势的分子机制．为了避免同类间的误杀,绿脓杆菌合成一种特异结合Tse2的抑制蛋白Tsi2来保护自己．序列分析显示,Tsi2是绿脓杆菌特有的一种新型类抗毒素蛋白．我们利用SAD方法成功地解析了Tsi2 1.8Å分辨率的晶体结构．Tsi2的三维结构采用一种规则的卷曲螺旋的结构特征,这是抗毒素分子中的一种全新的折叠方式,不同于经典的抗毒素分子在没有结合毒素分子状态下采用无规则构象的结构特征;二聚体是Tsi2的功能单位,二聚体内两个Tsi2单体通过广阔的疏水相互作用紧密结合,形成“夹子”状独特的二聚体组装方式;位于二聚体界面上的两个凹槽分别结合对称分子的两段螺旋,提供了Tsi2与Tse2结合可能的分子部位．该研究工作结果对于认识Tsi2抗毒素蛋白的分子本质,揭示其发挥抗毒素活性的结构基础,并为进一步开展Tse2-Tsi2复合物的结构与功能研究奠定了坚实的基础．     

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.     

铜绿假单胞菌中III型分泌系统受Rhl和PQS群体感应系统调节

摘要：【目的】研究铜绿假单胞菌中群体感应系统（Quorum sensing, QS）与III型分泌系统（Type III secretion system, T3SS）的关系。【方法】通过基因敲除的方法破坏铜绿假单胞菌QS系统相关基因,将T3SS相关基因exoS、exoY、exoT、exsD-pscA-L启动子-报道子luxCDABE融合体整合到野生型菌株及QS系统突变菌株的染色体组上,通过检测启动子活性,比较这些基因在不同菌株中的表达情况。【结果】研究结果表明,T3SS中的exoS与exoT在pqsR基因突变体中的表达有明显的增强,Rhl系统对这四种基因的表达具有抑制作用,而Las系统存在与否对T3SS基本没有影响。【结论】铜绿假单胞菌中的Rhl系统和奎诺酮信号（Pseudomonas Quinolone Signal, PQS）系统对T3SS相关基因的表达具有重要的调节作用。     

EscA is a crucial component of the type III secretion system of enteropathogenic Escherichia coli

The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.     

铜绿假单胞菌Ⅲ型分泌系统的分子调控机制

铜绿假单胞菌是临床上重要的革兰氏阴性条件致病菌.通过Ⅲ型分泌系统,铜绿假单胞菌将其毒力因子注入到真核宿主细胞内部,逃避宿主巨噬细胞的吞噬降解,引起宿主相应的病理变化,是铜绿假单胞菌感染致病的重要原因.本文在简单介绍铜绿假单胞菌Ⅲ型分泌系统组成和功能的基础上,主要对调控T3SS基因转录表达的分子机制的研究进展进行综述和讨论.