Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli

Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones. It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus. The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system. We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC). Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN. We showed by affinity chromatography that EscN and Tir bind CesT specifically. Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT. Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC. These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN.     

Sorting of early and late flagellar subunits after docking at the membrane ATPase of the type III export pathway

The bacterial flagellum assembles in a strict order, with structural subunits delivered to the growing flagellum by a type III export pathway. Early rod-and-hook subunits are exported before completion of the hook, at which point a subunit-specificity switch allows export of late filament subunits. This implies that in bacteria with multiple flagella at different stages of assembly, each export pathway can discriminate and sort unchaperoned early and chaperoned late subunits. To establish whether subunit sorting is distinct from subunit transition from the cytosol to the membrane, in particular docking at the membrane-associated FliI ATPase, the pathway was manipulated in vivo. When ATP hydrolysis by the FliI ATPase was disabled and when the pathway was locked into an early export state, both unchaperoned early and chaperoned late subunits stalled and accumulated at the inner membrane. Furthermore, a chaperone that attenuates late subunit export by stalling when docked at the wild-type ATPase also stalled at the ATPase in an early-locked pathway and inhibited export of early subunits in both native and early-locked pathways. These data indicate that the pathways for early and late subunits converge at the FliI ATPase, independent of ATP hydrolysis, before a distinct, separable sorting step. To ascertain the likely signals for sorting, the export of recombinant subunits was assayed. Late filament subunits unable to bind their chaperones were still sorted accurately, but chaperoned late subunits were directed through an early-locked pathway when fused to early subunit N-terminal export signal regions. Furthermore, while an early subunit signal directed export of a heterologous type III export substrate through both native and early-locked pathways, a late subunit signal only directed export via native pathways. These data suggest that subunits are distinguished not by late chaperones but by N-terminal export signals of the subunits themselves.     

Recognition of secretory proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis> requires signals in addition to the signal sequence and slow folding

Background  

The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway.     

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

Type III secretion (T3S), a protein export pathway common to Gram‐negative pathogens, comprises a trans‐envelope syringe, the injectisome, with a cytoplasm‐facing translocase channel. Exported substrates are chaperone‐delivered to the translocase, EscV in enteropathogenic Escherichia coli, and cross it in strict hierarchical manner, for example, first “translocators”, then “effectors”. We dissected T3S substrate targeting and hierarchical switching by reconstituting them in vitro using inverted inner membrane vesicles. EscV recruits and conformationally activates the tightly membrane‐associated pseudo‐effector SepL and its chaperone SepD. This renders SepL a high‐affinity receptor for translocator/chaperone pairs, recognizing specific chaperone signals. In a second, SepD‐coupled step, translocators docked on SepL become secreted. During translocator secretion, SepL/SepD suppress effector/chaperone binding to EscV and prevent premature effector secretion. Disengagement of the SepL/SepD switch directs EscV to dedicated effector export. These findings advance molecular understanding of T3S and reveal a novel mechanism for hierarchical trafficking regulation in protein secretion channels.     

EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.     

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways.     

Chaperone Recycling in Late-Stage Flagellar Assembly

The flagellum is a sophisticated nanomachine responsible for motility in Gram-negative bacteria. Flagellar assembly is a strictly choreographed process, in which the motor and export gate are formed first, followed by the extracellular propeller structure. Extracellular flagellar components are escorted to the export gate by dedicated molecular chaperones for secretion and self-assembly at the apex of the emerging structure. The detailed mechanisms of chaperone-substrate trafficking at the export gate remain poorly understood. Here, we structurally characterized the interaction of Salmonella enterica late-stage flagellar chaperones FliT and FlgN with the export controller protein FliJ. Previous studies showed that FliJ is absolutely required for flagellar assembly since its interaction with chaperone-client complexes controls substrate delivery to the export gate. Our biophysical and cell-based data show that FliT and FlgN bind FliJ cooperatively, with high affinity and on specific sites. Chaperone binding completely disrupts the FliJ coiled-coil structure and alters its interactions with the export gate. We propose that FliJ aids the release of substrates from the chaperone and forms the basis of chaperone recycling during late-stage flagellar assembly.     

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT‐interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C‐terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.     

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.     

Exported plasmodial J domain protein,PFE0055c,and PfHsp70-x form a specific co-chaperone-chaperone partnership

Plasmodium falciparum is a unicellular protozoan parasite and causative agent of a severe form of malaria in humans, accounting for very high worldwide fatality rates. At the molecular level, survival of the parasite within the human host is mediated by P. falciparum heat shock proteins (PfHsps) that provide protection during febrile episodes. The ATP-dependent chaperone activity of Hsp70 relies on the co-chaperone J domain protein (JDP), with which it forms a chaperone-co-chaperone complex. The exported P. falciparum JDP (PfJDP), PFA0660w, has been shown to stimulate the ATPase activity of the exported chaperone, PfHsp70-x. Furthermore, PFA0660w has been shown to associate with another exported PfJDP, PFE0055c, and PfHsp70-x in J-dots, highly mobile structures found in the infected erythrocyte cytosol. Therefore, the present study aims to conduct a structural and functional characterization of the full-length exported PfJDP, PFE0055c. Recombinant PFE0055c was successfully expressed and purified and found to stimulate the basal ATPase activity of PfHsp70-x to a greater extent than PFA0660w but, like PFA0660w, did not significantly stimulate the basal ATPase activity of human Hsp70. Small-molecule inhibition assays were conducted to determine the effect of known inhibitors of JDPs (chalcone, C86) and Hsp70 (benzothiazole rhodacyanines, JG231 and JG98) on the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. In this study, JG231 and JG98 were found to inhibit both the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. C86 only inhibited the PFE0055c-stimulated ATPase activity of PfHsp70-x, consistent with PFE0055c binding to PfHsp70-x through its J domain. This research has provided further insight into the molecular basis of the interaction between these exported plasmodial chaperones, which could inform future antimalarial drug discovery studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-020-01181-2.     

Conserved substrate binding by chaperones in the bacterial periplasm and the mitochondrial intermembrane space

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.     

Protein quality control in the bacterial periplasm

The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein.     

The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.     

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.     

EscO,a Functional and Structural Analog of the Flagellar FliJ Protein,Is a Positive Regulator of EscN ATPase Activity of the Enteropathogenic Escherichia coli Injectisome

Type III secretion systems (T3SSs) are multiprotein molecular devices used by many Gram-negative bacterial pathogens to translocate effector proteins into eukaryotic cells. A T3SS is also used for protein export in flagellar assembly, which promotes bacterial motility. The two systems are evolutionarily related, possessing highly conserved components in their export apparatuses. Enteropathogenic Escherichia coli (EPEC) employs a T3SS, encoded by genes in the locus of enterocyte effacement (LEE) pathogenicity island, to colonize the human intestine and cause diarrheal disease. In the present work, we investigated the role of the LEE-encoded EscO protein (previously Orf15 or EscA) in T3SS biogenesis. We show that EscO shares similar properties with the flagellar FliJ and the Yersinia YscO protein families. Our findings demonstrate that EscO is essential for secretion of all categories of T3SS substrates. Consistent with its central role in protein secretion, it was found to interact with the ATPase EscN and its negative regulator, EscL, of the export apparatus. Moreover, we show that EscO stimulates EscN enzymatic activity; however, it is unable to upregulate ATP hydrolysis in the presence of EscL. Remarkably, EscO partially restored the swimming defect of a Salmonella flagellar fliJ mutant and was able to stimulate the ATPase activity of FliI. Overall, our data indicate that EscO is the virulence counterpart of the flagellar FliJ protein.     

CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map

Map is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir-specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone-effector pairings, binding is apparent at the amino-terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp-2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

以SecA为靶点的新型抗绿脓杆菌药物细胞水平筛选模型的建立和应用

Sec途径(即分泌途径secretion pathway)是蛋白质转运的主要途径.其中,最为关键的组分之一是SecAATP酶,是蛋白质转运途径中的"动力泵",通过ATP的水解循环驱使蛋白质前体穿过细菌内膜,在细菌中是不可缺少的.我们推测抑制SecAATP酶活性的化合物.必然会在一定程度上抑制蛋白质的转运和分泌.通过绿脓杆菌与大肠杆菌SecA蛋白的互补作用,利用本实验室构建的高效表达SecA蛋白的基因工程菌,建立了SecA蛋白ATP酶活性抑制剂的细胞水平筛选模型.利用所纯化的绿脓杆菌SecA蛋白的ATP酶活测定体系,验证了所建立的细胞水平筛选模型具有一定的特异性.研究结果表明其中两个酯相组分在细胞水平和蛋白水平均具有活性,值得进行深入的研究.     

Structure and Functional Analysis of LptC, a Conserved Membrane Protein Involved in the Lipopolysaccharide Export Pathway in Escherichia coli

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.