Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa.

Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein. To determine the genetic relationship between the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by allelic exchange. A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS. Thirteen clones that cross-hybridized with the exoS probe were identified. One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS. Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of exoenzyme S are encoded by separate genes. In addition, this is the first report of the factor-activating-exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of exoenzyme S.     

Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa.

Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53). N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity. Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain. N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity. Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53. E385 was also identified as a potential active site residue of Exo53.     

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。     

Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions.

The exo loci of Rhizobium meliloti are necessary for the production of an acidic exopolysaccharide, EPS I, that is needed for alfalfa nodule invasion by strain Rm1021. We have isolated and characterized alkaline phosphatase fusions made with TnphoA in several exo loci of R. meliloti and used these fusions to examine the subcellular localization of exo gene products and the regulation of exo genes in free-living cells and in planta. In the course of this work, we isolated a new exo locus, exoT. We have obtained evidence that several of the exo loci may encode membrane proteins. The activity of TnphoA fusions in several exo loci is increased two- to fivefold in the presence of the regulatory mutations exoR95 and exoS96. While examining the regulation of the exo gens by exoR95 and exoS96, we found that certain classes of exo mutations are lethal in an exoR95 or exoS96 background unless a plasmid complementing the exo mutation is present. This result has possible implications for the role of these exo loci in EPS I biosynthesis. We have developed a method for staining nodules specifically for the alkaline phosphatase activity present in the inducing bacteria and used this method to show that an exoF::TnphoA fusion is expressed mainly in the invasion zone of the nodule.     

Recombinant Pseudomonas exoenzyme S and exoenzyme S from Pseudomonas aeruginosa DG1 share the ability to stimulate T lymphocyte proliferation.

Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have distinct characteristics, which has led to a controversy about their homology and their pathophysiologic consequences. We have been investigating the ability of exoenzyme S to activate T lymphocytes, and therefore performed studies to determine whether exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 and expressed in Pseudomonas aeruginosa PA103 or in E. coli BL21(DE3), could induce T lymphocyte activation and proliferation. Both preparations were able to activate T cells and induce lymphocyte proliferation at similar levels as measured by flow cytometry of surface-activation markers and DNA synthesis, respectively. Further, a monoclonal antibody raised against exoenzyme S from strain DG1 partially neutralized T cell activation induced by recombinant exoenzyme S and bound to it in an immunoblot suggesting that the epitope responsible for T cell activation is shared by exoenzyme S from strain DG1 and recombinant exoenzyme S. These data suggest that the two different preparations of exoenzyme S, despite biochemical differences, share the characteristic that is responsible for T lymphocyte activation.     

肉桂酸衍生物对铜绿假单胞菌PAO1 III型分泌系统的影响

【目的】铜绿假单胞菌是引起医院获得性感染最常见的条件致病菌,而III型分泌系统(Type III secretion system,TTSS)是其致病的主要因子之一。本文从合成的21个肉桂酸衍生物中筛选影响TTSS效应子(Effector)产生的化合物,并初步研究其作用机制。【方法】将TTSS效应子合成基因exoS的转录报告质粒pAT-exoS转入菌株PAO1中,获得PAO1(pAT-exoS)。待筛选的化合物与PAO1(pAT-exoS)菌株共培养6 h后,检测exoS基因的表达,从中筛选影响exoS基因表达的化合物。【结果】筛选结果表明：21个化合物中,3个化合物抑制exoS基因表达,2个化合物则促进exoS基因表达。此外,化合物TS128、TS143和TS160对菌株生长有明显的抑制作用。Western blot实验进一步证实筛选得到的化合物TS108、TS128和TS165可抑制ExoS的产生;化合物TS139和TS143则促进ExoS的产生。为进一步研究抑制剂的作用机理,过量表达TTSS主要的调控因子exsA基因可部分消除抑制剂TS108和TS165的抑制效果;而rsmZ rsmY双基因突变体PAO6421中添加抑制剂TS108和TS165并不能显著抑制exoS基因的表达,同样,抑制剂TS108和TS165也不影响受Gac/Rsm信号传导系统调控的群体感应信号分子的产生。【结论】抑制剂TS108和TS165的作用机制可能主要是影响esxA基因,从而影响exoS基因表达及蛋白产量。     

ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo

The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock-out strain, 388Δ exoS , were used in a bacterial and mammalian co-culture system as a model for the contact-dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP-ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins after exposure to ExoS-producing bacteria, but not after exposure to non-ExoS-producing bacteria. Examination of normal H-Ras, K-Ras and N-Ras by two-dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most extensively. ADP-ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H-, N- or K-Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts. Using Ras/Raf-1 co-immunoprecipitation after co-culture, it was found that exposure to ExoS-producing bacteria caused a decrease in the amount of Raf-1 associated with EGF-activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP-ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.     

The exoenzyme S regulon of Pseudomonas aeruginosa

Pseudomonas aeruginosa can cause severe life-threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P . aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P . aeruginosa may use a contact-mediated translocation mechanism to transfer anti-host factors directly into eukaryotic cells. Potential anti-host factors that may disrupt eukaryotic signal transduction through ADP-ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti-host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P . aeruginosa . It will be important to understand how the exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.     

Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells

Exoenzyme S of Pseudomonas aeruginosa is an ADP-ribosyltransferase, which is secreted via a type III-dependent secretion mechanism and has been demonstrated to exert cytotoxic effects on eukaryotic cells. Alignment studies predict that the amino-terminus of exoenzyme S has limited primary amino acid homology with the YopE cytotoxin of Yersinia, while biochemical studies have localized the FAS-dependent ADP-ribosyltransferase activity to the carboxyl-terminus. Thus, exoenzyme S could interfere with host cell physiology via several independent mechanisms. The goal of this study was to define the role of the ADP-ribosyltransferase domain in the modulation of eukaryotic cell physiology. The carboxyl-terminal 222 amino acids of exoenzyme S, which represent the FAS-dependent ADP-ribosyltransferase domain (termed deltaN222), and a point mutant, deltaN222-E381A, which possesses a 2000-fold reduction in the capacity to ADP-ribosylate, were transiently expressed in eukaryotic cells under the control of the immediate early CMV promoter. Lysates from cells transfected with deltaN222 expressed ADP-ribosyltransferase activity. Co-transfection of deltaN222, but not deltaN222-E381A, resulted in a decrease in the steady-state levels of two reporter proteins, green fluorescent protein and luciferase, in both CHO and Vero cells. In addition, transfection with deltaN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either deltaN222-E381A or control plasmid. Together, these data indicate that expression of the ADP-ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.     

Pseudomonas aeruginosa exoenzyme S requires a eukaryotic protein for ADP-ribosyltransferase activity

Pseudomonas aeruginosa exoenzyme S ADP-ribosylates several GTP-binding proteins of apparent Mr = 23,000-25,000. Exoenzyme S absolutely requires a soluble eukaryotic protein, which we have named FAS (Factor Activating exoenzyme S), in order to ADP-ribosylate all substrates. The rate of ADP-ribosylation of all exoenzyme S substrates increases linearly with time and with the FAS concentration. FAS is wide-spread in eukaryotes but appears to be absent from prokaryotes. We have estimated the molecular mass of the protein to be approximately 29,000 daltons and its pI to be 4.3-4.5. Several bacterial toxins share this sort of requirement for the presence of a eukaryotic protein for enzymic activity. In particular, FAS resembles ADP-ribosylation factor, a 21,000-dalton GTP-binding protein which performs an analogous function for cholera toxin. However, we can find no evidence that FAS binds GTP. In the presence of FAS, exoenzyme S ADP-ribosylates several proteins in lysates of P. aeruginosa. The requirement for a eukaryotic protein for enzymic activity, which is common to several bacterial toxins, may be a device to identify the eukaryotic environment and to ensure that the enzymes cannot function within and harm the toxin-producing bacteria.     

Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway

Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa . Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn 5  Tc insertion mutants were isolated which exhibited an exoenzyme S-deficient phenotype (388::Tn 5  Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn 5  Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb Eco RI fragment that is not contiguous with the exoenzyme S trans -regulatory operon. 388::Tn 5  Tc 469 and 550 mapped to a region downstream of the trans -regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn 5  Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniae Yop type III export apparatus. RNase-protection analysis of wild-type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa . Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co-ordinately regulated proteins.     

谷胱甘肽对铜绿假单胞菌exoS 和exoY基因的影响

摘要: 【目的】研究谷胱甘肽对铜绿假单胞菌exoS和exoY基因表达的影响。【方法】利用丁硫氨酸亚砜胺和马来酸二乙酯同时耗竭细胞内的谷胱甘肽,并构建包含被lacZGm破坏的谷胱甘肽合成酶基因的突变体。通过分别连有exoS 和exoY基因启动子的pMS402质粒上Lux报道子发光值大小检测exoS 和exoY基因表达变化情况。【结果】exoS和exoY基因的表达在用化学药品耗竭的细胞中或是在谷胱甘肽合成酶突变体中都降低。【结论】铜绿假单胞菌细胞内的谷胱甘肽可以促进exoS和exoY的表达。这将为进一步研究铜绿假单胞菌的感染以及致病性机理提供一定的理论基础。     

Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and flagellum production

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.     

Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon.

Extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa exoenzyme S regulon were compared to identify proteins coordinately secreted with ExoS. Data from amino-terminal sequence analysis of purified extracellular proteins were combined with data from nucleotide sequence analysis of loci linked to exoenzyme S production. We report the identification of P. aeruginosa homologs to proteins of Yersinia spp. that function as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism.     

ExoS and ExoT ADP ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting neutrophil apoptosis and bacterial survival

Pseudomonas aeruginosa is a leading cause of blinding corneal ulcers worldwide. To determine the role of type III secretion in the pathogenesis of P. aeruginosa keratitis, corneas of C57BL/6 mice were infected with P. aeruginosa strain PAO1 or PAK, which expresses ExoS, ExoT, and ExoY, but not ExoU. PAO1- and PAK-infected corneas developed severe disease with pronounced opacification and rapid bacterial growth. In contrast, corneas infected with ΔpscD or ΔpscJ mutants that cannot assemble a type III secretion system, or with mutants lacking the translocator proteins, do not develop clinical disease, and bacteria are rapidly killed by infiltrating neutrophils. Furthermore, survival of PAO1 and PAK strains in the cornea and development of corneal disease was impaired in ΔexoS, ΔexoT, and ΔexoST mutants of both strains, but not in a ΔexoY mutant. ΔexoST mutants were also rapidly killed in neutrophils in vitro and were impaired in their ability to promote neutrophil apoptosis in vivo compared with PAO1. Point mutations in the ADP ribosyltransferase (ADPR) regions of ExoS or ExoT also impaired proapoptotic activity in infected neutrophils, and exoST(ADPR-) mutants replicated the ΔexoST phenotype in vitro and in vivo, whereas mutations in rho-GTPase-activating protein showed the same phenotype as PAO1. Together, these findings demonstrate that the pathogenesis of P. aeruginosa keratitis in ExoS- and ExoT-producing strains is almost entirely due to their ADPR activities, which subvert the host response by targeting the antibacterial activity of infiltrating neutrophils.     

Rhizobium meliloti mutants that overproduce the R. meliloti acidic calcofluor-binding exopolysaccharide.

The acidic Calcofluor-binding exopolysaccharide of Rhizobium meliloti Rm1021 plays one or more critical roles in nodule invasion and possibly in nodule development. Two loci, exoR and exoS, that affect the regulation of synthesis of this exopolysaccharide were identified by screening for derivatives of strain Rm1021 that formed mucoid colonies that fluoresced extremely brightly under UV light when grown on medium containing Calcofluor. The exopolysaccharide produced in large quantities by the exoR95::Tn5 and exoS96::Tn5 strains was indistinguishable from that produced by the parental strain Rm1021, and its synthesis required the function of at least the exoA, exoB, and exoF genes. Both the exoR and exoS loci were located on the chromosome, and the exo96::Tn5 mutation was 84% linked to the trp-33 mutation by phi M12 transduction. Synthesis of the Calcofluor-binding exopolysaccharide by strain Rm1021 was greatly stimulated by starvation for ammonia. In contrast, the exoR95::Tn5 mutant produced high levels of exopolysaccharide regardless of the presence or absence of ammonia in the medium. The exoS96::Tn5 mutant produced elevated amounts of exopolysaccharide in the presence of ammonia, but higher amounts were observed after starvation for ammonia. The presence of either mutation increased the level of expression of exoF::TnphoA and exoP::TnphoA fusions (TnphoA is Tn5 IS50L::phoA). Analyses of results obtained when alfalfa seedlings were inoculated with the exoR95::Tn5 strain indicated that the mutant strain could not invade nodules. However, pseudorevertants that retained the original exoR95::Tn5 mutation but acquired unlinked suppressors so that they produced an approximately normal amount of exopolysaccharide were able to invade nodules and fix nitrogens. The exoS95::Tn5 strain formed Fix+ nodules, although some minor variability was observed.     

Bacillus licheniformis 749-C plasma membrane penicillinase, a hydrophobic polar protein

Plasma membrane penicillinase from Bacillus licheniformis 749/C is hydrophobic in nature, although it is virtually identical to its riydrophilic exoenzyme counterpart in amino acid composition and sequence. Unlike the exoenzyme, however, the purified membrane enzyme retains [³³P]phosphate and [³H]glycerol. By isoelectricfocusing the membrane enzyme is more acidic than the exoenzyme; it has a lower mobility in SDS gel electrophoresis, consistent with the presence of a very hydrophobic moiety. Unlike the exoenzyme, which binds no taurodeoxycholate, the membrane enzyme binds 10 molecules tightly and approximately 37 molecules in the presence of excess taurodeoxycholate (0.1% solution). The membrane enzyme is identical to the exoenzyme in its reaction with antibodies to exopenicillinase as determined by a radioimmune inhibition assay and immunodiffusion in agar. Heat stability studies indicate a slightly less stable conformation for the membrane enzyme, but this difference largely disappears in the presence of antibody to the exoenzyme. Conversion of membrane enzyme to exoenzyme has been achieved by brief treatment with trypsin, or by incubation of impure preparations at pH 9.0 in 25% potassium phosphate.Since the two forms of penicillinase are very similar in conformation, the hydrophobicity of the membrane form of the enzyme would seem to derive from combination with a hydrophobic moiety, probably phospholipid.