Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157:H7. (41/405)

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 microm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with lambda-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.  (+info)

Crystal structure of the quorum-sensing protein LuxS reveals a catalytic metal site. (42/405)

The ability of bacteria to regulate gene expression in response to changes in cell density is termed quorum sensing. This behavior involves the synthesis and recognition of extracellular, hormone-like compounds known as autoinducers. Here we report the structure of an autoinducer synthase, LuxS from Bacillus subtilis, at 1.6-A resolution (R(free) = 0.204; R(work) = 0.174). LuxS is a homodimeric enzyme with a novel fold that incorporates two identical tetrahedral metal-binding sites. This metal center is composed of a Zn(2+) atom coordinated by two histidines, a cysteine, and a solvent molecule, and is reminiscent of active sites found in several peptidases and amidases. Although the nature of the autoinducer synthesized by LuxS cannot be deduced from the crystal structure, features of the putative active site suggest that LuxS might catalyze hydrolytic, but not proteolytic, cleavage of a small substrate. Our analysis represents a test of structure-based functional assignment.  (+info)

Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS. (43/405)

Pyridoxal-5'-phosphate-dependent cysteine desulfurase (IscS) is an essential enzyme in the assembly of FeS clusters in bacteria as well as in the mitochondria of eukaryotes. Although FeS proteins are particularly important for the energy metabolism of amitochondrial anaerobic eukaryotes, there is no information about FeS cluster formation in these organisms. We identified and sequenced two IscS homologs of Trichomonas vaginalis (TviscS-1 and TviscS-2) and one of Giardia intestinalis (GiiscS). TviscS-1, TviscS-2, and GiiscS possess the typical conserved regions implicated in cysteine desulfurase activity. N-termini of TviscS-1 and TviscS-2 possess eight amino acid extensions, which resemble the N-terminal presequences that target proteins to hydrogenosomes in trichomonads. No presequence was evident in GiiscS from Giardia, an organism that apparently lacks hydrogenosmes or mitochondria. Phylogenetic analysis showed a close relationship among all eukaryotic IscS genes including those of amitochondriates. IscS of proteobacteria formed a sister group to the eukaryotic clade, suggesting that isc-related genes were present in the proteobacterial endosymbiotic ancestor of mitochondria and hydrogenosomes. NifS genes of nitrogen-fixing bacteria, which are IscS homologs required for specific formation of FeS clusters in nitrogenase, formed a more distant group. The phylogeny indicates the presence of a common mechanism for FeS cluster formation in mitochondriates as well as in amitochondriate eukaryotes. Furthermore, the analyses support a common origin of Trichomonas hydrogenosomes and mitochondria, as well as secondary loss of mitochondrion/hydrogenosome-like organelles in Giardia.  (+info)

Methioninase cancer gene therapy with selenomethionine as suicide prodrug substrate. (44/405)

In this study, we report a novel approach to gene-directed enzyme prodrug therapy for cancer. This gene therapy strategy exploits the toxic pro-oxidant property of methylselenol, which is released from selenomethionine (SeMET) by cancer cells with the adenoviral-delivered methionine alpha,gamma-lyase (MET) gene cloned from Pseudomonas putida. In MET-transduced tumor cells, the cytotoxicity of SeMET is increased up to 1000-fold compared with nontransduced cells. A strong bystander effect occurred because of methylselenol release from MET gene-transduced cells and uptake by surrounding tumor cells. Methylselenol damaged the mitochondria via oxidative stress and caused cytochrome c release into the cytosol, thereby activating the caspase cascade and apoptosis. Adenoviral MET-gene/SeMET treatment also inhibited tumor growth in rodents and significantly prolonged their survival. Recombinant adenovirus-encoding MET gene-SeMET treatment thereby offers a new paradigm for cancer gene therapy.  (+info)

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly. (45/405)

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.  (+info)

Intra- and interspecies regulation of gene expression by Actinobacillus actinomycetemcomitans LuxS. (46/405)

The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS(-), a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the luxS-regulated genes uvrB and hasF in this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron by A. actinomycetemcomitans and induce responses in other periodontal organisms in mixed-species oral biofilm.  (+info)

Transcription factor FnrP from Paracoccus denitrificans contains an iron-sulfur cluster and is activated by anoxia: identification of essential cysteine residues. (47/405)

The Paracoccus denitrificans transcription factor FnrP has been characterized using artificial FNR-dependent promoter-lacZ fusion plasmids in Escherichia coli. FnrP can activate both class I and class II FNR-dependent promoters in response to anoxia but shows a marked preference for the class II promoter, where the FNR binding site is centered at -41.5 with respect to the transcription start site. FnrP was found to be inactive in an iscS mutant in vivo, demonstrating a requirement for cysteine desulfurase activity to assemble an iron-sulfur cluster in FnrP. Accordingly, an iron-sulfur cluster could be reconstituted into the purified protein in vitro using cysteine desulfurase, ferrous ions, and cysteine. Thus, FnrP is a true orthologue of FNR from E. coli and switches on target genes in response to anoxia. Inactivation of FnrP by oxygen very likely involves the oxidative disassembly of an iron-sulfur cluster. Possible ligands for the iron-sulfur cluster were identified by substituting each of the seven cysteine residues with serine and characterizing the altered proteins in vivo. Four substituted proteins showed activities less than 5% of the wild type, and so identify the four cysteines (Cys-14, Cys-17, Cys-25, and Cys-113) that are most likely to be involved in cluster ligation. The effects of N-oxides, NO-releasing compounds and a nitrosating agent on FNR and FnrP activity were investigated in vivo using the reporter system. Both proteins are very sensitive to the inclusion of sodium nitroprusside (a source of NO(+)) in defined growth media but are only moderately sensitive to those sources of NO that were tested.  (+info)

Inhibition of gamma-glutamyl transpeptidase or cysteine S-conjugate beta-lyase activity blocks the nephrotoxicity of cisplatin in mice. (48/405)

Cisplatin is nephrotoxic. The mechanism underlying this organ-specific toxicity is unknown. We hypothesize that cisplatin is metabolized via a gamma-glutamyl transpeptidase (GGT) and cysteine S-conjugate beta-lyase-dependent pathway that has been shown to activate several haloalkenes to nephrotoxins. To test this hypothesis, we inhibited GGT and cysteine S-conjugate beta-lyase in C57BL/6 mice and analyzed the effect of the inhibitors on the nephrotoxicity of cisplatin. GGT was inhibited by pretreating the mice with acivicin. Cysteine S-conjugate beta-lyase was inhibited by aminooxyacetic acid (AOAA). Male C57BL/6 mice were treated with 15 mg/kg cisplatin (i.p.) and sacrificed on day 5. Half the mice treated with cisplatin alone died before sacrifice. The cisplatin-treated mice sacrificed at 5 days had significantly elevated levels of blood urea nitrogen (BUN). Histologic analysis revealed severe damage to the renal proximal tubules. Pretreatment with acivicin or AOAA protected the mice from the nephrotoxicity of cisplatin. None of the pretreated animals died before sacrifice. BUN levels and quantitative histologic analysis of the kidneys confirmed the protective effect of acivicin and AOAA. Platinum levels in the kidneys were not altered by acivicin or AOAA, indicating that neither affected the uptake of cisplatin into the kidney. Likewise, cisplatin-induced weight loss was not altered by acivicin or AOAA, suggesting that weight loss and nephrotoxicity are via distinct mechanisms. These data support the hypothesis that the nephrotoxicity of cisplatin is due to the metabolism of a platinum-glutathione conjugate by GGT and cysteine S-conjugate beta-lyase to a potent nephrotoxin.  (+info)