Citrobacter rodentium possesses a functional type II secretion system necessary for successful host infection.

Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens. To establish successful host infections these pathogens employ a plethora of virulence factors necessary to compete with the resident microbiota, and evade and subvert the host defenses. The type II secretion system (T2SS) is one such conserved molecular machine that allows for the delivery of effector proteins into the extracellular milieu. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of human intestinal disease caused by pathogenic Escherichia coli such as Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC). In this study, we determined that the C. rodentium genome encodes one T2SS and 22 potential T2SS-secreted protein effectors, as predicted via sequence homology. We demonstrated that this system was functional in vitro, identifying a role in intestinal mucin degradation allowing for its utilization as a carbon source, and promoting C. rodentium attachment to a mucus-producing colon cell line. During host infection, loss of the T2SS or associated effectors led to a significant colonization defect and lack of systemic spread. In mice susceptible to lethal infection, T2SS-deficient C. rodentium was strongly attenuated, resulting in reduced morbidity and mortality in infected hosts. Together these data highlight the important role of the T2SS and its effector repertoire during C. rodentium pathogenesis, aiding in successful host mucosal colonization.

Different organization of base excision repair of uracil in DNA in nuclei and mitochondria and selective upregulation of mitochondrial uracil-DNA glycosylase after oxidative stress.

Oxidative stress in the brain may cause neuro-degeneration, possibly due to DNA damage. Oxidative base lesions in DNA are mainly repaired by base excision repair (BER). The DNA glycosylases Nei-like DNA glycosylase 1 (NEIL1), Nei-like DNA glycosylase 2 (NEIL2), mitochondrial uracil-DNA glycosylase 1 (UNG1), nuclear uracil-DNA glycosylase 2 (UNG2) and endonuclease III-like 1 protein (NTH1) collectively remove most oxidized pyrimidines, while 8-oxoguanine-DNA glycosylase 1 (OGG1) removes oxidized purines. Although uracil is the main substrate of uracil-DNA glycosylases UNG1 and UNG2, these proteins also remove the oxidized cytosine derivatives isodialuric acid, alloxan and 5-hydroxyuracil. UNG1 and UNG2 have identical catalytic domain, but different N-terminal regions required for subcellular sorting. We demonstrate that mRNA for UNG1, but not UNG2, is increased after hydrogen peroxide, indicating regulatory effects of oxidative stress on mitochondrial BER. To examine the overall organization of uracil-BER in nuclei and mitochondria, we constructed cell lines expressing EYFP (enhanced yellow fluorescent protein) fused to UNG1 or UNG2. These were used to investigate the possible presence of multi-protein BER complexes in nuclei and mitochondria. Extracts from nuclei and mitochondria were both proficient in complete uracil-BER in vitro. BER assays with immunoprecipitates demonstrated that UNG2-EYFP, but not UNG1-EYFP, formed complexes that carried out complete BER. Although apurinic/apyrimidinic site endonuclease 1 (APE1) is highly enriched in nuclei relative to mitochondria, it was apparently the major AP-endonuclease required for BER in both organelles. APE2 is enriched in mitochondria, but its possible role in BER remains uncertain. These results demonstrate that nuclear and mitochondrial BER processes are differently organized. Furthermore, the upregulation of mRNA for mitochondrial UNG1 after oxidative stress indicates that it may have an important role in repair of oxidized pyrimidines.

Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major.

A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichia coli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi.

We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts. Further examination of the 5' flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain. The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis. The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context.

African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced.

[Prescription of platelet antiaggregants in secondary prevention of ischemic heart disease]. / Prescripción de antiagregantes plaquetarios en la prevención secundaria de la cardiopatía isquémica.

OBJECTIVE: To find out how many patients older than 64 years of age seen in a primary health care (PHC) centre receive antiplatelet drugs for secondary prevention of coronary heart disease (CHD), as well as by whom they are prescribed, which drug is chosen, and what are its contraindications, unwanted effects and motives for ending therapy. DESIGN: Description of all cases of CHD among patients older than 64, identified through the audit of clinical records. SETTING: Urban health care centre with 23,702 inhabitants, with 2,742 over 64, 2,660 of whom have clinical records. SUBJECTS: Patients over 64 with CHD, seen in the health centre within 1993. MEASUREMENTS AND INTERVENTIONS: Age, sex, type of CHD, therapy with a platelet aggregation inhibitor, drug used, dose, prescriptor, adverse events, contraindications. RESULTS: We identified 179 cases of CHD, a prevalence of 6.7%, of which 60.9% were male. 94 patients received an antiplatelet drug: aspirin (88.3%), dypiridamol and triflusal (5.3% each) and ticlopidine (1 case). 111 patients were adequately treated, including 84 given aspirin or ticlopidine, 12 patients in which therapy was ended due to adverse events, and 15 patients in which use of antiaggregant drugs was contraindicated. All prescriptions originating from general practitioners were for aspirin, while specialists prescribed other drugs in 11% of cases. CONCLUSION: Two-thirds of patients with CHD were correctly treated. Aspirin is the antiaggregant drug most frequently used, particularly among PHC physicians. Even low doses of aspirin were associated with interruptions of therapy due to adverse events.