A <Emphasis Type="Italic">Bacillus anthracis</Emphasis> system for acquisition of heme-bound iron

In Bacillus anthracis, an alternative to the use of chelating agents (siderophores) for acquiring iron consists in using a system of hemophores and transporter proteins for scavenging hemes from hemoglobin and delivering them into the bacterial cell. B. anthracis utilizes the siderophore-mediated mechanism of acquiring iron predominantly at the intracellular stage of its development, with the system of hemophores and transporter proteins being used at the extracellular stage of anthrax infection. The structure, genetic organization, and functions of the system for heme iron acquisition in B. anthracis, understanding of which may facilitate the development of new therapies for anthrax, are discussed.     

Antitrichomonal strains of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Parasporal inclusion proteins from a total of 816 Bacillus thuringiensis strains isolated in Japan were examined for antitrichomonal activity against Trichomonas vaginalis. Ten strains of B. thuringiensis inhibited the growth of T. vaginalis in 48 h cultures at 37 degrees C. Moreover, the B622 and B626 strains clearly showed trichomonacidal effects against T. vaginalis. The H antigen serotypes of both strains were identified as H13/29 (pakistani/amagiensis). The parasporal inclusion protein from both strains consisted of three major polypeptides of 77, 45 and 25 kDa.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

<Emphasis Type="Italic">chsZ</Emphasis>, a gene for a novel class of chitin synthase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

We cloned and characterized a novel Aspergillus oryzae chitin synthase gene, chsZ, encoding a polypeptide containing a new myosin motor-like domain in its N-terminal half. Alignment analysis revealed that ChsZ was less homologous to known class V enzymes, except for its probable chitin synthase conserved region in the C-terminal half. We also found a chsY gene and found that ChsY showed higher similarity to the class V enzymes than did ChsZ. Phylogenetic analysis clearly demonstrated that the A. oryzae ChsZ, together with Chs4 of Paracoccidioides brasiliensis and Chs6 of Ustilago maydis, formed a new subclass distinct from A. oryzae ChsY and known class V chitin synthases, including A. nidulans CsmA (ChsD) and A. fumigatus ChsE. In conclusion, we propose a new class, class VI chitin synthases, represented by A. oryzae ChsZ, P. brasiliensis Chs4 and U. maydis Chs6. Expression analysis suggested that the regulation of chsZ expression is distinct from that of chsY expression.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

Identification of the <Emphasis Type="Italic">het-r</Emphasis> vegetative incompatibility gene of <Emphasis Type="Italic">Podospora anserina</Emphasis> as a member of the fast evolving <Emphasis Type="Italic">HNWD</Emphasis> gene family

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. Sequence data reported here are available in the Genbank database under accession numbers FJ269240 and FJ269239 for het-r and het-R, respectively.     

Molecular typing of <Emphasis Type="Italic">Shigella</Emphasis> strains

Shigellosis remains a serious medical problem for many countries and is especially common in the developing world. In recent years, molecular-genetic methods have been increasingly replacing traditional phenotypic approaches in investigation of cases of shigellosis infection. This review discusses existing molecular-genetic methods of Shigella typing, the fields of their application, selection criteria in accordance with the goal of research, and their advantages and disadvantages.     

Immunogenicity and cross-reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> of proteoliposomes derived from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The only currently available vaccine against tuberculosis (TB) is Mycobacterium bovis Bacille Calmette-Guerin (BCG), which has inconsistent efficacy to protect against the disease in adults. M. tuberculosis (MTB) cell wall components have been implicated in the pathogenicity of TB and therefore have been a prime target for the identification and characterization of cell wall proteins with potential application in vaccine development. In this regard, proteoliposomes (PLs) derived from mycobacteria containing lipids and cell wall proteins could be potential vaccine candidates against TB. In the present study PLs derived from BCG were prepared. These homogeneous population of spherical microparticles was then immunized into Balb/c mice. Sera of immunized animals showed high IgG response and strong cross-reactivity against different MTB antigens.These results showed that BCG PLs could be potential vaccine candidates against TB.     

<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">aeruginosa</Emphasis> Biofilms in CF Infection

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.     

<Emphasis Type="Italic">KNQ1</Emphasis>, a<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis> gene encoding a drug efflux permease

Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells. In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene. The isolated gene exhibited 48.9% identity with the S. cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline-N-oxide and encoded a protein of 553 amino acids. When present in multicopy, it efficiently complemented the phenotype associated with the pdr5 or pdr1pdr3 mutations in S. cerevisiae. Overexpression of the KNQ1 gene in K. lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline-N-oxide, 3-aminotriazole, bifonazole and ketoconazole. The gene was assigned to K. lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide. Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.Communicated by K. Breunig     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

Evaluation of the BactiCard <Emphasis Type="Italic">Neisseria</Emphasis> for Identification of Pathogenic <Emphasis Type="Italic">Neisseria</Emphasis> Species and <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis>

The BactiCard Neisseria (Remel, USA) is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB), prolyl aminopeptidase (PRO), γ-glutamyl aminopeptidase (GLUT), and ?-galactosidase (BGAL). These substrates permit the identification of Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively. After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. IB and BGAL reactions are read for a blue-green color after 2 and 15 min, respectively. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. Identifications obtained with the BactiCard Neisseria were compared with those obtained using conventional procedures for 558 isolates in a blinded fashion. The BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates. The BactiCard Neisseria is an accurate and rapid system for identification of these microorganisms in the clinical laboratory. Electronic Publication     

Transmission of <Emphasis Type="Italic">Yersinia pestis</Emphasis> cultures with different plasmid content from <Emphasis Type="Italic">Xenopsylla cheopis</Emphasis> to <Emphasis Type="Italic">Calomys callosus</Emphasis>

Most Brazilian Yersinia pestis isolates display a typical plasmid profile composed of the three classical plasmids: pYV, pPst and pFra. However, some cultures lack at least one of these plasmids, while a few of them harbour atypical DNA bands of molecular weight ranging from 147 to 11.5 kb. To investigate whether Y. pestis displaying atypical plasmid content could be propagated among rodents in nature through flea bites, we carried out studies with fleas ( Xenopsylla cheopis) and rodents ( Calomys callosus) reared in the laboratory and five Y. pestis cultures differing in plasmid content. The results suggest that: (1) the single presence of pYV is not sufficient for the transmission of Y. pestis by fleas, (2) pPst is not essential for transmission, (3) two atypical DNA bands of molecular weight of 30 kb and >90 kb have no biological role, and (4) pFra is required for the transmission of Y. pestis by flea bites. Other studies are needed to determine whether this plasmid alone is sufficient for transmission.