Exploring the relationship between toxin and spore production in the human enteric pathogen Clostridium difficile

RESUMO: Clostridium difficile é presentemente a principal causa de doença gastrointestinal associada à utilização de antibióticos em adultos. C. difficile é uma bactéria Gram-positiva, obrigatoriamente anaeróbica, capaz de formar endósporos. Tem-se verificado um aumento dos casos de doença associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrência, em parte, devido à emergência de estirpes mais virulentas, mas também devido à má gestão do uso de antibióticos. C. difficile produz duas toxinas, TcdA e TcdB, que são os principais fatores de virulência e responsáveis pelos sintomas da doença. Estas são codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxigénio são essenciais para a transmissão do organismo e recorrência da doença. A expressão dos genes do PaLoc ocorre em células vegetativas, no final da fase de crescimento exponencial, e em células em esporulação. Neste trabalho construímos dois mutantes de eliminação em fase dos genes tcdR e tcdE. Mostrámos que a auto-regulação do gene tcdR não é significativa. No entanto, tcdR é sempre necessário para a expressão dos genes presentes no PaLoc. Trabalho anterior mostrou que, com a exceção de tcdC, os demais genes do PaLoc são expressos no pré-esporo. Mostrámos aqui que TcdA é detectada à superfície do esporo maduro e que a eliminação do tcdE não influencia a acumulação de TcdA no meio de cultura ou em associação às células ou ao esporo. Estas observações têm consequências para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser também um veículo para a entrega da toxina nos estágios iniciais da infecção, que TcdA possa ser libertada durante a germinação do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligação à mucosa do cólon.---------------------------ABSTRACT: Clostridium difficile is currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract. Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of “hypervirulent” strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence. The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation of tcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes. A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.

Proteocephalidea from Amazonian Freshwater Fishes: New Systematic Arrangement for the Species Described by Woodland as ANTHOBOTHRIUM (Tetraphyllidea)

The, taxonomic status of the Brazilian species Anthobothrium pristis, A. piramutab, A. mandubé and A. karuatayi has been reappraised. A. pristis from an elasmobranch in the Amazon, is confirmed as a member of the tetraphyllidean genus Anthobothrium, but the others, from siluroid teleosts, are transferred to Proteocephalidea. A. piramutab is transferred to Proteocephalus as a new combination. Gibsoniela n.g. is erected in Zygobothriinae for A. mandubé and Brayela n.g. is erected in a new subfamily, Brayelainae for A. Karuatayi.

Four new species of Wing Rolling Neodawnaria (Homoptera : Derbidae) from the Amazon Basin

Four new species of Neidawnaria are described, and compared with other members of the genus. Keys and geographical distribution are given for all nem species and the wings of two other species are illustrated.

Vocalizations of primary forest frog species in the Central Amazon.

The calls of 18 species of Amazonian forest frogs were recorded in 3 localities: the Tapajos National Park near Itaituba, the Reserva Ducke near Manaus, and the INPA-WWF reserves near Manaus. Structural and time parameters and sonographs of these calls including previously undescribed vocalization by 10 species are presented. Unlike open habitat species, several forest frong species characteriscally demonstrated one on more of the following temporal parameters: very low call rates, sporadic intervals, infrequent nights of calling and synchronized chorusing. It is hypothesized that predation has influenced the evolution of vocal behaviour in Amazonian forest frogs.

A new species of Abarema Pittier (Mimosaceae ingeae) from Amazonian Brazil.

Abarema piresii Barneby & Grimes is described from the Brazilian states of Amazonas and Pará. The species is illustrated an tis relationships are discussed. Two kindred species are incidentally transferred from Pithecellobium to Abarema as A. curvicarpa (Irwin) and A. moniliformis (Ducke) Barneby & Grimes.

A new species of Hibiscus from the Amazon basin.

A new species, Hibiscus amazonicus, is described from the Amazon Basin. Characters distinguishing the new species from its nearest relatives presented.

Cyperaceae of Tropical America: some new or critical species III

Discussed are the taxonomy of the Scleria arundinacea-latifoliacomplex, S. mitis, and S. myricocarpa, all controversial and widespread taxa in tropical America. Scleria cerradicola is described as new in the section Hymenolytrum and a range extension of a rare species, Scleria neogranatensis, is noted.

New and interesting species of Justicia(Acanthaceae) from Amazonia

A new Amazonian species of Justicia, J. zarucchii from the region oif the Rio Abacaxis is described, illustrated, and compared with J. schultesii and J. gutierrezii, its closest relatives.

Ficus species of Brazilian Amazonia and the Guianas

In order, to place the new taxa. of Ficus found in Amazonian Brazil and the Guianas (see Vázquez Avila, Berg & Kooy, Acta Amazonica, fallowing article in this volume) in a taxonomic and phytogeographic framework, a key to the taxa provisionally recognized for Amazonia and the Guianas is presented and fallowed by a concise treatment of these taxa.

A new species of Pithecellobiutn (MIMOSACEAE INGEAE) from Brazilian Amazonia

Phithecellobium prancei, related to T. simabifolium Benth. and P. spruceanum Benth., is described and figured from specimens collected on white sand savannas along rio Aracá in northern Amazonas, Brazil. Its relationships within sect. Samanea ser. Coriaceae Benth., a group due for generic segregation from Pithecellobium sens. lat., are discussed.

Reproductive biology of Bellucia (MELASTOMATACEAE)

The reproductive biology of five of the seven species of Bellucia (Melastomataceae), a genus of shrubs and small trees, was investigated in Amazonia. Sucessful fruit-set by Bellucia requires floral visitation by bees. The flowers are produced continuously all year, and are visited by a wide variety of female bees, the principal pollinators being Xylocopa, Centris, Ptilotopus, Epicharis, Eulaema, Bombus, and Oxaea. The floral attractants are color and the odor produced by the pollen, stamens, and petals; the reqard is pollen. Three species of Bellucia are self-incompatible. Indiscriminate visitor behavior and lack of phenological, morphological, or genetic barriers lead to hybridization between sympatric species of Bellucia, and no more than two species occupy the same habitat at anu one locality. Bellucia produces berries with numerous small seeds, and is dispersed by birds, bats, monkeys, tapirs, turtles, and ants. Seedling establisment requires full sunlight, and occurs on a variety of soil types. The reproductive strategy is interpreted as that of a pioneer species.

Lophopidae of the amazon basin with keys to new world genera and species (HOMOPTERA: FULGOROIDEA).

The two especies of Lophopidae found in the lowland Amazon Basin, Hesticus rufimanus, (walker) and H. sanguinifrons Muir, are redescribed and their geographical distributions given. Keys are provided to genera of New World lophopidae, and to species of Hesticus .

Distribution of Atta (Hymenoptera - Formicidae) in "terra-firme" rain forest of Central Amazonia: density, species composition and preliminary results on effects of forest fragmentation

One hundred and fourteen hectares of a "terra-fiirme" rain forest 70 km north of Manaus, Amazonas, Brazil, were surveyed for leaf-cutting ant colonies (Atta spp). One half of this area was in isolated forest fragments (surrounded by pastures or second growth) of two sizes: 1 and 10 ha. The other half was in non-isolated fragments (connected to a large parch of forest) of the same sizes. Only two species occured in this forest: Atta sexdens sexdens L. and A. cepfhalotes L. The first was the most abundant species with a mean density of 0.35 colonies per ha. The mean density of A. cephalotes colonies was 0.03 per ha. The density of colonies was not significantly different between the isolated fragments and the continuous forest. Furthermore, the species composition did not change with isolation. However, pre-isolation data and long term monitoring are necessary to conclude that the isolation of a forest fragment has no effect upon Atta colonies. The non-uniform spatial distribution of Atta colonics within the "terra-firme" forest must be taken into account when selecting conservation areas in the Amazon, in order to preserve this important group of ants together with their native habitat.

Transpiration rates for several woody successional species and for a pasture in the upper Amazon basin in Venezuela

Evapotranspiration rates for a eight month old tropical pasture were estimated using the Penman-Monteith equation. Transpiration rates for several woody secondary successional species and stump sprous in the pasture and conucos (farm sites) were measured using the tritiated water technique.The stuty area was located near the village of San Carlos de Rio Negro (1° 56' N, 67° 03' W) in southern Venezuela, near the confluence of the Casiquiare and the Rio Guania wich forms the Rio Negro. The terrain was gently rolling with the areas between the small ridges supporting Amazon caatina forests on spodosols, and higher never flooded areas (tierra firma) supporting a mixed species forest.Results indicated that for a one month period, ET loss (0.46 cm/day) from the pasture, including soil and root mat evaporation, was about 0.43 cm/day less than estimated from the adjacent undisturbed forest (0.89 cm/day). Pan A evaporation for the same time period was 0.64 cm/day. Transpiration rates for seed established species were significantly less (0.38 cm/day) than for stump sprouts (1.09 cm/day) of the primary forest in the pasture.

Brasilian Therevidae (diptera): a checklist and discriptions of species.

95 Specimens representing 12 Species in 5 genera form the basis of this paper. 6 new Species are described, namely: Henicomyia amazonica, n. sp. (Bnazil: Amazonas) , Brachylinga xanthoperra, n. sp. (Brazil:Roraima), Cyclotelus badicrusus, n. sp. (Brazil-.Amazonas; Ecuador: Pichinca), C. silacrusus, n. sp. (Brazil-.São Paulo), Penniverpa alvadusta. n. sp. (Brazil-.Amazonas) and P. alvatra, n. sp. (Brazil-.Roraima) . A check-list of Braziliam Therevidae, based on literature accounts and examined matenial, is ALSO provided. It lists 22 Species in 7 genera, one not formally described. New combinations, and new synonyms ore proposed.