Pathogenesis II: Fungal responses to host responses: interaction of host cells with fungi

Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures. The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules. Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival. Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity. Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules. The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization. Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic. It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation. For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence. Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell. These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface. The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper.

Patogenicidade cruzada de Ceratobasidium spp. do caquizeiro (Diospyros kaki) e do chá(Camellia sinensis) e reação de cultivares de caqui ao patógeno

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Divergence Between Sympatric Rice- and Maize-Infecting Populations of Rhizoctonia solani AG-1 IA from Latin America

The basidiomycetous fungus, Rhizoctonia solani anastomosis group (AG)-1 IA is a major pathogen in Latin America causing sheath blight (SB) of rice Particularly in Venezuela. the fungus also Causes banded leaf and sheath blight (BLSB) oil maize, which is considered all emerging disease problem where maize replaced traditional rice-cropping areas or is now planted in adjacent. fields Our goals in this study Were 10 elucidate (i) the effects of host specialization on gene flow between sympatric and allopatric rice and maize-infecting fungal populations and (ii) the reproductive mode of the fungus, looking for evidence of recombination in total, 375 isolates of R. solani AG1 IA sampled from three sympatric rice and maize fields in Venezuela (Porutuguesa State) and two allopatric rice fields from Colombia (Meta State) and Panama (Chiriqui State) were genotyped Using, 10 microsatellite loci Allopatric populations from Venezuela. Colombia. and Panama were significantly differentiated (Phi(ST), of 0 16 to 0 34). Partitioning of the genetic diversity indicated differentiation between sympatric populations from different host species, with 17% of the total genetic variation distributed between hosts while only 3 to 6% wits distributed geographically among the sympatric Venezuelan Fields We detected symmetrical historical migration between the rice- and the maize-infecting populations from Venezuela Rice- and maize-derived isolates were able to infect built rice and maize but were more aggressive Oil their original hosts, consistent with host specialization. Because the maize- and rice-infecting populations are still cross-pathogenic, we postulate that the genetic differentiation was relatively recent and mediated via a host shift. An isolation with nu.-ration analysis indicated that the maize-infecting population diverged from the rice-infecting population between 40 and 240 years ago Our findings also suggest that maize-infecting Populations have a mainly recombining reproductive system whereas the rice-infecting Populations have a Mixed reproductive system in Latin America

Pythium insidiosum: relato do primeiro caso de infecção humana no Brasil

A pitiose é causada por microorganismo aquático, fungo-símile, o Pythium insidiosum, patógeno de homens e animais. Observou-se um paciente com úlcera fagedênica no membro inferior, com exame anatomopatológico sugestivo de zigomicose, pouco sensível à terapêutica antifúngica, obtendo-se cura por meio de ampla exérese. A comprovação etiológica resultou de métodos moleculares, com amplificação e seqüenciamento de DNA de organismo isolado em ágar Sabouraud, observando-se 100% de analogia com seqüências de P. insidiosum depositadas no GenBank.

Virulence profiles of ten Paracoccidioides brasiliensis isolates obtained from armadillos (Dasypus novemcinctus)

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis ( PCM), the most important systemic mycosis in Latin America. The armadillo, Dasypus novemcinctus, has been confirmed as the primary natural reservoir of this fungus. Its geographic distribution is similar to that of human PCM. In this study, virulence profiles of 10 P. brasiliensis isolates from different armadillos and of two clinical isolates were tested in an experimental hamster model. Pathogenicity was evaluated by counting cfu and performing histopathological analysis in the testis, liver, spleen and lung. Circulating specific antibodies were measured using enzyme- linked immunosorbent assay ( ELISA). All isolates from armadillos were virulent in the model, with dissemination to many organs. The clinical isolates, which had long been stored in cultured collections, were less virulent. The isolates were classified into four virulence categories according to number of cfu per gram of tissue: very high, high, intermediate and low. This study confirms that armadillos harbor pathogenic genotypes of P. brasiliensis, probably the same ones that infect humans.

Clues to the presence of pathogenic fungi in certain environments

The presence of various pathogenic fungi in rather unsuspected hosts and environments has always attracted the attention of the scientific community. Reports on the putative role of animals in fungal infections of humans bear important consequences on public health as well as on the understanding of fungal ecology. Fungi are ubiquitous in nature and their great capacity for adaptation allows them to survive and indeed, to thrive, in plants, trees and other natural substrata. Nonetheless, we are just beginning to learn the significance that these diverse fungal habitats have on the increasing number of immunosuppressed individuals. The accidental or permanent presence of fungi in animals, plants, soils and watercourses should not be taken too lightly because they constitute the source where potential pathogens will be contracted. If those fungal habitats that carry the largest risks of exposure could be defined, if seasonal variations in the production of infectious propagules could be determined, and if their mode of transmission were to be assessed, it would be possible to develop protective measures in order to avoid human infection. Additionally, unsuspected avenues for the exploration of fungal survival strategies would be opened, thus enhancing our capacity to react properly to their advancing limits. This paper explores several ecological connections between human pathogenic fungi and certain animals, trees, waterways and degraded organic materials. The occurrence of such connections in highly endemic areas will hopefully furnish more precise clues to fungal habitats and allow the design of control programs aimed at avoiding human infection.

Influence of TLR-2 in the immune response in the infection induced by fungus Sporothrix schenckii

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Microscopic evidence supports the hypothesis of high cellulose degradation capacity by the symbiotic fungus of leaf-cutting ants

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis, a disease confined to Latin America and of marked importance in the endemic areas due to its frequency and severity. This species is considered to be clonal according to mycological criteria and has been shown to vary in virulence. To characterize natural genetic variation and reproductive mode in this fungus, we analyzed P. brasiliensis phylogenetically in search of cryptic species and possible recombination using concordance and nondiscordance of gene genealogies with respect to phylogenies of eight regions in five nuclear loci. Our data indicate that this fungus consists of at least three distinct, previously unrecognized species: S1 (species 1 with 38 isolates), PS2 (phylogenetic species 2 with six isolates), and PS3 (phylogenetic species 3 with 21 isolates). Genealogies of four of the regions studied strongly supported the PS2 clade, composed of five Brazilian and one Venezuelan isolate. The second clade, PS3, composed solely of 21 Colombian isolates, was strongly supported by the alpha-tubulin genealogy. The remaining 38 individuals formed S1. Two of the three lineages of P. brasiliensis, S1 and PS2, are sympatric across their range, suggesting barriers to gene flow other than geographic isolation. Our study provides the first evidence for possible sexual reproduction in P. brasiliensis S1, but does not rule it out in the other two species.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or internal proteins are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

ROLE of TLR2 and TLR4 on human neutrophil functions against Paracoccidioides brasiliensis

In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), studies have focused on the role of neutrophils that are involved in the primary response to the fungus. Neutrophil functions are regulated by pro- and anti-inflammatory cytokines. Molecular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll-like receptors (TLRs). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated by GM-CSF, IL-15, TNF-alpha or IFNgamma and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H(2)O(2) and IL-6, IL-8, TNFalpha and IL-10 production, by activating and challenging cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression, inducing an additional cytokine effect. on the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H(2)O(2) production; however, this process was not associated with TLR2 or TLR4. Neutrophil activation by GMCSF and TNF-alpha resulted in a significant increase of IL-8 production, while IL-15 and IFN-alpha have no effect. Pb18 also augmented IL-8 expression, inducing an additional effect to that of cytokines. None of the cytokines activated neutrophils by releasing IL-10. This cytokine was only detected after Pb18 challenge. Interestingly, IL-8 and IL-10 production involved TLR2 and mainly TLR4 modulation. The present results suggest that Pb18 interaction with neutrophils through TLR2 and TLR4 with consequent IL-8 and IL-10 production may be considered a pathogenic mechanism in paracoccidioidomycosis.

Role of TLR2 and TLR4 in Human Neutrophil Functions Against Paracoccidioides brasiliensis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efficacy of the nematode-trapping fungus Duddingtonia flagrans against infections by Haemonchus and Trichostrongylus species in lambs at pasture

The efficacy of the nematode-trapping fungus Duddingtonia flagrans against infections by trichostrongyle nematodes in sheep was assessed throughout 6 months. Twenty Ile de France lambs were divided into two groups (control and treated groups), which were kept in separate pastures. Animals of the treated group were fed with D. flagrans twice a week (Tuesdays and Fridays). Pellets were prepared with the fungus mycelia in liquid culture medium and contained approximately 20% fungus. They were mixed with the animals' diet at a concentration of 1 g pellet per 10 kg live weight. Faecal egg counts (FEC), packed cell volume (PCV), total serum protein and the animals' body weight were determined fortnightly from 7 October 2005 to 24 March 2006. Comparison of such parameters between groups showed no significant differences (P > 0.05), except on 10 February 2006, when the control group presented a higher mean FEC than the treated group (P < 0.05). Feeding sheep with pellets containing D. flagrans had no benefit to the prophylaxis of nematode infections under the experimental conditions used in the present study.

Substrate distribution in fungus chambers in nests of Atta bisphaerica Forel, 1908 (Hym., Formicidae)

Substrate distribution was studied in three adult colonies of the leaf-cutting ant Atta bisphaerica Forel, 1908 using dye. Some supply holes were mapped using baits made from small plastic straws impregnated with citric pulp. Two holes, equidistant from one another, were then selected in each nest. The colonies were excavated completely 24 h after bait placement. During excavation, fungus chambers were checked for dye. We observed that the dyed baits were distributed in all sectors and at all depths regardless of where the baits had been placed. This supported the hypothesis that toxic baits placed in a single supply hole are uniformly distributed throughout the colony.

Influence of physical and chemical factors during foraging and culture of the symbiont fungus in Atta sexdens rubropilosa (Hymenoptera : Formicidae)

The aim of this paper is to investigate the influence of physical and chemical factors on transport and use of substrate for Atta sexdens rubropilosa workers. Three types of rectangular fragments were used to study the physical influence factors: filter paper with paraffin, filter paper without paraffin and polyester film. To study the chemical factors, some fragments were impregnated with organic extract of orange albedo, others were soaked with soybean oil and for the remaining ones nothing was applied. The following parameters were evaluated: (i) attractiveness of substrate for transport and number of loading workers per treatment; (ii) foraged material incorporation; (iii) rejection by numbers of fragments deposited in the garbage or beside the fungus garden. All the polyester film fragments carried out to the fungus garden were subsequently rejected. We verified that chemical factors of the substrate were more quickly detected by the workers, whereas physical factors were used as a criterion in the decision-making to reject or accept the substrate collected.