The transported cations impose differences in the thermostability of the gastric H,K-ATPase. A kinetic analysis.

This work analyses the thermostability of a membrane protein, the gastric H,K-ATPase, by means of a detailed kinetic characterization of its inactivation process, which showed to exhibit first-order kinetics. We observed parallel time courses for the decrease of ATPase activity, the decrease of the autophosphorylation capacity and the loss of tertiary structure at 49 °C. Higher temperatures were required to induce a significant change in secondary structure. The correspondence between the kinetics of Trp fluorescence measured at 49 °C and the decrease of the residual activity after heating at that temperature, proves the irreversibility of the inactivation process. Inactivation proceeds at different rates in E1 or E2 conformations. The K+-induced E2 state exhibits a lower inactivation rate; the specific effect is exerted with a K0.5 similar to that found at 25 °C, providing a further inkling that K+ occlusion by the H,K-ATPase is not really favoured. Increasing [H+] from pH 8 to pH 7, which possibly shifts the protein to E1, produces a subtle destabilizing effect on the H,K-ATPase. We performed a prediction of potential intramolecular interactions and found that the differential stability between E1 and E2 may be mainly explained by the higher number of hydrophobic interactions in the α- and ß-subunits of E2 conformation.

The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1-E2 conformational changes assessed by eosin fluorescence measurements.

H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1-E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K â†’ E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that-during dephosphorylation-the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.

Analyses of the genetic diversity and population structures of Histoplasma capsulatum clinical isolates from Mexico, Guatemala, Colombia and Argentina, using a randomly amplified polymorphic DNA-PCR assay.

We studied the genetic diversity and the population structure of human isolates of Histoplasma capsulatum, the causative agent of histoplasmosis, using a randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assay to identify associations with the geographic distribution of isolates from Mexico, Guatemala, Colombia and Argentina. The RAPD-PCR pattern analyses revealed the genetic diversity by estimating the percentage of polymorphic loci, effective number of alleles, Shannon's index and heterozygosity. Population structure was identified by the index of association (IA) test. Thirty-seven isolates were studied and clustered into three groups by the unweighted pair-group method with arithmetic mean (UPGMA). Group I contained five subgroups based on geographic origin. The consistency of the UPGMA dendrogram was estimated by the cophenetic correlation coefficient (CCCr = 0.94, P = 0.001). Isolates from Mexico and Colombia presented higher genetic diversity than isolates from Argentina. Isolates from Guatemala grouped together with the reference strains from the United States of America and Panama. The IA values suggest the presence of a clonal population structure in the Argentinian H. capsulatum isolates and also validate the presence of recombining populations in the Colombian and Mexican isolates. These data contribute to the knowledge on the molecular epidemiology of histoplasmosis in Latin America.

Identification of Aspergillus tubingensis in a primary skin infection.

OBJECTIVE: Aspergillus section Nigri comprises a group of related species that include Aspergillus niger, A. welwitschiae, A. carbonarius, A. brasiliensis and A. tubingensis. Some of these species are morphologically very similar to A. niger but exhibit different patterns of susceptibility to antifungal agents; such is the case for A. tubingensis. Therefore, when diagnosing aspergillosis, it is important to identify the pathogen at the species level. This study aimed to identify the species of an Aspergillus spp. isolate (MM-82) obtained from a patient with a dermatosis localized to the right leg. MATERIALS AND METHODS: The MM-82 isolate was examined for macro- and microscopic morphology, conidia size and thermotolerance, and a phylogenetic analysis of a benA gene segment was performed for molecular identification. Susceptibility to antifungals was determined using antifungal microdilution according to the methodology of European Society of Clinical Microbiology and Infectious Diseases (EUCAST). RESULTS: Based on its phenotypic characteristics and the phylogenetic analysis of the sequence of a benA gene segment, the MM-82 isolate was identified as A. tubingensis. This fungus did not show resistance to antifungal agents commonly used for treatment. CONCLUSION: This study demonstrated that A. tubingensis can cause skin infection; this constitutes the first report of a case of aspergillosis caused by A. tubingensis in Mexico.

Identification of the source of histoplasmosis infection in two captive maras (Dolichotis patagonum) from the same colony by using molecular and immunologic assays.

Histoplasma capsulatum was isolated from the spleen of a first infected mara (Dolichotis patagonum) and from a second mara's liver and adrenal gland, both in the same colony at the Africam Safari, Puebla, Mexico. Studies of H. capsulatum isolates, using nested-PCR of a 100-kDa protein coding gene (Hcp100) fragment and a two-primer RAPD-PCR method, suggest that these isolates were spreading in the environment of the maras' enclosure. By using a Dot-ELISA method, sera from mice inoculated with three homogenates of soil samples from the maras' enclosed space developed positive brown spot reactions to a purified H. capsulatum antigen, which identified the probable source of the maras' infection.

Molecular typing of Histoplasma capsulatum isolated from infected bats, captured in Mexico.

The present paper represents data on the genetic polymorphism of 13 Histoplasma capsulatum isolates recovered from infected bats randomly captured in the Mexican states of Morelos, Puebla, and Oaxaca. The polymorphic DNA patterns were analyzed by two-primer RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) method. To amplify the fungal genome by PCR, the following primer arrangements were used: 5'-AACGCGCAAC-3' and 5'-AAGAGCCCGT-3'; 5'-AACGCGCAAC-3' and 5'-GTTTCCGCCC-3'; or 5'-AACGCGCAAC-3' and 5'-GCGATCCCCA-3'. A common polymorphic DNA pattern of H. capsulatum was revealed in different assays. This pattern is shared by 7 H. capsulatum isolates recovered from different specimens of nonmigratory bats (Artibeus hirsutus) captured in a cave in Morelos, by 5 isolates recovered from infected migratory bats (Leptonycteris nivalis) captured in Morelos and Puebla, and by 1 isolate from another migratory bat (L. curasoae) captured in Oaxaca. This polymorphic DNA pattern of H. capsulatum could represent fungal markers for the geographic areas studied, and considering its distribution in three different states of the Mexican Republic, the role of bats as responsible for H. capsulatum spreading in nature, in relation to their movements and migrations besides their shelter habits, is suggested. Analyses of DNA patterns of H. capsulatum isolated from infected bats, from clinical cases, and from blackbird excreta, have shown a major relatedness between bats and clinical isolates, in contrast to those isolates from bird excreta.

Relatedness analyses of Histoplasma capsulatum isolates from Mexican patients with AIDS-associated histoplasmosis by using histoplasmin electrophoretic profiles and randomly amplified polymorphic DNA patterns.

The present paper analyzes the histoplasmin electrophoretic profiles and the randomly amplified polymorphic DNA (RAPD) patterns of the fungus Histoplasma capsulatum isolated from Mexican patients with AIDS-associated histoplasmosis. Clinical isolates from Guatemala, Colombia, and Panama, as well as H. capsulatum isolates from different sources in nature, were also processed. All histoplasmin samples shared four antigenic fractions of 200, 49, 10.5, and 8.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their percentage of relatedness, based on SDS-PAGE histoplasmin electrophoretic image analysis, H. capsulatum isolates were divided in two groups: group A contained all AIDS-associated isolates studied and two human reference strains from Mexican histoplasmosis patients without AIDS; group B included bat guano, infected bat, and cock excreta isolates from the State of Guerrero, Mexico, plus three human histoplasmosis strains from Guatemala, Panama, and Colombia. Polymorphic DNA patterns evaluated by RAPD-PCR showed three major bands of 4.4, 3.2, and 2.3 kb in most H. capsulatum isolates studied. Four groups were related by DNA polymorphisms: group I was formed by most of the AIDS-associated H. capsulatum isolates studied, one human histoplasmosis strain from Colombia, two human reference strains from Mexican patients without AIDS, and one human histoplasmosis strain from Guatemala. Group II consisted of only a single strain from Panama. Group III included three strains: one from a Mexican patient with AIDS and two isolated from nature in Guerrero (cock excreta and bat guano). The last, group IV, consisted of only one strain isolated from an infected bat, captured in Guerrero. A tight relationship between phenotypic and genotypic characterization was observed, and both analyses could be useful tools for typing H. capsulatum from different sources and geographic origins.

Environmental conditions favoring bat infection with Histoplasma capsulatum in Mexican shelters.

Histoplasma capsulatum was isolated from gut, lung, liver, and spleen of 17 of 208 captured bats belonging to 6 different genera and species. Three of the 17 infected bats were from the State of Guerrero and 14 were from the State of Morelos. All were adult bats: 6 males (1 Pteronotus parnellii, 2 Natalus stramineus, 2 Artibeus hirsutus, and 1 Leptonycteris nivalis) and 11 females (1 Myotis californicus, 1 Mormoops megalophylla, 8 A. hirsutus, and 1 L. nivalis). High rates of bat infection with H. capsulatum were found in the monitored sites of the State of Morelos. Histoplasma infection of N. stramineus, A. hirsutus, and L. nivalis should be considered as the first records in the world. The fungus isolated from infected bats was identified by its typical mycelial-phase morphology and by its yeast-phase conversion. Exoantigen production confirmed the fungal identification by the presence of specific precipitation lines in double immunodiffusion assays using human immune serum. Histopathologic studies showed intracellular yeast-like cells compatible with H. capsulatum yeast-phase in tissues of several bats, especially in pulmonary (intra-alveolar and septal) macrophages, with none or minimal tissue reaction. In contrast to past reports, present data support a high risk of bat infection with H. capsulatum in Mexican cave environments.

Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum.

The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris-HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal D-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68kD and 180kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for beta-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues.

[Production of fungal antigens from local strains for the immunodiagnosis of mycoses in Mexico]. / Producción de antígenos fúngicos autóctonos en el inmunodiagnóstico de micosis en México.

Fungal antigens with good reactivity and specificity are essential for the immunodiagnosis of systemic mycoses. We give here data on crude and purified antigens of Histoplasma capsulatum, Coccidioidis immitis and Paracoccidioides brasiliensis from local strains and which are the more prevalent in Mexico. The crude antigens had good reactivity in precipitating and skin testing whereas the purified antigens (DPPC: deproteinized polysaccharide protein complex) had a higher specificity in more sensitive tests such as ELISA and complement fixation. Our efficiency analysis showed that the crude antigens are best for epidemiologic studies due to their low cost, easy handling and fast detection. The purified ones are suited to establish with more precision the diagnosis of systemic mycoses.

Two-dimensional immunoelectrophoresis of histoplasmin and a purified polysaccharide-protein antigen of Histoplasma capsulatum.

Crude histoplasmin and a polysaccharide-protein complex (PPC-histo) antigens obtained from culture filtrates of Histoplasma capsulatum were analyzed by single and tandem two-dimensional immunoelectrophoresis (TD-IEP) using a rabbit hyperimmune anti-histoplasmin polyvalent serum. Single TD-IEP showed 14 arc precipitates for histoplasmin. Continuity of arcs 2, 6, and 7, and 9 and 10 was observed, suggesting a different polymeric configuration of the same antigen. This was also confirmed in tandem TD-IEP of histoplasmin with homologous (PPC-histo) and heterologous PPC's from Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis. Tandem TD-IEP of histoplasmin and PPC-histo displayed a similar antigenic pattern to histoplasmin alone, being arcs 1 and 3 more evident and apparently present only in histoplasmin and PPC-histo. Tandem TD-IEP showed common antigens among the other heterologous fungal purified antigens, and seems useful to observe the multiplicity of antigens present in fungal preparations and to identify those precipitates (arcs 1 and 3) that are predominant in the purified preparation.

Immunosuppression transfer by spleen cells from young to adult mice previous to Histoplasma capsulatum infection.

The passive transfer of spleen cells from 1 month old mice into adult syngeneic mice, abrogates their resistance to histoplasmal infection. This suppressive state was detected in two cell populations, one non-adherent and another adherent with radioresistant characteristics. The transferred spleen cells were treated by different anti-sera: anti-theta, anti-adherent cells (produced in rabbits) and monoclonal anti-Thy 1.2 respectively. The irradiated and non-irradiated adult recipient mice were infected with Histoplasma yeasts utilizing the Lethal Dose50 for 1 month old mice. The infection course was determined by death percentage, the histoplasmosis murine signs and the number of the fungal colony forming units (CFU) from the infected spleens. The results of the anti-sera treatment suggest that non-adherent as well as adherent cells participate in the suppressive phenomena. A lower number of CFU was identified in infected animals which received cells treated with anti-Thy 1.2 anti-sera.

Relationship between age and cellular suppressive activity in resistance to Histoplasma capsulatum infection.

One-month-old and 1-year-old male BALB/c mice showed a lower resistance than 4.5-month-old mice to Histoplasma capsulatum infection. 4.5-month-old mice successfully resolved the infection when challenged with either a LD50 or LD100 for 1-month-old mice. A critical clinical course of experimental histoplasmosis was observed in 4.5-month-old syngeneic mice when spleen cells from 1-month-old BALB/c mice were transferred to them. Irradiated recipient mice, into which bone marrow and spleen cells were transferred, died when infected with the LD100 for 1-month-old mice. The same occurred with 4.5-month-old non-irradiated infected mice which received only spleen cells and with 1-month-old mice which were used as a control of infection. However, infected and non-transferred 4.5-month-old mice survived this dose. Thus, the adoptive transference of spleen cells from 1-month-old mice to 4.5-month-old mice suppressed the resistance of these adult mice to infection. Apparently, the transference of the suppressive state requires the presence of two cell populations, a non-adherent and an adherent and radioresistant cell present in the spleen of male 1-month-old mice.

Antigens from Histoplasma capsulatum and Blastomyces dermatitidis. I. Immunological comparative studies from polysaccharide-protein complexes of both fungi.

Certain groups of fungi share chemical structures which makes difficult isolation and differentiation of specific antigens by the usual methods of extraction and purification. Therefore, we have oriented our studies to the immunological and biochemical characterization of differences and similarities of molecular structures from fungi, etiologic agents of systemic mycoses, hoping to establish criteria for the utilization and handling of these antigens. A deproteinized polysaccharide-protein complex (D-PPC) was isolated from Histoplasma capsulatum and Blastomyces dermatitidis. The immunological studies with humoral tests indicate a variable cross reaction between antigens of both species. In immunodiffussion systems, the reaction was specific for each species using saline solution of phosphate butter solution, while using an agarose veronal system, the cross reactions were very evident. In addition, differences in cross reactions were obtained with immunoelectrophoresis, haemagglutination and complement fixation microtest. This variation to cross reaction responses suggest that these antigens (D-PPC) share common structures but at the same time must have some different component owned by each one of the fungi which makes them more specific than crude antigens.

Immunology of histoplasmosis: humoral and cellular activity from a polysaccharide-protein complex and its deproteinized fraction in experimentally immunized mice.

In a previous publication it was reported that a polysaccharide-protein complex (PPC), sensitive to beta-glucosidase, was isolated from Histoplasma capsulatum. This complex was strongly reactive in an agar gel diffusion assay with sera from patients with histoplasmosis, but was unreactive with sera from patients with coccidioidomycosis. Here, the studies with human sera have been expanded and attempts were made to determine the response of mice immunized with nonviable H. capsulatum or Coccidioides immitis to PPC or its deproteinized fraction (D-PPC) using more sensitive tests for antibody and including also test for cell-mediated immunity. Histoplasmin and coccidioidin were compared with PPC or its deproteinized fraction (D-PPC) in all assays. In a counterimmunoelectrophoresis (CIE) assay, PPC and D-PPC reacted only with sera from patients with histoplasmosis, whereas cross reactions were noted with histoplasmin and coccidioidin using heterologous sera. Cross-reaction were observed with all four antigen preparatins and both types of antisera using a microcomplement fixation assay. The assay for macrophage migration inhibitory factor (MIF) was also relatively nonspecific, in that inhibition occurred with cells from animals sensitized with Histoplasma or Coccidioides using both homologous and heterologous antigens. In the footpad assay, histoplasmin and coccidioidin were highly cross-reactive in animals sensitized with the heterologous fungus, but the PPC and D-PPC from H. capsulatum elicited significant reactions only in animals sensitized with Histoplasma.