Regulation of the plasma membrane potential in Pneumocystis carinii.

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.

Intranasal immunization confers protection against murine Pneumocystis carinii lung infection.

To evaluate the feasibility of mucosal immunization against Pneumocystis carinii (Pc) experimental infection, female BALB/c mice were intranasally immunized three times with soluble Pc antigens plus cholera toxin fraction B (Pc-CTB); control groups received either Pc antigen, CTB, or phosphate-buffered saline (PBS) alone. Two weeks after the last immunization, five animals from each group were sacrificed, and cellular and humoral immune responses were evaluated. The remaining five mice were CD4 depleted using a monoclonal antibody against mouse CD4 and inoculated with viable Pc. Significantly higher specific lymphoproliferative responses from tracheobronchial lymph node cells, immunoglobulin M (IgM) and IgG antibody levels in serum, and bronchoalveolar lavage (BAL)-derived IgA antibody concentrations were observed in the Pc-CTB group of mice relative to control groups (P < 0.01). Five weeks after challenge, no Pc organisms were observed in the lung smears of the Pc-CTB group, while the animals receiving antigen, adjuvant, or PBS had progressively higher numbers of Pc microorganisms. By Western blot analysis, a strongly reactive 55- to 60-kDa antigen was recognized by BAL IgA and by serum IgG. In summary, mucosal immunization elicited specific cellular and humoral immune responses and protected against Pc lung infection after immunosuppression.

Granulomatous hepatitis associated with Hepatozoon sp. meronts in a southern water snake (Nerodia fasciata pictiventris).

A wild-caught adult female southern water snake (Nerodia fasciata pictiventris) did poorly in captivity. A peripheral blood-film examination demonstrated numerous hemogregarines characterized as fusiform nondividing intraerythrocytic gametocytes. Xenodiagnostic typing in laboratory-reared mosquitoes demonstrated the parasite to be of the genus Hepatozoon. Gross and histopathologic examination of the liver demonstrated numerous granulomas centered on groups of one to six Hepatozoon sp. meronts, an unusual finding in naturally infected wild-caught snakes.

Immunological markers of disease progression in patients infected with the human immunodeficiency virus.

Identification of inexpensive and technically simple immunological tests useful in predicting the progression to AIDS in human immunodeficiency virus (HIV)-infected patients would be especially welcome in developing countries, in which 80% of HIV-infected patients reside and health budgets are low. In the current study, we evaluated CD4+ and total lymphocyte counts and the concentrations in serum of beta 2-microglobulin, p24 antigen, and immunoglobulin A (IgA) as predictors of disease progression in 74 Panamanian HIV-positive patients and 50 HIV-negative healthy individuals. Total lymphocyte and CD4(+)-cell counts for AIDS patients (1,451 +/- 811 cells/microliters, P < 0.001, and 238 +/- 392 cells/microliters, P < 0.0001, respectively and asymptomatic patients (2,393 +/- 664 cells/microliters, P > 0.05, and 784 +/- 475 cells/microliters, P < 0.001, respectively) were lower than those observed for healthy subjects (2,596 +/- 631 cells/microliters and 1,120 +/- 296 cells/microliters, respectively). The levels of beta 2-microglobulin and IgA in serum were significantly elevated in patients with AIDS (5.7 +/- 3.6mg/liter, P < 0.001, and 541 +/- 265 mg/dl, P < 0.0002, respectively) and asymptomatic infected subjects (3.4 +/- 2.1 mg/liter, P = 0.001, and 436 +/- 216 mg/dl, P < 0.0001, respectively) compared with the levels in healthy subjects (2.2 +/- 0.7 mg/liter and 204 +/- 113 mg/dl, respectively). Nonstatistically significant differences (P > 0.05) for concentrations of p24 antigen between asymptomatic infected patients (29 +/- 13 pg/ml) and AIDS patients (40 +/- 23 pg/ml) were observed. Total lymphocyte counts of 1,750 cells/microliters or less, CD4 counts of 200 cells/microliters or less, beta 2-microglobulin concentrations in serum of 4 mg/liter or higher, concentrations of IgA in serum of 450 mg/dl or higher, and the presence in serum of p24 antigen were correlated with elevated risks for developing AIDS. Monitoring both total lymphocytes and beta 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4(+)-cell evaluations and the costs associated with monitoring both total lymphocytes and beta 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4(+)-cell evaluations and the costs associated with monitoring the immune status of HIV-positive patients.

Lipid transfer from human epithelial cells to Pneumocystis carinii in vitro.

Pneumocystis carinii lipids are similar to host lipids, but it is not known if some of these lipids are acquired from host cells. The ability of P. carinii to incorporate a fluorescent fatty acid analogue (Bodipy-C12) was analyzed, the metabolism of the incorporated lipid by P. carinii was characterized, and lipid transfer from human alveolar epithelial cells (A549) to P. carinii was investigated. Both P. carinii and A549 cells incorporated exogenous Bodipy-C12 in a concentration-dependent manner. Biochemical analysis of labeled P. carinii revealed incorporation of Bodipy-C12 into complex lipid classes. Incubation of unlabeled P. carinii with Bodipy-C12-labeled A549 cells demonstrated lipid transfer to P. carinii, a process facilitated by attachment. These data suggest that P. carinii can incorporate and modify an exogenous fluorescent lipid. The observed transfer of lipid from A549 cells to P. carinii provides important insight into the interaction of this organism with alveolar epithelial cells.

Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.

Species-level identification of Acanthamoeba isolates is difficult and gives little or no indication of the isolate's pathogenicity. We identified two amplification-based genetic markers that were highly correlated with pathogenicity in Acanthamoeba spp. One marker, designed to amplify a 485-bp fragment of the small-subunit ribosomal RNA gene (ssrDNA), was preferentially amplified from the nonpathogenic strains; amplifications from the pathogenic strains yielded anomalous fragments of 650 and 900 bp. A second marker was developed on the basis of the anomalous 650-bp fragment. Primers to this sequence preferentially amplified a noncoding locus (called Ac6) only from the pathogenic strains. These two genetic markers may be useful for identification of pathogenic Acanthamoeba spp. strains.

An H(+)-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites.

Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS). Cytoplasmic pH (pHi) regulation in short-term-cultured P. carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein. With an extracellular pH of 7.4, the mean baseline pHi of P. carinii trophozoites was 7.40 +/- 0.10 (n = 8). This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+. Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM). In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations. In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), agents that were able to deplete significantly the intracellular ATP levels. Taken together, these results are consistent with an important role of an H(+)-ATPase similar to those found in other fungi in the regulation of pHi homoeostasis in P. carinii trophozoites.

Molecular differentiation of two new world saurian Plasmodium species in different host species using the polymerase chain reaction.

Several isolates of Plasmodium floridense obtained from naturally infected Anolis carolinensis and Anolis sagrei, and 2 isolates of Plasmodium chiricahuae obtained from Sceloporus jarrovi were characterized at the ribosomal DNA (rDNA) locus using the polymerase chain reaction and agarose gel electrophoresis. Enzymatic amplification of the rDNA locus from both Plasmodium species resulted in the generation of a 590-base pair (bp) DNA fragment. The results obtained with all isolates of P. floridense appeared as a doublet, with the second fragment being approximately 630 bp in size. Isolates of P. floridense obtained from A. carolinensis from ecologically different northern and southeastern Florida, and from A. sagrei a the same southeastern Florida site, were demonstrated to be molecularly similar. Plasmodium floridense and P. chiricahuae were molecularly distinct at the 18s rDNA locus, thus confirming their morphological and morphometrical distinction as taxonomic species. Anolis sagrei is a third natural host species for P. floridense in Florida.

Phylogenetic analysis of beta-tubulin sequences from amitochondrial protozoa.

It has been proposed that certain extant anaerobic protozoa are descended from organisms that diverged early in eukaryotic evolution prior to the acquisition of mitochondria. Among these are the extracellular parasites Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica, and the obligately intracellular microsporidia. Phylogenetic analysis of rRNA sequences from these amitochondrial organisms suggests that G. lamblia, T. vaginalis, and microsporidia are near the base of the eukaryotic tree, while E. histolytica clusters with mitochondria-containing species. However, since eukaryotes likely evolved by symbiotic associations, it is important to analyze other sequences which may have independent origins. Unlike ribosomes, microtubules appear to be unique to eukaryotes. Complete gene sequences for the beta-tubulin subunit of microtubules from T. vaginalis, E. histolytica, and the microsporidian Encephalitozoon hellem have recently been determined. Phylogenetic relationships among these, G. lamblia, and 20 additional beta-tubulins were analyzed by distance matrix and parsimony methods, using alpha- and gamma-tubulin outgroups. All analyses placed the E. histolytica sequence at the base of the beta-tubulin evolutionary tree. Similar results were obtained for E. histolytica alpha-tubulin using a less representative set of sequences. In contrast, the E. hellem sequence branched considerably higher, within the lineage containing animal and fungal beta-tubulins. Possible explanations are considered for these unexpected differences between the beta-tubulin and rRNA trees.

Characterization of the clinical and anatomical pathological changes associated with Hepatozoon mocassini infections in unnatural reptilian hosts.

Laboratory-reared Aedes aegypti mosquitoes were employed in the successful transmission of Hepatozoon mocassini from a cotton-mouth moccasin (Agkistrodon piscivorus leucostoma) to 3 lizard species (Sceloporus undulatus, Eumeces obsoletus and Sceloporus poinsetti). Marked to severe lethargy and anorexia developed in the S. undulatus, E. obsoletus and S. poinsetti at 15, 38, and 96 days postinfection (PI), respectively. All 3 lizards developed a leukocytosis and had increased plasma aspartate aminotransferase activity (AST) by 14 days PI. Multifocal random hepatocellular necrosis and intrahepatic aggregates of heterophils centered on mature H. mocassini meronts were demonstrated in all 3 lizards. The pulmonary interstitium was multifocally thickened by aggregates of heterophils centered on meronts. No comparable clinical or anatomical pathological changes were demonstrated in naturally infected snakes. The results of this study suggest that H. mocassini is capable of inducing necrotizing inflammatory by lesions in unnatural reptilian hosts.

Antiprotozoal activities of benzimidazoles and correlations with beta-tubulin sequence.

Benzimidazoles have been widely used since the 1960s as anthelmintic agents in veterinary and human medicine and as antifungal agents in agriculture. More recently, selected benzimidazole derivatives were shown to be active in vitro against two protozoan parasites, Trichomonas vaginalis and Giardia lamblia, and clinical studies with AIDS patients have suggested that microsporidia are susceptible as well. Here, we first present in vitro susceptibility data for T. vaginalis and G. lamblia using an expanded set of benzimidazole derivatives. Both parasites were highly susceptible to four derivatives, including mebendazole, flubendazole, and fenbendazole (50% inhibitory concentrations of 0.005 to 0.16 microgram/ml). These derivatives also had lethal activity that was time dependent: 90% of T. vaginalis cells failed to recover following a 20-h exposure to mebendazole at 0.17 microgram/ml. G. lamblia, but not T. vaginalis, was highly susceptible to five additional derivatives. Next, we examined in vitro activity of benzimidazoles against additional protozoan parasites: little or no activity was observed against Entamoeba histolytica, Leishmania major, and Acanthamoeba polyphaga. Since the microtubule protein beta-tubulin has been identified as the benzimidazole target in helminths and fungi, potential correlations between benzimidazole activity and beta-tubulin sequence were examined. This analysis included partial sequences (residues 108 to 259) from the organisms mentioned above, as well as the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi and the sporozoan Cryptosporidium parvum. beta-tubulin residues Glu-198 and, in particular, Phe-200 are strong predictors of benzimidazole susceptibility; both are present in Encephalitozoon spp. but absent in C. parvum.

PCR-based detection of arboviral RNA from mosquitoes homogenized in detergent.

An improved method for the extraction of viral RNAs was developed to facilitate the reverse transcription (RT)-PCR detection of mosquitoes infected with Western equine encephalitis virus or La Crosse virus. The solubilization method, which uses only EDTA and sodium dodecyl sulfate (SDS) followed by dilution of sample, allows accurate viral detection through the use of random hexamers for the RT followed by specific primers for the PCR. Identities of the reaction products were confirmed either by sequencing or restriction endonuclease digestion. Previous methods for the extraction of RNA for the coupled RT-PCR depended on combinations of guanidinium isothiocyanate, acid phenol, detergents and multiple centrifugations. Ideally, routine detection of viral RNAs for diagnostic purposes should bypass many of the above steps, while still providing a sensitive assay. Our level of detection is 1 infected mosquito in a group of 100.

Calcium enhances Acanthamoeba polyphaga binding to extracellular matrix proteins.

PURPOSE: To characterize better the ameba-host interactions that may be involved with the pathogenesis of Acanthamoeba keratitis, the role of calcium (Ca2+) on the binding of Acanthamoeba polyphaga to extracellular matrix proteins was examined in vitro. METHODS: The binding of a metabolically labeled A. polyphaga (CDC:0187:1) isolate from a case of human keratitis to collagen type IV, laminin, and fibronectin was assessed through a range of calcium concentrations in the external fluid. Binding to collagen IV was studied in detail, with and without other divalent cations and calcium channel modulators. RESULTS: Calcium increased binding in a dose-dependent manner, with significant effects at 0.1 to 1.0 microM and near-maximal effects at 1 to 100 microM, depending upon the matrix protein. Magnesium alone had no effect on ameba binding to collagen IV but suppressed the action of calcium. Strontium enhanced ameba binding, with maximal effect at 100 microM. The calcium channel antagonists nifedipine and diltiazem-HCl and a calcium channel activator, Bay-K8644, had no effect on the action of calcium. However, the inorganic calcium antagonists, lanthanum and cobalt, suppressed the effect of calcium. CONCLUSION: Low concentrations of calcium enhance the adhesion of A. polyphaga to extracellular matrix proteins. It remains uncertain whether calcium acts intracellularly or at the cell surface.

A rapid diagnostic assay for eastern equine encephalomyelitis viral RNA.

Eastern equine encephalomyelitis virus (EEEV) has been a low-frequency, but serious human and veterinary health problem. Increased frequency of this mosquito-borne virus is anticipated as wetlands are maintained and re-established. Control of EEEV has depended on mosquito abatement in response to increasing frequency of EEEV in the environment. A coupled reverse transcription/polymerase chain reaction assay was designed to rapidly, sensitively, and specifically detect EEEV RNA. The assay successfully detected the viral RNA in a single-blind study of a set of field samples composed of either pooled mosquitoes or bird tissue. These results suggest that it would be practical to use this assay for deciding when and where to implement mosquito abatement.

A ribosomal protein in Acanthamoeba polyphaga is conserved in eukaryotic nuclei, organelles and bacteria.

An EcoRI fragment from the mitochondrial DNA of Acanthamoeba polyphaga was cloned and partly sequenced, and the conceptual translation product of the open reading frame (partial sequence) was found to have similarities with rp114, a ribosomal protein. Phylogenetic analysis based on the amino acid (aa) sequences of this conserved protein resolved four branches that consisted of: (1) eubacteria and the chloroplasts of algae and higher plants, (2) ciliate mitochondria, (3) Acanthamoeba, and (4) archaebacteria and the nuclei of eukaryotes. The groupings based on the rp114 aa sequences were consistent with the phylogenies derived by rRNA analysis of these organisms.

Acanthamoeba binds to extracellular matrix proteins in vitro.

PURPOSE: To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro. METHODS: A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba. RESULTS: Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner. CONCLUSION: In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.