Edna S. Kaneshiro

Free-living amoebae, Legionella and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA

Legionella and Mycobacterium can proliferate within free-living amoebae (FLA) where they are protected from disinfectants at concentrations that can kill bacteria but not protozoa. Despite effective treatment of drinking water, microbes can enter water utility distribution systems (DS) and hence the plumbing within building premises. Additionally, biofilm formation may account for the persistence of microbes in the DS. In the present study a domestic water tap in north-central United States (USA) was sampled in March and September 2007 and analysed for FLA, Legionella and Mycobacterium. Identification of organisms was determined by growth on specific culture media, light and electron microscopy, and amplification of DNA probes specific for each organism. In both the spring and fall samples, amoebae, Legionella and Mycobacterium were detected. However, Acanthamoeba was prominent in the spring sample whereas Vahlkampfia and Naegleria were the amoebae detected in the autumn. Bacterial proliferation in laboratory cultures was noticeably enhanced in the presence of amoebae and biofilms rapidly formed in mixed amoebae and bacteria cultures. It is hypothesized that temperature affected the dynamics of FLA species population structure within the DS and that pathogenic bacteria that proliferate within FLA, which are themselves opportunistic pathogens, pose dual public health risks.

Microsporidian Infection Is Prevalent in Healthy People in Cameroon

ABSTRACT Most studies of opportunistic infections focus on those with weak immune systems, such as human immunodeficiency virus (HIV)/AIDS patients and children. However, there is a lack of information on these infectious agents in healthy people worldwide. In the present study, stool samples from both HIV patients and healthy people were examined to begin filling in this serious gap in the understanding of human microsporidiosis, particularly the enteric parasite Enterocytozoon bieneusi. Specimens were obtained from 191 individuals living in Yaoundé, the capital city of Cameroon, in sub-Sahara Africa, including 28 HIV-positive patients who also had tuberculosis (TB). E. bieneusi prevalence was 35.7% among the HIV+ TB patients, whereas it was only 24.0% among 25 HIV− TB patients in the same hospital. Unexpectedly, the prevalence (67.5%) of microsporidiosis was found to be even higher for 126 immunocompetent individuals than for those with TB (healthy people compared to HIV+ TB and HIV− TB patients; P < 0.001). The immunocompetent group included people ranging from 2 to 70 years of age living in four different neighborhoods in Yaoundé. The highest prevalence (81.5%) was among teenagers, and the highest mean infection score (+2.5) was among children. Additional studies of immunocompetent people in other parts of Cameroon, as well as in other countries, are needed to better understand microsporidiosis epidemiology. There is still much more to be learned about the natural history of microsporidia, the pathogenicity of different strains, and the role of enteric microsporidia as opportunistic infections in immunodeficient people.

Characterization of Pneumocystis carinii preparations developed for lipid analysis

Pneumocystis carinii organisms were isolated from viral antibody‐negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100‐1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non‐P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 108‐109 organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester‐propidium iodide assay, was 80–95%.

Evidence for the Presence of “Metabolic Sterols” in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

Mixed life cycle stages of rat‐derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas‐liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii, respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas‐liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost‐5‐en‐3‐ol), cholest‐5‐en‐3‐one, and β‐sitosterol (stigmast‐5‐en‐3‐ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C28 and C29 sterols with one or two double bonds, and a hydroxyl group at C‐3 (ergost‐5‐en‐3‐ol, ergost‐7‐en‐3‐ol, ergosta‐dien‐3‐ol, stigmast‐5‐en‐3‐ol, stigmast‐7‐en‐3‐ol and stigmasta‐dien‐3‐ol). Steryl esters of P. carinii, probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3‐hydroxy‐3‐methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own “metabolic sterols”

Quantitation of Absolute Pneumocystis carinii Nuclear DNA Content. Trophic and Cystic Forms Isolated from Infected Rat Lungs are Haploid Organisms

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4′,6‐diamidino‐2‐phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc‐28 temperature‐sensitive mutant cells, accumulated at the restrictive temperature (37° C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G‐2 phase) and S‐phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181‐stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A‐T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G‐2‐phase diploid nuclei) population was not detected in histograms of DB181‐ or DAPI‐stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organisms chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life‐cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms.

Inhibitors of Sterol Biosynthesis and Amphotericin B Reduce the Viability of Pneumocystis carinii f. sp.carinii

ABSTRACT Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derivedPneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Δ8 to Δ7 isomerase, andS-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Δ8 to Δ7isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearingP. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.

Changes in lipid composition during in vitro encystation and fatty acid desaturase activity of Giardia lamblia

Lipids of axenically-cultured Giardia lamblia trophozoites were compared with those of cells undergoing in vitro encystation. Although the lipid composition of the organisms grossly resembled those of low-bile or high-bile culture media, differences were clearly detected. Encysting trophozoites incubated in a high-bile medium for 24 h had a higher concentration of unsaturated fatty acids in the total cellular lipids than did nonencysting trophozoites. The organism, but not the medium, contained linoleate and linolenate, suggesting that G. lamblia desaturates oleate. The presence of a fatty acid desaturase activity in the organism was demonstrated by the conversion of a radiolabeled monounsaturated fatty acid (oleate) to radiolabeled polyunsaturated fatty acids. Triglycerides, a common form of storage lipids, were unusually low in G. lamblia, but steryl esters (which can also serve as reserves) were abundant. Steryl esters increased during encystation of G. lamblia. The changes observed in G. lamblia lipids (increased fatty acid unsaturation and the accumulation of storage lipids) are consistent with parasite differentiation into a cyst stage that is able to survive outside the host at reduced temperatures and reduced available nutrient resources. This study also demonstrated that G. lamblia not only has the capacity to de novo synthesize isoprenoid lipids (ubiquinone, prenylated proteins), but it can also metabolize fatty acids by the addition of double bonds.

Detection of ubiquinone in parasitic and free-living protozoa, including species devoid of mitochondria

Ubiquinone (coenzyme Q, CoQ) was analyzed and individual homologues quantified in 11 species of parasitic and free-living protozoa by a combination of thin-layer chromatography and high performance liquid chromatography. Fast atom bombardment ionization-mass spectrometry was used for the first time to confirm the identity of the fractionated CoQ homologues and proved to be a fast, gentle and convenient method for ubiquinone identification. Ubiquinone was detected in all organisms including those devoid of identifiable mitochondria. However, significantly lower levels of CoQ were present in those organisms lacking this respiratory organelle (5- to 50-fold lower in Entamoeba histolytica (CoQ9) and 15- to 350-fold for Giardia lamblia (CoQ9) and Tritrichomonas foetus (CoQ10)). Coenzyme Q9 was the predominant homologue in promastigotes of Leishmania donovani and Leishmania major. Lower amounts of CoQ8 and CoQ10 were also detected in L. donovani, and CoQ8 in L. major. Comparison of the in vitro cultivated promastigote and amastigote forms of Leishmania pifanoi and Leishmania amazonensis revealed CoQ9 to be the sole detectable ubiquinone homologue in the amastigote (macrophage) stage, whereas CoQ8 and CoQ10 were also present in the promastigotes (life cycle stage found in the insect gut) of L. pifanoi, and CoQ7 and CoQ8 in promastigotes of L. amazonensis. Interestingly, the total amounts of CoQ were similar in both forms of these organisms. The free-living ciliates, Tetrahymena thermophila and Paramecium tetraurelia contained CoQ8 as the predominant ubiquinone species and this homologue was also present in the isolated cilia from P. tetraurelia. The marine ciliate, Parauronema acutum contained CoQ7 as well as CoQ8. Comparison of xenosome-containing P. acutum with organisms lacking the symbiont revealed that twice the level of CoQ8 was present in cells grown with this cytoplasmic gram-negative bacterium. Results suggest that CoQ is ubiquitous amongst the protozoa, regardless of the presence of mitochondria, and may function in alternative roles to that of mitochondrial electron transport chain component.

The Pneumocystis carinii drug target S‐adenosyl‐L‐methionine:sterol C‐24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S‐adenosyl‐L‐methionine (SAM):C‐24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24‐methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24‐methylenelanosterol as substrates, the major reaction products were 24‐methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C‐24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C‐24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.

Effects of Atovaquone and Diospyrin-Based Drugs on the Cellular ATP of Pneumocystis carinii f. sp.carinii

ABSTRACT Atovaquone (also called Mepron, or 566C80) is a napthoquinone used for the treatment of infections caused by pathogens such asPlasmodium spp. and Pneumocystis carinii. The mechanism of action against the malarial parasite is the inhibition of dihydroorotate dehydrogenase (DHOD), a consequence of blocking electron transport by the drug. As an analog of ubiquinone (coenzyme Q [CoQ]), atovaquone irreversibly binds to the mitochondrial cytochrome bc1 complex; thus, electrons are not able to pass from dehydrogenase enzymes via CoQ to cytochrome c. Since DHOD is a critical enzyme in pyrimidine biosynthesis, and because the parasite cannot scavenge host pyrimidines, the drug is lethal to the organism. Oxygen consumption inP. carinii is inhibited by the drug; thus, electron transport has also been identified as the drug target in P. carinii. However, unlike Plasmodium DHOD, P. carinii DHOD is inhibited only at high atovaquone concentrations, suggesting that the organism may salvage host pyrimidines and that atovaquone exerts its primary effects on ATP biosynthesis. In the present study, the effect of atovaquone on ATP levels in P. carinii was measured directly from 1 to 6 h and then after 24, 48, and 72 h of exposure. The average 50% inhibitory concentration after 24 to 72 h of exposure was 1.5 μg/ml (4.2 μM). The kinetics of ATP depletion were in contrast to those of another family of naphthoquinone compounds, diospyrin and two of its derivatives. Whereas atovaquone reduced ATP levels within 1 h of exposure, the diospyrins required at least 48 h. After 72 h, the diospyrins were able to decrease ATP levels of P. carinii at nanomolar concentrations. These data indicate that although naphthoquinones inhibit the electron transport chain, the molecular targets in a given organism are likely to be distinct among members of this class of compounds.