Margaret S. Collins

Echinocandin treatment of pneumocystis pneumonia in rodent models depletes cysts leaving trophic burdens that cannot transmit the infection.

Fungi in the genus Pneumocystis cause pneumonia (PCP) in hosts with debilitated immune systems and are emerging as co-morbidity factors associated with chronic diseases such as COPD. Limited therapeutic choices and poor understanding of the life cycle are a result of the inability of these fungi to grow outside the mammalian lung. Within the alveolar lumen, Pneumocystis spp., appear to have a bi-phasic life cycle consisting of an asexual phase characterized by binary fission of trophic forms and a sexual cycle resulting in formation of cysts, but the life cycle stage that transmits the infection is not known. The cysts, but not the trophic forms, express β -1,3-D-glucan synthetase and contain abundant β -1,3-D-glucan. Here we show that therapeutic and prophylactic treatment of PCP with echinocandins, compounds which inhibit the synthesis of β -1,3-D-glucan, depleted cysts in rodent models of PCP, while sparing the trophic forms which remained in significant numbers. Survival was enhanced in the echincandin treated mice, likely due to the decreased β -1,3-D-glucan content in the lungs of treated mice and rats which coincided with reductions of cyst numbers, and dramatic remodeling of organism morphology. Strong evidence for the cyst as the agent of transmission was provided by the failure of anidulafungin-treated mice to transmit the infection. We show for the first time that withdrawal of anidulafungin treatment with continued immunosuppression permitted the repopulation of cyst forms. Treatment of PCP with an echinocandin alone will not likely result in eradication of infection and cessation of echinocandin treatment while the patient remains immunosuppressed could result in relapse. Importantly, the echinocandins provide novel and powerful chemical tools to probe the still poorly understood bi-phasic life cycle of this genus of fungal pathogens.

Structure-activity relationships of antitubercular nitroimidazoles. 2. Determinants of aerobic activity and quantitative structure-activity relationships.

The (S)-2-nitro-6-substituted 6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines have been extensively explored for their potential use as new antituberculars based on their excellent bactericidal properties on aerobic whole cells of Mycobacterium tuberculosis. An oxygen atom at the 2-position of the imidazole ring is required for aerobic activity. Here, we show that substitution of this oxygen by either nitrogen or sulfur yielded equipotent analogues. Acylating the amino series, oxidizing the thioether, or replacing the ether oxygen with carbon significantly reduced the potency of the compounds. Replacement of the benzylic oxygen at the 6-position by nitrogen slightly improved potency and facilitated exploration of the SAR in the more soluble 6-amino series. Significant improvements in potency were realized by extending the linker region between the 6-(S) position and the terminal hydrophobic aromatic substituent. A simple four-feature QSAR model was derived to rationalize MIC results in this series of bicyclic nitroimidazoles.

Biofilm formation by Pneumocystis spp.

ABSTRACT Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-β-d-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.

Early Acquisition of Pneumocystis carinii in Neonatal Rats as Evidenced by PCR and Oral Swabs

ABSTRACT The complete life cycle of Pneumocystis carinii has not been defined, but accumulating evidence suggests that the mammalian host may acquire this organism early in life. In the present study, the initial time of P. carinii acquisition was determined in rats by amplification of P. carinii DNA in oral swabs from seven sets of pups and dams and from fetal tissue obtained by cesarean section of three gravid female rats. DNA extracted from all samples was amplified by using PCR primers directed to the P. carinii mitochondrial large subunit rRNA. Amplicons were produced from 80% (28 of 35) of pups within 2 h after birth; from 97% (34 of 35) after 24 h, and in all of the serially sampled pups by 48 h. No P. carinii amplicons were produced from 48 fetuses or their placentae taken by cesarean section. Thus, P. carinii is acquired almost immediately after birth, and placental transmission occurs rarely, if ever, in rats.

Widespread Occurrence of Pneumocystis carinii in Commercial Rat Colonies Detected Using Targeted PCR and Oral Swabs

ABSTRACT The genus Pneumocystis contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization withPneumocystis becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of Pneumocystis in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of Pneumocystis in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provokePneumocystis growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence ofPneumocystis by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of Pneumocystis carinii f. sp. carinii. Upon receipt, 64% of rats were positive for P. carinii f. sp. carinii-specific antibodies, while P. carinii f. sp. carinii DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealedP. carinii f. sp. carinii DNA in all rat lungs, illustrating widespread occurrence of Pneumocystis in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of Pneumocystisexposure.

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.

Highly Active Anti-Pneumocystis carinii Compounds in a Library of Novel Piperazine-Linked Bisbenzamidines and Related Compounds

ABSTRACT Trimethoprim-sulfamethoxazole and pentamidine isethionate have been used extensively for the prophylaxis and therapy of pneumonia caused by Pneumocystis jirovecii. Problems associated with toxicity and potential emerging resistance for both therapies necessitate the development of safe and effective analogs or new treatment strategies. In the present study, a library of 36 compounds was synthesized by using the pentamidine molecule as the parent compound modified by a 1,4-piperazinediyl moiety as the central linker to restrict conformation flexibility. The compounds were evaluated for anti-Pneumocystis carinii activity in a bioluminescent ATP-driven assay. Four of the compounds were highly active, with 50% inhibitory concentration (IC50) values of <0.01 μg/ml; four had very marked activity (IC50 < 0.10 μg/ml); ten had marked activity (IC50 < 1.0 μg/ml); nine had moderate activity (IC50 < 10 μg/ml); one had slight activity (IC50 = 34.1 μg/ml); and the remaining eight did not demonstrate activity in this assay system. The high level of activity was specifically associated with an alkyl chain length of five to six carbons attached to one of the nitrogens of the bisamidinium groups. None of the highly active compounds and only one of the very marked compounds exhibited any toxicity when evaluated in three mammalian cell lines. The strategy of substitution of 1,4-piperazine-linked bisbenzamidines produced compounds with the highest level of activity observed in the ATP assay and holds great promise for the development of efficacious anti-P. carinii therapy.

Evidence for multiple sterol methyl transferase pathways in Pneumocystis carinii

The sterol composition of Pneumocystis carinii, an opportunistic pathogen responsible for life-threatening pneumonia in immunocompromised patients, was determined. Our purpose was to identify pathway-specific enzymes to impair using sterol biosynthesis inhibitors. Prior to this study, cholesterol 15 (ca. 80% of total sterols), lanosterol 1, and several phytosterols common to plants (sitosterol 31, 24α-ethyl and campesterol, 24α-methyl 30) were demonstrated in the fungus. In this investigation, we isolated all the previous sterols and many new compounds from P. carinii by culturing the microorganism in steroid-immunosuppressed rats. Thirty-one sterols were identified from the fungus (total sterol=100 fg/cell), and seven sterols were identified from rat chow. Unusual sterols in the fungus not present in the diet included, 24(28)-methylenelanosterol 2; 24(28)E-ethylidene lanosterol 3; 24(28)Z-ethylidene lanosterol 4; 24β-ethyllanosta-25(27)-dienol 5; 24β-ethylcholest-7-enol 6; 24β-ethylcholesterol 7; 24β-ethylcholesta-5,25(27)-dienol 8; 24-methyllanosta-7-enol 9; 24-methyldesmosterol 10; 24(28)-methylenecholest-7-enol 11; 24β-methylcholest-7-enol 12; and 24β-methylcholesterol 13. The structural relationships of the 24-alkyl groups in the sterol side chain were demonstrated chromatographically relative to authentic specimens, by MS and high-resolution 1H NMR. The hypothetical order of these compounds poses multiple phytosterol pathways that diverge from a common intermediate to generate 24β-methyl sterols: route 1, 1→2→11→12→13; route 2, 1→2→9→10→13; or 24β-ethyl sterols: route 3, 1→2→4→6→7; route 4, 1→2→5→8→7. Formation of 3 is considered to form an interrupted sterol pathway. Taken together, operation of distinct sterol methyl transferase (SMT) pathways that generate 24β-alkyl sterols in P. carinii with no counterpart in human biochemistry suggests a close taxonomic affinity with fungi and provides a basis for mechanism-based inactivation of SMI enzyme to treat Pneumocystis pneumonia.

Susceptibility of Pneumocystis to Echinocandins in Suspension and Biofilm Cultures

ABSTRACT The targeted inhibition of cyst but not trophic development by anidulafungin, caspofungin, and micafungin on Pneumocystis murina and Pneumocystis carinii in rodent models of Pneumocystis carinii pneumonia (PCP) was recently reported by us (M. T. Cushion et al., PLoS One 5:e8524, 2010). To better understand the effects of echinocandins on P. carinii, the same three compounds were evaluated in standard suspension and biofilm cultures supplemented with various concentrations of sera using the measurement of ATP as the indicator. In suspension cultures with 1 and 5% serum, anidulafungin was the most active compound but 10 and 20% serum abrogated the efficacy of all three echinocandins. Established biofilm cultures that included both the nonadherent and adherent phases were more resistant to micafungin than caspofungin regardless of serum concentration, while anidulafungin had significant activity at 1 and 5% serum concentrations. Nascent biofilms were mostly affected by anidulafungin in 1 and 5% serum, but none of the compounds showed significant activity in 20% serum. We show for the first time that (i) echinocandins differ in their abilities to deplete the ATP of Pneumocystis in biofilms and in suspension cultures, (ii) this variability mostly reflected the reported efficacies in animal models of infection, and (iii) high serum levels decreased the anti-Pneumocystis activities of the echinocandins in both in vitro systems.

PRIMACENES: novel non-cytotoxic primaquine-ferrocene conjugates with anti-Pneumocystis carinii activity

Primacenes, novel ferrocene–primaquine conjugates, were synthesized and screened for their antimalarial and anti-pneumocystis activity. Primacenes obtained by coupling primaquine amino acid derivatives to ferrocenoic acid were significantly active against Pneumocystis carinii and devoid of cytotoxicity, thus being more selective than the parent drug.