Allen B. Clarkson PhD, Associate Professor

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Parasites evolve unusual cellular and molecular processes to suit their purposes. Discovery and characterization of these processes serve our purposes by providing leads for selective therapies and, in some cases, provide explanations for the pathology the parasites produce. Recently the focus of this lab has been on the fungal parasite Pneumocystis which causes Pneumocystis pneumonia (PCP), the most common opportunistic infection associated with AIDS, cancer treatment and other immunosuppressive conditions. Area of interest include Pneumocystis polyamine metabolism, iron metabolism, and electron transport as well host response to the demands of the parasite.

Fig. 1 This electron micrograph shows a part of a lung alveolus and the adjacent blood capillary. The very dark structure is a red blood cell and above it is the air-filled capillary. Pneumocystis can be seen lining the alveolus where it blocks gas diffusion which is the major pathological consequence of PCP. Thickened septa and collagen deposition typical of PCP are also seen.

Fig. 2 Green-stained Pneumocystis is seen in a co-culture adhering to red-stained mammalian cells.

Polyamines are small positively charged molecules essential for all cells

Polyamines are simple small molecules with a straight chain carbon backbone, two primary amino groups and up to two secondary amino groups. Polyamines strongly associate with DNA, rise and fall with stages of the cell cycle and with the rate of cycling, substitute for metal cations for some enzymes, balance intracellular movement of calcium ions, and serve as second messengers. Despite their obvious importance, we lack a comprehensive understanding of their role in cells. Nevertheless, we and other researchers try to manipulate their metabolism to treat disease; the best success so far has been for parasitic infections.

Fig. 3 The three major polyamines.

Pneumocystiscan’t say no (to polyamine cycling)

Many parasites are faced with the challenge of multiply-fast-or-die and evolve simplified metabolic systems that allow for faster reproduction. However, this specialization can leave them less flexible when faced with deliberate biochemical challenges; i. e., targeted therapeutic agents. So it is with Pneumocystis, polyamines and drug design. Cells of mammals have exquisite mechanisms to control enzymes of polyamine metabolism by controlling transcription rate, transcript stability, translation rate, and protein stability as well as direct product feedback inhibition, allosteric control and production of a protein that binds specifically to the rate-controlling polyamine synthesis enzyme blocking its function. Our in vitro studies showed that Pneumocystis quickly becomes depleted of polyamines when their synthesis is blocked, but, when that block is relieved, they recover extremely quickly. Thus Pneumocystis is skilled at controlling polyamine synthesis but cannot control degradation. From these data, we reasoned that if we treated an infected host with a compound that inhibits polyamine synthesis of both host and parasite, the host would respond by strongly down-regulating degradation pathways thus conserving the polyamines on hand but the parasite would become depleted of polyamines and die. We further reasoned that it would be more important to keep the biosynthesis inhibitor constantly present than to ever achieve high concentrations. Using our animal model of Pneumocystis pneumonia, we tested and confirmed these predictions. The compound we used to block polyamine synthesis (difluoromethylornithine) had been a clinical investigational drug for PCP but is no longer used because efficacy was variable. A review of the clinical data showed that best efficacy was associated with slow steady infusion which exactly matches our biochemical and animal studies.

Pneumocystis can’t go it alone

Not only do parasites often drop metabolic control processes, sometimes they drop a pathway entirely and become dependent on their host for substances critical for their survival. This can help meet the multiply-fast-or-die demand but can leave them particularly vulnerable to medicinal chemists. We discovered that Pneumocystis has lost the ability to synthesize a metabolic intermediate involved in an extraordinary number of cell processes: S-adenosyl methionine (AdoMet, SAM, or SAMe). AdoMet is the major methyl donor used for DNA and RNA methylation and for synthesis of many lipids. It also interacts with folate metabolism and thus all reactions involving folates. The ability to make AdoMet is so critical that all cells examined can do this except for some Rickettsia (very simple and reduced parasitic bacteria), a few mutant cell lines, and Pneumocystis. We found that inclusion of AdoMet in the culture medium for Pneumocystis prolongs survival and allows us to perform in vitro experiments without the use of a mammalian feeder cell layer – something not possible before the AdoMet requirement was known. Pneumocystis demands AdoMet from its host to the degree that the plasma of infected experimental animals becomes = 98% AdoMet depleted. This depletion also occurs in humans with PCP and measurement of plasma AdoMet promises to be a diagnostic tool for infection and a measure of “response to therapy” since plasma AdoMet rises quickly once effective treatment is begun.

Nicotine is bad for Pneumocystis

Working with Dr. Merali’s laboratory in this Department, we are investigating a peculiar property of nicotine: selective suppression of the AdoMet content of lungs. We find that nicotine administered to immunosuppressed animals acts prophylactically against PCP. This is a prime example of taking advantage of an evolved loss of function by the parasite. We have some information on the mechanism by which nicotine causes this reduction in lung AdoMet and further information is actively being sought. Details are in Dr. Merali’s research description.

Host response to AdoMet scavenging by Pneumocystis

Currently we are studying the effect Pneumocystis AdoMet scavenging has on host polyamine and AdoMet metabolism. We are examining lungs of infected and non-infected animals for activities of relevant enzymes, for transcripts of genes coding for these enzymes using quantitative (“real time”) PCR and for the distribution of this mRNA in the different cell types using in situ hybridization of lung sections. Correlations are being sought between changes in these parameters and the intensity of Pneumocystis infection (measured by quantitative PCR of a parasite mitochondrial gene). Upon completion of this project, the same techniques will be used to study the effect of nicotine on host lung tissue to complement the proteomic studies being done by Dr. Merali’s lab.

Iron, an essential but dangerous cell component

The ready Fe+2/Fe+3 oxidation state cycling of iron makes it such a valuable enzyme cofactor that all cells have an absolute requirement for iron (the only known exceptions are Lactobacillus and Borrelia, the spirochete that causes Lyme disease). But this same property of iron must be carefully controlled because it also leads to generation of reactive oxygen species which are acutely toxic to cells. Organisms solve this with complex iron acquisition, transport, sequestration and storage systems that reduce free iron in cytoplasm and extracellular fluids to essentially undetectable levels but still provide a supply of iron when and where it is needed. The mammalian system is especially well developed and provides an additional advantage in making difficult for invading microbes to acquire the iron they need to multiply-fast-or-die.

Chelate iron: Spare the host and spoil the parasite

Despite the power and flexibility of the mammalian iron system, some diseases and some genetic variations cause the system to become overloaded and the excess iron becomes uncontrolled and thus toxic. To treat this, physicians use chelators which bind extraordinarily tightly to “free iron” (the toxic form) rendering it non-toxic and leading to elimination of some excess iron via urine and/or bile. We thought it possible that one such chelator, deferoxamine, might treat PCP by depriving the fungus of the iron it needs to multiply-fast-or-die. Deferoxamine was effective in experimental animals and a review of clinical records indicated that HIV-infected patients who were also being treated with deferoxamine for iron overload did not develop PCP. Thus at first our prediction seemed validated but then unexpected results led to an alternative explanation for how deferoxamine treats PCP and that led to a significant improvement in efficacy. The unexpected result came from an experiment designed to demonstrate that deferoxamine causes a nutritional deficiency for Pneumocystis. We reasoned that short term exposure in culture would have little effect since any nutritional deficiency would be corrected by removal of the chelator. But we were surprised that short and long term exposures were essentially equal in effect. Other experiments showed us that this large (MW 561) multiple-charged molecule penetrates Pneumocystis and binds to intracellular iron causing irreversible damage. These data led to the prediction that intense but short treatment would improve both efficacy and specificity. It did so remarkably. A low weekly dose delivered directly to the lungs as an aerosol provides the most effective means of blocking PCP development we have ever observed with any standard or experimental compound.

Fig. 4 Deferoxamine (a) binds to Fe+3 with such avidity to form feroxamine (b) that iron can be extracted from some iron-binding proteins. Mammalian cells tolerate deferoxamine well but Pneumocystis is irreversibly damaged.

Strange electron transport by Pneumocystis

The mitochondrial electron transport system that leads to ATP production is highly conserved across phyla but some parasites, some free-living fungi and some plants have a variation that involves a direct transfer of electrons to oxygen without involvement of cytochromes. This “alternative oxidase” system as used by parasites is another example paring down for “multiply fast or die” performance and another example of a vulnerable and selective target since this pathway does not exist in the host. Years ago we defined this pathway in African trypanosomes and demonstrated “proof of principle” as a drug target. We now have preliminary data showing alternative oxidase activity in Pneumocystis thus the likelihood of this enzyme being a feasible drug target for PCP.

Selected Publications

1. Baer K, Roosevelt M, Van Rooijen N, Clarkson Jr AB and Frevert U. Kupffer cells are obligatory for Plasmodium sporozoite infection of the liver. Cell. Microbiol. 2007 Feb:9(12);397-412.
2. Shivji M, Burger S, Moncada CA, Clarkson AB Jr, Merali S. Effect of nicotine on lung S-adenosylmethionine and development of Pneumocystis pneumonia. J Biol Chem. 2005 Apr;280(15):15219-28.
3. Merali S and Clarkson, AB Jr. Pneumocystis carinii and S-adenosylmethionine Metabolism. FEMS Microbiology Letters. 2004;237:179–186. (#J72814)
4. Skelly, M, Hoffman, J, Fabbri M, Holzman SR, Clarkson AB Jr. and Merali S. Changes in S-adenosylmethionine levels in the course of treatment of human Pneumocystis carinii pneumonia (PCP); a potential aid in rapid diagnosis. Lancet. 2003;361:1237.
5. Allen B. Clarkson, Jr., David Turkel-Parrella, Jonathan H. Williams, Lung Chi Chen, Terry Gordon and Salim Merali. Action of Deferoxamine Against Pneumocystis carinii. Antimicrobial Agents Chemotherapy. 2001;45:3560-3565. (#J47550)
6. Merali, S., Vargas, D., Franklin, M., Clarkson, A. B. Jr. S-Adenosylmethionine and Pneumocystis carinii. Journal of Biological Chemistry. 2000;275:14958-14963. (#J08960)
7. Merali, S., Saric´, M., Chin, K., Clarkson, A. B. Jr. The effect of a bis-benzyl polyamine analogue on Pneumocystis carinii. Antimicrobial Agents Chemotherapy. 2000;44:337-343.
8. Merali, S, Frevert, U., Williams, J., Chin, K., Bryan, R. and Clarkson, A. B. Continuous Axenic Cultivation of Pneumocystis carinii. Proceedings of the National Academy of Sciences USA. 1999;96: 2402-2407.
9. Chin K., Merali, S., Saric, M. and Clarkson, A.B.Jr. Continuous Infusion of DL-α-Difluoromethylornithine and Improved Efficacy Against a Rat Model of Pneumocystis carinii Pneumonia. Antimicrobial Agents Chemotherapy. 1996;40:2318-2320. (#J04552 )
10. Merali, S. Chin, K., Grady, R.W. and Clarkson, A.B. Trophozoite Elimination from a Rat Model of Pneumocystis carinii by Clinically Achievable Deferoxamine Plasma Concentrations. Antimicrobial Agents Chemotherapy. 1996;40:1298-130.
11. Clarkson, AB Jr. and Merali S. Polyamines, Iron and Pneumocystis carinii. To appear in 'Pneumocystis Pneumonia, Third Edition' edited by Walzer, P. and Cushion MT. In Press.