Beware of dead, wilted Artemisia plants

by lutgenp After some important research work had been done some 30 years ago on amino acids, the impact these molecules might have on a vast array of diseases has been neglected. But there is increasing evidence that the amino acid proline plays an important role in the virulence mechanism of human and mammalian pathogens. Different aminoacids have different roles in malaria. Arginine was a deadly weapon against malaria gametocytes. It was found that the Plasmodium parasite tries to extrude as much as possible of arginine from the infected erythrocyte into the extracellular medium, or to metabolize it into urea or ornithine, avoiding the aggressive NO produced by the enzyme i-NOS in the conversion of arginine. In preterm neonates arginine is synthesized from proline in the gut. Infants in intensive care have hypoarginemia compared to healthy infants. Arginine supplementation was shown to be beneficial. In murine schistosomiasis arginine is converted to proline by the parasite who needs it. Dunn MA, Rojkind M, Hait PK, Warren KS. Conversion of arginine to proline in murine schistosomiasis. Gastroenterology. 1978 Dec;75(6):1010-5. The two molecules, arginine and proline, appear not only to compete on the battlefied, but in the areas of high endemicity they even appear to trigger a genetic impact. In Sicily the area of Oristano was in the past a heavy malaria endemic area. To-days children in this area have a higher arginine genotype than those in the area of Nuoro where endemia was very light. Gloria-Bottini F, Meloni GF, Saccucci P and Bottini E. P 53 Codon 72 and Past Malaria Morbidity in Sardinia. Malar Chemoth Cont Elimin, 2013, 2-1 Bertolo RF, Brunton JA, Pencharz PB, Ball RO. Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs. Am J Physiol Endocrinol Metab. 2003;284:E915–E922 Over 40 years ago it was proposed that there is an evolutionary basis for the ubiquity of certain elements and many small molecules found in living systems. Certain biological molecules, the free amino acids proline, glycine, and arginine to name a few were found to accumulate in the cells of a large variety of water-stressed organisms. In general this class of molecules, known as osmolytes, has the ability to counter environmental water stress in plants and to protect cellular proteins against denaturation under these extreme conditions. These molecules are synthesized and accumulate in cells as a response to osmotically induced dehydration in the presence of high salt environments, where proline in particular has been shown to stimulate growth and respiration in bacteria in the presence of low water activities. Proline, in essence, acts as a “chaperone” to protein formation in these conditions. Madhab K. Chattopadhyay, Renée Kern, Michel-Yves Mistou, Abhaya M. Dandekar, Sandra L. Uratsu, and Gilbert Richarme, The Chemical Chaperone. Proline Relieves the Thermosensitivity of a dnaK Deletion Mutant at 42°C. doi: 10.1128/JB.186.23.8149-8152.2004 J. Bacteriol. December 2004 vol. 186 no. 23 8149-8152 Proline (and a number of other amino acids) has been found to function as an osmolyte in the cells of a large variety of water-stressed organisms (plant, animal, and bacterial systems). This may be related to the fact that proline is the most hydrophilic amino acid, remarkably, it is more soluble in water than hydroxyproline. Vitamin C transforms proline into hydroxyproline and collagen, which are essential additives in creams for smooth skin. An interesting observation is that in the case of Plasmodium falciparum resistance to certain antimalarials this resistance is accompanied by a 30fold increase of proline in the food vacuole, the concentration of all other amino acids remaining constant. Herman JD, Rice DP, Ribacke U, Silterra J, Deik AA, Moss EL, Broadbent KM, Neafsey DE, Desai MM, Clish CB, Mazitschek R, Wirth DF. A genomic and evolutionary approach reveals non-genetic drug resistance in malaria. Genome Biol. 2014;15(11):511. The attachment of merozoites to erythrocytes is receptor-mediated. Specific proteins mediate this attachment. The merozoite surface is also rich in proline. M E Perkins. Surface proteins of Plasmodium falciparum merozoites binding to the erythrocyte receptor, glycophorin. J. ExP. MED. Volume 160 September 1984 788-798 Another amino acid which is vital for Plasmodium is isoleucine. However human hemoglobin does not contain isoleucine. And since Plasmodium cannot synthesize isoleucine, it must acquire isoleucine from human serum. In vitro parasite growth is notably slower in medium containing low concentrations of isoleucine. Ledbetter, Shalon, "The Unconventional Amino Acid Starvation Response of the Malaria Parasite, Plasmodium falciparum" (2012). Thesis, Washington University of St Louis, 2012. When subjected to isoleucine starvation parasites only progress through the trophozoite stage. This explains why in human erythrocytes with mature Plasmodium falciparum trophozoites the rate of transport into the cells is 5-fold higher than in uninfected cells Martin RE, Kirk K Transport of the essential nutrient isoleucine in human erythrocytes infected with the malaria parasite Plasmodium falciparum. Blood. 2007 Mar 1;109(5):2217-24. Epub 2006 Oct 17. Proline alters antioxidant defenses. Roecker R, Junges GM, de Lima DD, da Cruz JG, Wyse AT, Dal Magro DD. Proline alters antioxidant enzyme defenses and lipoperoxidation in the erythrocytes and plasma of rats: in vitro and in vivo studies. Biol Trace Elem Res. 2012;147:172–179. In Eschericia coli it increases oxidative stress tolerance. Zhang L, Alfano JR, Becker DF. Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli. J Bacteriol. 2015 Feb;197(3):431-40. doi: 10.1128/JB.02282-14. In Toxoplasma gondii which is an apicomplexan like Plasmodium proline is de novo synthesised from glutamate. Schistosomiasis leads to the development of fibrosis and granuloma. In mice the incorporation of proline is trapped in liver granulomas. The source of the increased free proline is unknown. Increased hepatic sequestration of exogenous proline, increased synthesis of proline from argininine and glutamate are potentially important mechanisms. Dunn MA, Seifter S, Hait PK. Proline trapping in granulomas, the site of collagen biosynthesis in murine schistosomiasis. Hepatology. 1981 Jan-Feb;1(1):28-32. Proline is also released by the ova of Schistosoma mansoni. Isseroff H, Bock K, Owczarek A, Smith KR. Schistosomiasis: proline production and release by ova. J Parasitol. 1983 Apr;69(2):285-9. Free proline begins to increase about the 7th week of infection and reaches its maximum at the 8th to 9th week, when the granulamatous response to the Schistosoma mansoni eggs in the liver is most dominant. Tanabe M, Kaneko N, Takeuchi T. Schistosoma mansoni: higher free proline levels in the livers of infected mice. Exp Parasitol. 1991 Feb;72(2):134-44. Proline gives the symbionts of nematode worm and other parasites green light. Metabolic analysis and genetic knockouts confirm that pathogens can sense L-proline in blood. This not only serves as a wake-up call, activating secondary metabolite virulence factors, but also provides an energy source for a metabolic shift appropriate for adaptation to the host environment. Hematophagous insects like the mosquito Aedes aegypti use proline as nitrogen sink during blood meal digestion. Goldstrohm DA, Pennington JE, Wells MA The role of hemolymph proline as a nitrogen sink during blood meal digestion by the mosquito Aedes aegypti. J Insect Physiol. 2003 Feb;49(2):115-21 Proline levels which are usualy much higher in insects than in mammals, increase five-fold over unfed levels after a meal. Proteins derived from the amino acid are utilized to produce eggs. Trypanosoma cruzi is an excellent example of this behaviour. The mechanism of host cell invasion depends on parasite energy. Proline was shown to increase the infectivity, much more than glucose does. If proline is replaced by an analogue like T4C a dose-dependant retardation of epimastigote growth is noticed. Magdaleno A, Ahn IY, Paes LS, Silber AM. Actions of a proline analogue, L-thiazolidine-4-carboxylic acid (T4C), on Trypanosoma cruzi. PLoS One. 2009;4(2):e4534. doi: 10.1371/journal.pone.0004534. Leishmania donovani promastigotes also accumulate proline. Zilberstein D, Dwyer DM. Protonmotive force-driven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1716-20. Continuous culture of Leishmania tarentolae in the absence of proline is impossible. The proline-utilization pathway in Mycobacterium tuberculosis has recently been identified as an important factor in tuberculosis persistance in vivo. Lagautriere T1, Bashiri G1, Paterson NG1, Berney M2, Cook GM2, Baker EN1Characterization of the proline-utilization pathway in Mycobacterium tuberculosis through structural and functional studies. Acta Crystallogr D Biol Crystallogr. 2014 Apr;70(Pt 4):968-80. doi: 10.1107/S1399004713034391. The enzymes for the oxidation of proline to glutamate have been identified. Serrano H, Blanchard JS. Kinetic and isotopic characterization of L-proline dehydrogenase from Mycobacterium tuberculosis. Biochemistry. 2013 Jul 23;52(29):5009-15. doi: 10.1021/bi400338f. Mice lacking the gene for proline biosynthesis have attenuated forms of tuberculosis. Berney M, Weimar MR, Heikal A, Cook GM. Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia. Mol Microbiol. 2012 May;84(4):664-81. doi: 10.1111/j.1365-2958.2012.08053.x. For tick egg cultures, adding proline to the basal medium increases the multiplication rate of eggs 1.2-1.9 fold. Samish, M., Louloudes, S.J., Vaughn, J.L., Kurtti, T.J. and Munderloh, U.G. 1985. Promotion of tick cell growth by proline and fractions from tick eggs. Int. J. Parasitol. 15, 21-26. Straphylococus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Low-proline environments impair growth and in vivo survival of Staphylococcus aureus. Schwan WR, Wetzel KJ, Gomez TS, Stiles MA, Beitlich BD, Grunwald S. Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants. Microbiology. 2004 Apr;150 1055-61 For Helicobacter pylori, proline is the preferred respiratory substrate during colonization of the human stomach. Shelbi Christgen, Amanda Ramer-Tait and Don Becker, Exploring the Role of Proline Metabolism in Helicobacter Pathogenicity. 2015 The FASEB Journal vol. 29 no. 1 Supplement 573.49 Proline down-regulates CD4 in viral infections. Many viruses have proline rich domains (Herpes, Hepatititis, Influenza). Takeshi Yamada, Naotoshi Kaji, Takashi Odawara, Joe Chiba, Aikichi Iwamoto, and Yoshihiro Kitamura. Proline 78 Is Crucial for Human Immunodeficiency Virus Type 1 Nef To Down-Regulate Class I Human Leukocyte Antigen. Journal of Virology, 2003, 1589-1594 Some medicinal plants have an inhibitory effect on proline uptake. An Indian team studied the uptake in Candida albicans for 10 plants. Alium sativum gave the highest inhibition and Aloe vera among the lowest. Ch.Tanushree Das , Ritarani Das & R.C. Mohanty. Differential inhibitory effects of medicinal plant extracts on proline. Nature and Science 2010;8(9) This is in line with the known antimalarial properties of garlic and the absence of documented antimalarial properties for Aloe. It would be important to extend this study to other medicinal plants like Artemisia or Neem. Another tool is halofuginone, a halogenated derivative of febrifugine, which is the bioactive constituent in the Chang Shan herb that has long been used to treat malaria. It binds to proline which suggests that febrifugine might work by a similar mechanism Zhou H , Sun L , Yang XL , Schimmel P. ATP-directed capture of bioactive herbal-based medicine on human tRNA synthetase. Nature 2013, 494(7435):121-124 Zinc supply seems to be an easier approach! Zinc plays a key role in the metabolism of amino acids. It binds to the iNOS enzyme and favours the production of NO rather than downgrading arginine to urea by the arginase enzyme. This metal inhibits the transport of proline into cells. It also inhibits the exchange-exit reaction for other amino-acids. Yasuhiro ANRAKU, Fumiko GOTO and Emi KIN Transport of Sugars and Amino Acids in Bacteria XIII. Mechanism of Selective Inhibition of the Active Transport Reactions for Proline, Leucine, and Succinate by Zinc Ions. J Biochem 1975, 78, 149-157 Zinc also forms a stable complex with isoleucine. Brij B. Tewari, Ashish K. Tiwari. Interaction of isoleucine with metal ions in chemical and biolof gical systems. Revista Boliviana de Química. Volumen 32 Nº1. Año 2015 The zinc-proline salt complex is used as adjuvant in vaccines (Gerbu Biotecknik GmbH D69251 Gaiberg). It is thus not surprising that a pharmaceutical company patented this property (US 20050090480) and sels amino acid-zinc complexes as antimalarials. They claim that 5 to 10 µM of zinc complexes of proline inhibit growth of Plasmodium falciparum by about 100% and that these complexes are also effective against resistant strains. Proline becomes an important issue in the use of Artemisia annua and other medicinal plants against malaria. Plants growing under salt stress, cold stress or drought, and senescent plants contain higher concentrations of proline. The solubility of proline in water (154.56 g/100) is by far the highest of all amino acids. Its bioavailability consequently must be high. Proline is a promoter of beta-hematin (hemozoin) formation and could thus increase the severity of an infection. Uyen DT, Huy NT, Trang DT, Nhien NT, Oida T, Hirayama K, Harada S, Kamei K. Effects of amino acids on malarial heme crystallization. Biol Pharm Bull. 2008 Aug;31(8):1483-8. Artemisia annua, compared with several other Artemisia varieties has the highest proline production under drought stress. It does not present any morphological or anatomical drought-resistant traits. Y Wang, J Yu, Z Zhou. Artemisia dominant species succession relating to the soil moisture decrease in abandoned land. SpringerPlus 2016 5 :992 It is thus strongly recommended not to harvest dead, wilted leaves a few weeks or months after flowering.