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.
In a previous document published on www.malariaworld.org we have shown that another amino acid, 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. The two molecules are in competition and can be converted from one into the other. 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 (M Dunn et al., Gastroeneterology 1978, 75, 1010-15).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 (F Gloria-Bottini et al., Malar Chemoth Cont Elimin, 2013, 2-1).
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. 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.
PROLINE AND TROPICAL DISEASES
Our interest in proline was triggered when we found 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 (JD Herman et al., Genome Biology, 2014, 15 :511) . 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 (S Ledbetter, 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 (R Martin et al., Blood, 2007, 109-5)
Proline alters antioxidant defenses (R Roecker et al., Biol Trace Elem Res, 2 dec 2011). In Eschericia coli it increases oxidative stress tolerance (L Zhang et al., J Bacteriol, 2015, 197, 431-40). 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 (MA Dunn et al., Hepatology 1981, 1, 28-32).
Proline is also released by the ova of of Schistosoma mansoni (H Isseroff et al., J Parasit 1983, 69, 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 (M Tanabe et al, Exp Parasitol 1991 72, 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 (DA Goldstrohm et al., J Insect Physiol. 2003 49. 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 (A Magdaleno et al., PlosOne209, 4, e4534). Leishmania donovani promastigotes also accumulate proline (D Zilbertein et al., Proc Nat Acad Sci USA, 1985, 82, 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 (T Lagautriere et al., Acta Crystallograph D Biol Cristallogr 2014 70, 968-80). The enzymes for the oxidation of proline to glutamate have been identified (H Serrano et al., Biochemistry, 2013, 52-29). Mice lacking the gene for proline biosynthesis have attenuated forms of tuberculosis (M Berney et al., 2012, 84, 64-681).
For tick egg cultures, adding proline to the basal medium increases the multiplication rate of eggs 1.2-1.9 fold (M Samish et al., Internat J Parasitol 1985, 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 (WR Schwan et al., Microbiology. 2004 Apr;150 1055-61). For Helicobacter pylori, proline is the preferred respiratory substrate during colonization of the human stomach (Nakajima et al., 2008 Biomed Res 29, 9-18). Proline down-regulates CD4 in viral infections (T Yamada et al., Journal of Virology, 2003, 1589-1594). Many viruses have proline rich domains (Herpes, Hepatititis, Influenza).
HOW TO DEAL WITH THE NEGATIVE EFECTS OF PROLINE ON DISEASES ? ARTEMISIA and ZINC?
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 et al., Nature and Science, 2010, 8, 132-139). 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 (H Zhou et al., 2013, 494, 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 (B Hemens et al., J Biol Chem, 275, 48, 35786-91). Zinc easily builds a strong complex with zinc. This metal inhibits the transport of proline into cells. It also inhibits the exchange-exit reaction for other amino-acids (Y Anraku et al., J Biochem 1975, 78, 149-157). Zinc also forms a stable complex with isoleucine (Revista Boliviana de Quimica 2015, 31, 1-7). 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.
FUTURE PROSPECTS AND CONCERNS
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. Ist bioavailability consequently must be high. Proline is a promoter of beta-hematin (hemozoin) formation (DT Uyen et al., Biol Pharm Bull 2008. 31, 1483-88) and could thus increase the severity of an infection.