l-Serine, d- and l-proline and alanine as respiratory substrates of Helicobacter pylori: correlation between in vitro and in vivo amino acid levels

Abstract

Helicobacter pylori whole cells showed high rates of oxygen uptake with L-serine and L-proline as respiratory substrates, and somewhat lower rates with D-alanine and D-proline. These respiratory activities were inhibited by rotenone and antimycin A at low concentrations. Since pyruvate was produced from L-serine and D-and L-alanine in whole cells, the respiratory activities with these amino acids as substrates occurred via pyruvate. Whole cells showed 2,6-dichlorophenolindophenol (DCIP)-reducing activities with D-and L-proline and D-alanine as substrates, suggesting that hydrogen removed from these amino acids also participated in oxygen uptake by the whole cells. High amounts of L-proline, D-and L-alanine, and L-serine were present in H. pylori cells, and these amino acids also predominated in samples of human gastric juice. H. pylori seems to utilize D-and L-proline, D-alanine and L-serine as important energy sources in its habitat of the mucous layer of the stomach. INTRODUCTIONHelicobacter pylori is a Gram-negative bacterium associated with gastric inflammation and peptic ulcer disease and is a risk factor for gastric cancer (Blaser, 1990;Parsonnet et al., 1991Parsonnet et al., , 1994Rabeneck & Ransohoff, 1991). The natural habitat of H. pylori is the mucous layer of the human gastric epithelium, where populations are considered to persist for the lifetime of the host. H. pylori is a microaerophilic bacterium exhibiting a strict respiratory form of metabolism and oxidizing organic acids as an energy source. The carbohydrate utilized by H. pylori as the energy source has been reported to be only glucose . More recently the whole-genome analysis of H. pylori has supported these findings (Tomb et al., 1997). However, the incorporation of glucose from the culture medium was not influenced by inhibitors known to affect other bacterial glucose-related enzymes, such as glucose permease, and the K m value of its glucose transport was reported to be rather high, 4?8 mM (Burns & Mendz, 2001). In addition, glucose added to the culture medium composed of a mixture of amino acids was not utilized until the amino acids were significantly depleted Mendz & Hazell, 1995). These results suggested that glucose is not a preferred energy substrate of H. pylori. Candidates for the substrates of energy metabolism in this organism are thought to be organic acids such as pyruvate, D-lactate and succinate. Chang et al. (1995) reported that lower concentrations (25 mM) of pyruvate, D-lactate and succinate were rapidly oxidized, and the respiration rates were relatively high, suggesting that H. pylori cells may have adapted to utilizing these substrates in vivo. However, the oxygen uptake during lactate and pyruvate oxidation was insufficient for their complete oxidation to CO 2 and H 2 O via the citric acid cycle (Chang et al., 1995;Kelly et al., 2001). Thus, instead of organic acids, amino acids are considered to be other potential respiratory substrates and energy sources in H. pylori cells.Little study has been done on amino acids...

Possible involvement of put A gene in Helicobacter pylori colonization in the stomach and motility

“…The results showed that the Δput A mutant from the two strains did not display respiratory activity of L-proline because the mutant had lost the activity of proline dehydrogenase which is essential for proline respiration. H. pylori displays rather high respiratory activity with L-proline, and hydrogen derived from L-proline by L-proline dehydrogenase probably participates in ATP production via the respiratory chain of H. pylori as described previously (22). Interestingly, the Δput A from both strains had little motility and did not show swarming activity.…”

“…Cells were harvested at 0, 5, 10, 24 and 30 h, then centrifuged at 3,500 × g for 10 min at room temperature to obtain the cell-free supernatant. The amount of L-proline in the supernatant was assayed as described previously (22).…”

“…After 20 h cultivation, cells were harvested by centrifugation. The amounts of free-amino acids present within cells of both the wild-type and Δput A strains were assayed as described previously (22).…”

“…After 10 min incubation at 37°C, the cells were removed by centrifugation at 10,000 × g for 5 min and then A600 of the supernatant was measured using DU 640, Beckman, Fullerton, USA. An ε 600 of 21.6 mM −1 cm −1 was used as described previously (22).…”

“…We previously reported that H. pylori shows rather strong respiratory activity when amino acids such as L-proline, L-serine and L-alanine are used as respiratory substrates (22). We also reported that the gastric juice of patients infected with H. pylori contained rather large amounts of these amino acids, especially L-proline (22).…”