Abstract
Helicobacter pylori is cause of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. L-proline is a preferred energy source of the microaerophilic bacterium. Previous analyses revealed that HpputP and HpputA, the genes that are predicted to play a central role in proline metabolism as they encode for the proline transporter and proline dehydrogenase, respectively, are essential for stomach colonization. Here, the molecular basis of proline transport in H. pylori by HpPutP was investigated experimentally for the first time. Measuring radiolabeled substrate transport in H. pylori and E. coli heterologously expressing HpputP as well as in proteoliposomes reconstituted with HpPutP, we demonstrate that the observed proline transport in H. pylori is mediated by HpPutP. HpPutP is specific and exhibits a high affinity for L-proline. Notably, L-proline transport is exclusively dependent on Na+ as coupling ion, i.e., Na+/L-proline symport, reminiscent to the properties of PutP of E. coli even though H. pylori lives in a more acidic environment. Homology model-based structural comparisons and substitution analyses identified amino acids crucial for function. HpPutP-catalyzed proline uptake was efficiently inhibited by the known proline analogs 3,4-dehydro-D,L-proline and L-azetidine-2-carboxylic acid.
Highlights
Helicobacter pylori is a human pathogen, responsible for type B gastritis and peptic ulcers as well as for increasing the risk of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the stomach [1,2,3,4]
Based on the previously shown crucial role of the proline metabolism in H. pylori stomach colonization [9,11], we have investigated the functional properties of the predicted proline transporter HpPutP, product of gene hpp12_0049
We demonstrated that i) H. pylori P12 is able to transport L-proline, ii) transport of L-proline in cells grown in Brucella medium solely depends on hpp12_0049, iii) the isolated gene product, HpPutP, is sufficient to drive proline accumulation, iv) HpPutP is specific for L-proline, v) the smf is the driving force for transport, and vi) L-proline transport is obligatory coupled to the flux of Na+
Summary
Helicobacter pylori is a human pathogen, responsible for type B gastritis and peptic ulcers as well as for increasing the risk of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma of the stomach [1,2,3,4]. Amino acids (L-proline, L-serine, L-alanine) are efficiently oxidized by H. pylori, and in agreement with their occurrence in human gastric juice, are discussed as main respiratory substrates in the mucus of the stomach [7]. Wild-type and a putA mutant of the closely related human pathogen Helicobacter hepaticus displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed [12]. Taken together, these observations suggest that L-proline uptake and metabolism are of particular significance for physiology and virulence of Helicobacter strains
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