Dysfunction of the intestinal epithelial barrier plays an important role in the pathogenesis of several intestinal diseases, including celiac disease, inflammatory bowel disease, and irritable bowel syndrome.

The biogenesis of bacterial cell-envelope polysaccharides requires the translocation, across the plasma membrane, of sugar sub-units that are produced inside the cytoplasm. To this end, the hydrophilic sugars are anchored to a lipid phosphate carrier (undecaprenyl phosphate (C55-P)), yielding membrane intermediates which are translocated to the outer face of the membrane. Finally, the glycan moiety is transferred to a nascent acceptor polymer, releasing the carrier in the “inactive” undecaprenyl pyrophosphate (C55-PP) form. Thus, C55-P is generated through the dephosphorylation of C55-PP, itself arising from either de novo synthesis or recycling. Two types of integral membrane C55-PP phosphatases were described: BacA enzymes and a sub-group of PAP2 enzymes (type 2 phosphatidic acid phosphatases). The human pathogen Helicobacter pylori does not contain BacA homologue but has four membrane PAP2 proteins: LpxE, LpxF, HP0350 and HP0851. Here, we report the physiological role of HP0851, renamed HupA, via multiple and complementary approaches ranging from a detailed biochemical characterization to the assessment of its effect on cell envelope metabolism and microbe-host interactions. HupA displays a dual function as being the main C55-PP pyrophosphatase (UppP) and phosphatidylglycerol phosphate phosphatase (PGPase). Although not essential in vitro, HupA was essential in vivo for stomach colonization. In vitro, the remaining UppP activity was carried out by LpxE in addition to its lipid A 1-phosphate phosphatase activity. Both HupA and LpxE have crucial roles in the biosynthesis of several cell wall polysaccharides and thus constitute potential targets for new therapeutic strategies.

Ubiquitous PAP2 lipid phosphatases are involved in a wide array of central physiological functions. PgpB from Escherichia coli constitutes the archetype of this subfamily of membrane proteins. It displays a dual function by catalyzing the biosynthesis of two essential lipids, the phosphatidylglycerol (PG) and the undecaprenyl phosphate (C 55-P). C 55-P constitutes a lipid carrier allowing the translocation of peptidoglycan subunits across the plasma membrane. PG and C 55-p are synthesized in a redundant manner by PgpB and other PAP2 and/or unrelated membrane phosphatases. Here, we show that PgpB is the sole, among these multiple phosphatases, displaying this dual activity. The inactivation of PgpB does not confer any apparent growth defect, but its inactivation together with another PAP2 alters the cell envelope integrity increasing the susceptibility to small hydrophobic compounds. Evidence is also provided of an interplay between PAP2s and the peptidoglycan polymerase PBP1A. In contrast to PGP hydrolysis, which relies on a His/Asp/His catalytic triad of PgpB, the mechanism of C 55-PP hydrolysis appeared as only requiring the His/Asp diad, which led us to hypothesize distinct processes. Moreover, thermal stability analyses highlighted a substantial structural change upon phosphate binding by PgpB, supporting an induced-fit model of action. Undecaprenyl phosphate (C 55-P) plays an essential role in the biogenesis of bacterial envelope polysaccharides such as the peptidoglycan 1. It is used as a lipid carrier allowing the translocation of polymer subunits across the plasma membrane to the outer site, where the polymers are assembled 2 (Supplementary Fig. S1). The synthesis of C 55-P proceeds via the hydrolysis of its precursor, the undecaprenyl pyrophosphate (C 55-PP), itself being de novo synthesized at the cytosolic side of the plasma membrane or released during subunits polymerization at the outer side (Supplementary Fig. S1) 1. Four integral membrane enzymes catalyzing C 55-PP hydrolysis have been identified in Escherichia coli: BacA, PgpB, YbjG and LpxT (formerly YeiU), which belong to two unrelated protein families: BacA and Phosphatidic Acid Phosphatases of type 2 (PAP2) 3-5. None of these enzymes is essential for growth, but the simultaneous knockout of bacA, pgpB and ybjG genes elicits a lethal phenotype due to a default of C 55-P supply 4. These enzymes have their active sites oriented towards the periplasm, suggesting they may rather be involved in C 55-PP recycling 5-10. The mechanism of translocation of C 55-P back to the inner side of the membrane is yet unknown. The structure of BacA, which is reminiscent to that of transporters, raised the hypothesis that it may also catalyze the flip of C 55-P 7,10. The lack of a known cytoplasm-oriented C 55-PP phosphatase raised also the question as to whether BacA and PAP2s are required for the dephosphorylation of de novo synthesized C 55-PP (Supplementary Fig. S1). PgpB is involved in another essential metabolic pathway, i.e. the synthesis of phosphatidylglyc...