NAD(H)-mediated tetramerization controls the activity of Legionella pneumophila phospholipase PlaB

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires’ disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen–host interface.

Phospholipases are important enzymes in infectious disease pathogenesis involved in host modulation and damage. For example, some bacterial phospholipases, such as Pseudomonas aeruginosa ExoU, massively damage cells and contribute to host inflammatory response (1–4). Others, such as Legionella pneumophila VipD, facilitate bacterial intracellular replication by inhibiting phagosomal maturation, or as in the case of Listeria monocytogenes, phospholipases PlcA and PlcB are required for bacterial escape from the enclosing vacuole and for cell-to-cell spread (1, 5–8). Phospholipases have been assigned to different groups depending on the preferred cleavage site within their substrates. Phospholipases A (PLAs) and lysophospholipases A (LPLA) hydrolyze carboxyl ester bonds at the sn-1 or sn-2 position in phospholipids or lysophospholipids, respectively, and release fatty acids (1, 9). In L. pneumophila, a Gram-negative bacterium that causes Legionnaires’ disease, at least 15 genes encoding PLAs/LPLAs belonging to three families are found. Many of these are secreted to modulate the host cell but only one, PlaB, is uniquely presented at the bacterial surface (1, 10–13).In this study, we focused on PlaB, a hemolysin and virulence factor that promotes intracellular replication in macrophages by its PLA and LPLA activities (14–16). PlaB is also crucial for lung colonization and tissue destruction in guinea pig infections (13). It is the only characterized member of a recently discovered PLA family, and homologs are found in a wide array of mostly water-associated bacteria including the opportunistic pathogen P. aeruginosa (14, 15). Previous work suggested that PlaB is organized into two domains, namely an N-terminal phospholipase (amino acids 1 to ∼300) connected to a C-terminal domain (CTD) (amino acids ∼301 to 474) that is also essential for activity (15). The catalytic triad S85/D203/H251 of the N-terminal domain (NTD) of PlaB and its homologs is embedded in uncommon consensus motifs which are unique among lipases (15). The CTD is not related to formerly characterized proteins, but we have earlier shown that the last 15 amino acids of PlaB are necessary for activity, although their exact role is not understood (15, 16). Subcellular fractionation and proteinase K digests revealed that PlaB is associated with the outer membrane (OM) and exposed on the surface. However, due to the apparent lack of export signal sequences, lipid anchors, or transmembrane helices, determinants for export and membrane association have not yet been characterized and therefore still remain elusive (13, 14).PlaB represents a highly active PLA/LPLA of L. pneumophila and hydrolyzes lipids, such as phosphatidylcholine (PC) and phosphatidylglycerol (PG) found in the lung of the human host and in Legionella (14, 17–19). Hence, we reasoned that a control mechanism of enzyme activity may be crucial to prevent damage to the pathogen itself. Indeed, PlaB shows an extraordinary activation mechanism that requires protein deoligomerization. At higher PlaB concentrations, the enzyme occurs in an inactive tetrameric form, whereas it deoligomerizes in the lower nanomolar concentration range where it possesses its highest specific activity (16). However, the mechanism behind enzyme regulation and whether the resulting dimer or monomer represents the active form is not understood.To decipher the molecular basis for PlaB’s unusual activation and to gain insight on how it associates with the OM, we have determined its crystal structure. This allowed us to identify important structural features and, interestingly, revealed an NAD(H)-mediated tetramerization mechanism that controls activity.     

Micronutrient status in lactating mothers before and after introduction of fortified flour: cross-sectional surveys in Maela refugee camp

Background

Deficiency of micronutrients is common in refugee populations.

Objectives

Identify deficiencies and whether provided supplements and wheat flour fortified with 10 micronutrients impacts upon status among breast-feeding women from Maela refugee camp.

Methods

Two sequential cross-sectional studies were conducted in different groups of lactating mothers at 12?weeks postpartum. The first survey was before and the second 4–5?months after micronutrient fortified flour (MFF) had been provided to the camp (in addition to the regular food basket). Iron status and micronutrients were measured in serum, whole blood, and in breast milk samples.

Results

Iron and zinc deficiency and anemia were highly prevalent while low serum retinol and thiamine deficiency were rarely detected. Iron and zinc deficiency were associated with anemia, and their proportions were significantly lower after the introduction of MFF (21 vs. 35% with soluble transferrin receptor (sTfR) >8.5?mg/L, P?=?0.042, and 50 vs. 73% with serum zinc <0.66?mg/L, P?=?0.001). Serum sTfR, whole-blood thiamine diphosphate (TDP) and serum β-carotene were significant predictors (P?Conclusions High whole-blood TDP and breast milk thiamine reflected good compliance to provided thiamine; high prevalence of iron deficiency suggested insufficient dietary iron and low acceptance to ferrous sulfate supplements. MFF as an additional food ration in Maela refugee camp seemed to have an effect in reducing both iron and zinc deficiency postpartum.