Bacterial sensing via neuronal Nod2 regulates appetite and body temperature.

Gut bacteria influence brain functions and metabolism. We investigated whether this influence can be mediated by direct sensing of bacterial cell wall components by brain neurons. In mice, we found that bacterial peptidoglycan plays a major role in mediating gut-brain communication via the Nod2 receptor. Peptidoglycan-derived muropeptides reach the brain and alter the activity of a subset of brain neurons that express Nod2. Activation of Nod2 in hypothalamic inhibitory neurons is essential for proper appetite and body temperature control, primarily in females. This study identifies a microbe-sensing mechanism that regulates feeding behavior and host metabolism.

LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization.

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

Defective lytic transglycosylase disrupts cell morphogenesis by hindering cell wall de-O-acetylation in Neisseria meningitidis.

Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets.

Bacteria are surrounded by a tough yet flexible wall that protects the cell and serves as an anchor for several of the cell's structures. This cell wall contains a large mesh-like molecule called peptidoglycan made of many repeated building blocks. When a bacterial cell divides in two, it needs to make more of this material. Making peptidoglycan involves two different sets of enzymes working together: "polymerases" are the enzymes that link the individual building blocks to peptidoglycan, one after the other; while "lytic transglycosylases" are enzymes that modify the peptidoglycan to create space for the addition of new building blocks and for assemblies of proteins that must span the cell wall. Lytic transglycosylases are known to assemble with other proteins and enzymes to form the cell's peptidoglycan-modifying machinery, but it was not clear exactly what purpose they serve within these "enzyme complexes". It was also unclear whether these enzymes would be good targets for new antibiotics. To help answer these questions, Williams et al. looked at a lytic transglycoslyase called LtgA. This enzyme is originally from Neisseria meningitidis, a bacterium that can cause meningitis and life-threatening sepsis in humans. Williams et al. discovered that part of the enzyme's active site ­ the region of an enzyme where the chemical reaction takes ­ can switch from an ordered helix to a disordered, flexible loop. Bacteria were then genetically engineered to make a version of the enzyme that lacked this helix. These bacteria had weaker cell walls and were deformed; they were also less able to grow and divide, both in the laboratory and in a mouse model of infection. Further analysis showed that the deletion of the helix from the enzyme resulted in the peptidoglycan being modified much more than normal, which could likely explain their reduced virulence. Williams et al. also found that deleting the helix from LtgA interfered with the activity of a protein that interacts with this enzyme, called Ape1, which also contributed to the fragility of the cell wall. This shows that lytic transglycosylases assembled into enzyme complexes can alter the activities of other proteins in the complex. Together these findings show that researchers could target one enzyme in a complex in bacteria, and disrupt the activity of other proteins in that complex. This highlights the possibility of considering enzyme complexes as useful targets for new drugs, which is important considering the current problem of antibiotic resistance.

HupA, the main undecaprenyl pyrophosphate and phosphatidylglycerol phosphate phosphatase in Helicobacter pylori is essential for colonization of the stomach.

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.

Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex.

Bacterial cell wall biosynthesis is an essential process that requires the coordinated activity of peptidoglycan biosynthesis enzymes within multi-protein complexes involved in cell division (the "divisome") and lateral wall growth (the "elongasome"). MreC is a structural protein that serves as a platform during wall elongation, scaffolding other essential peptidoglycan biosynthesis macromolecules, such as penicillin-binding proteins. Despite the importance of these multi-partite complexes, details of their architecture have remained elusive due to the transitory nature of their interactions. Here, we present the crystal structures of the soluble PBP2:MreC core elongasome complex from Helicobacter pylori, and of uncomplexed PBP2. PBP2 recognizes the two-winged MreC molecule upon opening of its N-terminal region, revealing a hydrophobic zipper that serves as binding platform. The PBP2:MreC interface is essential both for protein recognition in vitro and maintenance of bacterial shape and growth. This work allows visualization as to how peptidoglycan machinery proteins are scaffolded, revealing interaction regions that could be targeted by tailored inhibitors.Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.