Malaria parasites replicate within a parasitophorous vacuole in red blood cells (RBCs). Progeny merozoites egress upon rupture of first the parasitophorous vacuole membrane (PVM), then poration and rupture of the RBC membrane (RBCM). Egress is protease-dependent , but none of the effector molecules that mediate membrane rupture have been identified and it is unknown how sequential rupture of the two membranes is controlled. Minutes before egress, the parasite serine protease SUB1 is discharged into the parasitophorous vacuole where it cleaves multiple substrates including SERA6, a putative cysteine protease. Here, we show that Plasmodium falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, PVM rupture and RBCM poration occur normally in SERA6-null parasites but RBCM rupture does not occur. Complementation studies show that SERA6 is an enzyme that requires processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage within the actin-binding domain of the major RBC cytoskeletal protein β-spectrin. We conclude that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes and culminates in RBCM disruption through rapid, precise, SERA6-mediated disassembly of the RBC cytoskeleton.

Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.

Most organisms are exposed to the genotoxic chemical formaldehyde, either from endogenous or environmental sources. Therefore, biology has evolved systems to perceive and detoxify formaldehyde. The frmRA(B) operon that is present in many bacteria represents one such system. The FrmR protein is a transcriptional repressor that is specifically inactivated in the presence of formaldehyde, permitting expression of the formaldehyde detoxification machinery (FrmA and FrmB, when the latter is present). The X-ray structure of the formaldehyde-treated Escherichia coli FrmR (EcFrmR) protein reveals the formation of methylene bridges that link adjacent Pro2 and Cys35 residues in the EcFrmR tetramer. Methylene bridge formation has profound effects on the pattern of surface charge of EcFrmR and combined with biochemical/biophysical data suggests a mechanistic model for formaldehyde-sensing and derepression of frmRA(B) expression in numerous bacterial species.

Summary The most severe form of human malaria is caused by Plasmodium falciparum . Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 ‘FIKK’ serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum , but not by Plasmodium knowlesi , which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identify unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis reveals that some FIKKs may act in the same pathways. Only deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival within the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.

P. 2018. van der Waals contact between nucleophile and transferring phosphorus is insufficient to achieve enzyme transition-state architecture. ACS Catalysis 8 (9) , pp.

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.

SummaryImidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.