Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii.

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.

Crystal structures of Mycobacterial MeaB and MMAA-like GTPases.

The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.

Structure of 3-ketoacyl-(acyl-carrier-protein) reductase from Rickettsia prowazekii at 2.25 Å resolution.

Rickettsia prowazekii, a parasitic Gram-negative bacterium, is in the second-highest biodefense category of pathogens of the National Institute of Allergy and Infectious Diseases, but only a handful of structures have been deposited in the PDB for this bacterium; to date, all of these have been solved by the SSGCID. Owing to its small genome (about 800 protein-coding genes), it relies on the host for many basic biosynthetic processes, hindering the identification of potential antipathogenic drug targets. However, like many bacteria and plants, its metabolism does depend upon the type II fatty-acid synthesis (FAS) pathway for lipogenesis, whereas the predominant form of fatty-acid biosynthesis in humans is via the type I pathway. Here, the structure of the third enzyme in the FAS pathway, 3-ketoacyl-(acyl-carrier-protein) reductase, is reported at a resolution of 2.25 Å. Its fold is highly similar to those of the existing structures from some well characterized pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei, but differs significantly from the analogous mammalian structure. Hence, drugs known to target the enzymes of pathogenic bacteria may serve as potential leads against Rickettsia, which is responsible for spotted fever and typhus and is found throughout the world.

The Rickettsia type IV secretion system: unrealized complexity mired by gene family expansion.

Many prokaryotes utilize type IV secretion systems (T4SSs) to translocate substrates (e.g. nucleoprotein, DNA, protein) across the cell envelope, and/or to elaborate surface structures (i.e. pili or adhesins). Among eight distinct T4SS classes, P-T4SSs are typified by the Agrobacterium tumefaciens vir T4SS, which is comprised of 12 scaffold components (VirB1-VirB11, VirD4). While most P-T4SSs include all 12 Vir proteins, some differ from the vir archetype by either containing additional scaffold components not analogous to Vir proteins or lacking one or more of the Vir proteins. In a special case, the Rickettsiales vir homolog (rvh) P-T4SS comprises unprecedented gene family expansion. rvh contains three families of gene duplications (rvhB9, rvhB8, rvhB4): RvhB9,8,4-I are conserved relative to equivalents in other P-T4SSs, while RvhB9,8,4-II have evolved atypical features that deviate substantially from other homologs. Furthermore, rvh contains five VirB6-like genes (rvhB6a-e), which are tandemly arrayed and contain large N- and C-terminal extensions. Our work herein focuses on the complexity underpinned by rvh gene family expansion. Furthermore, we describe an RvhB10 insertion, which occurs in a region that forms the T4SS pore. The significance of these curious properties to rvh structure and function is evaluated, shedding light on a highly complex T4SS.

Backbone chemical shift assignments for the sensor domain of the Burkholderia pseudomallei histidine kinase RisS: "missing" resonances at the dimer interface.

Using a deuterated sample, all the observable backbone (1)H(N), (15)N, (13)C(a), and (13)C' chemical shifts for the dimeric, periplasmic sensor domain of the Burkholderia pseudomallei histidine kinase RisS were assigned. Approximately one-fifth of the amide resonances are "missing" in the (1)H-(15)N HSQC spectrum and map primarily onto α-helices at the dimer interface observed in a crystal structure suggesting this region either undergoes intermediate timescale motion (µs-ms) and/or is heterogeneous.

Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it appears to lack three α-helices at the N-terminus and contains two amino acids in the allosteric pocket that make it appear more like the enzyme from the human host than that from the other parasite T. brucei. Thus, allosteric inhibitors of T. brucei UAP are unlikely to target Entamoeba UAPs.

Iron superoxide dismutases in eukaryotic pathogens: new insights from Apicomplexa and Trypanosoma structures.

Prior studies have highlighted the potential of superoxide dismutases as drug targets in eukaryotic pathogens. This report presents the structures of three iron-dependent superoxide dismutases (FeSODs) from Trypanosoma cruzi, Leishmania major and Babesia bovis. Comparison with existing structures from Plasmodium and other trypanosome isoforms shows a very conserved overall fold with subtle differences. In particular, structural data suggest that B. bovis FeSOD may display similar resistance to peroxynitrite-mediated inactivation via an intramolecular electron-transfer pathway as previously described in T. cruzi FeSOD isoform B, thus providing valuable information for structure-based drug design. Furthermore, lysine-acetylation results in T. cruzi indicate that acetylation occurs at a position close to that responsible for the regulation of acetylation-mediated activity in the human enzyme.

Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems.

UNLABELLED: Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some Bartonella species (vir, vbh, and trw). In a unique case, most rickettsial species encode a T4SS (rvh) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG Bartonella proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of trw and vir systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of Rickettsia typhi RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the rvh T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for Bartonella trw and vir T4SSs and structural for rvh T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium. IMPORTANCE: Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.

Selecting targets from eukaryotic parasites for structural genomics and drug discovery.

The selection of targets is the first step for any structural genomics project. The application of structural genomics approaches to drug discovery also starts with the selection of targets. Here, three protocols are described that were developed to select targets from eukaryotic pathogens. These protocols could also be applied to other drug discovery projects.