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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3, EccC3, and EccE3 and two copies of the EccD3 protein. In the structure, the protomers form a stable dimer that is consistent with assembly into a larger oligomer. The ESX-3 structure provides a framework for further study of these important bacterial transporters.

Heterogeneity in human immune responses is difficult to model in standard laboratory mice. To understand how host variation affects Bacillus Calmette Guerin-induced (BCG-induced) immunity against Mycobacterium tuberculosis, we studied 24 unique collaborative cross (CC) mouse strains, which differ primarily in the genes and alleles they inherit from founder strains. The CC strains were vaccinated with or without BCG and challenged with aerosolized M. tuberculosis. Since BCG protects only half of the CC strains tested, we concluded that host genetics has a major influence on BCG-induced immunity against M. tuberculosis infection, making it an important barrier to vaccine- mediated protection. Importantly, BCG efficacy is dissociable from inherent susceptibility to tuberculosis (TB). T cell immunity was extensively characterized to identify components associated with protection that were stimulated by BCG and recalled after M. tuberculosis infection. Although considerable diversity is observed, BCG has little impact on the composition of T cells in the lung after infection. Instead, variability is largely shaped by host genetics. BCG-elicited protection against TB correlated with changes in immune function. Thus, CC mice can be used to define correlates of protection and to identify vaccine strategies that protect a larger fraction of genetically diverse individuals instead of optimizing protection for a single genotype., Introduction The introduction of Bacillus Calmette Guerin (BCG) as a vaccine contributed significantly to tuberculosis (TB) control, which has threatened global health for millennia. As the only vaccine approved for [...]

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the invitro frameshift rate in a neutral HT at 100× the neutral substitution rate at 1.1 × 10−5 frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host. [ABSTRACT FROM AUTHOR]