Improving homology-directed repair efficiency in human stem cells.

The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7.

A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis.

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.

Large structural variants in KOLF2.1J are unlikely to compromise neurological disease modelling.

Gracia-Diaz and colleagues analysed high-density DNA microarray and whole genome sequencing (WGS) data from the KOLF2.1J 'reference' human induced pluripotent stem cell (hiPSC) line1, and report the presence of five high-confidence heterozygous copy number variants (CNVs) at least 100kbp in length2. Since three of these CNVs span coding genes, some of which have been associated with neurodevelopmental disease, the authors raise the concern that these CNVs may compromise the utility of KOLF2.1J for neurological disease modelling. We appreciate their thorough analysis and thoughtful interpretation, and agree that potential users of this line should be made aware of all cases where KOLF2.1J differs from the reference genome. However, we believe that the benefits from the widespread use of KOLF2.1J outweigh the potential risks that might arise from the identified CNVs.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.

The role of Rab GTPases in the transport of vacuoles containing Legionella pneumophila and Coxiella burnetii.

Intracellular pathogens survive in eukaryotic cells by evading a variety of host defences. To avoid degradation through the endocytic pathway, intracellular bacteria must adapt their phagosomes into protective compartments that promote bacterial replication. Legionella pneumophila and Coxiella burnetii are Gram-negative intracellular pathogens that remodel their phagosomes by co-opting components of the host cell, including Rab GTPases. L. pneumophila and C. burnetii are related phylogenetically and share an analogous type IV secretion system for delivering bacterial effectors into the host cell. Some of these effectors mimic eukaryotic biochemical activities to recruit and modify Rabs at the vacuole. In the present review, we cover how these bacterial species, which utilize divergent strategies to establish replicative vacuoles, use translocated proteins to manipulate host Rabs, as well as exploring which Rabs are implicated in vacuolar biogenesis in these two organisms.

Coxiella burnetii secretion systems.

The ability of bacteria to transport proteins across their membranes is integral for interaction with their environment. Distinct families of secretion systems mediate bacterial protein secretion. The human pathogen, Coxiella burnetii encodes components of the Sec-dependent secretion pathway, an export system used for type IV pilus assembly, and a complete type IV secretion system. The type IVB secretion system in C. burnetii is functionally analogous to the Legionella pneumophila Dot/Icm secretion system. Both L. pneumophila and C. burnetii require the Dot/Icm apparatus for intracellular replication. The Dot/Icm secretion system facilitates the translocation of many bacterial effector proteins across the bacterial and vacuole membranes to enter the host cytoplasm where the effector proteins mediate their specific activities to manipulate a variety of host cell processes. Several studies have identified cohorts of C. burnetii Dot/Icm effector proteins that are predicted to be involved in modulation of host cell functions. This chapter focuses specifically on these secretion systems and the role they may play during C. burnetii replication in eukaryotic host cells.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Genome-wide identification of Mycobacterium tuberculosis exported proteins with roles in intracellular growth.

The exported proteins of Mycobacterium tuberculosis that are localized at the bacterial cell surface or secreted into the environment are ideally situated to interact with host factors and to function in virulence. In this study, we constructed a novel ß-lactamase reporter transposon and used it directly in M. tuberculosis for genome-wide identification of exported proteins. From 177 ß-lactam-resistant transposon mutants, we identified 111 different exported proteins. The majority of these proteins have no known function, and for nearly half of the proteins, our demonstration that they are exported when fused to a ß-lactamase reporter is the first experimental proof of their extracytoplasmic localization. The transposon mutants in our banked library were of further value as a collection of mutants lacking individual exported proteins. By individually testing each of 111 mutants for growth in macrophages, six attenuated mutants with insertions in mce1A, mce1B, mce2F, rv0199, ctaC, and lppX were identified. Given that much of the M. tuberculosis genome encodes proteins of unknown function, our library of mapped transposon mutants is a valuable resource for efforts in functional genomics. This work also demonstrates the power of a ß-lactamase reporter transposon that could be applied similarly to other bacterial pathogens.

The Anaplasma phagocytophilum-occupied vacuole selectively recruits Rab-GTPases that are predominantly associated with recycling endosomes.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell-derived vacuole. The A. phagocytophilum-occupied vacuole (ApV) fails to mature along the endocytic pathway and is non-fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)-tagged Rab GTPases in A. phagocytophilum-infected HL-60 cells. GFP-Rab4A, GFP-Rab10, GFP-Rab11A, GFP-Rab14, RFP-Rab22A and GFP-Rab35, which regulate endocytic recycling, and GFP-Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome-associated GFP-Rabs and acquisition of GFP-Rab5, GFP-Rab7, and the lysosomal marker, LAMP-1. Wild-type and GTPase- deficient versions, but not GDP-restricted versions of GFP-Rab1, GFP-Rab4A and GFP-Rab11A, localize to the ApV. Strikingly, GFP-Rab10 recruitment to the ApV is guanine nucleotide-independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.

Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway.

Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.

Identification of functional Tat signal sequences in Mycobacterium tuberculosis proteins.

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated beta-lactamase, 'BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Deltatat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported.

Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.

The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a ß-lactamase enzyme (BlaM) fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into host cells after bacteria establish residency in a lysosome-derived organelle.

Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening.

UNLABELLED: Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication. IMPORTANCE: Q fever in humans is caused by the bacterium Coxiella burnetii. Infection with C. burnetii is marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets of C. burnetii infection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of the C. burnetii vacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.

The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1.

In bacteria, the majority of exported proteins are transported by the general Sec pathway from their site of synthesis in the cytoplasm across the cytoplasmic membrane. The essential SecA ATPase powers this Sec-mediated export. Mycobacteria possess two nonredundant SecA homologs: SecA1 and SecA2. In pathogenic Mycobacterium tuberculosis and the nonpathogenic model mycobacterium Mycobacterium smegmatis, SecA1 is essential for protein export and is the "housekeeping" SecA, whereas SecA2 is an accessory SecA that exports a specific subset of proteins. In M. tuberculosis the accessory SecA2 pathway plays a role in virulence. In this study, we uncovered basic properties of the mycobacterial SecA2 protein and its pathway for exporting select proteins. By constructing secA2 mutant alleles that encode proteins defective in ATP binding, we showed that ATP binding is required for SecA2 function. SecA2 mutant proteins unable to bind ATP were nonfunctional and dominant negative. By evaluating the subcellular distribution of each SecA, SecA1 was shown to be equally divided between cytosolic and cell envelope fractions, whereas SecA2 was predominantly localized to the cytosol. Finally, we showed that the canonical SecA1 has a role in the process of SecA2-dependent export. The accessory SecA2 export system is important to the physiology and virulence of mycobacteria. These studies help establish the mechanism of this new type of specialized protein export pathway.

Beta-lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells.

Mycobacterium tuberculosis is an intracellular pathogen that is able to avoid destruction by host immune defences. Exported proteins of M. tuberculosis, which include proteins localized to the bacterial surface or secreted into the extracellular environment, are ideally situated to interact with host factors. As a result, these proteins are attractive candidates for virulence factors, drug targets and vaccine components. Here we describe a beta-lactamase reporter system capable of identifying exported proteins of M. tuberculosis during growth in host cells. Because beta-lactams target bacterial cell-wall synthesis, beta-lactamases must be exported beyond the cytoplasm to protect against these drugs. When used in protein fusions, beta-lactamase can report on the subcellular location of another protein as measured by protection from beta-lactam antibiotics. Here we demonstrate that a truncated TEM-1 beta-lactamase lacking a signal sequence for export ('BlaTEM-1) can be used in this manner directly in a mutant strain of M. tuberculosis lacking the major beta-lactamase, BlaC. The 'BlaTEM-1 reporter conferred beta-lactam resistance when fused to both Sec and Tat export signal sequences. We further demonstrate that beta-lactamase fusion proteins report on protein export while M. tuberculosis is growing in THP-1 macrophage-like cells. This genetic system should facilitate the study of proteins exclusively exported in the host environment by intracellular M. tuberculosis.

The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases.

The twin-arginine translocation (Tat) pathway exports folded proteins across the bacterial cytoplasmic membrane and is responsible for the proper extracytoplasmic localization of proteins involved in a variety of cellular functions, including pathogenesis. The Mycobacterium tuberculosis and Mycobacterium smegmatis genomes contain open reading frames with homology to components of the Tat export system (TatABC) as well as potential Tat-exported proteins possessing N-terminal signal sequences with the characteristic twin-arginine motif. Due to the importance of exported virulence factors in the pathogenesis of M. tuberculosis and the limited understanding of mycobacterial protein export systems, we sought to determine the functional nature of the Tat export pathway in mycobacteria. Here we describe phenotypic analyses of DeltatatA and DeltatatC deletion mutants of M. smegmatis, which demonstrated that tatA and tatC encode components of a functional Tat system capable of exporting characteristic Tat substrates. Both mutants displayed a growth defect on agar medium and hypersensitivity to sodium dodecyl sulfate. The mutants were also defective in the export of active beta-lactamases of M. smegmatis (BlaS) and M. tuberculosis (BlaC), both of which possess twin-arginine signal sequences. The Tat-dependent nature of BlaC was further revealed by mutation of the twin-arginine motif. Finally, we demonstrated that replacement of the native signal sequence of BlaC with the predicted Tat signal sequences of M. tuberculosis phospholipase C proteins (PlcA and PlcB) resulted in the Tat-dependent export of an enzymatically active 'BlaC. Thus, 'BlaC can be used as a genetic reporter for Tat-dependent export in mycobacteria.

Involvement of Candida albicans NADH dehydrogenase complex I in filamentation.

The gene encoding the 51-kDa subunit of nicotinamide adenine dinucleotide (NADH) dehydrogenase complex I, a principal component of the mitochondrial electron transport chain, was cloned in Candida tropicalis. The homolog in C. albicans, CaNDH51, was identified, and each allele was successively disrupted by PCR-mediated gene disruption. Wild type, heterozygote, reintegrant, and homozygous null mutants grew as blastoconidia in rich medium containing 3% glucose, but the homozygous null mutant failed to grow in ethanol or acetate. When glucose concentration was varied from 1 mM (0.018%) to 200 mM (3.6%) in a basal salts medium, all strains grew equally well at all glucose concentrations; the wild-type strain, the heterozygote, and the reintegrant exhibited abundant germ tubes, pseudohyphae, and hyphae. In contrast, the ndh51/ndh51 strain failed to display any type of filamentous growth, even in glucose concentrations as low as 1 mM. These results suggest a previously unexplored relationship between mitochondrial electron transport and morphogenesis.

Use of antibiotic resistance analysis for representativeness testing of multiwatershed libraries.

The use of antibiotic resistance analysis (ARA) for microbial source tracking requires the generation of a library of isolates collected from known sources in the watershed. The size and composition of the library are critical in determining if it represents the diversity of patterns found in the watershed. This study was performed to determine the size that an ARA library needs to be to be representative of the watersheds for which it will be used and to determine if libraries from different watersheds can be merged to create multiwatershed libraries. Fecal samples from known human, domesticated, and wild animal sources were collected from six Virginia watersheds. From these samples, enterococci were isolated and tested by ARA. Based on cross-validation discriminant analysis, only the largest of the libraries (2,931 isolates) were found to be able to classify nonlibrary isolates as well as library isolates (i.e., were representative). Small libraries tended to have higher average rates of correct classification, but were much less able to correctly classify nonlibrary isolates. A merged multiwatershed library (6,587 isolates) was created and was found to be large enough to be representative of the isolates from the contributing watersheds. When isolates that were collected from the contributing watersheds approximately 1 year later were analyzed with the multiwatershed library, they were classified as well as the isolates in the library, suggesting that the resistance patterns are temporally stable for at least 1 year. The ability to obtain a representative, temporally stable library demonstrates that ARA can be used to identify sources of fecal pollution in natural waters.