Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

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 in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] 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.

Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug­drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical­genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo­relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical­genetic­environmental interactions that can be used to optimize drug­drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

A natural polymorphism of Mycobacterium tuberculosis in the esxH gene disrupts immunodomination by the TB10.4-specific CD8 T cell response.

CD8 T cells provide limited protection against Mycobacterium tuberculosis (Mtb) infection in the mouse model. As Mtb causes chronic infection in mice and humans, we hypothesize that Mtb impairs T cell responses as an immune evasion strategy. TB10.4 is an immunodominant antigen in people, nonhuman primates, and mice, which is encoded by the esxH gene. In C57BL/6 mice, 30-50% of pulmonary CD8 T cells recognize the TB10.44-11 epitope. However, TB10.4-specific CD8 T cells fail to recognize Mtb-infected macrophages. We speculate that Mtb elicits immunodominant CD8 T cell responses to antigens that are inefficiently presented by infected cells, thereby focusing CD8 T cells on nonprotective antigens. Here, we leverage naturally occurring polymorphisms in esxH, which frequently occur in lineage 1 strains, to test this "decoy hypothesis". Using the clinical isolate 667, which contains an EsxHA10T polymorphism, we observe a drastic change in the hierarchy of CD8 T cells. Using isogenic Erd.EsxHA10T and Erd.EsxHWT strains, we prove that this polymorphism alters the hierarchy of immunodominant CD8 T cell responses. Our data are best explained by immunodomination, a mechanism by which competition for APC leads to dominant responses suppressing subdominant responses. These results were surprising as the variant epitope can bind to H2-Kb and is recognized by TB10.4-specific CD8 T cells. The dramatic change in TB10.4-specific CD8 responses resulted from increased proteolytic degradation of A10T variant, which destroyed the TB10.44-11epitope. Importantly, this polymorphism affected T cell priming and recognition of infected cells. These data support a model in which nonprotective CD8 T cells become immunodominant and suppress subdominant responses. Thus, polymorphisms between clinical Mtb strains, and BCG or H37Rv sequence-based vaccines could lead to a mismatch between T cells that are primed by vaccines and the epitopes presented by infected cells. Reprograming host immune responses should be considered in the future design of vaccines.

N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

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.

Subpolar addition of new cell wall is directed by DivIVA in mycobacteria.

Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension.

High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7.

The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic Escherichia coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR(+) open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10-100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.

Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion.

This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O-island 51, a 14.93 kb cryptic prophage (CP-933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O-island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE-encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression.

Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function.

BACKGROUND: The human OXR1 gene belongs to a class of genes with conserved functions that protect cells from reactive oxygen species (ROS). The gene was found using a screen of a human cDNA library by its ability to suppress the spontaneous mutator phenotype of an E. coli mutH nth strain. The function of OXR1 is unknown. The human and yeast genes are induced by oxidative stress and targeted to the mitochondria; the yeast gene is required for resistance to hydrogen peroxide. Multiple spliced isoforms are expressed in a variety of human tissues, including brain. RESULTS: In this report, we use a papillation assay that measures spontaneous mutagenesis of an E. coli mutM mutY strain, a host defective for oxidative DNA repair. Papillation frequencies with this strain are dependent upon a G→T transversion in the lacZ gene (a mutation known to occur as a result of oxidative damage) and are suppressed by in vivo expression of human OXR1. N-terminal, C-terminal and internal deletions of the OXR1 gene were constructed and tested for suppression of the mutagenic phenotype of the mutM mutY strain. We find that the TLDc domain, encoded by the final four exons of the OXR1 gene, is not required for papillation suppression in E. coli. Instead, we show that the protein segment encoded by exon 8 of OXR1 is responsible for the suppression of oxidative damage in E. coli. CONCLUSION: The protein segment encoded by OXR1 exon 8 plays an important role in the anti-oxidative function of the human OXR1 protein. This result suggests that the TLDc domain, found in OXR1 exons 12-16 and common in many proteins with nuclear function, has an alternate (undefined) role other than oxidative repair.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7.

Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

Oligo-Mediated Recombineering and its Use for Making SNPs, Knockouts, Insertions, and Fusions in Mycobacterium tuberculosis.

Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over 10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This "recombineering" technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to) 500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events, including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.

The structure of the endogenous ESX-3 secretion system.

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.

Dam methyltransferase is required for stable lysogeny of the Shiga toxin (Stx2)-encoding bacteriophage 933W of enterohemorrhagic Escherichia coli O157:H7.

Shiga toxin 2 (Stx2), one of the principal virulence factors of enterohemorrhagic Escherichia coli, is encoded by 933W, a lambda-like prophage. 933W prophage induction contributes to Stx2 production, and here, we provide evidence that Dam methyltransferase is essential for maintenance of 933W lysogeny. Our findings are consistent with the idea that the 933W prophage has a relatively low threshold for induction, which may promote Stx2 production during infection.

The lambda Gam protein inhibits RecBCD binding to dsDNA ends.

Inactivation of the Escherichia coli RecBCD enzyme by the lambda Gam protein is an essential step that accompanies the lambda Red proteins for gene replacement using recombineering technology. It has been shown that Gam inhibits all the activities of RecBCD to the same extent. Nonetheless, some in vivo properties of recBCD mutants cannot be mimicked effectively by the expression of gam in vivo. An examination of the mechanism of Gam's inhibition of RecBCD was performed, and it was found that Gam inhibits the binding of RecBCD to double-stranded DNA ends, even if RecBCD is bound to DNA before its interaction with Gam. When ATP is added to the reaction to induce helicase activity, most of the reaction is inhibited by Gam, but residual amounts of unwinding are detected, despite a 40-fold excess of Gam/RecBCD. The same inhibitory effect of Gam was seen on RecBCD that had been modified by the P22 anti-RecBCD protein Abc2, though the inhibitory effect was diminished due to the tighter binding of Abc2-modified RecBCD to double-stranded DNA ends. These data suggest that cells containing Gam-expressing plasmids retain a small amount of uninhibited enzyme. Given the suspected instability of Gam in vivo, care must be taken when interpreting results from experiments containing Gam-inhibited RecBCD species. A revised model is proposed for Gam-induced radioresistance of E. coli to ionizing radiation.

ORBIT: a New Paradigm for Genetic Engineering of Mycobacterial Chromosomes.

Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacteriumtuberculosis and M. smegmatisIMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

λ Recombination and Recombineering.

The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

Mycobacterial recombineering.

The precise knockout or modification of Mycobacterium tuberculosis genes has been critical for the identification of functions important for the growth and pathogenicity of this important bacterium. Schemes have been previously described, using both non-replicating vectors and transducing particles, for the introduction of gene knockout substrates into M. tuberculosis, where the endogenous recombination systems of the host (both homologous and illegitimate) compete for transfer of the modified allele to the chromosome. Recombineering technologies, first introduced in laboratory and pathogenic strains of Escherichia coli over the last 16 years, have been developed for use in M. tuberculosis. Described in this chapter is the use of the mycobacterial Che9c phage RecET recombination system, which has been used to make gene knockouts, reporter fusions, promoter replacements, and single base pair modifications within the M. tuberculosis and M. smegmatis chromosomes at very high frequency. Higher success rates, in a shorter period of time, are routinely observed when recombineering is compared to previously described M. tuberculosis gene knockout protocols.

The Oxidative Stress Network of Mycobacterium tuberculosis Reveals Coordination between Radical Detoxification Systems.

M. tuberculosis (Mtb) survives a hostile environment within the host that is shaped in part by oxidative stress. The mechanisms used by Mtb to resist these stresses remain ill-defined because the complex combination of oxidants generated by host immunity is difficult to accurately recapitulate in vitro. We performed a genome-wide genetic interaction screen to comprehensively delineate oxidative stress resistance pathways necessary for Mtb to resist oxidation during infection. Our analysis predicted functional relationships between the superoxide-detoxifying enzyme (SodA), an integral membrane protein (DoxX), and a predicted thiol-oxidoreductase (SseA). Consistent with that, SodA, DoxX, and SseA form a membrane-associated oxidoreductase complex (MRC) that physically links radical detoxification with cytosolic thiol homeostasis. Loss of any MRC component correlated with defective recycling of mycothiol and accumulation of cellular oxidative damage. This previously uncharacterized coordination between oxygen radical detoxification and thiol homeostasis is required to overcome the oxidative environment Mtb encounters in the host.