Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCE: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.

Multidimensional Family Therapy for Justice-Involved Young Adults with Substance Use Disorders.

The present study explored the acceptability, feasibility, fidelity, and outcomes of a young adult adaptation of multidimensional family therapy (MDFT), an evidence-based family treatment originally developed for adolescents. Participants included 22 individuals between the ages of 19 to 25 who were enrolled in a criminal drug court program. MDFT was found to be feasible and was delivered with strong fidelity to young adults and their families. Participants reported high satisfaction with MDFT, and 95% completed treatment. Analyses revealed statistically significant decreases in substance use on all indicators from baseline to the 6-month follow-up. Significant improvements were also noted in vocational functioning, including a 73% increase in full-time employment from baseline to 6-month follow-up. Criminal justice outcomes included a significant decrease in legal risk, and 86% of study participants had no rearrests from baseline through the 18-month follow-up period. The article concludes with recommendations for implementing family-based interventions with young adults, as well as future research directions in this important area.

Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism, physiology and MAIT cell recognition, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.

Extraction and Separation of Mycobacterial Proteins.

The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery, elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from both whole-cell lysate and spent culture media are included. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.

Methods for Proteomic Analyses of Mycobacteria.

The use of proteomic technologies to characterize and study the proteome of mycobacteria has provided important information in terms of function, diversity, protein-protein interactions, and host-pathogen interactions in Mycobacterium spp. There are many different mass spectrometry methodologies that can be applied to proteomics studies of mycobacteria and microorganisms in general. Sample processing and appropriate study design are critical to generating high-quality data regardless of the mass spectrometry method applied. Appropriate study design relies on statistical rigor and data curation using bioinformatics approaches that are widely applicable regardless of the organism or system studied. Sample processing, on the other hand, is often a niched process specific to the physiology of the organism or system under investigation. Therefore, in this chapter, we will provide protocols for processing mycobacterial protein samples for the specific application of Top-down and Bottom-up proteomic analyses.

Deciphering the molecular basis of mycobacteria and lipoglycan recognition by the C-type lectin Dectin-2.

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

The N-terminal peptide moiety of the Mycobacterium tuberculosis 19 kDa lipoprotein harbors RP105-agonistic properties.

Radioprotective 105 kDa (RP105, CD180) is a member of the Toll-like receptor (TLR) family that interacts with TLR2 and facilitates recognition of mature lipoproteins expressed by Mycobacterium tuberculosis and Mycobacterium bovis BCG. In this study, we used synthetic lipopeptide analogs of the M. tuberculosis 19 kDa lipoprotein to define structural characteristics that promote RP105-mediated host cell responses. A tripalmitoylated lipopeptide composed of the first 16 N-terminal amino acids of the M. tuberculosis 19 kDa lipoprotein induced RP105-dependent TNF and IL-6 production by macrophages. Di- and tripalmitoylated variants of this lipopeptide elicited an equivalent RP105-dependent response, indicating that while the lipid moiety is required for macrophage activation, it is not a determinant of RP105 dependency. Instead, substitution of two polar threonine residues at positions 7 and 8 with nonpolar alanine residues resulted in reduced RP105 dependency. These results strongly suggest that the amino acid composition of the M. tuberculosis 19 kDa lipoprotein, and likely other mycobacterial lipoproteins, is a key determinant of RP105 agonism.

Boosting BCG-primed responses with a subunit Apa vaccine during the waning phase improves immunity and imparts protection against Mycobacterium tuberculosis.

Heterologous prime-boosting has emerged as a powerful vaccination approach against tuberculosis. However, optimal timing to boost BCG-immunity using subunit vaccines remains unclear in clinical trials. Here, we followed the adhesin Apa-specific T-cell responses in BCG-primed mice and investigated its BCG-booster potential. The Apa-specific T-cell response peaked 32-52 weeks after parenteral or mucosal BCG-priming but waned significantly by 78 weeks. A subunit-Apa-boost during the contraction-phase of BCG-response had a greater effect on the magnitude and functional quality of specific cellular and humoral responses compared to a boost at the peak of BCG-response. The cellular response increased following mucosal BCG-prime-Apa-subunit-boost strategy compared to Apa-subunit-prime-BCG-boost approach. However, parenteral BCG-prime-Apa-subunit-boost by a homologous route was the most effective strategy in-terms of enhancing specific T-cell responses during waning in the lung and spleen. Two Apa-boosters markedly improved waning BCG-immunity and significantly reduced Mycobacterium tuberculosis burdens post-challenge. Our results highlight the challenges of optimization of prime-boost regimens in mice where BCG drives persistent immune-activation and suggest that boosting with a heterologous vaccine may be ideal once the specific persisting effector responses are contracted. Our results have important implications for design of prime-boost regimens against tuberculosis in humans.

RP105 Engages Phosphatidylinositol 3-Kinase p110δ To Facilitate the Trafficking and Secretion of Cytokines in Macrophages during Mycobacterial Infection.

Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105(-/-) macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105(-/-) macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105(-/-) macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA-mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105(-/-) macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105(-/-) macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton's tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria.

Fractionation and analysis of mycobacterial proteins.

The extraction and isolation of native bacterial proteins continue to be valuable technical pursuits in order to understand bacterial physiology, screen for virulence determinants, and describe antigens. In this chapter, methods for the manipulation of whole mycobacterial cells are described in detail. Specifically, the concentration of spent culture filtrate media is described in order to permit separation of soluble, secreted proteins; several discrete separation techniques, including precipitation of protein mixtures with ammonium sulfate and separation of proteins by hydrophobic chromatography are also provided. Similarly, the generation of whole cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol enriched proteins is described. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided, as well as a separation technique (isoelectric focusing) that can be applied to separate hydrophobic proteins. Lastly, two commonly used analytical techniques, in-gel digestion of proteins for LC-MS and analysis of intact proteins by MALDI-ToF MS, are provided for rapid analysis of discrete proteins within subcellular or chromatographic fractions. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins. In addition, a number of these methods may be applied to extract and analyze mycobacterial proteins from cell lines and host derived samples.

O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Assessment of vaccine testing at three laboratories using the guinea pig model of tuberculosis.

The guinea pig model of tuberculosis is used extensively in different locations to assess the efficacy of novel tuberculosis vaccines during pre-clinical development. Two key assays are used to measure protection against virulent challenge: a 30 day post-infection assessment of mycobacterial burden and long-term post-infection survival and pathology analysis. To determine the consistency and robustness of the guinea pig model for testing vaccines, a comparative assessment between three sites that are currently involved in testing tuberculosis vaccines from external providers was performed. Each site was asked to test two "subunit" type vaccines in their routine animal model as if testing vaccines from a provider. All sites performed a 30 day study, and one site also performed a long-term survival/pathology study. Despite some differences in experimental approach between the sites, such as the origin of the Mycobacterium tuberculosis strain and the type of aerosol exposure device used to infect the animals and the source of the guinea pigs, the data obtained between sites were consistent in regard to the ability of each "vaccine" tested to reduce the mycobacterial burden. The observations also showed that there was good concurrence between the results of short-term and long-term studies. This validation exercise means that efficacy data can be compared between sites.

Three protein cocktails mediate delayed-type hypersensitivity responses indistinguishable from that elicited by purified protein derivative in the guinea pig model of Mycobacterium tuberculosis infection.

Purified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosis infection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4(+)/CD8(+) T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4(+) and CD8(+) T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosis infection.

Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis.

Live Mycobacterium tuberculosis persists in macrophage phagosomes by interfering with phagolysosome biogenesis. Here, using four-dimensional microscopy and in vitro assays, we report the principal difference between phagosomes containing live and dead mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal constituents, is retained on phagosomes harboring dead mycobacteria but is continuously eliminated from phagosomes with live bacilli. We show that the exclusion of PI3P from live mycobacterial phagosomes can be only transiently reversed by Ca2+ fluxes, and that live M. tuberculosis secretes a lipid phosphatase, SapM, that hydrolyzes PI3P, inhibits phagosome-late endosome fusion in vitro, and contributes to inhibition of phagosomal maturation.

Behavioral differences among rainbow trout clonal lines.

Salmonid fish show considerable geographical variation in morphology, physiology, and behavior. Understanding the genetic mechanisms underlying this variation could be useful for enhancing aquaculture stocks, identifying unique populations for conservation, and determining the genetic factors underlying natural adaptation and domestication. As a first step toward the genetic dissection of salmonid behavioral diversity, we examined variation in behavior patterns among four clonal lines of rainbow trout ( Oncorhynchus mykiss ) derived from geographically diverse source populations with different domestication histories. Clonal lines were crossed with two outbred (i.e., not homozygous) females, and the resulting progenies were reared and tested under identical conditions. Clonal line had significant genetic effects on mean swim level, hiding, foraging, startle response, and aggression level. Multiple comparisons suggest that domestication history of the source populations had a strong influence on these behavior patterns. Progeny of two clonal lines derived from populations reared in captivity for over 100 years exhibited reductions in predator avoidance behavior patterns and increases in aggression compared to progeny of two clonal lines from more recently domesticated populations. These results will facilitate future investigation of the genetic factors underlying population variation in these behavior patterns influenced by domestication.