Carolina Trujillo

University of the Mexican Valley · Docencia

Significance Efforts to improve tuberculosis therapy include optimizing multidrug regimens to take advantage of drug–drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed libra...

Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene “vulnerabil...

Studying latent Mycobacterium tuberculosis (Mtb) infection has been limited by the lack of a suitable mouse model. We discovered that transient depletion of biotin protein ligase (BPL) and thioredoxin reductase (TrxB2) results in latent infections during which Mtb cannot be detected but that relapse in a subset of mice. The immune requirements for...

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 drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundl...

Studying latent Mycobacterium tuberculosis (Mtb) infection has been limited by the lack of a suitable mouse model. We discovered that transient depletion of protein biotin ligase (BPL) and thioredoxin reductase (TrxB2) results in latent infections during which Mtb cannot be detected but that relapse in a subset of mice. The immune requirements for...

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline responsive TetR- tetO unit. To minimize fluctuation...

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 whi...

Tuberculosis is a massive global burden and Mycobacterium tuberculosis is increasingly resistant to first- and second-line drugs. There is an acute need for new anti-mycobacterial drugs with novel targets. We previously evaluated a series of 2-aminothiazoles with activity against Mycobacterium tuberculosis. In this study, we identify the glycolytic...

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds ag...

Significance A better understanding of essential processes in Mycobacterium tuberculosis ( Mtb ) is required for the development of new chemotherapeutics. Isocitrate lyase (ICL) and malate synthase (MS) function in the glyoxylate shunt, a pathway required by Mtb to metabolize fatty acids (FAs). Here, we demonstrate that Mtb MS enables growth and su...

Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon...

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and gluco...

Generation of M. tuberculosis otsB2 gene deletion mutants. (A) Organization of the otsB2 locus in M. tuberculosis wild-type as well as in a marked otsB2 gene deletion mutant. The sizes of relevant fragments as well as the location of the probe used for Southern analyses are indicated. WT, wild-type; (u), unmarked locus; γδres, res-sites of the γδ-r...

Strains of M. tuberculosis H37Rv used in this study. Mutants were generated by allelic exchange employing specialized transduction as described in S1 Text. Abbreviations: WT, wild-type; Kanr, kanamycin resistant; Hygr, hygromycin resistant; Apra, apramycin resistant; u, unmarked mutant. (PDF)

Generation of M. tuberculosis c-otsB2-4×tetO gene knock-in mutants. (A) Organization of the otsB2 locus in M. tuberculosis wild-type as well as in a c-otsB2-4×tetO gene knock-in mutant. The sizes of relevant fragments as well as the location of the probe used for Southern analyses are indicated. WT, wild-type; (u), unmarked locus; hyg, hygromycin r...

Generation of a marked and unmarked M. tuberculosis otsA gene deletion mutants. (A) Organization of the otsA locus in M. tuberculosis wild-type as well as in a marked and unmarked otsA gene deletion mutant. The sizes of relevant fragments as well as the location of the probe used for Southern analyses are indicated. WT, wild-type; (u), unmarked loc...

Exogenous T6P has no toxic effect on M. tuberculosis cells. (A) WT cells were grown in liquid medium containing increasing concentrations of T6P, revealing no growth inhibitory effect. (B) Cells of the conditional M. tuberculosis c-otsB2-tet-on mutant were cultivated in liquid medium containing increasing concentrations of ATc either in presence or...

Arginine supplementation does not rescue growth of the conditional M. tuberculosis c-otsB2-tet-on mutant under silencing conditions. Cells of the conditional M. tuberculosis c-otsB2-tet-on mutant were cultivated in liquid medium containing increasing concentrations of ATc either in presence or absence of 1 mM arginine. ATc-dependent growth was not...

Anhydrotetracycline- (ATc-) dependent growth of the conditional M. tuberculosis ΔpanCD c-otsB2-tet-on mutant. Growth of three independent clones was measured using the resazurin microplate assay. (TIF)

Differentially essential genes in the M. tuberculosis ΔotsA mutant compared to wild-type. In order to identify those genes in the M. tuberculosis ΔotsA mutant background whose inactivation cannot be rescued by supplementation with trehalose, i.e. those genes that are essential in context of otsA deletion both in absence and presence of trehalose bu...

Differential gene expression of the DNA-damage-responsive genes in induced and partially silenced cells of the conditional M. tuberculosis c-otsB2-tet-on mutant. Cells were cultivated either in presence of 200 ng/ml (100% growth relative to WT) or 30 ng/ml ATc (ca. 30% residual growth relative to WT) for 7 days. rRNA-depleted samples were analyzed...

Most abundant transcripts in induced and partially silenced cells of the conditional M. tuberculosis c-otsB2-tet-on mutant. Cells were cultivated in presence of 200 ng/ml (100% growth relative to WT) or 30 ng/ml ATc (ca. 30% residual growth relative to WT) for 7 days. rRNA-depleted samples were analyzed by RNAseq. RPKM, reads per kilobase of transc...

Drug susceptibility of induced and partially silenced cells of the conditional M. tuberculosis c-otsB2-tet-on mutant. Cells of the conditional M. tuberculosis c-otsB2-tet-on mutant were either induced in presence of 200 ng/ml or partially silenced in presence of 30 ng/ml ATc and incubated with the indicated concentrations of either rifampicin, ison...

Independent silencing experiment to reproduce the bacteriostatic effect of otsB2 silencing during the acute infection phase in mice. Mice were infected with the conditional M. tuberculosis c-otsB2-tet-on mutant via the aerosol route. Mice received doxycycline via the mouse chow to induce otsB2 in the conditional M. tuberculosis c-otsB2-tet-on mutan...

Supplementary Material and Methods. (PDF)

Oligonucleotides used for generation of allelic exchange substrates. Resulting phages listed here were used for generation of gene deletion or knock-in mutants of M. tuberculosis H37Rv listed in S3 Table by specialized transduction as described in S1 Text. (PDF)

Oligonucleotides used for qRT-PCR of M. tuberculosis transcripts. (PDF)

RNAseq analysis of the stress response elicited by trehalose-6-phosphate accumulation. (XLSX)

Mycobacterium tuberculosis relies on its own ability to biosynthesise coenzyme A in order to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optim...

Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb’s thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis...

Susceptibility of partially TrxB2-depleted Mtb to oxidative stress. (A) Immunoblot analysis of TrxB2 in protein extracts prepared from cultures used in (B). (B) TrxB2-TetON-tetO-1C mutant was cultured in the absence of atc for 3 days to decrease TrxB2 expression. Mtb strains were then exposed to 0.25 mM plumbagin for 5 h, to 5.4 mM H2O2 for 4 h or...

Semiquantitative immunoblot analysis of TrxB2 in TrxB2-DUC. Immunoblot of protein extracts from TrxB2-DUC treated with atc for 6, 24 and 48 hrs. Serially diluted H37Rv lysate was used to determine the limit of detection of TrxB2. Eno serves as loading control. (PDF)

TrxB2 depletion in Mtb causes cell elongation. Representative images of H37Rv and TrxB2-DUC treated or not with atc for 4 days. Samples were examined with bright-field microscopy. Data processing was performed with ImageJ. (PDF)

DTT treatment does not affect atc-mediated TrxB2 depletion in TrxB2-DUC. Immunoblot of protein extracts from TrxB2-DUC with different treatment as indicated. Blot was probed with TrxB2-specific and Eno-specific (loading control) antisera. (PDF)

Model for the activities of thioredoxin reductase in Mtb. Mtb’s thioredoxin system is composed of thioredoxin reductase (encoded by trxB2), thioredoxin (Trx) and NADPH. TrxB2 catalyzes the disulfide-thiol exchange of thioredoxins and thioredoxin-like proteins using electrons from NADPH. Thioredoxins and thioredoxin-like proteins are then able to re...

Construction of TrxB2-DUC. To generate the dual control (DUC) mutant, the trxB2-trxC plasmid located in the attL5 site of ΔtrxB2::P750-trxB2-trxC was replaced with a plasmid containing DAS-tagged trxB2 expressed from the tet-operator containing promoter P750, trxC with its native promoter, and reverse tet repressor with a constitutive promoter. In...

NadE depletion does not cause detectable lysis of Mtb. Immunoblot analysis of DlaT, Eno, and Ag85B from culture filtrates of H37Rv, TrxB2-DUC and NadE-DUC treated or not with atc for 6 days. (PDF)

DTT mitigates the transcriptional impact of TrxB2 depletion. Heat-map representation of expression level of fold-changes of selected genes in response to atc and DTT treatment. mRNA abundances in TrxB2-DUC treated with atc, DTT or both were compared to those in untreated TrxB2-DUC. One-way ANOVA was used for group comparison (n = 3 per group), with...

TrxB2 is required for growth of Mtb. (A) Map of the trxB2-trxC genomic region in H37Rv and ΔtrxB2::P750-trxB2-trxC. To construct ΔtrxB2::P750-trxB2-trxC, we first generated a merodiploid strain by integrating a second copy of the trxB2-trxC operon into the attL5 site. Then trxB2 and the first 4 bps of trxC, which overlap with trxB2, were replaced w...

Silencing TrxB2 during the acute phase of infection prevented pulmonary pathology in mice. Gross pathology (A) and H&E staining (B) of lung tissue sections from infected mice receiving doxy-containing food starting from day 0, day 10 or not treated. Lungs were isolated on day 35 and 56 post-infection. Scale bar, 1.0 mm. (PDF)

Enzymes in the pantothenate and coenzyme A (CoA) biosynthesis pathways have attracted considerable interest as potential targets for the development of drugs against a number of human pathogens, including M. tuberculosis . While potent inhibitors have been developed against M. tuberculosis pantothenate synthase and pantothenate kinase, these have f...

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. De...

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. De...

Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mut...

Significance The mechanisms that enable Mycobacterium tuberculosis , the causative agent of tuberculosis, to resist drug treatment and survive the immune response are poorly understood. In this study we discovered that M. tuberculosis produces the protein channel protein with necrosis-inducing toxin (CpnT), which forms a channel in the outer membra...

Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of gluc...

Quantification of ppgK and glkA (Rv0650) transcript levels in wt Mtb and mutants. Absolute mRNA amounts were determined by quantitative real time PCR. Serial dilutions of defined copy numbers of the Mtb chromosome were included for each real-time PCR to generate standard curves, which were used to calculate the absolute copy number of each gene. Da...

Comparison of Mtb's glucokinases, GLKA and PPGK. (A) Amino acid sequence alignment of PPGK and GLKA. The overall sequence identity is 22%. (B) Schematic representation of PPGK and GLKA. Residues involved in glucose binding are indicated in red with corresponding residue number shown below. Grey boxes represent ATP binding domains and the catalytic...

KatG and SodA amounts in cell extracts. KatG and SodA were detected by immunoblotting in lysates from wt, ΔppgK, ΔglkA and ΔppgKΔglkA. (TIFF)

Sequence of the qTags used in this study. Sequences that bind or correspond to the primers and probes used for real-timer PCR are shown in red, blue, and brown. (TIF)

Bacterial strains and plasmids. (DOC)

TaqMan probes and primers. (DOC)

Mycobacterium tuberculosis (Mtb) represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags) and their use...