Harim Won

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in… Show more

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

Authors: Harim I. Won, Samuel Zinga, Olga Kandror, Tatos Akopian, Ian D. Wolf, Jessica T. P. Schweber, Ernst W. Schmid, Michael C. Chao, Maya Waldor, Eric J. Rubin & Junhao Zhu Show less

Although prokaryotic organisms lack traditional organelles, they must still organize cellular structures in space and time, challenges that different species solve differently. To systematically define the subcellular architecture of mycobacteria, we perform high-throughput imaging of a library of fluorescently tagged proteins expressed in Mycobacterium smegmatis and develop a customized computational pipeline, MOMIA and GEMATRIA, to analyze these data. Our results establish a spatial… Show more

Although prokaryotic organisms lack traditional organelles, they must still organize cellular structures in space and time, challenges that different species solve differently. To systematically define the subcellular architecture of mycobacteria, we perform high-throughput imaging of a library of fluorescently tagged proteins expressed in Mycobacterium smegmatis and develop a customized computational pipeline, MOMIA and GEMATRIA, to analyze these data. Our results establish a spatial organization network of over 700 conserved mycobacterial proteins and reveal a coherent localization pattern for many proteins of known function, including those in translation, energy metabolism, cell growth and division, as well as proteins of unknown function. Furthermore, our pipeline exploits morphologic proxies to enable a pseudo-temporal approximation of protein localization and identifies previously uncharacterized cell-cycle-dependent dynamics of essential mycobacterial proteins. Collectively, these data provide a systems perspective on the subcellular organization of mycobacteria and provide tools for the analysis of bacteria with non-standard growth characteristics.

Authors: Junhao Zhu, Ian D. Wolf, Charles L. Dulberger, Harim I. Won, Jemila C. Kester, Julius A. Judd, Samantha E. Wirth, Ryan R. Clark, Yawei Li, Yuan Luo, Todd A. Gray, Joseph T. Wade, Keith M. Derbyshire, Sarah M. Fortune, Eric J. Rubin Show less

Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux towards acetate fermentation, a phenomenon known as overflow metabolism or the bacterial ‘Crabtree effect’. Numerous studies of the major acetate-generating pathway, the Pta-AckA, revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to… Show more

Under conditions of glucose excess, aerobically growing bacteria predominantly direct carbon flux towards acetate fermentation, a phenomenon known as overflow metabolism or the bacterial ‘Crabtree effect’. Numerous studies of the major acetate-generating pathway, the Pta-AckA, revealed its important role in bacterial fitness through the control of central metabolism to sustain balanced growth and cellular homeostasis. In this work, we highlight the contribution of the Pta-AckA pathway to fitness of the spore-forming bacterium, Bacillus anthracis. We demonstrate that disruption of the Pta-AckA pathway causes a drastic growth reduction in the mutants and alters the metabolic and energy status of the cells. Our results revealed that inactivation of the Pta-AckA pathway increases the glucose consumption rate, affects intracellular ATP, NAD+ and NADH levels and leads to a metabolic block at the pyruvate and acetyl-CoA nodes. Consequently, accumulation of intracellular acetyl-CoA and pyruvate forces bacteria to direct carbon into the TCA and/or glyoxylate cycles as well as fatty acid and poly(3‐hydroxybutyrate) (PHB) biosynthesis pathways. Notably, the presence of phosphate butyryltransferase in B. anthracis partially compensates for the loss of phosphotransacetylase activity. Furthermore, overexpression of the ptb gene not only eliminates the negative impact of the pta mutation on B. anthracis fitness, but also restores normal growth in the pta mutant of the non-butyrate-producing bacterium, Staphylococcus aureus. Taken together, the results of this study demonstrate the importance of the Pta-AckA pathway for B. anthracis fitness by revealing its critical contribution to the maintenance of metabolic homeostasis during aerobic growth under conditions of carbon overflow. Show less

Toxoplasma gondii is a globally distributed apicomplexan parasite and the causative agent of toxoplasmosis in humans. While pharmaceuticals exist to combat acute infection, they can produce serious adverse reactions, demonstrating a need for enhanced therapies. KG8 is a benzoquinone acyl hydrazone chemotype identified from a previous chemical screen for which we previously showed in vitro and in vivo efficacy against T. gondii. However, the genetic target and mechanism of action of KG8 remain… Show more

Toxoplasma gondii is a globally distributed apicomplexan parasite and the causative agent of toxoplasmosis in humans. While pharmaceuticals exist to combat acute infection, they can produce serious adverse reactions, demonstrating a need for enhanced therapies. KG8 is a benzoquinone acyl hydrazone chemotype identified from a previous chemical screen for which we previously showed in vitro and in vivo efficacy against T. gondii. However, the genetic target and mechanism of action of KG8 remain unknown. To investigate potential targets, we generated resistant T. gondii lines by chemical mutagenesis followed by in vitro selection. Whole-genome sequencing of resistant clones revealed a P207S mutation in the gene encoding rhoptry organelle protein 1 (ROP1) in addition to two lesser resistance-conferring mutations in the genes for rhoptry organelle protein 8 (ROP8) and a putative ADP/ATP carrier protein (TGGT1_237700). Expressing ROP1P207S in parental parasites was sufficient to confer significant (10.3-fold increased half-maximal effective concentration [EC50]) KG8 resistance. After generating a library of mutants carrying hypermutated rop1 alleles followed by KG8 pressure, we sequenced the most resistant clonal isolate (>16.9-fold increased EC50) and found independent recapitulation of the P207S mutation, along with three additional mutations in the same region. We also demonstrate that a rop1 knockout strain is insensitive to KG8. These data implicate ROP1 as a putative resistance gene of KG8. This work further identifies a compound that can be used in future studies to better understand ROP1 function and highlights this novel chemotype as a potential scaffold for the development of improved T. gondii therapeutics. Show less

Burying beetles (Nicrophorus spp.) are among the relatively few insects that provide parental care while not belonging to the eusocial insects such as ants or bees. This behavior incurs energy costs as evidenced by immune deficits and shorter life-spans in reproducing beetles. In the absence of an assembled transcriptome, relatively little is known concerning the molecular biology of these beetles. This work details the assembly and analysis of the Nicrophorus orbicollis transcriptome at… Show more

Burying beetles (Nicrophorus spp.) are among the relatively few insects that provide parental care while not belonging to the eusocial insects such as ants or bees. This behavior incurs energy costs as evidenced by immune deficits and shorter life-spans in reproducing beetles. In the absence of an assembled transcriptome, relatively little is known concerning the molecular biology of these beetles. This work details the assembly and analysis of the Nicrophorus orbicollis transcriptome at multiple developmental stages. RNA-Seq reads were obtained by next-generation sequencing and the transcriptome was assembled using the Trinity assembler. Validation of the assembly was performed by functional characterization using Gene Ontology (GO), Eukaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Differential expression analysis highlights developmental stage-specific expression patterns, and immunity-related transcripts are discussed. The data presented provides a valuable molecular resource to aid further investigation into immunocompetence throughout this organism's sexual development. Show less

Additional Authors: Nicolas Gouin, Alan S. Kolok, Paul H. Davis
Trichomycterus areolatus is an endemic species of pencil catfish that inhabits the riffles and rapids of many freshwater ecosystems of Chile. Despite its unique adaptation to Chile's high gradient watersheds and therefore potential application in the investigation of ecosystem integrity and environmental contamination, relatively little is known regarding the molecular biology of this environmental sentinel. Here, we detail the… Show more

Additional Authors: Nicolas Gouin, Alan S. Kolok, Paul H. Davis
Trichomycterus areolatus is an endemic species of pencil catfish that inhabits the riffles and rapids of many freshwater ecosystems of Chile. Despite its unique adaptation to Chile's high gradient watersheds and therefore potential application in the investigation of ecosystem integrity and environmental contamination, relatively little is known regarding the molecular biology of this environmental sentinel. Here, we detail the assembly of the Trichomycterus areolatus transcriptome, a molecular resource for the study of this organism and its molecular response to the environment. RNA-Seq reads were obtained by next-generation sequencing with an Illumina® platform and processed using PRINSEQ. The transcriptome assembly was performed using TRINITY assembler. Transcriptome validation was performed by functional characterization with KOG, KEGG, and GO analyses. Additionally, differential expression analysis highlights sex-specific expression patterns, and a list of endocrine and oxidative stress related transcripts are included. Show less