Biology subjects

Tsunemoto, H.

Publications and source records attributed to Tsunemoto, H..

6 recordsLinked to original sources

bioRxiv2021

Machine Learning of Pseudomonas aeruginosa transcriptomes identifies independently modulated sets of genes associated with known transcriptional regulators

The transcriptional regulatory network (TRN) of Pseudomonas aeruginosa plays a critical role in coordinating numerous cellular processes. We extracted and quality controlled all publicly available RNA-sequencing datasets for P. aeruginosa to find 281 high-quality transcriptomes. We produced 83 new RNAseq data sets under critical conditions to generate a comprehensive compendium of 364 transcriptomes. We used this compendium to reconstruct the TRN of P. aeruginosa using independent component analysis (ICA). We identified 104 independently modulated sets of genes (called iModulons), among which 81 (78%) reflect the effects of known transcriptional regulators. We show that iModulons: 1) play an important role in defining the genomic boundaries of biosynthetic gene clusters (BGCs); 2) show increased expression of the BGCs and associated secretion systems in conditions that emulate cystic fibrosis (CF); 3) show the presence of a novel BGC named RiPP (bacteriocin producer) which might have a role in worsening CF outcomes; 4) exhibit the interplay of amino acid metabolism regulation and central metabolism across carbon sources, and 5) clustered according to their activity changes to define iron and sulfur stimulons. Finally, we compare the iModulons of P. aeruginosa with those of E. coli to observe conserved regulons across two gram negative species. This comprehensive TRN framework covers almost every aspect of the transcriptional regulatory machinery in P. aeruginosa, and thus could prove foundational for future research of its physiological functions.

bioinformatics↗

bioRxiv2021

Bacterial Cytological Profiling Identifies Rhodanine-containing PAINS Analogs as Specific Inhibitors of E. coli Thymidylate Kinase in Vivo

In this study, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules inhibiting the growth of E. coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite their structural similarity to related PAINS. A genetic analysis of resistant mutants revealed that thymidylate kinase (an enzyme essential for DNA synthesis) is an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by assays of in vitro activity as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the lowest IC50 in vitro and MIC in vivo displayed the greatest specificity for the inhibition of DNA replication in E. coli, despite containing a rhodamine moiety. While its generally thought that PAINS cannot be developed as antibiotics, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics, and it showcases novel inhibitors of E. coli thymidylate kinase. ImportanceWe demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotics in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and non-specific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

microbiology↗

bioRxiv2020

Characterizing the response of Acinetobacter baumannii ATCC 17978 to azithromycin in multiple in vitro growth conditions

Multi-drug resistant (MDR) Acinetobacter baumannii is one of the most concerning pathogens in hospital infections. A. baumannii is categorized as an "Urgent Threat" by the U.S. Centers for Disease Control and the highest priority pathogen by the World Health Organization due to its propensity for broad antibiotic resistance and its associated high mortality rates. New treatment options are urgently needed for MDR A. baumannii infections. Our prior studies have demonstrated an unappreciated utility of the macrolide azithromycin (AZM) against MDR A. baumannii in tissue-culture medium. This finding is all the more surprising since AZM has no appreciable activity against A. baumannii in standard bacteriological media. The basis for this media-dependent activity of AZM against A. baumannii is not fully defined. In this study, we utilize a variety of techniques (growth dynamics, bacterial cytological profiling, RNA sequencing, and LC/MS) to profile the response of MDR A. baumannii to AZM in both standard bacteriological and more physiological relevant mammalian tissue-culture medium. [Table 1]

microbiology↗

bioRxiv2020

Identifying the effect of vancomycin on HA-MRSA strains using bacteriological and physiological media

Healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) USA100 strains are of major concern due to their evolving antibiotic resistant. They are resistant to a broad class of antibiotics like macrolides, aminoglycosides, fluoroquinolones, and many more. The selection of appropriate antibiotic susceptibility examination media is very important. Thus, we use bacteriological (CA-MHB) as well as physiological (R10LB) media to determine the effect of vancomycin on USA100 strains. The study includes the profiling behaviour of HA-MRSA USA100 D592 and D712 strains in the presence of vancomycin through various high-throughput assays. The US100 D592 and D712 strains were characterized at sub-inhibitory concentrations through growth curves, RNA sequencing, bacterial cytological profiling, and exo-metabolomics high throughput experiments. The study reveals the vancomycin resistance behavior of USA100 strains in dual media conditions using wide-ranging experiments.

microbiology↗

bioRxiv2020

Profiling the effect of nafcillin on HA-MRSA D592 using bacteriological and physiological media

Staphylococcus aureus is a leading human pathogen associated with both hospital-acquired and community-acquired infections. The bacterium has steadily gained resistance to {beta}-lactams and other important first-line antibiotics culminating in its categorization as an urgent threat by the U.S. Centers for Disease Control and Prevention. Observations of a varying response to antimicrobial exposure as a function of media type has revealed that clinical susceptibility testing performed in standard bacteriological media might not adequately represent pharmacological responses in the patient. Such observations have encouraged research designed to identify media types that more closely mimic the in vivo environment. In this study, we examine the response of a hospital-acquired USA100 lineage methicillin-resistant, vancomycin-intermediate S. aureus (MRSA/VISA) strain (D592) to nafcillin in a bacteriological compared to a more physiological tissue culture-based medium. We performed multi-dimensional analysis including growth and bacterial cytological profiling, RNA sequencing, and exo-metabolomics measurements (both HPLC and LC/MS) to shed light on the media-dependent activity of the commonly prescribed {beta}-lactam antibiotic nafcillin. O_TBL View this table: org.highwire.dtl.DTLVardef@85b95borg.highwire.dtl.DTLVardef@14c1504org.highwire.dtl.DTLVardef@1f7e976org.highwire.dtl.DTLVardef@100598dorg.highwire.dtl.DTLVardef@1f770e9_HPS_FORMAT_FIGEXP M_TBL C_TBL

microbiology↗

bioRxiv2020

Revealing 29 sets of independently modulated genes in Staphylococcus aureus, their regulators and role in key physiological responses

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its Transcriptional Regulatory Network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying Independent Component Analysis (ICA) to a compendium of 108 RNAseq expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed (1) high confidence associations between 21 i-modulons and known regulators; (2) an association between an i-modulon and {sigma}S, whose regulatory role was previously undefined; (3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR and Vim-3, (4) the roles of three key transcription factors (codY, Fur and ccpA) in coordinating the metabolic and regulatory networks; and (5) a low dimensional representation, involving the function of few transcription factors, of changes in gene expression between two laboratory media (RPMI, CAMHB) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation. Significance StatementStaphylococcus aureus infections impose an immense burden on the healthcare system. To establish a successful infection in a hostile host environment, S. aureus must coordinate its gene expression to respond to a wide array of challenges. This balancing act is largely orchestrated by the Transcriptional Regulatory Network (TRN). Here, we present a model of 29 independently modulated sets of genes that form the basis for a segment of the TRN in clinical USA300 strains of S. aureus. Using this model, we demonstrate the concerted role of various cellular systems (e.g. metabolism, virulence and stress response) underlying key physiological responses, including response during blood infection.

microbiology↗