Biology subjects

Morishige, Y.

Publications and source records attributed to Morishige, Y..

6 recordsLinked to original sources

bioRxiv2026

Transposon insertion sequencing identifies genetic determinants of intrinsic rifamycin resistance in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to many antimicrobial agents, including rifampicin, a frontline anti-tuberculosis drug, severely limiting treatment options. Here, we used transposon insertion sequencing (Tn-Seq) to perform a genomewide screen to identify genes required for intrinsic rifampicin resistance in M. abscessus. We uncovered candidate genes that confer intrinsic resistance to the rifamycin-class antibiotics, rifampicin and rifabutin. The genes we identified included previously reported genes such as arr, helR, and MAB_2807. By comparing our results with the rifampicin intrinsic resistance gene in Mycobacterium tuberculosis, we found that the mechanisms underlying rifampicin intrinsic resistance were distinct between the two species. The contribution of seven representative candidate genes to rifampicin resistance was confirmed by characterizing targeted gene deletion or transposon insertion mutants. Among these determinants, MAB_2807 was identified as a major efflux-based contributor to rifamycin resistance, and its disruption increased intracellular rifampicin accumulation. In addition to known resistance determinants, Tn-Seq revealed contributions from many genes involved in cell envelope processes to rifamycin resistance. Guided by this genetic signature, we evaluated the combined effects of cell wall-targeting antimicrobial agents with rifamycins. We found that rifamycins displayed selective synergistic interactions with specific {beta}-lactam antibiotics. Interestingly, we found that rifamycin exposure altered cell envelope ultrastructure and increased the accumulation of the peptidoglycan precursor UDP-N-acetylglucosamine, suggesting that rifampicin perturbs envelope-associated metabolic homeostasis. These findings highlight rifamycin-induced vulnerable cellular processes that may inform rational combination strategies. ImportanceRifamycins are cornerstone antibiotics for the treatment of mycobacterial infections, yet they are ineffective against Mycobacterium abscessus. Although several key resistance mechanisms have been described, the full genetic basis of intrinsic rifamycin resistance remains to be elucidated. Here, we used transposon insertion sequencing to systematically identify genes that contribute to intrinsic rifamycin resistance in M. abscessus. Because recent studies have shown that chemical modification of rifamycins can overcome specific intrinsic resistance mechanisms, a comprehensive understanding of these determinants could facilitate the development of next-generation rifamycins with improved activity against M. abscessus. In addition, we unexpectedly found that rifamycin exposure alters cell envelope structure, the intracellular levels of a key metabolic intermediate in peptidoglycan biosynthesis, and susceptibility to certain {beta}-lactams in M. abscessus. These findings may help guide the rational development of combination therapeutic strategies with next-generation rifamycins.

microbiology↗

bioRxiv2025

Concentration-dependent suppression of macrolide resistance in Mycobacterium avium by combination therapy: an improved in vitro time-kill assay and PK/PD modelling study

ObjectivesMacrolides are key drugs in the treatment for Mycobacterium avium pulmonary disease, and combination chemotherapy is essential to preventing macrolide resistance. However, the concentrations of concomitant agents required to suppress resistance emergence remain undefined. This study aimed to quantify the concentration-dependent effects of companion drugs on macrolide resistance using an improved time-kill assay and pharmacokinetic/pharmacodynamic (PK/PD) modelling approach. MethodsTime-kill assays were performed on M. avium ATCC 700898 using azithromycin alone or in combination with ethambutol, rifampicin, amikacin, or clofazimine. Total and resistant bacterial populations were quantified over 28 days. A pharmacodynamic (PD) model describing the dynamics of susceptible and resistant subpopulations was linked to a pharmacokinetic (PK) model incorporating alveolar macrophage concentrations to simulate resistance and bactericidal rates under various dosing regimens. ResultsMacrolide resistance most frequently emerged at 2-4x the minimum inhibitory concentration (MIC) of azithromycin, whereas resistance was less frequent at 8-16x MIC or sub-MIC levels. The addition of companion drugs suppressed resistance emergence even at sub-MIC levels. PK/PD simulations demonstrated that standard-dose ethambutol effectively prevented macrolide resistance, whereas rifampicin provided minimal protection. More frequent dosing schedules enhanced resistance suppression compared with once-daily regimens. ConclusionThis study defines the concentration-dependent contributions of concomitant agents to macrolide resistance suppression in M. avium and establishes a quantitative PK/PD framework for evaluating resistance emergence. These findings provide mechanistic insights to support the optimization of combination dosing strategies for M. avium pulmonary disease.

pharmacology and toxicology↗

bioRxiv2024

Functional genomics reveals the mechanism of hypoxic adaptation in nontuberculous mycobacteria

Mycobacterium intracellulare is a major etiological agent of the recently expanding Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD). Therapeutic regimens that include a combination of macrolides and antituberculous drugs have been implemented with limited success. To identify novel targets for drug development that accommodate the genomic diversity of M. avium-intracellulare, we subjected eight clinical MAC-PD isolates and the type strain ATCC13950 to genome-wide profiling to comprehensively identify universally essential functions by transposon sequencing (TnSeq). Among these strains, we identified 131 shared essential or growth-defect-associated genes by TnSeq. Unlike the type strain, the clinical strains showed increased requirements for genes involved in gluconeogenesis and the type VII secretion system under standard growth conditions, the same genes required for hypoxic pellicle-type biofilm formation in ATCC13950. Consistent with the central role of hypoxia in the evolution of M. intracellulare, the clinical MAC-PD strains showed more rapid adaptation to hypoxic growth than the type strain. Importantly, the increased requirements of hypoxic fitness genes were confirmed in a mouse lung infection model. These findings confirm the concordant genetic requirements under hypoxic conditions in vitro and hypoxia-related conditions in vivo, and highlight the importance of using clinical strains and host-relevant growth conditions to identify high-value targets for drug development.

microbiology↗

bioRxiv2022

Visual Perception of 3D Space and Shape in Time - Part III: 2D Shape Recognition by Log-Scaling

Human vision has a remarkable ability to recognize complex 3D objects such as faces that appear with any size and 3D orientations at any 3D location. If we initially memorize a face only with a normalized size and viewed from directly head on, the direct comparison between the one-sized memory and a new incoming image would demand tremendous mental frame translations in 7D. How can we perform such a demanding task so promptly and reliably as we experience the objects in the world around us? Intriguingly, our primary visual cortex exhibits a 2D retinotopy with a log-polar coordinate system, where scaling up/down of shape is converted to linear frame translation. As a result, mental scaling can be performed by linearly translating the memory or the perceptual image until they overlap with each other. According to our new model of NHT (Neural Holography Tomography), alpha brainwaves traveling at a constant speed can conduct this linear translation. With this scheme, every scaling up/down by a factor of two should take the same amount of extra mental time to recognize a smaller/larger face. To test this hypothesis, we designed a reaction time (RT) experiment, where participants were first asked to memorize sets of unfamiliar faces with a given specific size (4{degrees} or 8{degrees}). Following the memorization phase, similar stimuli with a wide range of sizes (from 1{degrees} to 32{degrees}) were presented, and RTs were recorded. As predicted, the increase in RT was proportional to the scaling factor in the log scale. Furthermore, we observed that RTs were fastest for 8{degrees} faces even if the memorized face was 4{degrees}. This supports our hypothesis that we always memorize faces at the exact size of ~8 {degrees}. To our surprise, the increases in RT were also consistent with the mentally-estimated depth sensation, which indicates that the apparent size of the recognized face can create a proper depth sensation.

neuroscience↗

bioRxiv2021

Bovine serum albumin as a resuscitation promoting factor for viable but non-culturable Mycobacterium tuberculosis via the activation of protein kinase A-dependent cellular processes

We investigated the mechanisms underlying the fetal bovine serum (FBS)-induced reactivation of the viable but non-culturable (VBNC) state of Mycobacterium tuberculosis (Mtb) using the H37Rv strain. We found that bovine serum albumin (BSA), a major component of FBS, could reactivate the VBNC state of Mtb cells without other reactivation-promoting agents. Human serum albumin also had a restorative effect similar to BSA, but mouse serum albumin and egg-white albumin (ovalbumin) did not, suggesting that a common protein structure between bovine and human serum albumin is essential for reactivation. In addition, antioxidative agents, such as N-acetyl-L-cysteine, showed no restorative effect, suggesting that the restoration of culturability might not be due to the antioxidative property of BSA. BSA-mediated reactivation is inhibited by H89 and staurosporine. These inhibitors are known to inhibit multiple protein kinases, including serine/threonine protein kinases, in Mtb, suggesting the involvement of mycobacterial protein kinases in cell division during reactivation. These findings provide new insights into the mechanisms of Mtb reactivation, and may contribute to the development of novel strategies for the treatment of latent tuberculosis. ImportanceReactivation of dormant, including viable but non-culturable (VBNC) Mycobacterium tuberculosis (Mtb) cells, represents a critical step in the progression from latent infection to active tuberculosis. However, the molecular mechanisms that trigger this transition are not yet fully understood. We herein report that bovine serum albumin (BSA), a major component of fetal bovine serum, can restore the culturability of VBNC Mtb cells. Human serum albumin showed a similar effect, whereas mouse and egg-white albumins did not, suggesting that the specific structural features shared between bovine and human albumins are essential for reactivation. The lack of reactivation by the antioxidant N-acetyl-L-cysteine indicates that the mechanism is not related to redox balance. Inhibition of BSA-induced reactivation by H89 and staurosporine implicates mycobacterial serine/threonine protein kinases in this process. These findings highlight the previously unrecognized role of albumin-kinase signaling in Mtb reactivation and suggest new molecular targets for preventing tuberculosis relapse.

microbiology↗

bioRxiv2018

Six species of non-tuberculosis mycobacteria carry non-identical 16S rRNA gene copies

Non-tuberculosis mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze the 16S rRNA gene sequences of six NTM species, Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. The approach facilitated the identification of two distinct gene copies in each species. The two M. cosmeticum genes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified in M. hodleri, M. flavescens, and M. xenopi. M. pallens had a difference in four nucleotides and M. crocinum in 23. Hence, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies.\n\nImportanceThe presence of multiple 16S rRNA gene copies with nucleotide polymorphisms represents a challenge for species identification using 16S rRNA as the target sequence. Our analysis was focused on six NTM species, M. cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. As a result, we generated the full-length sequences of two non-identical 16S rRNA copies for each NTM species. The data will be helpful for the sequence analysis of specimens or other samples.

bioinformatics↗