Chidi Akusobi

All Publications

• Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus. PLoS pathogens McGowen, K., Funck, T., Wang, X., Zinga, S., Wolf, I. D., Akusobi, C., Denkinger, C. M., Rubin, E. J., Sullivan, M. R. 2025; 21 (4): e1013027

  Abstract

  Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

  View details for DOI 10.1371/journal.ppat.1013027

  View details for PubMedID 40208857

  View details for PubMedCentralID PMC12017575

• Durlobactam in combination with β-lactams to combat Mycobacterium abscessus. Antimicrobial agents and chemotherapy Shin, E., Dousa, K. M., Taracila, M. A., Bethel, C. R., Nantongo, M., Nguyen, D. C., Akusobi, C., Kurz, S. G., Plummer, M. S., Daley, C. L., Holland, S. M., Rubin, E. J., Bulitta, J. B., Boom, W. H., Kreiswirth, B. N., Bonomo, R. A. 2025; 69 (2): e0117424

  Abstract

  Mycobacterium abscessus (Mab) presents significant clinical challenges. This study evaluated the synergistic effects of a β-lactam and β-lactamase inhibitor combination against Mab and explored the underlying mechanisms. Synergy was assessed through MIC tests and time-kill studies, and binding affinities of nine β-lactams and BLIs to eight target receptors (L,D-transpeptidases [LDT] 1-5, D,D-carboxypeptidase, penicillin-binding protein [PBP] B, and PBP-lipo) were assessed using mass spectrometry and kinetic studies. Thermal stability and morphological changes were determined. Imipenem demonstrated high binding affinity to LDTs and PBPs, with extremely low inhibition constants (Ki,app; ≤0.002 mg/L for LDT1-2, ≤0.6 mg/L for PBPs), while cephalosporins, sulopenem, tebipenem, and amoxicillin exhibited moderate to low binding affinity. Durlobactam inactivated BlaMab and LDT/PBPs more potently than avibactam. The Ki,apps of durlobactam for PBP B, PBP-lipo, and LDT2 were below clinically achievable unbound concentrations, while avibactam's Ki,app for LDT/PBPs exceeded the clinical concentrations. Single β-lactam treatments resulted in minimal killing (~1 log10 reduction). Although avibactam yielded no effect, combinations with avibactam showed a significant reduction (~4 log10 CFU/mL). Durlobactam alone showed ~2 log10 reduction, and when combined with imipenem or two β-lactams, durlobactam achieved near-eradication of Mab, surpassing the current therapy (amikacin + clarithromycin + imipenem/cefoxitin). Inactivation of PBP-lipo by sulopenem, imipenem, durlobactam, and amoxicillin (with avibactam) led to morphological changes, showing filaments. This study demonstrates the mechanistic basis of combinations therapy, particularly imipenem + durlobactam, in overcoming β-lactam resistance in Mab.

  View details for DOI 10.1128/aac.01174-24

  View details for PubMedID 39714147

  View details for PubMedCentralID PMC11823594

• Transposon-sequencing across multiple Mycobacterium abscessus isolates reveals significant functional genomic diversity among strains. mBio Akusobi, C., Choudhery, S., Benghomari, B. S., Wolf, I. D., Singhvi, S., Ioerger, T. R., Rubin, E. J. 2025; 16 (2): e0337624

  Abstract

  Mycobacterium abscessus (Mab) is a clinically significant pathogen and a highly genetically diverse species due to its large accessory genome. The functional consequence of this diversity remains unknown mainly because, to date, functional genomic studies in Mab have been primarily performed on reference strains. Given the growing public health threat of Mab infections, understanding the functional genomic differences among Mab clinical isolates can provide more insight into how its genetic diversity influences gene essentiality, clinically relevant phenotypes, and importantly, potential drug targets. To determine the functional genomic diversity among Mab strains, we conducted transposon-sequencing (TnSeq) on 21 genetically diverse clinical isolates, including 15 M. abscessus subsp. abscessus isolates and 6 M. abscessus subsp. massiliense isolates, cataloging all the essential and non-essential genes in each strain. Pan-genome analysis revealed a core set of 3,845 genes and a large accessory genome of 11,507. We identified 259 core essential genes across the 21 clinical isolates and 425 differentially required genes, representing ~10% of the Mab core genome. We also identified genes whose requirements were subspecies, lineage, and isolate-specific. Finally, by correlating TnSeq profiles, we identified 19 previously uncharacterized genetic networks in Mab. Altogether, we find that Mab clinical isolates are not only genetically diverse but functionally diverse as well.This study investigates the genetic diversity of Mycobacterium abscessus (Mab), a bacteria known for causing difficult-to-treat infections. Researchers performed transposon-sequencing (TnSeq) on 21 different clinical isolates of Mab to identify essential and non-essential genes in each strain. Through this analysis, they identified core genes required for growth across all strains. Interestingly, they also identified genes whose requirement for growth or "essentiality" were subspecies, lineage, and isolate-specific. This study reveals that Mab's genetic diversity translates into significant functional differences among clinical isolates. Insights from this paper lay essential groundwork for future studies exploring the biological and clinical implications of genetic diversity in Mab clinical isolates. Understanding this diversity could guide targeted therapies and offer new insights into managing infections caused by Mab, a growing public health concern.

  View details for DOI 10.1128/mbio.03376-24

  View details for PubMedID 39745363

  View details for PubMedCentralID PMC11796383

• Efflux pumps and membrane permeability contribute to intrinsic antibiotic resistance in Mycobacterium abscessus. bioRxiv : the preprint server for biology McGowen, K., Funck, T., Wang, X., Zinga, S., Wolf, I. D., Akusobi, C. C., Denkinger, C. M., Rubin, E. J., Sullivan, M. R. 2024

  Abstract

  Mycobacterium abscessus is a pulmonary pathogen that exhibits intrinsic resistance to antibiotics, but the factors driving this resistance are incompletely understood. Insufficient intracellular drug accumulation could explain broad-spectrum resistance, but whether antibiotics fail to accumulate in M. abscessus and the mechanisms required for drug exclusion remain poorly understood. We measured antibiotic accumulation in M. abscessus using mass spectrometry and found a wide range of drug accumulation across clinically relevant antibiotics. Of these compounds, linezolid accumulates the least, suggesting that inadequate uptake impacts its efficacy. We utilized transposon mutagenesis screening to identify genes that cause linezolid resistance and found multiple transporters that promote membrane permeability or efflux, including an uncharacterized, M. abscessus-specific protein that effluxes linezolid and several chemically related antibiotics. This demonstrates that membrane permeability and drug efflux are critical mechanisms of antibiotic resistance in M. abscessus and suggests that targeting membrane transporters could potentiate the efficacy of certain antibiotics.

  View details for DOI 10.1101/2024.08.23.609441

  View details for PubMedID 39229117

  View details for PubMedCentralID PMC11370614

• Mycobacterium abscessus Cluster in Cardiac Surgery Patients Potentially Attributable to a Commercial Water Purification System. Annals of internal medicine Klompas, M., Akusobi, C., Boyer, J., Woolley, A., Wolf, I. D., Tucker, R., Rhee, C., Fiumara, K., Pearson, M., Morris, C. A., Rubin, E., Baker, M. A. 2023; 176 (3): 333-339

  Abstract

  Nontuberculous mycobacteria are water-avid pathogens that are associated with nosocomial infections.To describe the analysis and mitigation of a cluster of Mycobacterium abscessus infections in cardiac surgery patients.Descriptive study.Brigham and Women's Hospital, Boston, Massachusetts.Four cardiac surgery patients.Commonalities among cases were sought, potential sources were cultured, patient and environmental specimens were sequenced, and possible sources were abated.Description of the cluster, investigation, and mitigation.Whole-genome sequencing confirmed homology among clinical isolates. Patients were admitted during different periods to different rooms but on the same floor. There were no common operating rooms, ventilators, heater-cooler devices, or dialysis machines. Environmental cultures were notable for heavy mycobacterial growth in ice and water machines on the cluster unit but little or no growth in ice and water machines in the hospital's other 2 inpatient towers or in shower and sink faucet water in any of the hospital's 3 inpatient towers. Whole-genome sequencing confirmed the presence of a genetically identical element in ice and water machine and patient specimens. Investigation of the plumbing system revealed a commercial water purifier with charcoal filters and an ultraviolet irradiation unit leading to the ice and water machines in the cluster tower but not the hospital's other inpatient towers. Chlorine was present at normal levels in municipal source water but was undetectable downstream from the purification unit. There were no further cases after high-risk patients were switched to sterile and distilled water, ice and water machine maintenance was intensified, and the commercial purification system was decommissioned.Transmission pathways were not clearly characterized.Well-intentioned efforts to modify water management systems may inadvertently increase infection risk for vulnerable patients.National Institutes of Health.

  View details for DOI 10.7326/M22-3306

  View details for PubMedID 36877966

• Biotin-dependent cell envelope remodelling is required for Mycobacterium abscessus survival in lung infection. Nature microbiology Sullivan, M. R., McGowen, K., Liu, Q., Akusobi, C., Young, D. C., Mayfield, J. A., Raman, S., Wolf, I. D., Moody, D. B., Aldrich, C. C., Muir, A., Rubin, E. J. 2023; 8 (3): 481-497

  Abstract

  Mycobacterium abscessus is an emerging pathogen causing lung infection predominantly in patients with underlying structural abnormalities or lung disease and is resistant to most frontline antibiotics. As the pathogenic mechanisms of M. abscessus in the context of the lung are not well-understood, we developed an infection model using air-liquid interface culture and performed a transposon mutagenesis and sequencing screen to identify genes differentially required for bacterial survival in the lung. Biotin cofactor synthesis was required for M. abscessus growth due to increased intracellular biotin demand, while pharmacological inhibition of biotin synthesis prevented bacterial proliferation. Biotin was required for fatty acid remodelling, which increased cell envelope fluidity and promoted M. abscessus survival in the alkaline lung environment. Together, these results indicate that biotin-dependent fatty acid remodelling plays a critical role in pathogenic adaptation to the lung niche, suggesting that biotin synthesis and fatty acid metabolism might provide therapeutic targets for treatment of M. abscessus infection.

  View details for DOI 10.1038/s41564-022-01307-5

  View details for PubMedID 36658396

  View details for PubMedCentralID PMC9992005

• Unusual prophages in Mycobacterium abscessus genomes and strain variations in phage susceptibilities. PloS one Amarh, E. D., Dedrick, R. M., Garlena, R. A., Russell, D. A., Gauthier, C. H., Aull, H. G., Abad, L., Jacobs-Sera, D., Akusobi, C., Rubin, E. J., Hatfull, G. F. 2023; 18 (2): e0281769

  Abstract

  Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections.

  View details for DOI 10.1371/journal.pone.0281769

  View details for PubMedID 36795728

  View details for PubMedCentralID PMC9934374

• Transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity. eLife Akusobi, C., Benghomari, B. S., Zhu, J., Wolf, I. D., Singhvi, S., Dulberger, C. L., Ioerger, T. R., Rubin, E. J. 2022; 11

  Abstract

  Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab-specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

  View details for DOI 10.7554/eLife.71947

  View details for PubMedID 35659317

  View details for PubMedCentralID PMC9170245

• Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets. mSystems Choudhery, S., Brown, A. J., Akusobi, C., Rubin, E. J., Sassetti, C. M., Ioerger, T. R. 2021; 6 (5): e0087621

  Abstract

  TnSeq is a widely used methodology for determining gene essentiality, conditional fitness, and genetic interactions in bacteria. The Himar1 transposon is restricted to insertions at TA dinucleotides, but otherwise, few site-specific biases have been identified. As a result, most analytical approaches assume that insertions are expected to be randomly distributed among TA sites in nonessential regions. However, through analysis of Himar1 transposon libraries in Mycobacterium tuberculosis, we demonstrate that there are site-specific biases that affect the frequency of insertion of the Himar1 transposon at different TA sites. We use machine learning and statistical models to characterize patterns in the nucleotides surrounding TA sites that correlate with high or low insertion counts. We then develop a quantitative model based on these patterns that can be used to predict the expected counts at each TA site based on nucleotide context, which can explain up to half of the variance in insertion counts. We show that these insertion preferences exist in Himar1 TnSeq data sets from other mycobacterial and nonmycobacterial species. We present an improved method for identification of essential genes, called TTN-Fitness, that can better distinguish true biological fitness effects by comparing observed counts to expected counts based on our site-specific model of insertion preferences. Compared to previous essentiality methods, TTN-Fitness can make finer distinctions among genes whose disruption causes a fitness defect (or advantage), separating them out from the large pool of nonessentials, and is able to classify many smaller genes (with few TA sites) that were previously characterized as uncertain. IMPORTANCE When using the Himar1 transposon to create transposon insertion mutant libraries, it is known that the transposon is restricted to insertions at TA dinucleotide sites throughout the genome, and the absence of insertions is used to infer which genes are essential (or conditionally essential) in a bacterial organism. It is widely assumed that insertions in nonessential regions are otherwise random, and this assumption is used as the basis of several methods for statistical analysis of TnSeq data. In this paper, we show that the nucleotide sequence surrounding TA sites influences the magnitude of insertions, and these Himar1 insertion preferences (sequence biases) can partially explain why some sites have higher counts than others. We use this predictive model to make improved estimates of the fitness effects of genes, which help make finer distinctions of the phenotype and biological consequences of disruption of nonessential genes.

  View details for DOI 10.1128/mSystems.00876-21

  View details for PubMedID 34665010

  View details for PubMedCentralID PMC8525568

• Audio Interview: Delivering Covid-19 Vaccines to Minority Communities. The New England journal of medicine Rubin, E. J., Baden, L. R., Del Rio, C., Akusobi, C., Morrissey, S. 2021; 384 (13): e60

  View details for DOI 10.1056/NEJMe2105496

  View details for PubMedID 33789019

• Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111. Viruses Lin, A. E., Diehl, W. E., Cai, Y., Finch, C. L., Akusobi, C., Kirchdoerfer, R. N., Bollinger, L., Schaffner, S. F., Brown, E. A., Saphire, E. O., Andersen, K. G., Kuhn, J. H., Luban, J., Sabeti, P. C. 2020; 12 (1)

  Abstract

  For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013‒2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP's roles in protein structure, virion budding, and transcription and replication.

  View details for DOI 10.3390/v12010105

  View details for PubMedID 31952352

  View details for PubMedCentralID PMC7019320

• Parallel Evolution of Host-Attachment Proteins in Phage PP01 Populations Adapting to Escherichia coli O157:H7. Pharmaceuticals (Basel, Switzerland) Akusobi, C., Chan, B. K., Williams, E. S., Wertz, J. E., Turner, P. E. 2018; 11 (2)

  Abstract

  The emergence of antibiotic resistance has sparked interest in phage therapy, which uses virulent phages as antibacterial agents. Bacteriophage PP01 has been studied for potential bio-control of Escherichia coli O157:H7, its natural host, but in the laboratory, PP01 can be inefficient at killing this bacterium. Thus, the goal of this study was to improve the therapeutic potential of PP01 through short-term experimental evolution. Four replicate populations of PP01 were serially passaged 21 times on non-evolving E. coli O157:H7 with the prediction that the evolved phage populations would adsorb faster and more efficiently kill the host bacteria. Dead-cell adsorption assays and in vitro killing assays confirmed that evolved viruses improved their adsorption ability on E. coli O157:H7, and adapted to kill host bacteria faster than the wildtype ancestor. Sequencing of candidate tail-fiber genes revealed that the phage populations evolved in parallel; the lineages shared two point mutations in gp38 that encodes a host recognition protein, and surprisingly shared a ~600 bp deletion in gp37 that encodes the distal tail fibers. In contrast, no mutations were observed in the gp12 gene encoding PP01’s short tail fibers. We discuss the functional role of the observed mutations, including the possible adaptive role of the evolved deletions. This study demonstrates how experimental evolution can be used to select for viral traits that improve phage attack of an important bacterial pathogen, and that the molecular targets of selection include loci contributing to cell attachment and phage virulence.

  View details for DOI 10.3390/ph11020060

  View details for PubMedID 29925767

  View details for PubMedCentralID PMC6027323

• The bacterial Type III toxin-antitoxin system, ToxIN, is a dynamic protein-RNA complex with stability-dependent antiviral abortive infection activity. Scientific reports Short, F. L., Akusobi, C., Broadhurst, W. R., Salmond, G. P. 2018; 8 (1): 1013

  Abstract

  Bacteria have evolved numerous defense systems to protect themselves from viral (bacteriophage) infection. The ToxIN system of Pectobacterium atrosepticum is a Type III toxin-antitoxin complex and "altruistic suicide" anti-phage system, which kills phage-infected cells through the release of a ribonuclease toxin, ToxN. ToxN is counteracted by a co-transcribed antitoxic RNA pseudoknot, ToxI, which self-assembles with ToxN into an inactive 3 ToxI:3 ToxN complex in vitro. However it is not known whether this complex is predominant in vivo, or how the complex is disassembled following infection to trigger a lethal, "altruistic" response. In this study, we characterise ToxI turnover and folding, and explore the link between complex stability and anti-phage activity, with a view to understanding events that lead to ToxN-mediated suicide following phage infection. We present evidence that ToxN constantly cleaves fresh ToxI in vivo rather than staying associated with pre-processed antitoxin, and that the ToxI antitoxin can partially fold spontaneously using conserved nucleotides. We also show that reducing the stability of the ToxIN complex can increase the strength of the antiviral response in a phage-dependent manner. Based on this information, we propose a revised model for ToxN inhibition, complex assembly and activation by infecting bacteriophage.

  View details for DOI 10.1038/s41598-017-18696-x

  View details for PubMedID 29343718

  View details for PubMedCentralID PMC5772629

• Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems. Frontiers in microbiology Chen, B., Akusobi, C., Fang, X., Salmond, G. P. 2017; 8: 1006

  Abstract

  Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous "escape" mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5-10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxINPa.

  View details for DOI 10.3389/fmicb.2017.01006

  View details for PubMedID 28620370

  View details for PubMedCentralID PMC5449768