Mycobacterium abscessus (Mab), a rapidly growing mycobacterial species with intrinsic and acquired resistance to multiple antibiotics, is an emerging public health concern. The rise in clinical cases of treatment-refractory infections of M. abscessus has propelled its research toward novel therapeutic approaches. The number of publications entitled “ Mycobacterium abscessus” has increased by ~300% over the last decade, of which the majority of studies exploring the fundamental biology and pathogenesis of Mab have used the reference strain ATCC19977. However, whole-genome sequence analyses, combined with transposon-seq based functional genomics, reveal an open pan-genome with significant variations in the essential genes across ATCC19977 and clinical isolates. These new discoveries demand a careful selection of strains and growth conditions in experimental design. In this minireview, we discuss these challenges and propose a framework for future M. abscessus studies in silico , including a new web-based resource for pangenome analysis, in vitro, and in animal models.

Lipomannan (LM) and lipoarabinomannan (LAM) are important components of the cell envelope of all mycobacteria that have been extensively studied for their roles in mycobacterial physiology and host-pathogen interactions. Despite the considerable progress made in deciphering the structure and biosynthesis of these lipoglycans over the last few decades, some of the key steps leading to their assembly and export to the cell surface remain ill-defined. We report on the characterization of a conserved and essential polyprenyl phosphate mannose-dependent mannosyltransferase named MptB, involved in the initial steps of the elongation of the mannan domain of LM and LAM from a phosphatidylinositol mannoside (PIM) anchor. Genetic silencing of mptB in Mycobacterium smegmatis led to the arrest of LM, LAM and PIM synthesis beyond di-mannosylated forms of these glycolipids. In cell-free assays, mptB overexpression led to the increased production of tetra-mannosylated forms of PIMs by M. smegmatis membranes, whereas reduced mptB expression resulted in the dramatically decreased synthesis of phosphatidylinositol tri-, tetra- and hexa-mannosides. Together with structural modeling predictions, the results of these assays support MptB as the α-(1,6)-mannosyltransferase elongating the mannan backbone of LM from a di- and/or tri-mannosylated PIM primer.

Novel treatment strategies are urgently needed to combat Mycobacterium avium complex (MAC) pulmonary disease (PD). Animal models are important for screening therapeutic strategies, but their ability to reproduce human-like immunopathology and impaired respiratory function is poorly characterised. We modelled chronic lung infection in BALB/c mice over 20 weeks with three isolates of MAC (MAC101, MAC104 and MAC2285R) to compare bacterial growth, histological injury, immune cellular dynamics and respiratory function. We found that MAC101 caused a proliferative infection over 20 weeks, associated with a strong adaptive response, progressive granulomatous inflammation and increasing respiratory effort. For MAC104, lung bacterial burden rose initially but fell after week 12, accompanied by increased regulatory T-cell response and stabilisation of pathological and respiratory changes. By contrast, MAC2285R caused a low-virulence, non-proliferative infection associated with a strong myeloid cell response, modest histopathological change and increased respiratory effort. Immune cell dynamics in chronic murine MAC-PD correlate with bacterial burden and pathology and are strongly MAC-isolate dependent. These findings provide a spectrum of quantifiable and clinically relevant disease outcomes to facilitate the preclinical screening of novel antimicrobial and host-directed therapies for MAC-PD.

High-dimensional phenotypic screens of bacterial loss-of-function mutant libraries have determined gene-gene connections and specific phenotypes for thousands of bacterial genes in many species, but deciphering the underlying mechanisms remains decidedly low-throughput. Here, we demonstrate the utility of proteome-wide AI-based protein-protein interaction (PPI) predictions for overcoming this gap by using pooled-AlphaFold3 to assess all ∼1.3 million possible pairwise interactions in the proteome of Mycobacterium leprae. We identify ∼2,000 strong and intermediate PPIs that underlie a significant fraction of phenotypes and gene-gene connections observed in large-scale chemical genomics screens from Mycobacterium tuberculosis, Mycobacterium smegmatis, and Corynebacterium glutamicum . This combined approach predicts specific functions for dozens of previously uncharacterized core, conserved, and essential mycobacterial proteins. We highlight new information derived from the study, including insights into mycobacterial envelope assembly, peptidoglycan remodeling, and new modulators of the central dogma enzymes RNA polymerase and DNA gyrase. These data establish combined pooled-AlphaFold3 PPI prediction and high-throughput genomics approach as the gold standard for large-scale characterization of protein function.

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment and is largely due to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts with Rv3035, forming a stable heterodimer that associates with the cell envelope biosynthesis protein AftB. We report the structures of Rv3035 alone and in complex with FecB and identify critical residues for complex formation and function. Coessentiality and genetic interaction analyses support a functional link between FecB, Rv3035, and AftB, an arabinofuranosyltransferase that synthesizes arabinogalactan and lipoarabinomannan. Loss of FecB or Rv3035 disrupted AftB-mediated arabinan synthesis, suggesting that these proteins support AftB's enzymatic activity. FecB is required for Mtb virulence in mice, underscoring its physiological relevance. These findings highlight FecB, Rv3035, and AftB as promising therapeutic targets.

SUMMARYThe mycobacterial cell envelope, one of the most complex membranes found in bacteria, plays a major role in bacterial pathogenesis, virulence, and antimicrobial resistance. Biogenesis and modeling of this cell envelope are heavily influenced by the mycobacterial membrane protein large (MmpL) family of transporters due to their ability to export fatty acids and lipid components. Select MmpL transporters can also function as siderophore exporters to help regulate the acquisition of iron, which is critical for mycobacterial survival. Additionally, certain MmpLs can participate in active efflux of antimycobacterial drugs, directly contributing to antimicrobial resistance. Given the physiological significance of these MmpL membrane proteins and their potential to serve as important antimycobacterial targets, questions regarding their functional roles, cellular assemblies, interactions, and regulation need to be fully addressed. In this review, we summarize our current knowledge on the structures and functions of these MmpL transporters. It is our hope that researchers in the field will continue to build upon these efforts and apply various structural, biophysical, and biochemical methodologies to fully elucidate how MmpL transporters coordinate to participate in cell envelope biogenesis, cell elongation and division, and antimicrobial resistance.

De novo fatty acid synthesis produces the acyl units needed to generate phospholipids, lipoproteins, enzyme prosthetic factors, polyketides, and mycolic acids in mycobacterium tuberculosis (Mtb). Here, we identified sALT629, a butoxyphenyl-tetrazole-acetamide compound that inhibits de novo lipid synthesis in Mtb. This compound disrupts the Mtb lipidome and prevents incorporation of metabolic tracers into acyl chains of Mtb lipids. Unexpectedly, we also found that sALT629 treatment significantly depleted triacylglycerol (TAG) pools as a metabolic compensation mechanism when de novo fatty acid synthesis was inhibited. Resistance to sALT629 was mediated by loss of function mutations in HadC, the non-essential hydroxyacyl- acyl carrier protein -dehydratase subunit involved in the synthesis of long-chain oxygenated mycolic acids. Inactivating HadC rescued sALT629-mediated inhibition by sustaining TAG pools to fulfill Mtb's biosynthetic demand for acyl chains. Lastly, loss of function HadC resistance mutations resulted in cell wall perturbations that confer fitness defects in vitro and in vivo suggesting that this specific resistance mechanism is unlikely to arise in Mtb in a clinical setting. Significance. Having effective antibiotics to treat tuberculosis underpins our ability to control this disease. The spread of antibiotic-resistant tuberculosis has prompted a need to identify new drug candidates with new mechanisms of action. Here we describe an antitubercular that targets de novo fatty acid synthesis in Mtb, a critical process required to generate multiple essential lipid and lipid-based factors in the bacteria. One resistance mechanism to this inhibitor is associated with perturbations to mycolic acid synthesis resulting in Mtb attenuation. These findings demonstrate that de novo fatty acid synthesis in Mtb is an actionable drug target, and uncovered a compensatory metabolic resistance network between TAGs and mycolic acids.

Mycobacterium avium complex (MAC) is driving a global rise in pulmonary disease (MAC-PD) characterised by chronic infection, granulomatous inflammation and impaired respiratory function. Better animal models are needed to screen candidate therapies targeting bacteria and immune-mediated tissue injury. The C3HeB/FeJ mouse was previously reported to model necrotic granulomatous lung infection in MAC-PD following infection with a low-dose inoculum of the clinical isolate MAC2285R. We investigated whether this model was reproducible with variations in MAC strain and inoculating dose. Six-week-old female C3HeB/FeJ mice were infected intratracheally with a clinical isolate of MAC (MAC2285R) or reference strains (MAC104 or MAC101). Mice were culled at 4-weekly intervals post-infection until week 12. Lungs, spleen and liver were harvested for bacterial burden enumeration and histological examination. Whole body plethysmography (WBP) was performed weekly to measure changes in respiratory function (Buxco system). C3HeB/FeJ mice infected with low dose inoculum of MAC2285R infection exhibited increasing bacterial lung infection for 8 weeks (p < 0.05), followed by stable lung burden from weeks 8–12. High dose inoculum resulted in stable lung bacterial burden over 12 weeks. Histological analysis revealed only mild inflammatory changes in both low and high dose inoculum groups at weeks 4, 8 and 12 post-infection, with no evidence of necrotising or non-necrotising granulomatous inflammation. Surrogate measures of respiratory effort (frequency, tidal volume, inspiratory and expiratory flow rates) were increased in mice with high dose inoculum compared to uninfected controls (p < 0.001), but not low dose inoculum. Similar findings on lung bacterial burden and histological analysis were found in mice infected with low- and high-dose inoculum of MAC104 and MAC101. MAC104 infection caused greater changes in respiratory function, whereas MAC101 did not significantly affect breathing patterns. The C3HeB/FeJ mouse is susceptible to chronic MAC infection from intratracheal infection with reference and clinical isolates, but this was not associated with severe granulomatous inflammation as previously reported. A low dose inoculum generated a proliferative lung infection, whereas high dose inoculum resulted in chronic, stable lung bacterial burden. Mice with high-dose inoculum MAC2285R and MAC104 infection also displayed evidence of increased respiratory effort.

Mycobacterium abscessus is a rapidly growing nontuberculous Mycobacterium causing severe pulmonary infections, especially in immunocompromised individuals and patients with underlying lung conditions like cystic fibrosis (CF). While rifamycins are the pillar of tuberculosis treatment, their efficacy against M. abscessus lung disease is severely compromised by intrabacterial ADP-ribosylation. Additionally, rifamycins induce cytochrome P450 3A4 (CYP3A4), a major human drug-metabolizing enzyme, further limiting their use in patients with comorbidities that require treatment with CYP3A4 substrates such as CF and HIV coinfection. We chemically reengineered rifabutin to enhance its potency against M. abscessus by blocking intrabacterial inactivation and eliminate drug-drug interactions by removing induction of CYP3A4 gene expression. We have designed and profiled a series of C25-substituted derivatives resistant to intracellular inactivation and lacking CYP3A4 induction, while retaining excellent pharmacological properties. Against Mycobacterium tuberculosis, devoid of ADP-ribosyltransferase, the frontrunners are equipotent to rifabutin, suggesting superior clinical utility since they no longer come with the drug interaction liability typical of rifamycins. Prioritized compounds demonstrated superior antibacterial activity against a panel of M. abscessus clinical isolates, were highly bactericidal against replicating and drug-tolerant nonreplicating bacteria in caseum surrogate and were active against intracellular bacteria. As single agents, these rifamycins were as effective as a standard-of-care four-drug combination in a murine model of M. abscessus lung infection.

Leprosy, caused primarily by Mycobacterium leprae, is considered a disease introduced into the Americas during European colonization. However, the recent discovery of a second pathogen causing leprosy, M. lepromatosis, mainly found in the Americas, challenges this view. Here, we show that M. lepromatosis infected humans in the Americas before European contact. By screening 389 ancient and 408 contemporary samples, we have expanded the genetic data available for the species. Phylogenetic analyses revealed distinct human-infecting clades of M. lepromatosis, with one dominating North America since colonial times. The presence of millennia-old strains in North and South America indicates that M. lepromatosis may have been widespread during the Late Holocene, demonstrating that M. lepromatosis leprosy has a long-standing history in the Americas before European arrival.

Like other tuberculous and nontuberculous mycobacterial pathogens of human lung such as Mycobacterium tuberculosis and M. abscessus, M. avium is likely exposed to a variety of stressors during infection, including hypoxic conditions inside activated macrophages and in the avascular necrotic regions of granulomas. How M. avium survives hypoxic stress to establish a chronic infection is currently not well understood. Using RNA-sequencing, we here show that M. avium grown under progressive microaerophilic conditions activates more than 4-fold a subset of 16 genes, the expression of 13 of which is dependent on the two-component system regulator DosRS. A subset of M. avium DosR regulon genes was confirmed to also be activated upon exposure to nitric oxide. Although a second sensor kinase besides DosS has been proposed to function with the transcriptional regulator DosR in M. avium, we show that this other kinase cannot compensate for a deficiency in DosS. Loss of dosRS expression in M. avium led to a significant reduction in viability under hypoxia that was more marked at acidic than at neutral pH. Unlike the situation in M. abscessus, however, loss of DosRS did not significantly impact the ability of M. avium to establish a drug tolerant state in vitro or form biofilms under host relevant conditions. Collectively, these results are suggestive of a lesser impact of DosRS on the ability of M. avium to develop antibiotic tolerance compared to other nontuberculous mycobacteria. The M. avium dosRS mutant further showed no signs of virulence attenuation in murine macrophages and in chronically infected immunocompetent BALB/c mice.

Mycobacterium abscessus (MAB), a rapidly growing non-tuberculous mycobacterium, is becoming increasingly recognized as a significant pathogen affecting humans. These bacteria particularly impact individuals with cystic fibrosis (CF), non-CF bronchiectasis, and compromised immune systems. Treating pulmonary infections with MAB is challenging due to the bacteria's inherent and acquired resistance to many antibiotics, including most anti-tuberculosis antibiotics. Antibiotic therapy of MAB infection is lengthy, involves multiple oral and parenteral administered drugs, induces significant toxicity, and, on many occasions, fails to cure. Consequently, developing more effective antibiotics has become a high priority. Preclinical studies to evaluate antibiotic efficacy against MAB are challenging because they fail to establish a progressive and sustained pulmonary infection in commonly used animal models. To address this issue, the course of MAB pulmonary infection was evaluated in 15 immunocompetent or deficient mouse strains. We report bacterial burden and histopathology and classify the models according to their ability to clear or sustain progressive infection beyond 28 days. We also examined the potential of these models for drug screening. Our findings provide a foundation for selecting suitable mouse models of pulmonary MAB infection for drug discovery.

Mycobacterium abscessus is a major human pathogen, mostly infecting people with pre-existing lung conditions, such as cystic fibrosis. The production of glycopeptidolipids (GPL) is a major determinant of virulence of this bacterium, with clinical isolates that lack GPL generally exhibiting more aggressive clinical behavior. The current paradigm is that GPL production is abolished in vivo via irreversible, spontaneous mutations taking place as part of in-host evolution. Little is known about the mechanisms or extent to which GPL production may be regulated. Here, we describe an unusual TetR-like transcription factor of M. abscessus, mab_1638, that appears to be a strong positive regulator of the entire GPL biosynthesis and export gene cluster through a combination of direct and indirect mechanisms. The inactivation of mab_1638 abolished GPL production, leading to stable rough colony morphology and increased virulence in infection models, characteristics of rough, non-GPL producers. Transcriptome analysis found that the mab_1638 mutant had 118 differentially expressed genes, including the GPL locus and a second, recently described GPL-like locus that produces a related glycosylated lipopeptide called GP8L. Chromatin immunoprecipitation and sequencing revealed a consensus inverted-repeat DNA sequence motif, characteristic of genes regulated by mab_1638. Together, these findings found that mab_1638 encodes a transcription factor required for GPL production and, therefore, has a profound effect on virulence traits. We propose naming this gene GPL regulator 1 (gplR1). This finding raises the important possibility that M. abscessus strains appearing smooth in laboratory growth conditions may nonetheless downregulate GPL-cluster genes in other conditions, including in-patient conditions, and thus acquire the phenotypic characteristics of rough strains.IMPORTANCEMycobacterium abscessus is an important human pathogen, causing disease that is difficult to treat. M. abscessus strains have been observed to have two distinct colony morphologies, smooth and rough, which substantially impact clinical presentation. Rough strains are associated with later-stage, more severe disease and are more virulent in animal models. Smooth morphology is conferred by a molecule called glycopeptidolipid in the outer cell envelope, and rough morphology is known to occur when mutations inactivate genes required for glycopeptidolipid biosynthesis. Little is known about the possibility that glycopeptidolipid production could be regulated. Here, we have identified a transcription factor that is required for glycopeptidolipid biosynthesis, indicating that glycopeptidolipid production is indeed a regulated process and raising the important possibility that strains exhibiting smooth morphology in the lab may downregulate GPL production in the human host, thereby acquiring the virulence properties of rough strains.

Mycobacterium abscessus is one of the leading causes of pulmonary infections caused by non-tuberculous mycobacteria. The ability of M. abscessus to establish a chronic infection in the lung relies on a series of adaptive mutations impacting, in part, global regulators and cell envelope biosynthetic enzymes. One of the genes under strong evolutionary pressure during host adaptation is ubiA, which participates in the elaboration of the arabinan domains of two major cell envelope polysaccharides: arabinogalactan (AG) and lipoarabinomannan (LAM). We here show that patient-derived UbiA mutations not only cause alterations in the AG, LAM, and mycolic acid contents of M. abscessus but also tend to render the bacterium more prone to forming biofilms while evading uptake by innate immune cells and enhancing their pro-inflammatory properties. The fact that the effects of UbiA mutations on the physiology and pathogenicity of M. abscessus were impacted by the rough or smooth morphotype of the strain suggests that the timing of their selection relative to morphotype switching may be key to their ability to promote chronic persistence in the host.IMPORTANCEMultidrug-resistant pulmonary infections caused by Mycobacterium abscessus and subspecies are increasing in the U.S.A. and globally. Little is known of the mechanisms of pathogenicity of these microorganisms. We have identified single-nucleotide polymorphisms (SNPs) in a gene involved in the biosynthesis of two major cell envelope polysaccharides, arabinogalactan and lipoarabinomannan, in lung-adapted isolates from 13 patients. Introduction of these individual SNPs in a reference M. abscessus strain allowed us to study their impact on the physiology of the bacterium and its interactions with immune cells. The significance of our work is in identifying some of the mechanisms used by M. abscessus to colonize and persist in the human lung, which will facilitate the early detection of potentially more virulent clinical isolates and lead to new therapeutic strategies. Our findings may further have broader biomedical impacts, as the ubiA gene is conserved in other tuberculous and non-tuberculous mycobacterial pathogens.

Methylglucose lipopolysaccharides (MGLPs) are small cytoplasmic polysaccharides produced by mycobacteria and a few Nocardia species. Although their ability to form 1:1 complexes with long-chain fatty acids and acyl-CoAs in vitro has long suggested that they play a role in fatty acid and lipid metabolism, direct evidence for this assumption has been waiting for the availability of mutants with well-defined MGLP contents and fatty acyl-binding properties. Here, we report on the generation of eight mycobacterial mutants with various defects in the biosynthesis of MGLPs. The characterization of the MGLP content of these mutants provides the most comprehensive picture of the MGLP biosynthetic machinery to date. Importantly, the successful generation of three mutants in which MGLP synthesis is abolished indicates that, despite their conservation in mycobacteria, MGLPs are not essential for growth under standard laboratory conditions. Analysis of the fatty acyl-binding properties of truncated forms of MGLPs produced by some mutants provided unexpected new insight into the structural determinants governing the ability of MGLPs to form complexes with long acyl-CoAs calling for a revision of the structural model of these lipopolysaccharides. The characterization of the MGLP produced by the different mutants further unveiled previously unknown cation-binding properties associated with acylated forms of these polysaccharides. The availability of well-defined Mycobacterium tuberculosis MGLP mutants opens the way to a precise assessment of the role of these unique cytoplasmic lipopolysaccharides in the physiology and virulence of this vital bacterial pathogen.

ABSTRACT Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis , an essential inner membrane transporter of the resistance–nodulation–division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro , inside macrophages, and in M. tuberculosis- infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections. IMPORTANCE MmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.

Mycobacterium abscessus is increasingly recognized as the causative agent of chronic pulmonary infections in humans. One of the genes found to be under strong evolutionary pressure during adaptation of M. abscessus to the human lung is embC which encodes an arabinosyltransferase required for the biosynthesis of the cell envelope lipoglycan, lipoarabinomannan (LAM). To assess the impact of patient-derived embC mutations on the physiology and virulence of M. abscessus, mutations were introduced in the isogenic background of M. abscessus ATCC 19977 and the resulting strains probed for phenotypic changes in a variety of in vitro and host cell-based assays relevant to infection. We show that patient-derived mutational variations in EmbC result in an unexpectedly large number of changes in the physiology of M. abscessus, and its interactions with innate immune cells. Not only did the mutants produce previously unknown forms of LAM with a truncated arabinan domain and 3-linked oligomannoside chains, they also displayed significantly altered cording, sliding motility, and biofilm-forming capacities. The mutants further differed from wild-type M. abscessus in their ability to replicate and induce inflammatory responses in human monocyte-derived macrophages and epithelial cells. The fact that different embC mutations were associated with distinct physiologic and pathogenic outcomes indicates that structural alterations in LAM caused by nonsynonymous nucleotide polymorphisms in embC may be a rapid, one-step, way for M. abscessus to generate broad-spectrum diversity beneficial to survival within the heterogeneous and constantly evolving environment of the infected human airway.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly recognized as an important pathogen of the human lung, disproportionally affecting people with cystic fibrosis (CF) and other susceptible individuals with non-CF bronchiectasis and compromised immune functions. M. abscessus infections are extremely difficult to treat due to intrinsic resistance to many antibiotics, including most anti-tuberculous drugs. Current standard-of-care chemotherapy is long, includes multiple oral and parenteral repurposed drugs, and is associated with significant toxicity. The development of more effective oral antibiotics to treat M. abscessus infections has thus emerged as a high priority. While murine models have proven instrumental in predicting the efficacy of therapeutic treatments for M. tuberculosis infections, the preclinical evaluation of drugs against M. abscessus infections has proven more challenging due to the difficulty of establishing a progressive, sustained, pulmonary infection with this pathogen in mice. To address this issue, a series of three workshops were hosted in 2023 by the Cystic Fibrosis Foundation (CFF) and the National Institute of Allergy and Infectious Diseases (NIAID) to review the current murine models of M. abscessus infections, discuss current challenges and identify priorities toward establishing validated and globally harmonized preclinical models. This paper summarizes the key points from these workshops. The hope is that the recommendations that emerged from this exercise will facilitate the implementation of informative murine models of therapeutic efficacy testing across laboratories, improve reproducibility from lab-to-lab and accelerate preclinical-to-clinical translation.