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.

Abstract Introduction Infections by Mycobacterium abscessus (MAB), a saprophytic bacterium, are rising globally. While most people exposed do not get infected, immunocompromised individuals, or those with lung conditions like cystic fibrosis, can develop pulmonary MAB disease (MAB-PD). Our research shows GM-CSF is crucial for early pulmonary MAB clearance. GM-CSF is vital for alveolar macrophage (AM) and granulocyte maturation. We hypothesize that both AM and neutrophils are needed to control bacterial growth in early infection stages. Methods We measured the bacterial load in the lungs of immunocompetent C57BL/6 mice depleted of neutrophils and/or AM. To demonstrate that AM are essential for MAB clearance, we depleted AM using clodronate liposomes and neutrophils, combined with two monoclonal antibodies (anti-Ly6G and anti-Ly6C). After depletion, mice were exposed to a MAB aerosol. Mice were euthanized, and lungs, blood, bone marrow, and bronchoalveolar lavage were collected. Bacterial burden in the lungs was assessed, and the presence or absence of AMs and neutrophils during infection was determined by flow cytometry. Results We monitored the frequency of AM and neutrophils in the lungs using flow cytometry at 3, 10, and 15 days post-infection (DPI). While control mice (untreated) and mice depleted of AM or neutrophils showed a significant reduction in bacterial burden in the lungs at 10 and 15 days post-infection, mice depleted of both AM and neutrophils were unable to control the infection by day 10. However, their bacterial load was reduced by day 15. Conclusion Our preliminary results demonstrate the essential, cooperative roles of both AM and neutrophils in the early containment and control of MAB infection within the pulmonary environment. We are currently employing multi-parameter flow cytometry to analyze potential compensatory cellular mechanisms in secondary lymphoid organs, specifically the bone marrow and blood, following the depletion of these key subsets. Funding Source RO1 AI155922 Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

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.

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.

The Nix-TB clinical trial evaluated a new 6 month regimen containing three oral drugs; bedaquiline (B), pretomanid (Pa), and linezolid (L) (BPaL regimen) for the treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug-resistant or extensively drug-resistant TB participants were cured but many patients also developed severe adverse events (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile, but it lacks oral bioavailability. Here, we propose to replace L in the BPaL regimen with spectinamide (S) administered via inhalation and we demonstrate that inhaled spectinamide 1599, combined with BPa ––BPaS regimen––has similar efficacy to that of the BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effects in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested the development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL and BPa, but not the BPaS treatment, also decreased myeloid to erythroid ratio suggesting the S in the BPaS regimen was able to recover this effect. Moreover, the BPaL also increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen without L-associated AEs.

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.

IntroductionChronic pulmonary infection with Mycobacterium abscessus (M. abscessus) is a significant cause of morbidity and mortality in people with cystic fibrosis (CF). Developing an animal model of M. abscessus pulmonary infection, especially under CF conditions, is essential to understanding clinical pulmonary M. abscessus infection. βENaC transgenic mice are known to develop spontaneous CF-like disease characterized by airway mucus obstruction and inflammation. The aim of this study was to evaluate the suitability of βENaC mice as a preclinical model and characterize their respiratory function during M. abscessus lung infection.MethodsMice received an intrapulmonary aerosol of M. abscessus using a high-pressure syringe device (Penn-Century) for subsequent characterization of disease progression and respiratory function. Whole body unrestrained plethysmography (WBP) data was collected to monitor lung function and endpoints determined organ bacterial burden and associated pathology.ResultsEndpoint CFU data in the lung and spleen showed that there was no significant difference in bacterial clearance between βENaC and WT mice. WBP data showed an impairment in overall respiratory function during and after M. abscessus infection in both strains of mice. Interestingly, even in wildtype control mice, lung dysfunction persisted after bacterial clearance.DiscussionEven with CF-like features, the βENaC transgenic mice cleared M. abscessus at a similar rate than WT mice, however, the associated respiratory monitoring revealed that there are long-term implications of M. abscessus lung exposure. The clear decline in respiratory function, even after M. abscessus clearance, suggests that WBP coupled animal modeling provides important insight that is relevant to disease burden and treatment efficacy. The M. abscessus clearance in the βENaC mice may help improve the fields understanding of CF-modulated immune deficiencies in M. abscessus pulmonary infection.

The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.