While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

Objectives: Slow-growing nontuberculous mycobacteria (NTMs) are highly prevalent and routinely cause opportunistic intracellular infectious disease in immunocompromised hosts. Methods: The activity of the triple combination of antibiotics, clarithromycin (CLR), rifabutin (RFB), and clofazimine (CFZ), was evaluated and compared with the activity of single antibiotics as well as with double combinations in an in vitro biofilm assay and an in vivo murine model of Mycobacterium avium subsp. hominissuis (M. avium) lung infection. Results: Treatment of 1-week-old biofilms with the triple combination exerted the strongest effect of all (0.12 ± 0.5 × 10⁷ CFU/mL) in reducing bacterial growth as compared to the untreated (5.20 ± 0.5 × 10⁷/mL) or any other combination (≥0.75 ± 0.6 × 10⁷/mL) by 7 days. The treatment of mice intranasally infected with M. avium with either CLR and CFZ or the triple combination provided the greatest reduction in CLR-sensitive M. avium bacterial counts in both the lung and spleen compared to any single antibiotic or remaining double combination by 4 weeks posttreatment. After 4 weeks of treatment with the triple combination, there were no resistant colonies detected in mice infected with a CLR-resistant strain. No clear relationships between treatment and spleen or lung organ weights were apparent after triple combination treatment. Conclusions: The biofilm assay data and mouse disease model efficacy results support the further investigation of the triple-antibiotic combination.

Mycobacterium avium subsp. hominissuis (M. avium) is an opportunistic environmental pathogen that typically infects patients with existing lung conditions such as cystic fibrosis or COPD. Pulmonary M. avium infection generates peribronchial granulomas that contain infected macrophages and multinucleated giant cells (MGCs). While granuloma formation with MGCs is a common feature of mycobacterial infection, the role of MGCs within the granulomas as well as in the host-pathogen interaction is poorly understood. To shed light on the role of MGCs, we established a novel in vitro model utilizing THP-1 cells stimulated with a combination of IFN-γ and TNF-α. In this study, we show that MGCs can take up M. avium, which replicates intracellularly before leaving the cell. Bacteria that escape the MGC exhibit a highly invasive phenotype, which warrants further evaluation. Characterization of MGCs with transmission electron microscopy revealed an accumulation of cytoplasmic lipid droplets, autophagic activity, and multiple nuclei. Autophagy markers are upregulated in both uninfected and infected MGCs early in infection, measured by RT-qPCR analysis of Beclin-1 and LC3. Inhibition of autophagy with siRNA significantly reduced M. avium survival significantly in THP-1 macrophages. Depletion of host cholesterol and sphingomyelin in MGCs also resulted in decreased survival of M. avium. These processes potentially contribute to the formation of a supportive intracellular environment for the pathogen. Collectively, our results suggest that M. avium is adapted to replicate in MGCs and utilize them as a springboard for local spread.

Background/Objectives: Mycobacterium avium subsp. hominissuis is a cause of disseminated infections in immunosuppressed patients and pulmonary infections in individuals with chronic lung conditions. The treatment of this infection is prolonged, and ultimately associated with failure in a large percentage of patients. Previous studies have determined that the bacteria respond to antibiotic contact, altering pathways affecting metabolic and other functions, as well as structural aspects. Methods: Based on this assumption and the fact that the medication used to treat M. avium infection may encounter the target bacterium at different sequential times, we evaluated the effect of three antibiotics, clarithromycin, ethambutol and rifabutin, delivered together or separately in different orders. Results: The results showed no difference regarding the response of the bacterium to treatment with the three antibiotics when in planktonic state. However, in either the biofilm phenotype or inside macrophages, different orders of exposure to antibiotics led to different responses. In macrophages, the sequence of exposure when ethambutol was given first, followed by clarithromycin and rifabutin, was associated with bacterial count decrease, similar to the simultaneous exposure to the three antibiotics, whereas the other combinations showed a decreased effect. Conclusions: In summary, the study highlights an important aspect of bacterial response to combination therapies, and more specifically, the response of M. avium to therapy. The use of a cell wall-active antibiotic first appears to be linked to an improvement in therapy efficacy.