Triple-Antibiotic Combination Exerts Effective Activity against Mycobacterium avium subsp. hominissuis Biofilm and Airway Infection in an In Vivo Murine Model.

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 abscessus infection results in decrease of oxidative metabolism of lung airways cells and relaxation of the epithelial mucosal tight junctions.

Mycobacterium abscessus complex is a group of environmental pathogens that recently have been isolated more from patients with underlying lung diseases, such and COPD, bronchiectasis, and cystic fibrosis. The mechanisms involved in the pathogenesis of these diseases have only recently been investigated. Infection is associated with biofilm formation on the airway mucosa, invasion of the mucosal epithelial cells and a time-dependent impairment of the integrity of the monolayer. Using electron microscopy, it was shown that Mycobacterium abscessus induced lesions of the cell surface structures. Tight junction proteins claudin-1 and occludin-1 have increased transcription in cells exposed to Mycobacterium abscessus, in contrast to cells exposed to Mycobacterium avium. Infection of A549 alveolar epithelial cells by Mycobacterium abscessus reduced the oxidative metabolism of the cell, without inducing necrosis. A transposon library screen identified mutants that do not alter the metabolism of the A549 cells.Once the bacterium crosses the epithelial barrier, it may encounter sub-epithelial macrophages. Select mutants were used for infection assays to determine their effects on membrane integrity. Translocated select mutants were attenuated in macrophages compared to wild type Mycobacterium abscessus. In summary, the dynamics of Mycobacterium abscessus infection appears to be different from other non-tuberculous mycobacteria (NTMs). Future studies will attempt to address the mechanism involved in airway membrane lesions.

MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.

Virulent Mycobacterium avium subspecies hominissuis subverts macrophages during early stages of infection.

Virulent non-tuberculous Mycobacteria (NTMs) successfully reside and multiply within the phagosomes of phagocytic cells such as monocytes and macrophages. Macrophages play a very important role in the innate clearance of intracellular pathogens including NTMs. Attenuated Mycobacterium avium subsp. hominissuis 100 enters macrophages but is incapable of escaping these cells via canonical mycobacteria escape mechanisms. Alternatively, virulent Mycobacterium avium subsp. hominissuis 104 and Mycobacterium abscessus subsp. abscessus are able to modify macrophages to suit their growth, survival and ultimately escape from macrophages, while non-virulent Mycobacterium smegmatis is readily killed by macrophages. In this study we focused on early infection of macrophages with NTMs to determine the phenotypic response of macrophages, M1 or M2 differentiation, and phosphorylation alterations that can affect cellular response to invading bacteria. Our findings indicate that infection of the macrophage with MAH 100 and M. smegmatis favours the development of M1 macrophage, a pro-inflammatory phenotype associated with the killing of intracellular pathogens, while infection of the macrophage with MAH 104 and M. abscessus favoured the development of M2 macrophage, an anti-inflammatory phenotype associated with the healing process. Interference with the host post-translational mechanisms, such as protein phosphorylation, is a key strategy used by many intracellular bacterial pathogens to modulate macrophage phenotype and subvert macrophage function. By comparing protein phosphorylation patterns of infected macrophages, we observed that uptake of both MAH 100 and M. smegmatis resulted in MARCKS-related protein phosphorylation, which has been associated with macrophage activation. In contrast, in macrophages infected with MAH 104 and M. abscessus, methionine adenosyltransferase IIß, an enzyme that catalyses the biosynthesis of S-adenosylmethionine, a methyl donor for DNA methylation. Inhibition of DNA methylation with 5-aza-2 deoxycytidine, significantly impaired the survival of MAH 104 in macrophages. Our findings suggest that the virulent MAH 104 and M. abscessus enhance its survival in the macrophage possibly through interference with the epigenome responses.

Mycobacterium avium subsp. hominissuis (MAH) Microaggregate induction of host innate immunity is linked to biofilm formation.

Bacterial aggregation is a strategy employed by many pathogens to establish infection. Mycobacterium avium subsp. hominissuis (MAH) undergoes a phenotypic change, microaggregation, when exposed to the respiratory epithelium. We therefore compared how non-aggregated bacteria, or planktonic, and microaggregated MAH can establish lung infections by evaluating mucosal epithelial cell and phagocytic cell responses. It was determined that human mucosal lung epithelial cells recognition of MAH occurs through toll-like receptors 1 and 2. MAPK 1/3 is phosphorylated at 30 min post infection, and active at the transcriptional level 2 h post infection for both phenotypes. Microaggregate infected BEAS-2B cells up-regulated CCL5, IL-1ß, and TNF-α cDNA, while planktonic infected cells only up-regulated IL-1ß cDNA at 2 h post infection. Microaggregates are associated with increased uptake by macrophages after 1 h compared to planktonic bacteria (8.83% vs. 5.00%, P < 0.05). In addition, the microaggregate phenotype, when internalized by macrophages, had reduced growth compared to planktonic bacteria, which increased when the host cells were exposed to microaggregate supernatant, obtained from the incubation of MAH with HEp-2 cells. Moreover, microaggregate supernatant stimulated biofilm formation by planktonic and microaggregated bacteria. Microaggregate supernatant also induces the production of both pro- and anti-inflammatory cytokines, which was suppressed following MAH infection. The results suggest that epithelial recognition occurs during MAH infection, and the microaggregate phenotype stimulates an inflammatory response. The initial bacterial interaction with the mucosal epithelium and development of the microaggregate phenotype has a role in pathogenesis, allowing for more robust biofilm formation and infection establishment.

Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation.

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.