Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Immunological and microbial shifts in the aging rhesus macaque lung during nontuberculous mycobacterial infection.

Nontuberculous mycobacteria (NTM) are environmentally ubiquitous organisms that predominately cause NTM pulmonary disease (NTMPD) in individuals over the age of 65. The incidence of NTMPD has increased in the U.S., exceeding that of Mycobacterium tuberculosis. However, the mechanisms leading to higher susceptibility and severity of NTMPD with aging are poorly defined in part due to the lack of animal models that accurately recapitulate human disease. Here, we compared bacterial load, microbial communities, and host responses longitudinally between three young (two female and one male) and two aged (two female) rhesus macaques inoculated with Mycobacterium avium subsp. hominissuis (MAH) in the right caudal lobe. Unilateral infection resulted in a low bacterial load in both young and aged animals confined to the infected side. Although a robust inflammatory response was only observed in the inoculated lung, immune cell infiltration and antigen-specific T cells were detected in both lungs. Computed tomography, gross pathology, and histopathology revealed increased disease severity and persistence of bacterial DNA in aged animals. Additional analyses showed the translocation of gut and oral-pharyngeal bacterial DNA into the lower respiratory microbiome. Finally, single-cell RNA sequencing revealed a heightened inflammatory response to MAH infection by alveolar macrophages in aged animals. These data are consistent with the model that increased disease severity in the aged is mediated by a dysregulated macrophage response that may be sustained through persistent antigen presence. IMPORTANCE: Nontuberculous mycobacteria (NTM) are emerging as pathogens of high consequence, as cases of NTM pulmonary disease (NTMPD) have exceeded those of Mycobacterium tuberculosis. NTMPD can be debilitating, particularly in patients over 65 years of age, as it causes chronic cough and fatigue requiring prolonged treatments with antibiotics. The underlying mechanisms of this increased disease severity with age are poorly understood, hampering the development of therapeutics and vaccines. Here, we use a rhesus macaque model to investigate the impact of age on host-NTM interactions. This work shows that aging is associated with increased disease severity and bacterial persistence in aged rhesus macaques, thus providing a preclinical model to develop and test novel therapeutics and interventions.

Positive Culture of Atypical Mycobacterium Avium Following Revision Total Knee Arthroplasty.

CASE: We report a rare case of mycobacterial periprosthetic joint infection (PJI) after primary total knee arthroplasty 14 years earlier. Progressive knee pain over three years with a negative PJI infectious workup led to revision total knee arthroplasty. A surprising result was isolation of Mycobacterium avium from tissue cultures taken at time of revision surgery. After six months of antibiotic treatment, the patient is alive with well- functioning pain-free TKA at over one-year follow-up. CONCLUSION: Periprosthetic joint infection can present acutely or chronically years following total knee arthroplasty. Depending on the infecting organism, patients can present with sepsis, or a more indolent slower course that mimics aseptic loosening. In the absence of positive pre-operative labs and cultures, and based on the Musculoskeletal Infection Society (MSIS) criteria, aseptic loosening is a diagnosis of exclusion. An atypical infectious organism should be considered a possible cause and may require specialized cultures of operative specimens.

Diagnostic advancements: Isolating Mycobacterium avium ssp. paratuberculosis and unveiling its molecular identity with nested-PCR.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis, which is currently prevalent in many parts of Iran and produces severe economic loss. It is hence necessary to identify and isolate the animals infected with this bacterium, so this research aimed to isolate MAP from milk and fecal samples of ELISA-positive animals and determine the molecular identity of isolates. After performing ELISA on 3,700 bovine blood samples, 115 samples of milk and feces were taken from ELISA-positive cattle and were cultured on Herald's egg yolk medium with and without mycobactin-J and then the acid-fastness of positive samples was determined using Ziehl-Neelsen staining. The 16S rRNA-PCR test was performed after DNA extraction to determine the molecular identity of isolates. Primers IS6110 and IS901 were employed to ensure that the isolates were not related to members of M. tuberculosis complex and  M. avium, respectively. Primer IS900 was also used to determine the molecular identity of MAP isolates. Also, expression levels of MAP-related genes (IS900, ISMAP02, F57, MAP2191, MAP4027) were evaluated via qPCR. Finally, positive samples were confirmed based on the Nested-PCR. Results showed that a total of 9 isolates were obtained from the culture of 90 ELISA-positive samples. The results revealed that all grown samples were positive for acid-fastness. The 16S rRNA-PCR test revealed the 543 bp band, which confirms the presence of Mycobacterium in the samples. The PCR test with Primer IS900 generated the 398 bp fragment in the first step and the 298 bp fragment in the second step, indicating the presence of MAP in samples. Also, relative expression analysis revealed that MAP-related genes were significantly higher in ELIZA-positive samples than in negative ones. Based on the study findings, it can be concluded that MAP-infected animals can be identified by ELISA. In addition, mycobacterium can be isolated by culturing the samples on appropriate media and then its molecular identity can be determined by using nested-PCR.

Mavintramycin A is a promising antibiotic for treating Mycobacterium avium complex infectious disease.

Mycobacterium avium complex (MAC) is a serious disease that is mainly caused by infection with the non-tuberculous mycobacteria (NTM), Mycobacterium avium and Mycobacterium intracellulare. Seven new compounds, designated mavintramycins A-G (1-7), were isolated along with structurally related compounds, including amicetin (9) and plicacetin (10), from the culture broth of Streptomyces sp. OPMA40551 as anti-MAC compounds that were active against M. avium and M. intracellulare. Among them, mavintramycin A showed the most potent and selective inhibition of M. avium and M. intracellulare. Furthermore, mavintramycin A was active against more than 40 clinically isolated M. avium, including multidrug-resistant strains, and inhibited the growth of M. avium in a persistent infection cell model using THP-1 macrophages. Mavintramycin A also exhibited in vivo efficacy in silkworm and mouse infection assays with NTM. An experiment to elucidate its mechanism of action revealed that mavintramycin A inhibits protein synthesis by binding to 23S ribosomal RNA in NTM. Mavintramycin A, with a different chemical structure from those of clinically used agents, is a promising drug candidate for the treatment of MAC infectious disease.

Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

Evaluation of nucleotide MALDI-TOF-MS for the identification of Mycobacterium species.

Background: The accurate identification of the Mycobacterium tuberculosis complex (MTBC) and different nontuberculous mycobacteria (NTM) species is crucial for the timely diagnosis of NTM infections and for reducing poor prognoses. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been extensively used for microbial identification with high accuracy and throughput. However, its efficacy for Mycobacterium species identification has been less studied. The objective of this study was to evaluate the performance of nucleotide MALDI-TOF-MS for Mycobacterium species identification. Methods: A total of 933 clinical Mycobacterium isolates were preliminarily identified as NTM by the MPB64 test. These isolates were identified by nucleotide MALDI-TOF-MS and Sanger sequencing. The performance of nucleotide MALDI-TOF MS for identifying various Mycobacterium species was analyzed based on Sanger sequencing as the gold standard. Results: The total correct detection rate of all 933 clinical Mycobacterium isolates using nucleotide MALDI-TOF-MS was 91.64% (855/933), and mixed infections were detected in 18.65% (174/933) of the samples. The correct detection rates for Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, MTBC, Mycobacterium gordonae, and Mycobacterium massiliense were 99.32% (585/589), 100% (86/86), 98.46% (64/65), 94.59% (35/37), 100.00% (34/34), 95.65% (22/23), and 100% (19/19), respectively. For the identification of the MTBC, M. intracellulare, M. abscessus, M. kansasii, M. avium, M. gordonae, and M. massiliense, nucleotide MALDI-TOF-MS and Sanger sequencing results were in good agreement (k > 0.7). Conclusion: In conclusion, nucleotide MALDI-TOF-MS is a promising approach for identifying MTBC and the most common clinical NTM species.

Sudapyridine (WX-081) antibacterial activity against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium chelonae in vitro and in vivo.

Sudapyridine (WX-081) is a structural analog of bedaquiline (BDQ), which shows anti-tuberculosis and non-tuberculous mycobacteria (NTM) activities but, unlike BDQ, did not prolong QT interval in animal model studies. This study evaluated the antibacterial activity of this novel compound against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium chelonae in vitro and in vivo. The minimum inhibitory concentration (MIC) of WX-081 against three kinds of non-tuberculous mycobacteria (NTM) clinical strains was determined using microplate-based alamarBlue assay (MABA), and the antibacterial activity of WX-081 against NTM in J774A.1 cells and mice was evaluated. MIC ranges of WX-081 against clinical strains of M. avium and M. abscessus were 0.05-0.94 µg/mL, 0.88-7.22 µg/mL (M. abscessus subsp. abscessus), and 0.22-8.67 µg/mL (M. abscessus subsp. massiliense), respectively, which were slightly higher than those of BDQ. For M. avium, M. abscessus, and M. chelonae, WX-081 can reduce the intracellular bacterial load by 0.13-1.18, 0.18-1.50, and 0.17-1.03 log10 colony forming units (CFU)/mL, respectively, in a concentration-dependent manner. WX-081 has bactericidal activity against three NTM species in mice. WX-081 exhibited anti-NTM activity to the same extent as BDQ both in vivo and in vitro. WX-081 is a promising clinical candidate and should be studied further in clinical trials. IMPORTANCE: Due to the rapidly increased cases globally, non-tuberculous mycobacteria (NTM) disease has become a significant public health problem. NTM accounted for 11.57% of all mycobacterial isolates in China, with a high detection rate of Mycobacterium abscessus, Mycobacterium avium, and Mycobacterium chelonae during 2000-2019. Treatment of NTM infection is often challenging, as natural resistance to most antibiotics is quite common among different NTM species. Hence, identifying highly active anti-NTM agents is a priority for potent regimen establishment. The pursuit of new drugs to treat multidrug-resistant tuberculosis may also identify some agents with strong activity against NTM. Sudapyridine (WX-081) is a structural analog of bedaquiline (BDQ), which was developed to retain the anti-tuberculosis efficacy but eliminates the severe side effects of BDQ. This study initially evaluated the antimicrobial activity of this novel compound against M. avium, M. abscessus, and M. chelonae in vitro, in macrophages and mice, respectively.

Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

The pan-genome of Mycobacterium avium subsp. paratuberculosis (Map) confirms ancestral lineage and reveals gene rearrangements within Map Type S.

BACKGROUND: To date genomic studies on Map have concentrated on Type C strains with only a few Type S strains included for comparison. In this study the entire pan-genome of 261 Map genomes (205 Type C, 52 Type S and 4 Type B) and 7 Mycobacterium avium complex (Mac) genomes were analysed to identify genomic similarities and differences between the strains and provide more insight into the evolutionary relationship within this Mycobacterial species. RESULTS: Our analysis of the core genome of all the Map isolates identified two distinct lineages, Type S and Type C Map that is consistent with previous phylogenetic studies of Map. Pan-genome analysis revealed that Map has a larger accessory genome than Mycobacterium avium subsp. avium (Maa) and Type C Map has a larger accessory genome than Type S Map. In addition, we found large rearrangements within Type S strains of Map and little to none in Type C and Type B strains. There were 50 core genes identified that were unique to Type S Map and there were no unique core genes identified between Type B and Type C Map strains. In Type C Map we identified an additional CE10 CAZyme class which was identified as an alpha/beta hydrolase and an additional polyketide and non-ribosomal peptide synthetase cluster. Consistent with previous analysis no plasmids and only incomplete prophages were identified in the genomes of Map. There were 45 hypothetical CRISPR elements identified with no associated cas genes. CONCLUSION: This is the most comprehensive comparison of the genomic content of Map isolates to date and included the closing of eight Map genomes. The analysis revealed that there is greater variation in gene synteny within Type S strains when compared to Type C indicating that the Type C Map strain emerged after Type S. Further analysis of Type C and Type B genomes revealed that they are structurally similar with little to no genetic variation and that Type B Map may be a distinct clade within Type C Map and not a different strain type of Map. The evolutionary lineage of Maa and Map was confirmed as emerging after M. hominissuis.

The role of rifampicin within the treatment of Mycobacterium avium pulmonary disease.

Rifampicin is recommended for the treatment of Mycobacterium avium complex pulmonary disease alongside azithromycin and ethambutol. We evaluated the azithromycin-ethambutol backbone with and without rifampicin in an intracellular hollow fiber model and performed RNA sequencing to study the differences in adaptation. In an in vitro hollow fiber experiment, we simulated epithelial lining fluid pharmacokinetic profiles of the recommended 3-drug (rifampicin, ethambutol, and azithromycin) or a 2-drug (ethambutol and azithromycin) treatment. THP-1 cells infected with M. avium ATCC700898 were exposed to these regimens for 21 days. We determined intra- and extra-cellular bacterial load- and THP-1 cell densities on days 0, 3, 7, 14, and 21, alongside RNA sequencing. The emergence of macrolide resistance was studied by inoculating intra- and extra-cellular fractions of azithromycin-containing Middlebrook 7H10 agar plates. Complete pharmacokinetic profiles were determined at days 0 and 21. Both therapies maintained stasis of both intra- and extra-cellular bacterial populations for 3 days, whilst regrowth coinciding with the emergence of a macrolide-resistant subpopulation was seen after 7 days. THP-1 cell density remained static. Similar transcriptional profiles were observed for both therapies that were minimally influenced by exposure duration. Transcriptional response was slightly larger during 2-drug treatment. Rifampicin did not add to the antimycobacterial effect to the 2-drug therapy or suppression of emergence resistance. RNA transcription was not greatly altered by the addition of rifampicin, which may be due to strong transcriptional influence of azithromycin and host cells. This questions the role of rifampicin in the currently recommended therapy. These findings should be confirmed in clinical trials.

Cure of mycobacterium avium keratitis caused by trauma in elderly: case report.

We report a case of Mycobacterium avium keratitis, first detected in the laboratory, which is from an 81-year-old female patient with a 13-year history of recurrent keratitis after eye injuries. This patient underwent anterior chamber irrigation of the right eye, and autologous conjunctival flap covering plus medication, and the corneal ulcer healed. She recovered well after continuing with the medication for half a year. The patient was not immune-compromised. Complex eye diseases such as blurred vision and cataracts caused by advanced age, delayed symptoms caused by slow growth of Mycobacterium avium and low-grade inflammation, difficulty with laboratory culture, repeated medication use, and repeated illnesses were the main reasons for the prolonged failure to clarify the etiology of this case. Morphological examination provided important clues for the initial discovery of pathogenic bacteria. Remind to pay attention to the morphology of Mycobacterium under Gram staining and Rui's Giemsa staining. Acid fast staining and Deoxyribonucleic Acid(DNA) microarray chip method can be used as indicators for rapid diagnosis of Mycobacterium species.

Shift of human pathogen community composition and their potential human health risk after supply suspension in tap water.

Water supply suspension-restoration can occur frequently due to the overhauling of civil infrastructure in developing countries and the shutdown of commercial buildings during the pandemic. For comprehensive insights into the effects of water supply suspension-restoration, this study characterized the variations of the pathogen community composition of the tap water and their infection risk under different water supply scenarios. Metagenomic sequencing revealed a significant change of the human pathogen profiles, among which the most dominant pathogen changed from Pseudomonas aeruginosa (4.91%) to Acinetobacter johnsonii (0.59%). Furthermore, absolute quantification of pathogens by propidium-monoazide-qPCR revealed that the abundance of the three typical pathogens (Pseudomonas aeruginosa, Mycobacterium avium and Salmonella sp.) showed an increase of 2.44 log to 3.60 log immediately after water supply suspension-restoration and did not return to the normal level even after 2-h supply restoration, except for Pseudomonas aeruginosa. Quantitative microbial risk assessment suggested the infection risks of the three pathogens arising from direct utilization of tap water under stable water supply, including dermal exposure and oral intake, were all above the threshold of 10-4, and evidently increased after water supply suspension-restoration. This study warns us against the risk induced by the pathogens in tap water, especially after water supply suspension-restoration.

Identification of peptides presented through the MHC-II of dendritic cells stimulated with Mycobacterium avium.

Mycobacterium avium (M. avium) represents a species of concern, because of its ability to modulate the host's innate immune response, and therefore influence trajectory of adaptative immunity. Since eradicative response against mycobacteria, and M. tuberculosis/M. avium, relies on peptides actively presented on a Major Histocompatibility complex-II (MHC-II) context, we assessed paradoxical stimulation of Dendritic Cell resulting on immature immunophenotype characterized by membrane minor increase of MHC-II and CD40 despite of high expression of the pro-inflammatory tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in supernatants. Identification of M. avium leucine rich peptides forming short α-helices shutting down Type 1T helper (Th1), contribute to the understanding of immune evasion of an increasingly prevalent pathogen, and may provide a basis for future immunotherapy to infectious and non-infectious disease.

The potential role of drug transporters and amikacin modifying enzymes in M. avium.

OBJECTIVES: Mycobacterium avium (M. avium) complex bacteria cause opportunistic infections in humans. Treatment yields cure rates of 60% and consists of a macrolide, a rifamycin, and ethambutol, and in severe cases, amikacin. Mechanisms of antibiotic tolerance remain mostly unknown. Therefore, we studied the contribution of efflux and amikacin modification to antibiotic susceptibility. METHODS: We characterised M. avium ABC transporters and studied their expression together with other transporters following exposure to clarithromycin, amikacin, ethambutol, and rifampicin. We determined the effect of combining the efflux pump inhibitors berberine, verapamil and CCCP (carbonyl cyanide m-chlorophenyl hydrazone), to study the role of efflux on susceptibility. Finally, we studied the modification of amikacin by M. avium using metabolomic analysis. RESULTS: Clustering shows conservation between M. avium and M. tuberculosis and transporters from most bacterial subfamilies (2-6, 7a/b, 10-12) were found. The largest number of transporter encoding genes was up-regulated after clarithromycin exposure, and the least following amikacin exposure. Only berberine increased the susceptibility to clarithromycin. Finally, because of the limited effect of amikacin on transporter expression, we studied amikacin modification and showed that M. avium, in contrast to M. abscessus, is not able to modify amikacin. CONCLUSION: We show that M. avium carries ABC transporters from all major families important for antibiotic efflux, including homologues shown to have affinity for drugs included in treatment. Efflux inhibition in M. avium can increase susceptibility, but this effect is efflux pump inhibitor- and antibiotic-specific. Finally, the lack of amikacin modifying activity in M. avium is important for its activity.

Long non-coding RNA-XLOC_002383 enhances the inhibitory effects of THP-1 macrophages on Mycobacterium avium and functions as a competing endogenous RNA by sponging miR-146a-5p to target TRAF6.

The morbidity associated with infection by Mycobacterium avium (M. avium), a type of non-tuberculous mycobacteria (NTM), has increased in recent years due to infections that are easily missed, and thus, difficult to diagnose and treat. Here, we reported that miR-146a-5p was highly expressed, and XLOC_002383 and TRAF6 were downregulated in a time- and MOI-dependent manner in THP-1 macrophages infected with M. avium. In macrophages obtained from peripheral blood mononuclear cells, the expression levels of XLOC_002383 and TRAF6 were also decreased, and miR-146a-5p expression was increased following 24 h of infection with M. avium. miR-146a-5p was a target of XLOC_002383 and TRAF6 mRNA was a target of miR-146a-5p, and XLOC_002383 regulated TRAF6 expression by adsorbing miR-146a-5p, and further increased IL-6, TNF-α, IL-1ß and iNOS levels in THP-1 macrophages. The results of qPCR and CFU assays indicated that XLOC_002383 decreased the intracellular M. avium loads. Overall, the present study demonstrated that XLOC_002383 may function as a competing endogenous RNA and interacts with miR-146a-5p to increase THP-1 macrophage inflammatory factors and microbicidal mediators iNOS. This enhanced the inhibitory effects of THP-1 macrophages on M. avium, which improved the understanding of the pathogenesis and host defenses in the process of NTM infectious diseases.

Protective RNA nanovaccines against Mycobacterium avium subspecies hominissuis.

The induction of an effective immune response is critical for the success of mRNA-based therapeutics. Here, we developed a nanoadjuvant system compromised of Quil-A and DOTAP (dioleoyl 3 trimethylammonium propane), hence named QTAP, for the efficient delivery of mRNA vaccine constructs into cells. Electron microscopy indicated that the complexation of mRNA with QTAP forms nanoparticles with an average size of 75 nm and which have ~90% encapsulation efficiency. The incorporation of pseudouridine-modified mRNA resulted in higher transfection efficiency and protein translation with low cytotoxicity than unmodified mRNA. When QTAP-mRNA or QTAP alone transfected macrophages, pro-inflammatory pathways (e.g., NLRP3, NF-kb, and MyD88) were upregulated, an indication of macrophage activation. In C57Bl/6 mice, QTAP nanovaccines encoding Ag85B and Hsp70 transcripts (QTAP-85B+H70) were able to elicit robust IgG antibody and IFN- É£, TNF-α, IL-2, and IL-17 cytokines responses. Following aerosol challenge with a clinical isolate of M. avium ss. hominissuis (M.ah), a significant reduction of mycobacterial counts was observed in lungs and spleens of only immunized animals at both 4- and 8-weeks post-challenge. As expected, reduced levels of M. ah were associated with diminished histological lesions and robust cell-mediated immunity. Interestingly, polyfunctional T-cells expressing IFN- É£, IL-2, and TNF- α were detected at 8 but not 4 weeks post-challenge. Overall, our analysis indicated that QTAP is a highly efficient transfection agent and could improve the immunogenicity of mRNA vaccines against pulmonary M. ah, an infection of significant public health importance, especially to the elderly and to those who are immune compromised.

Metabolic pathways that permit Mycobacterium avium subsp. hominissuis to transition to different environments encountered within the host during infection.

Introduction: M. avium subsp. hominissuis (M. avium) is an intracellular, facultative bacterium known to colonize and infect the human host through ingestion or respiratory inhalation. The majority of pulmonary infections occur in association with pre- existing lung diseases, such as bronchiectasis, cystic fibrosis, or chronic obstructive pulmonary disease. M. avium is also acquired by the gastrointestinal route in immunocompromised individuals such as human immunodeficiency virus HIV-1 patients leading to disseminated disease. A hallmark of M. avium pulmonary infections is the ability of pathogen to form biofilms. In addition, M. avium can reside within granulomas of low oxygen and limited nutrient conditions while establishing a persistent niche through metabolic adaptations. Methods: Bacterial metabolic pathways used by M. avium within the host environment, however, are poorly understood. In this study, we analyzed M. avium proteome with a focus on core metabolic pathways expressed in the anaerobic, biofilm and aerobic conditions and that can be used by the pathogen to transition from one environment to another. Results: Overall, 3,715 common proteins were identified between all studied conditions and proteins with increased synthesis over the of the level of expression in aerobic condition were selected for analysis of in specific metabolic pathways. The data obtained from the M. avium proteome of biofilm phenotype demonstrates in enrichment of metabolic pathways involved in the fatty acid metabolism and biosynthesis of aromatic amino acid and cofactors. Here, we also highlight the importance of chloroalkene degradation pathway and anaerobic fermentationthat enhance during the transition of M. avium from aerobic to anaerobic condition. It was also found that the production of fumarate and succinate by MAV_0927, a conserved hypothetical protein, is essential for M. avium survival and for withstanding the stress condition in biofilm. In addition, the participation of regulatory genes/proteins such as the TetR family MAV_5151 appear to be necessary for M. avium survival under biofilm and anaerobic conditions. Conclusion: Collectively, our data reveal important core metabolic pathways that M. avium utilize under different stress conditions that allow the pathogen to survive in diverse host environments.