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.

Induction of killing of Mycobacterium avium subsp. hominissuis in macrophages by cytokine stimulated innate-like lymphoid cells is negatively affected by the pathogen.

Mycobacterium avium subsp. hominissuis (MAH) is a common environmental bacterium that causes infection in immunocompromised patients such as those with HIV/AIDS, or patients with chronic lung disease such as cystic fibrosis. There are many strains of MAH with varying levels of virulence. Infection with MAH strains 100 and 104 has been associated with different immune responses in mice and outcome of the disease. While MAH 100 infection tends to be cleared from mice, MAH 104 is virulent and grows in host tissue. What is currently unknown are the mechanisms related to this difference in host defense and virulence. Our hypothesis is that differences in circulating innate lymphocytes response are associated with increased protection from infection. Innate lymphoid cells (ILC) are lymphoid cells with an important role in regulation of innate immune systems. ILCs can be categorized into three subpopulations ILC1, ILC2, and ILC3 based on their cytokine production and regulatory transcription factors. Investigation was carried out on how macrophage anti-MAH response change depending on activation by primary mouse lymphocytes activated with IL-12, IL-33, and IL-23, triggering differentiation into ILC-like subpopulations. Our results do not affirm the role of any one ILC subpopulation in macrophage anti-M. avium ability. Our findings instead support the conclusion that MAH infection of macrophages suppresses the stimulatory function of ILCs.

Pyruvate Supports RET-Dependent Mitochondrial ROS Production to Control Mycobacterium avium Infection in Human Primary Macrophages.

Macrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium (M. avium) infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we showed that macrophages infected with M. avium switch to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle volume or changes in the energy charge. Instead, we found that pyruvate import contributes to hyperpolarization of mitochondria in infected cells and increases production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduces the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.

The Tumor Necrosis Factor Alpha and Interleukin 6 Auto-paracrine Signaling Loop Controls Mycobacterium avium Infection via Induction of IRF1/IRG1 in Human Primary Macrophages.

Macrophages sense and respond to pathogens by induction of antimicrobial and inflammatory programs to alert other immune cells and eliminate the infectious threat. We have previously identified the transcription factor IRF1 to be consistently activated in macrophages during Mycobacterium avium infection, but its precise role during infection is not clear. Here, we show that tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) autocrine/paracrine signaling contributes to controlling the intracellular growth of M. avium in human primary macrophages through activation of IRF1 nuclear translocation and expression of IRG1, a mitochondrial enzyme that produces the antimicrobial metabolite itaconate. Small interfering RNA (siRNA)-mediated knockdown of IRF1 or IRG1 increased the mycobacterial load, whereas exogenously provided itaconate was bacteriostatic at high concentrations. While the overall level of endogenous itaconate was low in M. avium-infected macrophages, the repositioning of mitochondria to M. avium phagosomes suggests a mechanism by which itaconate can be delivered directly to M. avium phagosomes in sufficient quantities to inhibit growth. Using mRNA hybridization, we further show that uninfected bystander cells actively contribute to the resolution of infection by producing IL-6 and TNF-α, which, via paracrine signaling, activate IRF1/IRG1 and strengthen the antimicrobial activity of infected macrophages. This mechanism contributes to the understanding of why patients on anti-inflammatory treatment, e.g., with tocilizumab or infliximab, can be more susceptible to mycobacterial disease. IMPORTANCE The prevalence of lung diseases caused by nontuberculous mycobacteria, such as Mycobacterium avium, is increasing in countries where tuberculosis is not endemic, most likely because of an aging population that is immunocompromised from underlying disease or immunosuppressive therapy. Our study contributes to the understanding of mycobacterial survival and killing in human macrophages and, more broadly, to the impact of immunometabolism during infection. We show evidence of an antimicrobial program in human primary macrophages where activation of the transcription factor IRF1 and expression of the mitochondrial enzyme IRG1 restrict the intracellular growth of M. avium, possibly by directed delivery of itaconate to M. avium phagosomes. The study also sheds light on why patients on immunosuppressive therapy are more susceptible to mycobacterial infections, since TNF-α and IL-6 contribute to driving the described antimycobacterial program.

Anti-TNF-α agents Modulate SARS-CoV-2 Receptors and Increase the Risk of Infection Through Notch-1 Signaling.

Although millions of patients with underlining conditions are treated primarily with anti-TNF-α agents, little is known about the safety of this standard therapy during the coronavirus disease-2019 (COVID-19) pandemic. In this study, we investigated the effect of anti-TNF-α monoclonal antibodies on the cellular entry mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and increasing the risk of COVID-19 development. We focused on the expression of angiotensin-converting enzyme II (ACE2), type II transmembrane serine proteases (TMPRSS2)/TNF-α converting enzyme (TACE) ratio. We also investigated the involvement of Notch-1 signaling and its downstream influence on IL-6, myeloid cell leukemia sequence-1(MCL-1) in the anti-TNF-α mode of action and increased the susceptibility to Mycobacterium avium subspecies paratuberculosis (MAP) infection. Surprisingly, anti-TNF-α downregulated ACE2 expression by 0.46-fold and increased TMPRSS2/TACE ratio by 44% in THP-1 macrophages. Treatment of macrophages with rIL-6 also downregulated ACE2 and increased TMPRSS2/TACE ratio by 54%. Interestingly, anti-TNF-α treatment upregulated Notch-1, IL-6, and MCL-1 by 1.3, 1.2, and 1.9-fold, respectively, and increased viability and burden of MAP infection in macrophages. Blocking Notch signaling doubled ACE2 expression, decreased TMPRSS2/TACE ratio by 38%, and reduced MAP viability by 56%. In a small group of patients, ACE2 level was significantly lower in the plasma from rheumatoid arthritis (RA) patients on anti-TNF-α treatment compared to healthy control. The data in this critical study demonstrated that through Notch-1/IL-6 signaling, anti-TNF-α agents decreased ACE2 expression and shedding through TMPRSS2/TACE modulation and increased the susceptibility to infection. Overall, this study warns against anti-TNF-α therapy in some patients with underlining inflammatory conditions during the COVID-19 pandemic. The findings should impact current guidelines regarding treatment decisions of patients on anti-TNF-α during the COVID-19 pandemic.

H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection.

During infections, the host redistributes iron in order to starve pathogens from this nutrient. Several proteins are involved in iron absorption, transport, and storage. Ferritin is the most important iron storage protein. It is composed of variable proportions of two peptides, the L- and H-ferritins (FTL and FTH). We previously showed that macrophages increase their expression of FTH1 when they are infected in vitro with Mycobacterium avium, without a significant increase in FTL. In this work, we investigated the role of macrophage FTH1 in M. avium infection in vivo. We found that mice deficient in FTH1 in myeloid cells are more resistant to M. avium infection, presenting lower bacterial loads and lower levels of proinflammatory cytokines than wild-type littermates, due to the lower levels of available iron in the tissues. Importantly, we also found that FTH1 produced by myeloid cells in response to infection may be found in circulation and that it plays a key role in iron redistribution. Specifically, in the absence of FTH1 in myeloid cells, increased expression of ferroportin is observed in liver granulomas and increased iron accumulation occurs in hepatocytes. These results highlight the importance of FTH1 expression in myeloid cells for iron redistribution during infection.

Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

No effect of inoculation site and injection device on the skin test response of red deer to the intradermal injection of Mycobacterium avium-derived purified protein derivative (PPD).

Mycobacterial diseases are important health issues in farmed deer. The single intradermal tuberculin test is the standard test for tuberculosis testing in deer. We studied two factors which might influence the response of deer to skin testing: the inoculation site and the injection device. Deer included in this study were 2.5 years old farmed red deer (Cervus elaphus) hinds (n = 80). Two areas of 3 cm × 3 cm were shaved at the left side of the neck. Site A (SA) was situated about 10 cm caudal to the head, while site B (SB) was 10 cm caudal to SA. All hinds received at the same time two 0.1 ml inoculations of Mycobacterium avium derived purified protein derivative (aPPD). One inoculation was made by syringe and the other one with the needle-free syringe Dermojet. To test the inoculation site effect, half of the animals were inoculated by Dermojet in SA and by syringe in SB to compare with the inoculation in SA by syringe and Dermojet in SB in the other half. No differences were recorded for the injection device nor for the inoculation site. Ten hinds had a skinfold increase larger than 30 tenths of mm by any injection device and inoculation site. Seven (9%) and 6 (8%) hinds were classified as positive by syringe and Dermojet, and at the anterior or posterior inoculation site, respectively. The distribution of skinfold thickness increases did not differ by injection device. Our findings support the needle-free Dermojet syringe as a suitable tool for skin-testing in red deer and suggest no relevant effect of the position of the inoculation site along the neck in red deer.

Levels of miR-125a-5p are altered in Mycobacterium avium-infected macrophages and associate with the triggering of an autophagic response.

Macrophages are major pathogen-killing cells. Mycobacteria can represent a serious threat to human health, in particular Mycobacterium tuberculosis and, less so, the opportunistic Mycobacterium avium. They can cause disseminated infections because of their capacity to survive and proliferate within macrophage phagolysosomes. Accumulating evidence indicates that the regulation of miRNA expression is implicated in the mechanisms of defense of macrophages against mycobacterial infections. Nevertheless, the precise contribution of miRNAs is largely unknown. The present study analyzes the expression profile of miRNAs during M. avium infection of macrophages by means of microarrays. We detected that the levels of 23 miRNAs were significantly changed ≥2.5-fold 24 h after M. avium infection. In particular, MiR-125a-5p was found to be highly expressed as part of the known immunological response of macrophages to bacterial or viral infections. MiR-125a-5p overexpression inhibited the expression of target signal transducers and activators of transcription 3 (STAT3) in THP-1 cells. Conversely, inhibitors of miR-125a-5p had the opposite effect. Silencing of STAT3 significantly enhanced the level of autophagy in both uninfected and M. avium-infected cells. Overexpression of miR-125a-5p significantly increased autophagy and decreased M. avium survival within THP-1 cells. Instead, co-transfection with miR-125a-5p mimic and a human STAT3 expressing construct reversed the effects: autophagy decreased and intracellular bactericidal survival was improved. Taken together, our findings indicate that miR-125a-5p participates in the regulation of innate host defenses by targeting STAT3 and enhancing autophagy levels. The results reported here contribute to a better understanding of host defense mechanisms against mycobacterial infections and offer some clues about their control.

Fate of internalized Campylobacter jejuni and Mycobacterium avium from encysted and excysted Acanthamoeba polyphaga.

Association of the water- and foodborne pathogen Campylobacter jejuni with free-living Acanthamoeba spp. trophozoites enhances C. jejuni survival and resistance to biocides and starvation. When facing less than optimal environmental conditions, however, the Acanthamoeba spp. host can temporarily transform from trophozoite to cyst and back to trophozoite, calling the survival of the internalized symbiont and resulting public health risk into question. Studies investigating internalized C. jejuni survival after A. castellanii trophozoite transformation have neither been able to detect its presence inside the Acanthamoeba cyst after encystation nor to confirm its presence upon excystation of trophozoites through culture-based techniques. The purpose of this study was to detect C. jejuni and Mycobacterium avium recovered from A. polyphaga trophozoites after co-culture and induction of trophozoite encystation using three different encystation methods (Neff's medium, McMillen's medium and refrigeration), as well as after cyst excystation. Internalized M. avium was used as a positive control, since studies have consistently detected the organism after co-culture and after host excystation. Concentrations of C. jejuni in A. polyphaga trophozoites were 4.5 × 105 CFU/ml, but it was not detected by PCR or culture post-encystation. This supports the hypothesis that C. jejuni may be digested during encystation of the amoebae. M. avium was recovered at a mean concentration of 1.9 × 104 from co-cultured trophozoites and 4.4 × 101 CFU/ml after excystation. The results also suggest that M. avium recovery post-excystation was statistically significantly different based on which encystation method was used, ranging from 1.3 × 101 for Neff's medium to 5.4 × 101 CFU/ml for refrigeration. No M. avium was recovered from A. polyphaga cysts when trophozoites were encysted by McMillen's medium. Since C. jejuni internalized in cysts would be more likely to survive harsh environmental conditions and disinfection, a better understanding of potential symbioses between free-living amoebae and campylobacters in drinking water distribution systems and food processing environments is needed to protect public health. Future co-culture experiments examining survival of internalized C. jejuni should carefully consider the encystation media used, and include molecular detection tools to falsify the hypothesis that C. jejuni may be present in a viable but not culturable state.

Genetic Variation/Evolution and Differential Host Responses Resulting from In-Patient Adaptation of Mycobacterium avium.

Members of the Mycobacterium avium complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within M.avium isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that M. avium accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted M. avium strains. The study revealed the rapid genetic evolution of M. avium in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates.

Aerosolization of Mycobacterium avium and Mycobacterium abscessus from a household ultrasonic humidifier.

PURPOSE: To measure the aerosolization of Mycobacterium avium subspecies hominissuis and Mycobacterium abscessus subspecies abscessus from ultrasonic humidifiers. METHODOLOGY: An ultrasonic humidifier was filled with sterile tap water and inoculated with water-acclimated cells of either the M. avium or M. abscessus strains to achieve a range of densities similar to those of mycobacteria found in drinking waters. During operation of the humidifier, aerosols were collected using an Andersen 6-Stage Cascade Sampler. RESULTS: Cells of the M. avium and M. abscessus strains were readily aerosolized and recovered in particles (1-5 µm diameter); small enough to enter the furthest reaches of the human lung. Aerosolization of M. abscessus was significantly reduced in the presence of a normal drinking water bacterial flora. Significantly greater numbers of M. avium cells were aerosolized from high-density suspensions (1200 c.f.u. ml-1), than from low-density (120 c.f.u. ml-1) and very low-density (12 c.f.u. ml-1) suspensions. CONCLUSIONS: This report documents the potential for M. avium subspecies hominissuis and M. abscessus subspecies abscessus cells in drinking water to be aerosolized from one type of portable humidifier; an ultrasonic humidifier. Care should be taken in using an ultrasonic humidifier where an individual at risk for mycobacterial pulmonary disease could be exposed.

Mycobacteria produce proteins involved in biofilm formation and growth-affecting processes.

The aim of this study was to determine the effect of mycobacterial proteins on mycobacterial biofilm formation and growth processes. We separated growth-affecting proteins (GEPs) from wild type of Mycobacterium bovis and ATCC strain of Mycobacterium avium subsp. avium. Our results showed that these mycobacteria-secreted GEPs are involved in biofilm formation, growth stimulatory, and inhibitory processes. Our findings suggest that GEP stimulated M. avium subsp. avium growth in vitro. Stimulation process was observed in mycobacteria affected with GEP extracted from M. avium subsp. avium. We found that both GEPs inhibited the growth of the M. bovis. Optical density measurement and visual analysis confirm that GEP plays an important role in biofilm formation process. Most of M. bovis GEP are associated with the type VII secretion and general secretion pathways. Our results contribute to a better understanding of the mechanisms underlying mycobacterial biofilm formation and growth-affecting processes and better characterization of mycobacterial proteins and their functions. It is noteworthy that this finding represents the first demonstration of GEP-mediated growth effects on a solid and liquid medium.

Response of the respiratory mucosal cells to mycobacterium avium subsp. Hominissuis microaggregate.

Mycobacterium avium: subsp. hominissuis (MAH) is an opportunistic pathogen that commonly infects immunocompromised individuals. Recently, we described an invasive phenotypic change MAH undergoes when incubated with lung airway epithelial host cells for 24 h, which is accompanied with microaggregate formation in vitro. The microaggregate phenotype also resulted in higher colonization in the lungs of mice early during infection. Previously, we identified genes highly regulated during microaggregate formation and further characterized the function of two highly upregulated bacterial proteins, mycobacterial binding protein-1 (MBP-1) and mycobacterial inversion protein-1 (MIP-1), which were found to be involved in binding and invasion of the respiratory mucosa. While these studies are valuable in understanding the pathogenesis of MAH, they primarily investigated the bacteria during microaggregate infection without commenting on the differences in the host response to microaggregate and planktonic infection. The bacteria-host interaction between microaggregates and epithelial cells was examined in a variety of assays. Using a transwell polarized epithelial cell model, microaggregates translocated through the monolayer more efficiently than planktonic bacteria at set timepoints. In addition, during infection with microaggregate and planktonic bacteria, host phosphorylated proteins were identified revealing differences in immune response, glutathione synthesis, and apoptosis. The host immune response was further investigated by measuring pro-inflammatory cytokine secretion during microaggregate and planktonic infection of BEAS-2B bronchial epithelial cells. The epithelial cells secreted more CCL5 during infection with microaggregates suggesting that this chemokine may play an important role during microaggregate invasion. Subsequent experiments showed that microaggregates are formed more efficiently in the presence of CCL5, suggesting that MAH had evolved a strategy to use the host response in its benefit. Collectively, this study establishes the different nature of infection by planktonic bacteria and microaggregates.

Cattle infection response network and its functional modules.

BACKGROUND: Weighted Gene Co-expression Network analysis, a powerful technique used to extract co-expressed gene pattern from mRNA expression data, was constructed to infer common immune strategies used by cattle in response to five different bacterial species (Escherichia coli, Mycobacterium avium, Mycobacterium bovis, Salmonella and Staphylococcus aureus) and a protozoa (Trypanosoma Congolense) using 604 publicly available gene expression microarrays from 12 cattle infection experiments. RESULTS: A total of 14,999 transcripts that were differentially expressed (DE) in at least three different infection experiments were consolidated into 15 modules that contained between 43 and 4441 transcripts. The high number of shared DE transcripts between the different types of infections indicated that there were potentially common immune strategies used in response to these infections. The number of transcripts in the identified modules varied in response to different infections. Fourteen modules showed a strong functional enrichment for specific GO/pathway terms related to "immune system process" (71%), "metabolic process" (71%), "growth and developmental process" (64%) and "signaling pathways" (50%), which demonstrated the close interconnection between these biological pathways in response to different infections. The largest module in the network had several over-represented GO/pathway terms related to different aspects of lipid metabolism and genes in this module were down-regulated for the most part during various infections. Significant negative correlations between this module's eigengene values, three immune related modules in the network, and close interconnection between their hub genes, might indicate the potential co-regulation of these modules during different infections in bovine. In addition, the potential function of 93 genes with no functional annotation was inferred based on neighbor analysis and functional uniformity among associated genes. Several hypothetical genes were differentially expressed during experimental infections, which might indicate their important role in cattle response to different infections. CONCLUSIONS: We identified several biological pathways involved in immune response to different infections in cattle. These findings provide rich information for experimental biologists to design experiments, interpret experimental results, and develop novel hypothesis on immune response to different infections in cattle.

The Voltage-Dependent Anion Channels (VDAC) of Mycobacterium avium phagosome are associated with bacterial survival and lipid export in macrophages.

Mycobacterium avium subsp. hominissuis is associated with infection of immunocompromised individuals as well as patients with chronic lung disease. M. avium infects macrophages and actively interfere with the host killing machinery such as apoptosis and autophagy. Bacteria alter the normal endosomal trafficking, prevent the maturation of phagosomes and modify many signaling pathways inside of the macrophage by secreting effector molecules into the cytoplasm. To investigate whether M. avium needs to attach to the internal surface of the vacuole membrane before releasing efferent molecules, vacuole membrane proteins were purified and binding to the surface molecules present in intracellular bacteria was evaluated. The voltage-dependent anion channels (VDAC) were identified as components of M. avium vacuoles in macrophages. M. avium mmpL4 proteins were found to bind to VDAC-1 protein. The inactivation of VDAC-1 function either by pharmacological means or siRNA lead to significant decrease of M. avium survival. Although, we could not establish a role of VDAC channels in the transport of known secreted M. avium proteins, we demonstrated that the porin channels are associated with the export of bacterial cell wall lipids outside of vacuole. Suppression of the host phagosomal transport systems and the pathogen transporter may serve as therapeutic targets for infectious diseases.

Systemic mycobacteriosis caused by 'Mycobacterium avium subspecies hominissuis' in a 14-month-old Japanese black beef steer.

A 14-month-old Japanese black beef steer presented with severe chronic diarrhea and emaciation and was euthanized. Postmortem examination showed thickened and corrugated intestinal mucosa and enlarged granulomatous mesenteric lymph nodes with caseating necrosis. Numerous epithelioid cells and multinucleated giant cells infiltrated in the lamina propria and the submucosal tissue of the intestines. These cells were also observed in the systemic organs. Many acid-fast bacilli were detected in the cytoplasm of these cells and were identified as 'Mycobacterium avium subsp. hominissuis' (Mah) on the basis of the results of molecular examinations and immunohistochemistry. These findings indicate that Mah can cause systemic mycobacteriosis, and this unique infection needs to be distinguished from Johne's disease and tuberculosis in cattle.

Effects of nutritional and ambient oxygen condition on biofilm formation in Mycobacterium avium subsp. hominissuis via altered glycolipid expression.

Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of nontuberculous mycobacteriosis, the representative case of environment-related infectious diseases the incidence of which is increasing in industrialized countries. MAH is found in biofilm in drinking water distribution system and residential environments. We investigated the effect of gaseous and nutritional conditions, and the role of glycopeptidolipids (GPLs) on biofilm-like pellicle formation in MAH. Pellicle formation was observed under 5% oxygen in Middlebrook 7H9 broth containing 0.2% glycerol and 10% albumin-dextrose-catalase enrichment but not under normoxia or in nutrient-poor media. An analysis of 17 environmental isolates revealed that hypoxia (5% oxygen) preferentially enhanced pellicle formation both in plastic plates and in glass tubes, compared with hypercapnia (5% carbon dioxide). Wild-type strains (WT) developed much thicker pellicles than GPL-deficient rough mutants (RM). WT bacterial cells distributed randomly and individually in contrast to that RM cells positioned linearly in a definite order. Exogenous supplementation of GPLs thickened the pellicles of RM, resulting in a similar morphological pattern to WT. These data suggest a significant implication of eutrophication and hypoxia in biofilm-like pellicle formation, and a functional role of GPLs on development of pellicles in MAH.

Comparative genome analyses of Mycobacterium avium reveal genomic features of its subspecies and strains that cause progression of pulmonary disease.

Pulmonary disease caused by nontuberculous mycobacteria (NTM) is increasing worldwide. Mycobacterium avium is the most clinically significant NTM species in humans and animals, and comprises four subspecies: M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS), M. avium subsp. paratuberculosis (MAP), and M. avium subsp. hominissuis (MAH). To improve our understanding of the genetic landscape and diversity of M. avium and its role in disease, we performed a comparative genome analysis of 79 M. avium strains. Our analysis demonstrated that MAH is an open pan-genome species. Phylogenetic analysis based on single nucleotide variants showed that MAH had the highest degree of sequence variability among the subspecies, and MAH strains isolated in Japan and those isolated abroad possessed distinct phylogenetic features. Furthermore, MAP strains, MAS and MAA strains isolated from birds, and many MAH strains that cause the progression of pulmonary disease were grouped in each specific cluster. Comparative genome analysis revealed the presence of genetic elements specific to each lineage, which are thought to be acquired via horizontal gene transfer during the evolutionary process, and identified potential genetic determinants accounting for the pathogenic and host range characteristics of M. avium.

Mycobacterium avium MAV2054 protein induces macrophage apoptosis by targeting mitochondria and reduces intracellular bacterial growth.

Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.