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.

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.

Quantitative analysis of Mycobacterium avium subsp. hominissuis proteome in response to antibiotics and during exposure to different environmental conditions.

Mycobacterium avium subsp. hominissuis (MAH) belongs to the clinically important non-tuberculous mycobacterial group that infects immunocompromised patients and individuals with underling lung conditions. The need for prolonged therapy is a major challenge of MAH treatment, influencing the development of persistent and drug-resistant infections. The reason why bactericidal drugs take several months to eliminate MAH is unknown. To investigate MAH proteome remodeling under aerobic, anaerobic and biofilm conditions (as it is encountered in patient lungs) and identify metabolic changes potentially associated with bacterial persistent state, we performed the relative protein quantitative analysis using Tandem Mass Tag Mass Spectrometry sequencing. MAH was exposed to amikacin (4 µg/ml) and clarithromycin (16 µg/ml) under aerobic, anaerobic or biofilm condition for 24 h and the response was compared with bacterial proteomics of the corresponding conditions. Overall, 4000 proteins were identified out of 5313 MAH proteome of across all experimental groups. Numerous sets of de novo synthesized proteins belonging to metabolic pathways not evidenced in aerobic condition were found commonly enriched in both anaerobic and biofilm conditions, including pantothenate and CoA biosynthesis, glycerolipid metabolism, nitrogen metabolism and chloroalkene degradation, known to be associated with bacterial tolerance in M. tuberculosis. The common pathways observed in anaerobic and biofilm conditions following drug treatments were peptidoglycan biosynthesis, glycerophospholipid metabolism and protein export. The LprB lipoprotein, highly synthesized in MAH biofilms during drug treatments and shown to be essential for M. tuberculosis virulence and survival in vivo, was selected and overexpressed in MAH. Results demonstrate that LprB is secreted in MAH biofilms and the overexpression clone is more tolerant to antimicrobials than the wild-type strain. Our study identified promising metabolic pathways that can be targeted to prevent the bacterial tolerance mechanism and, subsequently, reduce the length of MAH therapy.

Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin.

Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

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.

Mycobacterium tuberculosis Proteome Response to Antituberculosis Compounds Reveals Metabolic "Escape" Pathways That Prolong Bacterial Survival.

Tuberculosis (TB) continues to be one of the most common bacterial infectious diseases and is the leading cause of death in many parts of the world. A major limitation of TB therapy is slow killing of the infecting organism, increasing the risk for the development of a tolerance phenotype and drug resistance. Studies indicate that Mycobacterium tuberculosis takes several days to be killed upon treatment with lethal concentrations of antibiotics both in vitro and in vivo To investigate how metabolic remodeling can enable transient bacterial survival during exposure to bactericidal concentrations of compounds, M. tuberculosis strain H37Rv was exposed to twice the MIC of isoniazid, rifampin, moxifloxacin, mefloquine, or bedaquiline for 24 h, 48 h, 4 days, and 6 days, and the bacterial proteomic response was analyzed using quantitative shotgun mass spectrometry. Numerous sets of de novo bacterial proteins were identified over the 6-day treatment. Network analysis and comparisons between the drug treatment groups revealed several shared sets of predominant proteins and enzymes simultaneously belonging to a number of diverse pathways. Overexpression of some of these proteins in the nonpathogenic Mycobacterium smegmatis extended bacterial survival upon exposure to bactericidal concentrations of antimicrobials, and inactivation of some proteins in M. tuberculosis prevented the pathogen from escaping the fast killing in vitro and in macrophages, as well. Our biology-driven approach identified promising bacterial metabolic pathways and enzymes that might be targeted by novel drugs to reduce the length of tuberculosis therapy.

Identification of Mycobacterium avium subsp. hominissuis secreted proteins using an in vitro system mimicking the phagosomal environment.

BACKGROUND: Mycobacterium avium subsp. hominissuis is a common intracellular pathogen that infects patients with HIV/AIDS and cause lung infection in patients with underlying lung pathology. M.avium preferably infects macrophages and uses diverse mechanisms to alter phagosome maturation. Once in the macrophage, the pathogen can alter the host cellular defenses by secreting proteins into the cytosol of host cells, but despite considerable research, only a few secreted effector proteins have been identified. We hypothesized that the environmental cues inside the phagosome can trigger bacterial protein secretion. To identify M. avium secretome within the phagosome, we utilized a previously established in vitro system that mimics the metal ion concentrations and pH of the M. avium phagosome. RESULTS: M. avium was exposed to phagosome metal concentrations for different time points and exported proteins were profiled and analyzed against bacterial proteins secreted in the culture medium. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 46 were unique to bacteria incubated in the metal mixture. Ten of potential effectors were selected and secretion of these proteins was monitored within M. avium infected mononuclear phagocytic cells using the beta-lactamase FRET-based reporter system. In addition, pull-down assay was performed for secreted calmodulin-like protein MAV_1356 protein to evaluate for eukaryotic target. All examined M. avium proteins were secreted into the macrophage cytosol, and gene expression analysis suggested that the metal environment likely stimulates secretion of pre-made proteins. Further investigation of bacterial secreted MAV_1356 protein, lead to the observation that the MAV_1356 interacts with the host proteins Annexin A1 and Protein S100-A8. CONCLUSIONS: We established an in vitro system for the study if proteins secreted intracellularly, and revealed that the metal mixture mimicking the concentration of metals in the phagosome environment, triggers protein secretion.

The environment of "Mycobacterium avium subsp. hominissuis" microaggregates induces synthesis of small proteins associated with efficient infection of respiratory epithelial cells.

"Mycobacterium avium subsp. hominissuis" is an opportunistic environmental pathogen that causes respiratory illness in immunocompromised patients, such as those with cystic fibrosis as well as other chronic respiratory diseases. Currently, there is no efficient approach to prevent or treat M. avium subsp. hominissuis infection in the lungs. During initial colonization of the airways, M. avium subsp. hominissuis forms microaggregates composed of 3 to 20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregate-binding protein 1 (MBP-1) (MAV_3013) is highly expressed during microaggregate formation. When expressed in noninvasive Mycobacterium smegmatis, MBP-1 increased the ability of the bacteria to bind to HEp-2 epithelial cells. Using anti-MBP-1 immune serum, microaggregate binding to HEp-2 cells was significantly reduced. By far-Western blotting, and verified by coimmunoprecipitation, we observed that MBP-1 interacts with the host cytoskeletal protein vimentin. As visualized by confocal microscopy, microaggregates, as well as MBP-1, induced vimentin polymerization at the site of bacterium-host cell contact. Binding of microaggregates to HEp-2 cells was inhibited by treatment with an antivimentin antibody, suggesting that MBP-1 expression is important for M. avium subsp. hominissuis adherence to the host cell. MBP-1 immune serum significantly inhibited M. avium subsp. hominissuis infection throughout the respiratory tracts of mice. This study characterizes a pathogenic mechanism utilized by M. avium subsp. hominissuis to bind and invade the host respiratory epithelium, suggesting new potential targets for the development of antivirulence therapy.

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model.

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFß. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.

Mycobacterium avium biofilm attenuates mononuclear phagocyte function by triggering hyperstimulation and apoptosis during early infection.

Mycobacterium avium subsp. hominissuis is an opportunistic human pathogen that has been shown to form biofilm in vitro and in vivo. Biofilm formation in vivo appears to be associated with infections in the respiratory tract of the host. The reasoning behind how M. avium subsp. hominissuis biofilm is allowed to establish and persist without being cleared by the innate immune system is currently unknown. To identify the mechanism responsible for this, we developed an in vitro model using THP-1 human mononuclear phagocytes cocultured with established M. avium subsp. hominissuis biofilm and surveyed various aspects of the interaction, including phagocyte stimulation and response, bacterial killing, and apoptosis. M. avium subsp. hominissuis biofilm triggered robust tumor necrosis factor alpha (TNF-α) release from THP-1 cells as well as superoxide and nitric oxide production. Surprisingly, the hyperstimulated phagocytes did not effectively eliminate the cells of the biofilm, even when prestimulated with gamma interferon (IFN-γ) or TNF-α or cocultured with natural killer cells (which have been shown to induce anti-M. avium subsp. hominissuis activity when added to THP-1 cells infected with planktonic M. avium subsp. hominissuis). Time-lapse microscopy and the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay determined that contact with the M. avium subsp. hominissuis biofilm led to early, widespread onset of apoptosis, which is not seen until much later in planktonic M. avium subsp. hominissuis infection. Blocking TNF-α or TNF-R1 during interaction with the biofilm significantly reduced THP-1 apoptosis but did not lead to elimination of M. avium subsp. hominissuis. Our data collectively indicate that M. avium subsp. hominissuis biofilm induces TNF-α-driven hyperstimulation and apoptosis of surveilling phagocytes, which prevents clearance of the biofilm by cells of the innate immune system and allows the biofilm-associated infection to persist.

Identification of Mycobacterium avium genes expressed during in vivo infection and the role of the oligopeptide transporter OppA in virulence.

Mycobacterium avium causes disseminated disease in patients with AIDS and other immunosuppressive conditions and pulmonary infections in individuals with chronic lung diseases. Much still need to be learn about the mechanisms of M. avium pathogenesis. Using a mouse model of disseminated M. avium disease, we applied an in vivo expression technology system and identified M. avium genes up-regulated in different organs of mice during early stage of infection. The M. avium oppA gene, involved in an active transport of oligopeptides across the cell membrane, was found highly expressed in lung, liver and spleen of mice. Mutation in the transport domain of the oppA gene resulted in bacterial attenuation in both macrophages and in mice. Using protein-protein interaction assay, it was determined that two hypothetical small proteins, MAV_2941 (73aa) and MAV_4320 (45aa), interact with OppA. MAV_2941 was shown to be secreted by the bacterium into the macrophage cytoplasm. Mutations in MAV_2941 was associated with significant impairment of growth in macrophages. Understanding the mechanisms involved in the functions of MAV_2941 and MAV_4320 is warranted.

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 tuberculosis FadD18 Promotes Proinflammatory Cytokine Secretion to Inhibit the Intracellular Survival of Bacillus Calmette-Guérin.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

No holes barred: invasion of the intestinal mucosa by Mycobacterium avium subsp. paratuberculosis.

The infection biology of Mycobacterium avium subsp. paratuberculosis has recently crystallized, with added details surrounding intestinal invasion. The involvement of pathogen-derived effector proteins such as the major membrane protein, oxidoreductase, and fibronectin attachment proteins have been uncovered. Mutations constructed in this pathogen have also shed light on genes needed for invasion. The host cell types that are susceptible to invasion have been defined, along with their transcriptional response. Recent details have given a new appreciation for the dynamic interplay between the host and bacterium that occurs at the outset of infection. An initial look at the global expression pathways of the host has shown a circumvention of the cell communication pathway by M. avium subsp. paratuberculosis, which loosens the integrity of the tight junctions. We now know that M. avium subsp. paratuberculosis activates the epithelial layer and also actively recruits macrophages to the site of infection. These notable findings are summarized along with added mechanistic details of the early infection model. We conclude by proposing critical next steps to further elucidate the process of M. avium subsp. paratuberculosis invasion.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Surface-exposed proteins of pathogenic mycobacteria and the role of cu-zn superoxide dismutase in macrophages and neutrophil survival.

Pathogenic mycobacteria are important agents causing human disease. Mycobacterium avium subsp. hominissuis (M. avium) is a species of recalcitrant environmental pathogen. The bacterium forms robust biofilms that allow it to colonize and persist in austere environments, such as residential and commercial water systems. M. avium is also an opportunistic pathogen that is a significant source of mortality for immune-compromised individuals. Proteins exposed at the bacterial surface play a central role in mediating the relationship between the bacterium and its environment. The processes underlying both biofilm formation and pathogenesis are directly dependent on this essential subset of the bacterial proteome. Therefore, the characterization of the surface-exposed proteome is an important step towards an improved understanding of the mycobacterial biology and pathogenesis. Here we examined the complement of surface exposed proteins from Mycobacterium avium 104, a clinical isolate and reference strain of Mycobacterium avium subsp. hominissuis. To profile the surface-exposed proteins of viable M. avium 104, bacteria were covalently labeled with a membrane impermeable biotinylation reagent and labeled proteins were affinity purified via the biotin-streptavidin interaction. The results provide a helpful snapshot of the surface-exposed proteome of this frequently utilized reference strain of M. avium. A Cu-Zn SOD knockout mutant, MAV_2043, a surface identified protein, was evaluated regarding its role in the survival in both macrophages and neutrophils.

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.

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.