Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from Wolbachia determine M1 macrophage activation and killing of Leishmania protozoans.

Leishmaniases are severe vector-borne diseases affecting humans and animals, caused by Leishmania protozoans. Over one billion people and millions of dogs live in endemic areas for leishmaniases and are at risk of infection. Immune polarization plays a major role in determining the outcome of Leishmania infections: hosts displaying M1-polarized macrophages are protected, while those biased on the M2 side acquire a chronic infection that could develop into a deadly disease. The identification of the factors involved in M1 polarization is essential for the design of therapeutic and prophylactic interventions, including vaccines. Infection by the filarial nematode Dirofilaria immitis could be one of the factors that interfere with leishmaniasis in dogs. Indeed, filarial nematodes induce a partial skew of the immune response towards M1, likely caused by their bacterial endosymbionts, Wolbachia. Here we have examined the potential of AsaiaWSP, a bacterium engineered for the expression of the Wolbachia surface protein (WSP), as an inductor of M1 macrophage activation and Leishmania killing. Macrophages stimulated with AsaiaWSP displayed a strong leishmanicidal activity, comparable to that determined by the choice-drug amphotericin B. Additionally, AsaiaWSP determined the expression of markers of classical macrophage activation, including M1 cytokines, ROS and NO, and an increase in phagocytosis activity. Asaia not expressing WSP also induced macrophage activation, although at a lower extent compared to AsaiaWSP. In summary, the results of the present study confirm the immunostimulating properties of WSP highlighting a potential therapeutic efficacy against Leishmania parasites. Furthermore, Asaia was designed as a delivery system for WSP, thus developing a novel type of immunomodulating agent, worthy of being investigated for immuno-prophylaxis and -therapy of leishmaniases and other diseases that could be subverted by M1 macrophage activation.

Enhanced production and immunomodulatory activity of levan from the acetic acid bacterium, Tanticharoenia sakaeratensis.

Levan is a fructose polymer with ß-(2 â†’ 6) glycosidic linkages. It is produced by several microorganisms, and due to its potential biotechnological and industrial applications, various levan-producing bacteria with different levels of production efficiencies have been reported. We investigated the levan-producing ability of the acetic acid bacterium, Tanticharoenia sakaeratensis. The exopolysaccharides produced by the bacterium under a sucrose environment were characterized as levan by FT-IR, and 1H and 13C NMR. The molecular weight of levan thus produced range from 1.0 × 105-6.8 × 105 Da. The maximum yield of levan from T. sakaeratensis is 24.7 g·L-1 in a liquid medium containing 20% (w/v) sucrose and incubated at 37 °C, 250 RPM for 35 h. The levan produced by T. sakaeratensis can promote nitric oxide production in RAW264.7 macrophage cells in a concentration-dependent manner, suggesting it has immunomodulatory effects. Our study reveals that T. sakaeratensis can be potentially employed as a new source of levan for industrial applications.

Interaction Between Familial Transmission and a Constitutively Active Immune System Shapes Gut Microbiota in Drosophila melanogaster.

Resident gut bacteria are constantly influencing the immune system, yet the role of the immune system in shaping microbiota composition during an organism's life span has remained unclear. Experiments in mice have been inconclusive due to differences in husbandry schemes that led to conflicting results. We used Drosophila as a genetically tractable system with a simpler gut bacterial population structure streamlined genetic backgrounds and established cross schemes to address this issue. We found that, depending on their genetic background, young flies had microbiota of different diversities that converged with age to the same Acetobacteraceae-dominated pattern in healthy flies. This pattern was accelerated in immune-compromised flies with higher bacterial load and gut cell death. Nevertheless, immune-compromised flies resembled their genetic background, indicating that familial transmission was the main force regulating gut microbiota. In contrast, flies with a constitutively active immune system had microbiota readily distinguishable from their genetic background with the introduction and establishment of previously undetectable bacterial families. This indicated the influence of immunity over familial transmission. Moreover, hyperactive immunity and increased enterocyte death resulted in the highest bacterial load observed starting from early adulthood. Cohousing experiments showed that the microenvironment also played an important role in the structure of the microbiota where flies with constitutive immunity defined the gut microbiota of their cohabitants. Our data show that, in Drosophila, constitutively active immunity shapes the structure and density of gut microbiota.

An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.

There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.

Persistence of the bacterial pathogen Granulibacter bethesdensis in chronic granulomatous disease monocytes and macrophages lacking a functional NADPH oxidase.

Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-γ to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients.

Serologic reactivity to the emerging pathogen Granulibacter bethesdensis.

BACKGROUND: Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown. METHODS: Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH. CONCLUSIONS: These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.