Rothia from the Human Nose Inhibit Moraxella catarrhalis Colonization with a Secreted Peptidoglycan Endopeptidase.

Moraxella catarrhalis is found almost exclusively within the human respiratory tract. This pathobiont is associated with ear infections and the development of respiratory illnesses, including allergies and asthma. Given the limited ecological distribution of M. catarrhalis, we hypothesized that we could leverage the nasal microbiomes of healthy children without M. catarrhalis to identify bacteria that may represent potential sources of therapeutics. Rothia was more abundant in the noses of healthy children compared to children with cold symptoms and M. catarrhalis. We cultured Rothia from nasal samples and determined that most isolates of Rothia dentocariosa and "Rothia similmucilaginosa" were able to fully inhibit the growth of M. catarrhalis in vitro, whereas isolates of Rothia aeria varied in their ability to inhibit M. catarrhalis. Using comparative genomics and proteomics, we identified a putative peptidoglycan hydrolase called secreted antigen A (SagA). This protein was present at higher relative abundance in the secreted proteomes of R. dentocariosa and R. similmucilaginosa than in those from non-inhibitory R. aeria, suggesting that it may be involved in M. catarrhalis inhibition. We produced SagA from R. similmucilaginosa in Escherichia coli and confirmed its ability to degrade M. catarrhalis peptidoglycan and inhibit its growth. We then demonstrated that R. aeria and R. similmucilaginosa reduced M. catarrhalis levels in an air-liquid interface culture model of the respiratory epithelium. Together, our results suggest that Rothia restricts M. catarrhalis colonization of the human respiratory tract in vivo. IMPORTANCE Moraxella catarrhalis is a pathobiont of the respiratory tract, responsible for ear infections in children and wheezing illnesses in children and adults with chronic respiratory diseases. Detection of M. catarrhalis during wheezing episodes in early life is associated with the development of persistent asthma. There are currently no effective vaccines for M. catarrhalis, and most clinical isolates are resistant to the commonly prescribed antibiotics amoxicillin and penicillin. Given the limited niche of M. catarrhalis, we hypothesized that other nasal bacteria have evolved mechanisms to compete against M. catarrhalis. We found that Rothia are associated with the nasal microbiomes of healthy children without Moraxella. Next, we demonstrated that Rothia inhibit M. catarrhalis in vitro and on airway cells. We identified an enzyme produced by Rothia called SagA that degrades M. catarrhalis peptidoglycan and inhibits its growth. We suggest that Rothia or SagA could be developed as highly specific therapeutics against M. catarrhalis.

Biogeography of Bacterial Communities and Specialized Metabolism in Human Aerodigestive Tract Microbiomes.

The aerodigestive tract (ADT) is the primary portal through which pathogens and other invading microbes enter the body. As the direct interface with the environment, we hypothesize that the ADT microbiota possess biosynthetic gene clusters (BGCs) for antibiotics and other specialized metabolites to compete with both endogenous and exogenous microbes. From 1,214 bacterial genomes, representing 136 genera and 387 species that colonize the ADT, we identified 3,895 BGCs. To determine the distribution of BGCs and bacteria in different ADT sites, we aligned 1,424 metagenomes, from nine different ADT sites, onto the predicted BGCs. We show that alpha diversity varies across the ADT and that each site is associated with distinct bacterial communities and BGCs. We identify specific BGC families enriched in the buccal mucosa, external naris, gingiva, and tongue dorsum despite these sites harboring closely related bacteria. We reveal BGC enrichment patterns indicative of the ecology at each site. For instance, aryl polyene and resorcinol BGCs are enriched in the gingiva and tongue, which are colonized by many anaerobes. In addition, we find that streptococci colonizing the tongue and cheek possess different ribosomally synthesized and posttranslationally modified peptide BGCs. Finally, we highlight bacterial genera with BGCs but are underexplored for specialized metabolism and demonstrate the bioactivity of Actinomyces against other bacteria, including human pathogens. Together, our results demonstrate that specialized metabolism in the ADT is extensive and that by exploring these microbiomes further, we will better understand the ecology and biogeography of this system and identify new bioactive natural products. IMPORTANCE Bacteria produce specialized metabolites to compete with other microbes. Though the biological activities of many specialized metabolites have been determined, our understanding of their ecology is limited, particularly within the human microbiome. As the aerodigestive tract (ADT) faces the external environment, bacteria colonizing this tract must compete both among themselves and with invading microbes, including human pathogens. We analyzed the genomes of ADT bacteria to identify biosynthetic gene clusters (BGCs) for specialized metabolites. We found that the majority of ADT BGCs are uncharacterized and the metabolites they encode are unknown. We mapped the distribution of BGCs across the ADT and determined that each site is associated with its own distinct bacterial community and BGCs. By further characterizing these BGCs, we will inform our understanding of ecology and biogeography across the ADT, and we may uncover new specialized metabolites, including antibiotics.

IL12 Abrogates Calcineurin-Dependent Immune Evasion during Leukemia Progression.

Exploitation of the immune system has emerged as an important therapeutic strategy for acute lymphoblastic leukemia (ALL). However, the mechanisms of immune evasion during leukemia progression remain poorly understood. We sought to understand the role of calcineurin in ALL and observed that depletion of calcineurin B (CnB) in leukemia cells dramatically prolongs survival in immune-competent but not immune-deficient recipients. Immune-competent recipients were protected from challenge with leukemia if they were first immunized with CnB-deficient leukemia, suggesting robust adaptive immunity. In the bone marrow (BM), recipients of CnB-deficient leukemia harbored expanded T-cell populations as compared with controls. Gene expression analyses of leukemia cells extracted from the BM identified Cn-dependent significant changes in the expression of immunoregulatory genes. Increased secretion of IL12 from CnB-deficient leukemia cells was sufficient to induce T-cell activation ex vivo, an effect that was abolished when IL12 was neutralized. Strikingly, recombinant IL12 prolonged survival of mice challenged with highly aggressive B-ALL. Moreover, gene expression analyses from children with ALL showed that patients with higher expression of either IL12A or IL12B exhibited prolonged survival. These data suggest that leukemia cells are dependent upon calcineurin for immune evasion by restricting the regulation of proinflammatory genes, particularly IL12. SIGNIFICANCE: This report implicates calcineurin as an intracellular signaling molecule responsible for immune evasion during leukemia progression and raises the prospect of re-examining IL12 as a therapeutic in leukemia.