Select streptococci can degrade Candida mannan to facilitate growth

Background: Multiple studies have found that streptococci have a synergistic relationship with Candida species, but the details of these interactions are still being discovered. Candida species are covered by mannan, a polymer of mannose, which could serve as a carbon source for certain microbes. We hypothesized that streptococci that possess mannan-degrading glycosyl hydrolases would also be able to enzymatically cleave mannose residues, which could serve as a primary carbohydrate source to support growth. Methods & Results: We analyzed 90 streptococci genomes to predict the capability of streptococci to transport and utilize mannose and to degrade diverse mannose-linkages found on mannan. The genome analysis revealed mannose transporters and downstream pathways in most streptococci, but only <50% of streptococci harbored the glycosyl hydrolases required for mannan degradation. To confirm the ability of streptococci to use mannose or mannan, we grew 6 representative streptococci in a chemically defined media lacking glucose supplemented with mannose, yeast extract or purified mannan isolated from Candida and Saccharomyces strains. Although all tested Streptococcus strains could use mannose, S. salivarius and S. agalactiae , which did not possess mannan-degrading glycosyl hydrolases, could not use yeast extract or mannan to enhance their growth. In contrast, we found that S. mitis , S. parasanguinis, S. sanguinis , and S. pyogenes possessed the necessary glycosyl hydrolases to use yeast extract and isolated mannan, which promoted robust growth. Conclusions : Our data indicate that several streptococci are capable of degrading fungal mannans and harvesting mannose for energy. Importance: This work highlights a previously undescribed aspect of streptococcal- Candida interactions. Our work identifies that certain streptococci possess the enzymes required to degrade mannan and through this mechanism, they can release mannose residues from the cell wall of fungal species and use them as a nutrient source. We speculate that streptococci that can degrade fungal mannan may have a competitive advantage for colonization. This finding has broad implications for human health as streptococci and Candida are found at multiple body sites.

The people behind the papers – Masaki Kinoshita, Toshihiro Kobayashi, Hiroshi Nagashima, Ramiro Alberio and Austin Smith

The ability to derive and maintain pluripotent stem cells (PSCs) from livestock species in defined media conditions will contribute to many new research avenues, including comparative embryology and xenotransplantation. In a new paper in Development, Masaki Kinoshita, Toshihiro Kobayashi, Hiroshi Nagashima, Ramiro Alberio, Austin Smith and colleagues describe their three-component medium, which supports long-term propagation of PSCs in the absence of feeders or serum factors. We caught up with the authors to find out more about their research and their future plans.

De novo biosynthesis of para-nitro-L-phenylalanine in Escherichia coli

Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 µM in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block.HighlightsPara-nitro-L-phenylalanine (pN-Phe) is a valuable small molecule for its applications in genetic code expansion.We establish de novo biosynthesis of pN-Phe from glucose in E. coli, which is also the first example of a de novo pathway design for an unnatural but commonly used non-standard amino acid.We show the first use of an N-oxygenase enzyme in the de novo synthesis of a nitroaromatic product.Screening of natural N-oxygenases and strain engineering resulted in final pN-Phe titers of 820 ± 130 µM in shake flask experiments with rich defined media.

Preparation of Single Cell Suspension from Human Lung Tissue v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from human lung sections of about two grams. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Isolation of Nucleated Cells from Whole Blood v1

This protocol describes a method for the isolation of pan-lymphocytes and pan-myeloid cells from human whole blood. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Lymph Node Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from the epithelial layer and the lamina propria of human gut sections of about one gram of tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples. This protocol can be used for any section of the intestinal tract from duodenum to distal colon.

Preparation of Single Cell Suspension from Human Spleen Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of a Single Cell Suspension from Bronchoalevolar Lavage v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from lavage fluid collected from human lung. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.