Comprehensive comparative genomics reveals over 50 phyla of free-living and pathogenic bacteria are associated with diverse members of the amoebozoa.

The Amoebozoa, a group containing predominantly amoeboid unicellular protists has been shown to play an important ecological role in controlling environmental bacteria. Amoebozoans not only graze bacteria but also serve as a safe niche for bacterial replication and harbor endosymbiotic bacteria including dangerous human pathogens. Despite their importance, only a few lineages of Amoebozoa have been studied in this regard. In this research, we conducted a comprehensive genomic and transcriptomic study with expansive taxon sampling by including representatives from the three known clades of the Amoebozoa. We used culture independent whole culture and single cell genomics/transcriptomics to investigate the association of bacteria with diverse amoebozoans. Relative to current published evidence, we recovered the largest number of bacterial phyla (64) and human pathogen genera (51) associated with the Amoebozoa. Using single cell genomics/transcriptomics we were able to determine up to 24 potential endosymbiotic bacterial phyla, some potentially endosymbionts. This includes the majority of multi-drug resistant pathogens designated as major public health threats. Our study demonstrates amoebozoans are associated with many more phylogenetically diverse bacterial phyla than previously recognized. It also shows that all amoebozoans are capable of harboring far more dangerous human pathogens than presently documented, making them of primal public health concern.

Transglutaminases Are Active in Perivascular Adipose Tissue.

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model to test interactions of amines and TGs. We hypothesized that transglutaminases are active in PVAT. Real time RT-PCR determined that Sprague Dawley rat aortic, superior mesenteric artery (SMA), and mesenteric resistance vessel (MR) PVATs express TG2 and blood coagulation Factor-XIII (FXIII) mRNA. Consistent with this, immunohistochemical analyses support that these PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs when in a catalyzing calcium solution. Both TG2 and FXIII were active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT. Finally, experimentally added NE competitively inhibited incorporation of BAP into MRPVAT adipocytes. Further studies to determine the identity of amidated proteins will give insight into how these enzymes contribute to functions of PVAT and, ultimately, blood pressure.