Searching for tryptase in the RBL-2H3 mast cell model: Preparation for comparative mast cell toxicology studies with zebrafish.

Mast cells comprise a physiologically and toxicologically important cell type that is ubiquitous among species and tissues. Mast cells undergo degranulation, in which characteristic intracellular granules fuse with the plasma membrane and release many bioactive substances, such as enzymes ß-hexosaminidase and tryptase. Activity of mast cells in the toxicology model organism, zebrafish, has been monitored via tryptase release and cleavage of substrate N-α-benzoyl-dl-Arg-p-nitroanilide (BAPNA). An extensively used in vitro mast cell model for studying toxicant mechanisms is the RBL-2H3 cell line. However, instead of tryptase, granule contents such as ß-hexosaminidase have usually been employed as RBL-2H3 degranulation markers. To align RBL-2H3 cell toxicological studies to in vivo mast cell studies using zebrafish, we aimed to develop an RBL-2H3 tryptase assay. Unexpectedly, we discovered that tryptase release from RBL-2H3 cells is not detectable, using BAPNA substrate, despite optimized assay that can detect as little as 1 ng tryptase. Additional studies performed with another substrate, tosyl-Gly-Pro-Lys-pNA, and with an enzyme-linked immunosorbent assay, revealed a lack of tryptase protein released from stimulated RBL-2H3 cells. Furthermore, none of the eight rat tryptase genes (Tpsb2, Tpsab1, Tpsg1, Prss34, Gzmk, Gzma, Prss29, Prss41) is expressed in RBL-2H3 cells, even though all are found in RBL-2H3 genomic DNA and even though ß-hexosaminidase mRNA is constitutively expressed. Therefore, mast cell researchers should utilize ß-hexosaminidase or another reliable marker for RBL-2H3 degranulation studies, not tryptase. Comparative toxicity testing in RBL-2H3 cells in vitro and in zebrafish mast cells in vivo will require use of a degranulation reporter different from tryptase.

The LCP Family Protein, Psr, Is Required for Cell Wall Integrity and Virulence in Streptococcus agalactiae.

A robust cell envelope is the first line of protection for an infecting pathogen when encountering the immune defense of its host. In Gram-positive organisms, LytR-CpsA-Psr (LCP) family proteins play a major role in the synthesis and assembly of the cell envelope. While these proteins could be considered for potential new drug targets, not enough is known about how they function to support the integrity of the cell wall. Streptococcus agalactiae (group B streptococcus or GBS) is known to encode at least three LCP family proteins, including CpsA, LytR (BrpA) and Psr. Using strains of GBS that have mutations in two of the three LCP proteins, we were able to determine a role for these proteins in GBS cell wall integrity. The results presented here demonstrate that the absence of Psr results in a decreased growth rate, decreased viability over time, inconsistent cocci morphology and diminished cell wall integrity, as well as an increased penicillin susceptibility, decreased capsule levels and attenuation in virulence in a zebrafish model of infectious disease. A strain that is missing two of the LCP family proteins, CpsA and Psr, exhibits an increase in these defective phenotypes, indicating that CpsA and Psr are partially redundant in function.