A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens.

Carsten Østergaard Frederiksen,Miika Leppänen,Estefania Perez-Calvo,Rual Lopez-Ulibarri,Lars Kobberøe Skov,Esben Gjerløff Wedebye Schmidt,Marianne Thorup Cohn,Mikkel Klausen,Kirk Matthew Schnorr,Steen Buskov,Ilari Maasilta

doi:10.1093/jimb/kuab008

Abstract

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Highlights

The gastrointestinal tract of animals is home to a complex microbial ecosystem that live in close interaction with host cells
Visual qualitative effects of muramidase peptidoglycan hydrolysis were investigated by helium ion microscopy for the first time
Bacterial rod and cocci shapes were recognizable, respectively, for L. gallinarum and E. gallinarum preparations– whereas peptidoglycan from the other three preparations were fragmented to a level where cocci or rod shapes were not visible

Summary

Introduction

The gastrointestinal tract of animals is home to a complex microbial ecosystem that live in close interaction with host cells. Attempts to quantify metabolic status of bacteria in the gastrointestinal tract have been reported for humans (faeces), Syrian hamsters (caeca), Arctic ground squirrels (caeca), and Rex rabbits (stomach, jejunum, ileum, colon, caecum) using flow cytometry and live/dead PCR. Caeca samples from Syrian hamsters and Arctic ground squirrels contained between 72 and 81% intact cells, 4 and 9% damaged cells, and 10 and 20% dead cells (Hatton et al, 2017; Sonoyama et al, 2009; Stevenson et al, 2014) Another study used a live/dead PCR approach to measure live and dead cells in Rex rabbits, and noted that 1–3% live cells were found in the foregut (stomach, jejunum, ileum), 25% in the caecum, and 19% in the colon. Injured cells are not quantified by live/dead PCR (Fu et al, 2018)

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Results

Conclusion