Bacterial Debris – Peptidoglycans and Their Influence on Gut Functionality

Introduction

Although bacteria are too small to be seen without the aid of a microscope, their abundance by mass has been estimated to be 1,166 times larger than the mass of all humans. All animals contain populations of bacteria on outer and inner body surfaces such as the skin and the gastrointestinal tract, with the gastrointestinal tract being by far the most densely populated. It has been estimated that cattle, sheep, goats, pigs, chickens, ducks and turkey contain between 2.1 × 10¹⁰ and 3.2 × 10¹¹ bacteria per gram of intestinal wet contents (Bar-On, Phillips and Milo, 2018; Whitman, Coleman and Wiebe, 1998).

The bacterial ecosystem in the gastrointestinal tract closely interact with the host and has received tremendous attention by researchers in recent decades. Despite all the mechanisms discovered to date the field remains at the beginning of an era with respect to solutions that can manipulate the ecosystem to the benefit of gastrointestinal functionality. One number illustrating the complexity of the bacterial gastrointestinal community is that it contains an estimated 150 times more unique genes than its host (Cani et al., 2019).

In an early report of bacterial gut ecosystem manipulation, Italian anatomist and surgeon Acquapendente (1537-1619) coined “transfaunation” when he transferred gastrointestinal contents from healthy to sick animals to improve gastrointestinal functionality. Many animals practice coprophagia, leading to transfer of beneficial and pathogenic bacterial populations between animals (de Groot et al., 2017).

The most widely used research tools to describe changes in the bacterial ecosystem (which bacteria are present) and metabolic potential (which genes are present) are based on sequencing the phylogenetic marker gene 16S ribosomal RNA or genome DNA. These tools are extremely powerful but fail to answer basic questions about physiological status or if the bacteria are dead or alive (Maurice, Haiser and Turnbaugh, 2013; Ben-Amor et al., 2005). Another limitation of the widely applied sequencing tools is that they cannot quantitate the components of bacterial cell debris in the gut. The differentiation between live cells, injured cells, dead cell and cell debris is important because each unity will interact with the host differently.

Live cells produce metabolites that can interact with the host, two examples being short chain fatty acids and branched chain amino acids. Another way live cells interact with metabolism is through the release of bacterial cell structure components during cell division that can affect host metabolism by interacting with the immune system. Here, two examples are lipopolysaccharides (endotoxin) and peptidoglycan. Upon bacterial death, molecules are released that otherwise are hidden in the cytoplasm or cell wall of the living cell. Many of these molecules are unique to bacteria and recognized by the innate immune system. Well known examples are LPS, peptidoglycan, teichoic acid and unmethylated CpG DNA motifs (Stewart-Tull, 1980; Akira, Uematsu and Takeuchi, 2006). The impact of dead bacterial components on host response have been demonstrated for both gram-negative and gram-positive bacteria through changes in body temperature as a response to intravenous infusion in rabbits. In one example with gram-positive bacteria (that does not have the potent pyrogen LPS in the cell wall), fever developed to a similar level for either dead or live bacteria (Kluger and Matthew, 2015).

Abundance of live and dead bacteria in the gastrointestinal tract

Only six studies have reported the relative abundance of live and dead cells in the gastrointestinal tract (Figure 1). Two human studies have looked at fecal sample live/dead/injured cell ratio by fluorescence activated cell sorting. The first study from 2005 found 49% intact cells 19% damaged cell and 32 % dead cells. The second study found 56% intact cells, 27% damaged cells and 17% dead cells, and observed a wide range of variation between individuals. Dead bacterial % was in the range of 6% - 41% (Maurice, Haiser and Turnbaugh, 2013; Ben-Amor et al., 2005). Cecal samples from Syrian hamsters and arctic ground squirrels contained 72% - 81% intact cells, 4% - 9% damaged cells, and 10% - 20% dead cells ((Hatton et al., 2017; Stevenson, Duddleston and Buck, 2014; Sonoyama et al., 2009). Yet 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 cecum and 19% in the colon. Injured cells are not quantified by live/dead PCR (Fu et al., 2018).

Figure 1 Literature estimates of percent live, injured and dead cell in intestinal samples from human, hamster, squirrel and rabbit. Green bars show % live cells, yellow bars show percent injured cells and grey cells show % dead cell as estimated by flow cytometry (human/hamster/squirrel) and live/dead PCR (rabbit)

DNA independent methods are needed to quantify amounts of debris from cells that are decomposed to a level where DNA is no longer is contained within the cell wall. One major component of bacterial cell debris is the key structural cell wall polymer peptidoglycan. Peptidoglycan’s role for the bacterial cell is to protect cell integrity against tugor pressure, and build cell shape as well as being an anchor point for other cell envelope components such as proteins and teichoic acid (Vollmer, 2008). As peptidoglycan and some of its buildings blocks are uniquely found in the bacterial cell walls, it also functions as a host immune system recognition target (Stewart-Tull, 1980). The uniqueness of peptidoglycan and, more specifically, the sugar component muramic acid – has made it a biomarker target to estimate bacterial abundance in soil, as well as in cattle feces (Joergensen, 2018; Jost et al., 2013).

Quantification of soluble and total peptidoglycan in the gastrointestinal tract

Literature reports on the physiological effects of enzymatic hydrolysis of peptidoglycan from cell debris in the gastrointestinal tract of chickens prompted us to determine if muramic acid could be used as an indicator of peptidoglycan hydrolysis (Lichtenberg et al., 2017; Goodarzi Boroojeni et al., 2019; Sais et al., 2019). Peptidoglycan is the only source of muramic acid in the intestinal tract.

Hydrolyzed peptidoglycan in vitro is more soluble than intact peptidoglycan. The principle of measuring enzyme hydrolysis by quantifying substrate solubilization in digesta samples has successfully measured the effect of carbohydrases (Choct et al., 2004; Rosenthal and Dziarski, 1994).

Briefly, total muramic acid was determined in freeze dried digesta samples similarly as in soil science (Joergensen, 2018). In addition, soluble muramic acid was measured in the supernatant from a slurry of freeze dried digesta from the sample used for determination of total muramic acid. The ratio of soluble peptidoglycan as a % of total was used as a measure of peptidoglycan hydrolysis.

Analysis of chicken intestinal samples from the crop, jejunum and caecum confirmed that a supplemented muramidase (dose 45,000 LSU(F)/kg) increased the relative abundance of soluble peptidoglycan compared to intact peptidoglycan (Figure 2). In crop, soluble peptidoglycan as a % of total increased from 37% (n=12) in control group to 46% (n=9) in muramidase supplemented group (n=9). In jejunum, soluble peptidoglycan as a % of total increased from 32% (n=11) in the control group to 66% (n=11) in the muramidase supplemented group. In cecum, soluble peptidoglycan as a % of total increased from 9% (n=10) in control group to 16% (n=12) in muramidase supplemented group.

Figure 2 Soluble peptidoglycan, % of total, in chicken crop, jejunum and caecum intestinal samples. Muramidase supplemented diets have significant higher soluble peptidoglycan, % of total. Bars with different letters A and B are significant different, within each intestinal segment in a student’s t-test (p = 0.05).

Soluble peptidoglycan, % of total, in chicken crop, jejunum and caecum intestinal samples. Muramidase supplemented diets have significant higher soluble peptidoglycan, % of total. Bars with different letters A and B are significant different, within each intestinal segment in a student’s t-test (p = 0.05).

A first step towards understanding the role of the bacterial macromolecule peptidoglycan in the gastrointestinal tract is to develop analytical methods for its quantification. Here, we find that muramic acid in the digesta can estimate the ratio between soluble and total peptidoglycan in the gut. This provides new knowledge about the interaction of peptidoglycan hydrolysis with gastrointestinal functionality and animal physiology.

Published in the proceedings of the Animal Nutrition Conference of Canada 2020. For information on the event, past and future editions, check out https://animalnutritionconference.ca/.