Intracellular glycogen accumulation by human gut commensals as a niche adaptation trait

ABSTRACT

The human gut microbiota is a key contributor to host metabolism and physiology, thereby impacting in various ways on host health. This complex microbial community has developed many metabolic strategies to colonize, persist and survive in the gastrointestinal environment. In this regard, intracellular glycogen accumulation has been associated with important physiological functions in several bacterial species, including gut commensals. However, the role of glycogen storage in shaping the composition and functionality of the gut microbiota offers a novel perspective in gut microbiome research. Here, we review what is known about the enzymatic machinery and regulation of glycogen metabolism in selected enteric bacteria, while we also discuss its potential impact on colonization and adaptation to the gastrointestinal tract. Furthermore, we survey the presence of such glycogen biosynthesis pathways in gut metagenomic data to highlight the relevance of this metabolic trait in enhancing survival in the highly competitive and dynamic gut ecosystem.

GRAPHICAL ABSTRACT

KEYWORDS:

• glycogen metabolism
• commensal bacteria
• gut microbiota
• colonization factors
• bacteria-host interactions
• microbiome
• carbohydrate-active enzymes

Introduction

The human gastrointestinal tract (GIT) contains a vast, complex and dynamic microbial community, termed the gut microbiota.^1,2 This microbial population acts as a key contributor to host metabolism and physiology, and for this reason impacts on host health.^3–5 For example, elements of the gut microbiota stimulate proliferation and differentiation of intestinal epithelial cells (IECs) assuring efficient nutrient absorption, thereby positively contributing to human metabolism.⁶ Other beneficial metabolic functions of the gut microbiota include: (i) regulation of iron, copper, magnesium and manganese availability,^7,8 (ii) the fermentative conversion of non-digestible food components such as dietary fibers into short chain fatty acids (SCFAs), that act as an energy source for IECs and elicit immune modulatory effects on T_regs and resident macrophages, (iii) the degradation of toxic compounds, and (iv) the production of vitamins (for example vitamin K and certain B vitamins).^9–12 Furthermore, the presence of commensal bacteria has been shown to provide a barrier to intestinal infections.^13,14

Gut microbiota studies are currently highly topical in the fields of microbiology, biotechnology, immunology and clinical medicine, while they have also raised awareness of the importance of intestinal health among the general public. Numerous primary research publications and reviews have covered aspects of the gut microbiota and its potential role in supporting and promoting human health, especially during early life,¹⁵ a period during which the microbiota clearly contributes to the development of the infant gut and its associated immune system.^16–20 The gut microbiota is also known to impact on the risk of contracting certain diseases that are typically associated with adult life, such as cardiometabolic disorders, inflammatory bowel diseases, neuropsychiatric diseases and cancer.³ Therefore, study of the gut microbiota and its associated host benefits holds a lot of promise in terms of promoting human health and well-being.

The human colon is one of the most densely populated microbial habitats known.^21,22 Bacteria make up the majority of living microbes present in the human GIT and by inference are believed to be the main contributors to the metabolic activities that take place in the gut.^23,24 Bacterial gut inhabitants mainly belong to the phyla Bacteroidota, Bacillota, Pseudomonadota, Actynomicetota, Fusobacteriota and Verrucomicrobiota.^23,25,26 It is generally accepted that microbial colonization of the GIT commences at birth; the composition of this early human gut microbiota demonstrates remarkable dynamism that gradually develops complex metabolic functions.²⁷ In contrast, in adults the microbiota is more stable and resilient, though may fluctuate in response to environmental factors, while maintaining core taxa for many years.²⁸

Determining the forces that shape our gut microbiota is fundamental to our understanding of the functioning of these intestinal communities.² Central to this goal is gaining detailed information on the ecology of our gut microbiota and the mechanisms by which the individual components of this microbiota first establish themselves and then persist in their particular niche.²⁹ The intracellular accumulation of glucose-containing polymers, such as glycogen, is an energy storage that has been associated with important physiological functions in several bacterial species. However, its role in influencing the presence and functionality of (specific components of) the gut microbiota in its habitat has not yet been thoroughly addressed.

Here, we critically review recent insights into the enzymatic machinery that certain gut bacteria use to accumulate and subsequently degrade intracellular glycogen, and discuss how this metabolic ability may facilitate colonization of the gut environment. We furthermore perform an in silico survey for the presence of genes that encode glycogen biosynthesis-associated enzymes in gut metagenomic libraries to highlight the prevalence of this metabolic trait as a possible fitness marker in the highly competitive and dynamic gut niche.

Human gut microbiota and its metabolic adaptation to its natural environment

Commensal bacteria inhabiting the gut ecosystem are believed to require specific adaptations to this particular ecological niche²³. The establishment of intricate and dynamic interactions between the human host and its inhabiting microbes is necessary for these gut commensals to survive within the GIT, obtain nutrients and reproduce, and is believed to be beneficial for both partners³⁰. The GIT is rich in macromolecules that can serve as nutrients for these gut microorganisms and therefore represent a secure habitat at a constant and optimal temperature where microbes can establish and multiply³¹. Although many members of the gut microbiota cooperate, there is also intense competition for space and resources within the GIT.³⁰

It is generally accepted that host diet is one of the main drivers that shape gut microbiota composition.^17,32,33 Put in simple terms, available nutrients determine whether or not a microorganism is able to establish and persist in the gut.^29,34 Carbohydrate fermentation is a core activity of the human gut microbiota, as carbohydrates represent a major energy and carbon source in the gut.³⁵ The mammalian GIT has different compartments with diverse nutrient and environmental conditions and, consequently, different microbial communities. The small intestine houses microorganisms that utilize simple sugars, while in the colon, the bacterial inhabitants are more specialized toward complex dietary fiber fermentation²⁹ and also host glycans such as mucin-associated carbohydrates.³⁶ Accordingly, a plant-based, polysaccharide-rich diet favors expansion of those organisms that can metabolize such dietary fibers either directly or through cross-feeding activities³⁷. Bacterial glycan fermentation generally produces beneficial metabolites in the form of SCFAs⁹. Acetate, propionate, and butyrate are the three main SCFAs, and each of these play different roles in human physiology.^9,38–40

Metabolic adaptation describes the ability to metabolically switch from one substrate to another, a property that is essential for the fitness and survival of microorganisms in competitive environments such as the GIT.^41,42 In the mammalian gut environment, nutrient availability is constantly fluctuating and saccharidic substrates that support bacterial growth are not continually available and if so, they are typically present in limiting amounts.⁴³ The model bacterium Escherichia coli, as an example of a gut commensal, exhibits metabolic adaptation through its ability to use a wide range of dietary sugars in order to ensure durable gut colonization.^42–44 Similarly, gut bacteria belonging to the genera Bacteroides, Bifidobacterium and Ruminococcus are capable of degrading complex carbohydrates.^45,46 For example, in a complex nutrient environment, such as that resulting from a high fiber diet, human gut-associated Bacteroides species can switch their metabolism to suit available glycan substrates. However, a microorganism can still persist in the gut if it is able to utilize some of the available nutrients more efficiently than its competitors.²⁹

In order to cope with life in a highly competitive nutrient-limiting or nutrient-fluctuating environment, certain bacteria, including gut commensals, have developed the ability to create intracellular energy stores, such as glycogen. This polymer is accumulated under certain conditions thanks to a dedicated enzymatic machinery to serve as a major energy reserve in bacteria that may underpin successful gut colonization and survival.^43,47,48

Box 1: Colonization factors in gut commensal bacteria

To bestow beneficial activities on a human host through host-microbe mutualistic interactions, symbionts and probiotic commensals need to establish themselves, at least temporarily, in the gut environment.⁴⁹ Thus, investigation of the mechanisms that enable these microbes to colonize, stay alive and be metabolically active in the GIT is of key importance in order to fully understand their functions.⁵⁰ Apart from the ability of gut bacteria to metabolize dietary or host-derived compounds, other colonization factors have been described (Figure 1). These include mechanisms such as amino acid decarboxylases or the F₀F₁-ATPase, which allow bacteria to tolerate fluctuating and low pH (ranging from 6.5 to 7.5 in the mouth to 1.5–6 in the stomach, and from 5.7 to 7.4 in the small intestine, cecum and colon).^51,52 In the small intestine, bacteria need to survive exposure to toxic bile, a fluid which is predominantly composed of primary and conjugated bile acids (BAs), and which acts as a potent detergent, essential for fat digestion and absorption.⁵³ Many gut bacterial inhabitants use hydrolytic enzymes to protect themselves from BA toxicity.^54–58 Production of surface exopolysaccharides (EPS), biofilms and efflux pumps have also been associated with providing bile tolerance in some bacterial species.^59–62

Figure 1. Colonization Factors in gut commensal bacteria.

Schematic representation of some of the molecular and cellular mechanisms that commensal bacteria evolved to survive in the gastrointestinal tract. (Top left panel) To tolerate the wide range of pH during the transit through different gut locations, bacteria use an ATP-driven proton pump to keep an optimum cytoplasmic pH. (Bottom left panel) Surface exopolysaccharide (EPS), and pili produced by several commensal bacteria have been shown to be essential for their adherence and colonization in the GIT. (Right panel) Bile is synthesized in the liver and then secreted into the duodenum. Bile is made of primary bile acids (BAs) and is toxic to bacteria. To protect themselves from BA-mediated toxicity several commensal bacteria use hydrolytic enzymes to deconjugate BAs.

Adhesion to intestinal epithelial cells, mucus, or components of the extracellular matrix represents an additional important bacterial colonization factor.^63–66 Adhesion to the intestinal epithelium is considered crucial for commensal bacteria to facilitate gut establishment, thereby competing with and thus limiting colonization by intestinal pathogens.⁶⁷ EPS appears to play an important role in bacterial interaction with the intestinal mucosa, although the precise mechanism of this interplay is still poorly understood.⁶⁸ The presence of EPS surrounding the bacterial surface can reduce adherence to intestinal cells thereby negatively impacting on intestinal colonization.^69–72 whereas others have demonstrated that EPS-producing bacteria are better at tolerating the gastrointestinal transit and can persist longer in the GIT.^59,73 In addition to EPS, other extracellular structures, such as pili are implicated in adhesion processes.^74,75 For example, sortase-mediated and Tad (tight adherence) pili have been identified in several species of commensal bacteria, including lactobacilli and bifidobacteria, and shown to be important for their adherence, gut colonization and host cell proliferation.^76–82

Glycogen storage by members of the gut microbiota

Energy storage in the form of branched glucose polymers is clearly an ancient ability and may have been present in the last universal common ancestor of all present life.⁸³ Glycogen and other related polyglucose compounds share a common structure composed of glucose monomers that are connected by α-1,4- and α-1,6-linkages, and are among the preferred carbon sources of many gut commensals. Various bacteria in the GIT environment possess the enzymatic machinery to utilize these dietary polysaccharides as an extracellular carbon and energy source, as has been addressed in several reviews.^84,85 Certain bacteria, employing products of the degradation of these polysaccharides, or other carbohydrates, are capable of synthesizing de novo glycogen-like polymers intracellularly (glycogen, starch or maltodextrin).⁸⁶ However, relatively little attention has been paid to the physiological role of these intracellular energy storage polymers, generally referred to as glycogen, or the molecular pathways and the specific enzymes required for intracellular biosynthesis and subsequent degradation. Herein, we discuss intracellular glycogen metabolism under the terms glycogen accumulation/degradation to describe such processes, unless otherwise indicated.

Bacterially produced glycogen is a water-soluble polymer consisting of chains of α-1,4-linked glucose units that contain regular α-1,6-linked branches⁸⁷ with an estimated average molecular weight between 10⁷ and 10⁸ Daltons.^48,88 Glycogen branching is important for fast response to metabolic needs, because both synthesis as well as degradation of the glycogen polymer occur from the non-reducing ends of the α-1,4 chains. Thus, highly branched glycogen has a higher number of “ends” per volume, while branching also increases glycogen solubility.⁸³ Due to its large molecular mass, high solubility and highly branched structure, glycogen represents an efficient form of energy and carbon storage with little effect on the internal osmolarity of cells.^86,88

Several early studies have described accumulation of glycogen in bacteria. This characteristic was thoroughly studied in E. coli.^89,90 Using spectrophotometry, Levine and colleagues discovered that under certain cultivation conditions a characteristic set of absorption bands which appeared in the infrared spectrum of this enteric bacterium, were due to a glycogen-like polysaccharide.⁹¹ These authors also showed marked variability in intracellular glycogen levels among different enteric strains and upon growth on different carbon sources,⁹¹ with E. coli even accumulating intracellular glycogen granules.⁸⁹ A study carried out in Enterobacter aerogenes (previously Aerobacter aerogenes) demonstrated that utilization of degradation products of intracellular storage polymers supports bacterial survival under certain challenging conditions in vitro.⁹² Another classic publication provides further information on the role of glycogen as a bacterial energy and carbon reserve, though being dependent on the bacterial species and nature of the carbon and energy source in the environment.⁹³ In Bacteroides fragilis, the intracellular polysaccharide was shown to be degraded rapidly when glucose was in short supply and fatty acids accumulated in the medium.⁹⁴ Thus, the concept of intracellular polysaccharide accumulation in (gut-associated) bacteria was established in the early 60s.^95,96

At that time, findings suggested that certain microorganisms, e.g. Pseudomonas aeruginosa, do not synthesize any specific reserve compound.⁹⁷ More recently, it has been conclusively established that the ability to produce and accumulate intracellular α-glucan polysaccharides such as glycogen represents a feature of many, but not all bacteria.⁹⁸ Nonetheless, intracellular glycogen accumulation has been observed in more than 50 bacterial species, including Gram-positive and Gram-negative bacteria, as well as archaebacteria.^48,87 Indeed, glycogen storage is ubiquitous among enteric bacteria, such as En. aerogenes, E. coli, Lactobacillus acidophilus, Edwardsiella tarda, Hafnia alvei, several species of Bacillus, and Streptococcus, and various ruminal bacteria (Preiss 2006) (Table 1). Furthermore, some of these glycogen-accumulating bacteria are gut pathogens, such as Clostridium botulinum, several species of Salmonella and Vibrio cholerae. As we will discuss below, glycogen accumulation in both pathogenic and nonpathogenic bacteria is associated with increased gut survival and tolerance to gut-associated stress conditions.

Table 1. Intracellular accumulation of glycogen reported in gut-related bacteria.

	Habitat	Genome size (Mb)^a	GC (%)^b	Role of intracellular glycogen storage	References
Acetivibrio cellulolyticus (synonym Hungateiclostridium cellulolyticum)	Sewage	6.16	35.5	Survival in starvation conditions	Patel and Breuil (1981)^Citation99
Aeromonas hydrophila	Aquatic environments; animal and human (gut) pathogen	4.74	61.5	Survival in low carbohydrate environment	Shaw and Squires (1980)^Citation100
Aerobacter aerogenes (Enterobacter aerogenes, Klebsiella aerogenes)	Human gut (opportunistic pathogen)	5.28	54.8	Survival in unfavorable environments	Strange et al. (1961)^Citation92,Citation101
Bacillus megaterium	Soil; human gut	5.34	38.1	Carbon and energy reserve	Barry et al. (1952)^Citation102
Bacillus subtilis	Soil; water; vegetables; human and animal gut	4.46	43.4	Sporulation	Kiel et al. (1964)^Citation103
Bacteroides fragilis	Mammal gut (human opportunistic pathogen);	5.21	43.2	Carbon and energy supply under low carbohydrate level conditions	Lindner et al (1979)^Citation94
Clostridium botulinum	Soil aquatic sediments; improperly preserved foods; animal and human gut (pathogen)	4.39	28	ND	Whyte and Strasdine (1972)^Citation104
Corynebacterium glutamicum	Soil; some species of Corynebacterium are members of the human gut microbiota	3.31	53.8	Cell shape and osmoprotection	Seibold et al., (2007), Seibold and Eikmanns (2013), Seibold et al., (2011)^Citation47,Citation105,Citation106
Escherichia coli	Gut of humans and other warm- blooded animals (some species are pathogenic)	4.64	50.8	Energy maintenance during metabolic adaptation and colonization	Holme et al., (1956), Fung et al., (2013), Morin et al., (2017), Jones et al., (2008), Wang et al., (2019), Wang et al., (2020)^Citation107–109
Fibrobacter succinogenes	Rumen	3.84	48		Stewart et al., (1981), Gaudet et al., (1992)^Citation110,Citation111
Lactobacillus acidophilus	Human oral, gastrointestinal, and vaginal microbiota	2.01	34.7	Gut fitness and retention	Goh and Klaenhammer (2013), Goh and Klaenhammer (2014)^Citation86,Citation112
Megasphaera elsdenii	Rumen	2.5	52.8	ND	Cheng et al., (1973)^Citation113
Prevotella ruminicola	Rumen, reported in swine cecum	3.62	47.7	Survival mechanism during periods of carbon starvation	Lou et al., (1997)^Citation114
Ruminococcus albus	Rumen	3.69	44.2	ND	Cheng et al., (1977)^Citation115
Salmonella enterica	Gut pathogen	4.86	52.2	Biofilm formation and virulence	Bonafonte et al., (2000), Levine et al., (1953)^Citation91,Citation116
Selenomonas ruminantium	Rumen	3	50.7	Energy source in starvation conditions	Kamio et al., (1981), Wallace (1980)^Citation117,Citation118
Shigella dysenteriae	Contaminated food and water, human gut pathogen	4.75	50.9	ND	Levine et al., (1953)^Citation91
Streptococcus agalactie	Human skin and mucous membranes (can be pathogenic)	2.28	35.8	Metabolic adaptation to stress conditions	McFarland et al., (1984)^Citation119
Streptococcus mitis	Human oral cavity and digestive tract; throat and nasopharynx	2.17	40	Survival	van Houte and Jansen (1970)^Citation120
Vibrio cholerae	Human gut pathogen	4.08	47.7	Environmental persistence and host transmission	Bourassa and Camilli (2009)^Citation121

a Genome size (Mb) of the reference genome of each specie.

b GC (%) of the reference genome of each specie.

Biosynthesis and degradation of glycogen

Glycogen metabolism allows microorganisms to employ glucose polymers as a dedicated carbon and energy storage facility that can be mobilized for future purposes. This metabolic property relies on an enzymatic machinery responsible for glycogen biosynthesis and subsequent degradation, along with regulatory mechanisms in order to store or recruit glucose moieties depending on cellular needs.

In E. coli, Bacillus megaterium and other bacteria, glycogen is synthesized during times when carbon is abundant while other nutrients (e.g. nitrogen or phosphate) are limiting.^122,123 However, in Fibrobacter succinogenes glycogen synthesis may take place during any growth phase.¹¹¹ Glycogen accumulation in L. acidophilus is carbon source dependent.¹¹² In contrast, glycogen degradation occurs when carbon sources become limiting.^43,124 Under such circumstances, release of glucose units from glycogen by specific enzymes provide carbon resources to central metabolism.¹²⁵ Therefore, analysis of the enzymatic functions involved in intracellular glycogen synthesis/degradation and their mechanisms of regulation are important to understand how and when microbes implement this apparent survival strategy.

Most of the enzymes involved in glycogen metabolism in bacteria belong to glycosyl transferase or glycosyl hydrolase families, that contain specific carbohydrate-binding modules^87,88,126 (Table 2). The classical glycogen biosynthetic pathway in bacteria, such as E. coli and L. acidophilus, involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyl transferase (GlgC), ADP-glucose-specific glycogen synthase (GlgA) and branching enzyme (GlgB). The enzymes GlgP (glycogen phosphorylase) and GlgX (debranching enzyme) are implicated in glycogen degradation (Table 2 and Figure 2).

Figure 2. Glycogen metabolism pathways in gut commensal bacteria.

Phosphoglucomutase (Pgm) converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC in the presence of ATP and Mg²⁺. GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan with the concomitant release of ADP. Then, GlgB cleaves off portions of this α-1,4-glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen structure. Glycogen catabolism is mediated by glycogen phosphorylase (GlgP) which removes glucose units from the non-reducing ends of the glycogen/maltodextrin molecules with concomitant phosphorylation, thereby liberating glucose 1-phosphate. At the same time, a debranching enzyme (GlgX) cleaves the α-1,6-bonds of the limit dextrins generated by GlgP releasing maltodextrins that can be further metabolized by GlgP. An alternative pathway employs trehalose or maltose to produce maltose 1-phosphate that is used by GlgE to elongate the glucan chain. Furthermore, enzymes involved in maltose/maltodextrin metabolism may play a role in glycogen synthesis and degradation. MalQ produces maltodextrins, using maltose or malto-oligosaccharides as substrates, and then GlgB can form glycogen from these linear maltodextrins. These maltodextrins can be cleaved by MalP liberating glucose-1-phosphate.

Table 2. Biosynthetic and degradative enzymes in the metabolism of glycogen.

Protein	Activity	EC number	GH/GT family*	Motif**
Pgm	Phosphoglucomutase	EC 5.4.2.2	^______
GlgC	Glucose-1-phosphate adenylyltransferase	EC 2.7.7.27	^______
GlgA	Glycogen synthase	EC 2.4.1.11	GT5
GlgB	1,4-alpha-glucan branching enzyme	EC 2.4.1.18	GH13, GH57
GlgP	Glycogen phoshorylase	EC 2.4.1.1	GT35
GlgX	Glycogen debranching enzyme	EC 3.2.1.68	GH13
GlgE	Alpha-1,4-glucan:maltose-1-phosphate maltosyltransferase	EC 2.4.99.16	GH13
MalQ	4-alpha-glucanotransferase (amylomaltase)	EC 2.4.1.25	GH77
MalP	Maltodextrin phosphorylase	EC 2.4.1.1	GT35

*GH Glycoside hydrolase; GT Glycoside transferase.

**PGM phosphoglucomutase; NTP_trans nucleotidyltransferase; Glyco_trans glucosyl transferase; CBM_48 carbohydrate binding module family 48; α-amylase; glyco_hydro glycoside hydrolase

Studies conducted on the gut bacterium E. coli were pivotal to elucidate the glycogen metabolic pathway that was described in detail by Preiss.^122,127 Extracellular glucose is taken up and converted to glucose-6-phosphate by a bacterial phosphoenol-phosphotransferase system (PEP-PTS). Alternative sugars that can be converted to glucose-6-phosphate may also be used. Pgm converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC.¹²⁷ GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan with the concomitant release of ADP. GlgB subsequently cleaves off portions of this α-1,4-glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen branching structure.⁸⁶ Some bacteria possess a regulatory subunit of GlgC, called GlgD,¹¹² where GlgC itself acts as the catalytic subunit. Glycogen catabolism is mediated by glycogen phosphorylase (GlgP) which removes glucose units from the non-reducing ends of the glycogen/maltodextrin molecules liberating glucose 1-phosphate.¹²⁸ At the same time an isoamylase-type debranching enzyme (GlgX) cleaves the α-1,6-bonds of the limit dextrins generated by GlgP (typically 3–5 glucosyl residues in length) releasing maltodextrins^86,129 that can be further metabolized by GlgP (Figure 2). The glucose-1-phosphate produced can be transformed into glucose-6-phosphate by Pgm and shuttled into the central carbon metabolic pathways, e.g. glycolysis or bifid shunt, the latter defined as the central and unique metabolic pathway for carbohydrate fermentation employed by bifidobacteria.¹³⁰ This model represents the prevalent bacterial pathway of intracellular glycogen synthesis and degradation.⁹⁸

It has been reported that certain microorganisms, such as species belonging to the genus Streptomyces, possess an alternative route for intracellular α-glucan biosynthesis, called the GlgE pathway. This alternative pathway employs trehalose or maltose as precursors for the disaccharide α-maltose 1-phosphate. A dedicated maltosyltransferase (GlgE) uses the latter molecule as the building block to elongate glucan chains^131,132 (Figure 2). Furthermore, it has been described that maltose metabolic enzymes play a role in glycogen synthesis and degradation in E. coli.¹³³ Briefly, the action of an amylomaltase (MalQ) on maltose or maltooligosaccharides can lead to the formation of maltodextrins¹³³ and a maltodextrin phosphorylase (MalP) releases glucose 1-phosphate from glycogen molecules⁹⁰ (Figure 2). Generally, the glycogen metabolism genes are organized in a single gene cluster in most bacteria known to accumulate glycogen (see section below).

Presence and distribution of glycogen metabolism genes in human gut commensals

Genomics approaches have been crucial to reveal the complex interactions between a host and its resident bacteria and to unravel gut colonization strategies and microbiota functionality⁴⁶. A previous survey of 55 bacterial genomes from different environments, such as plant, animals and humans, revealed that about half of these bacterial species contain an apparently full set of glycogen metabolic genes.^125,126 This finding was later substantiated by another study of 1202 deduced bacterial proteomes.¹³⁴ Based on recent genomic analyses, intact glycogen metabolic pathways are present in bacterial species adaptable to diverse environments and possessing flexible lifestyles.^98,134 In contrast, a significant proportion of bacteria with parasitic behavior lack this glycogen biosynthetic ability.¹²⁶ Taking advantage of the increasing number of available gut metagenomic datasets, we first examined the abundance of the glycogen metabolism pathway among selected members of the human gut community, using an existing computational method¹³⁵ across 1,267 gut metagenomes from American, European, and Chinese subjects. We found evidence for the presence of this pathway in all metagenomes surveyed (minimum abundance = 1 copy/1000 cells), suggesting that this appears to be a core activity of the human gut microbiota (unpublished data).

We then analyzed the occurrence of putative glycogen gene products in 70 representatives of the different genera using the Catalog of Reference Genomes From the Human (gut) Microbiome¹³⁶. We employed BLASTP analysis (cutoff values: E-value <0.00001, at least 20% identity across at least 50% of sequence length) and functional annotation profiling efforts to establish the occurrence of specific glycogen biosynthetic and degradative enzymes among gut bacteria (as outlined in Figure 2). Interestingly, this in silico analysis revealed that the majority of assessed members (~80%) of the gut microbiome, including components of the major bacterial phylogenetic divisions of the human gut microbiota, are predicted to harbor the metabolic genes involved in intracellular glycogen synthesis/breakdown, suggesting that this trait is a core function of such microbes from the gut environment. This analysis also revealed that the orthologous proteins are variably distributed across species, where certain enzymes seem to be more specific while others are present among all members. Therefore, there are organisms completely lacking glg homologs, organisms harboring partial sets of such homologs, and organisms harboring all known glg gene homologs, while some members of the latter two groups harbor more than one copy of a particular glg gene homolog.⁹⁸

Remarkably, all members of Bacteriodetes lack GlgC though it may be that they use other proteins such as MalQ or UDP-glucose adenylyltransferase to generate the glycogen polysaccharide. Alternatively, Bacteroidetes members may not encode an E. coli-type GlgC enzyme, yet possess an enzyme that is more similar to human glycogen enzymes as has been described for other bacteria.⁸³ Indeed, the glycogen synthesis pathway is functional in Bacteroides fragilis since accumulation of an intracellular polysaccharide formed from glucose has been demonstrated in 27 Bacteroides fragilis strains.⁹⁴ Figure 3 shows that GlgE is not common in the adult human gut microbiome since only a relatively small number of species, for example members of the genus Bifidobacterium, encode this enzyme. Transcriptome analysis has revealed that the expression of genes predicted to encode proteins participating in glycogen metabolism are up-regulated under particular circumstances in E. coli and L. acidophilus.^88,112 Nonetheless, bifidobacterial species that are not associated with the mammalian GIT lack genes encoding glycogen metabolism.¹³⁷ Certain bacterial species that have been found in fecal samples, for example Pediococcus acidilactici, Weissella paramesenteroides, Gordonibacter pamelaeae, Dialister succinatiphilus, and Leuconostoc mesenteroides subsp. cremoris, among others, do not encode Glg proteins and thus do not appear to be able to synthesize intracellular glycogen. Some of these species may in fact have a food origin and therefore may not belong to the autochthonous gut microbiota population.¹³⁸ The glg genes also appear to be absent from the genome of Streptococcus infantarius subsp. infantarius ATCC BAA-102, which is associated with colorectal cancer, or the pathogenic strains Helicobacter pylori 83 and Enterococcus faecalis EnGen0107 (Figure 3). As mentioned above, certain bacterial species contain more than one homologue of a particular glg gene, particularly in the case of glgB and glgP. Most notable examples are Blautia obeum ATCC 29,174 which possesses two homologues for glgC, glgD, glgP and glgX, and three glgB homologues, or Dorea longicatena DSM 13814 which contains two homologues for each of the glgC, glgD, glgB and glgP genes. In these species with several copies of a given glg gene, some of the copies represent homologs from different species (rather than being paralogs; data not shown). This peculiarity has been noticed previously in a study of glycogen metabolism genes in gammaproteobacterial species and may be a genetic adaptation to increase intracellular glycogen production/degradation rates or represent glycogen production under distinct circumstances by a specific set of glg genes.⁹⁸

Figure 3. Presence/Absence of predicted glycogen biosynthetic and degradative enzymes in gut bacteria.

Heatmap representing the distribution across gut-related strains of proteins homologous to the enzymes involved in glycogen metabolism, using the protein sequences of GlgC, GlgA, GlgB, GlgP, GlgX and MalQ) from E. coli SE11, GlgD from Lactobacillus acidophilus ATCC 4796 and GlgE from Corynebacterium glutamicum DSM 20300 as query. Gene products from 70 representative strain genomes available on line from the Human (gut) Microbiome.¹³⁶ The significant homology of 20% identity over 50% of protein length are represented in the matrix, employing a color code that represents the degree of identity. Members of the same phylum are indicated by the same color. The numbers indicate copies of a given protein. MalP and GlgP present a high identity of protein sequence and the BlastP analysis retrieved the same results using both sequences as query.

Genetic organization of glycogen genes

Bacterial genes associated with the same metabolic pathway are often clustered.^98,139 In E. coli, this cluster includes the glgC, glgA and glgB genes, plus two genes involved in glycogen degradation, glgX and glgP.^127,140 The gene arrangement of these glg genes is glgBXCAP which is also conserved in all species harboring glg genes in the orders Enterobacteriales and Pasteurellales.⁹⁸ Clustering of genes involved in glycogen metabolism has also been described for other bacteria, e.g. Bacillus stearothermophilus and B. subtilis, Lactiplantibacillus plantarum and Vibrio spp. among others.^{86,98,103,141} In the probiotic bacterium L. acidophilus the glycogen metabolic pathway is encoded by a 11.7-kb chromosomal region consisting of glgBCDAP-amy-pgm, while glg operons in other lactobacilli have the apparently conserved gene arrangement glgBCDAP^86,112 (Figure 4). Notably, in the gut commensals surveyed in this review there are exceptions to this single glg gene cluster. For example in Holdemanella filiformis and Erysipelatoclostridium ramosum these genes are organized in two clusters, glgCDA and glgBPX. Curiously, in the species Bacteroides intestinalis, Bacteroides finegoldii, Citrobacter youngae, Parabacteroides goldsteinii and Bifidobacterium longum subsp. longum, among others, the glg genes are scattered across the chromosome (Figure 4).

Figure 4. Genetic organization of glycogen genes in gut commensal bacteria.

Schematic representation of the gene arrangements of some bacteria, as indicated, containing glycogen genes identified in this review. The genes with identical function are represented by the same color.

Thus, the presence of complete glycogen pathways, and their tight regulation, in human gut commensals is likely reflecting the importance of this metabolism in the gut ecosystem (see Box 2 and next section).

Box 2: Regulation of glycogen metabolism

Glycogen metabolism is closely interconnected with various metabolic processes as reviewed by Wilson and colleagues.⁸⁸ Thus, not surprisingly, it is subject to complex transcriptional, post-transcriptional and allosteric regulation as described below. The reader should be aware that most of the findings related to regulatory mechanisms of glycogen metabolism were generated in E. coli and will be covered as such in the following paragraphs unless otherwise indicated.

At a transcriptional level, glg genes are generally co-ordinately expressed within various regulons, and their expression is triggered by signals related to nutrient starvation. Carbon catabolite repression (CCR) is a transcriptional regulatory mechanism aimed at ensuring sequential utilization of more preferred carbon sources through repression of genes required for the metabolism of less preferred carbon compounds.¹⁴² This repression has been associated with activation of glycogen metabolism upon glucose shortage in several organisms¹⁴² including E. coli, Salmonella spp., Streptococcus suis, Corynebacterium glutamicum, Mycobacterium spp., or V. cholerae.⁴⁷ So-called catabolite responsive elements (cre) are located upstream glg genes in various organisms, which indicates CCR-mediated regulation.¹⁴³ Glucose and other nutrient depletion signals also trigger the stringent response, which re-programmes metabolic processes allowing bacteria to adapt and survive adverse conditions.¹⁴⁴ In E. coli the stringent and general stress responses are major determinants of intracellular glycogen content.¹⁴⁵ Under a range of stress factors and nutrient limitations, the alarmone ppGpp, a small nucleotide that acts as a global transcriptional regulator in bacteria, is a well-established positive regulator of the glycogen biosynthetic genes in E. coli.^146–148 However, glycogen accumulation is independent of (p)ppGpp or cre elements in various organisms, suggesting that these genes are subject to an alternative control mechanism.¹⁴⁵ Alternative sigma factors have been implicated in the transcriptional control of glg genes in E. coli, Bacillus subtilis and C. difficile.^103,149,150

At post-transcriptional level, glycogen storage is negatively regulated by the carbon storage regulator CsrA, a small-RNA binding protein that inhibits expression of targeted genes^151,152. CsrA controls a wide variety of cellular processes including some that affect interactions of enteric species with mammalian hosts, such as central carbon metabolism, biofilm formation, virulence and motility, among others^42,153,154. At the enzymatic level, allosteric mechanisms of regulation have been described for GlgC and GlgP, which catalyze key steps in the biosynthesis and catabolism of intracellular glycogen, respectively. Specifically, these enzymes are generally activated by metabolites that reflect signals of high carbon and energy content such as phosphoenolpyruvate, glucose-6-phosphate, fructose-1,6-bisphosphate or pyruvate, whereas they are inhibited by metabolites reminiscent of low metabolic energy levels like AMP, ADP and/or Pi^{141,145,155–159}. While different levels of allosteric regulation for these enzymes have been described in various organisms^141,157, allosteric regulation of GlgC is considered a main control point of glycogen accumulation in early stationary phase cells¹⁶⁰.

Roles of glycogen storage in gut bacteria

Environmental stresses in the GIT impose physiological challenges upon bacteria¹⁶¹ which must adapt in response to such adverse conditions²⁹ (see Box 1). In this regard, glycogen accumulation is a common feature across phylogenetically divergent species, and appears to represent a bacterial adaptation to nutrient (or other) limitation in the gut.

What follows below is an in-depth analysis of available evidence, obtained from in vitro studies simulating the gut environment and in vivo studies employing animal models. All of which point toward glycogen metabolism as a widespread and fitness-enhancing trait among gut commensals.

The role of glycogen accumulation in simulated gut conditions

Glycogen is a stored energy resource linked to important physiological functions in bacteria as discussed below. Transient and resident gut commensals, including those administered as adjunct cultures in fermented foods or those provided as probiotic supplements, must cope with a range of challenges during their transit along the GIT that serially enforce strong selective pressures hampering bacterial survival and establishment in the lower gut ecosystem.^162,163 Glycogen accumulation has been linked to (long-term) survival where it can offer an energy source when the gut environment cannot.⁸⁸ However, the role of glycogen metabolism as a specific strategy for gut commensals to survive and thrive in their natural habitat has not received much scientific attention. Next, laboratory in vitro experiments simulating various stresses are shown to provide valuable insights to understand processes that occur in the mammalian gut¹⁶⁴.

Glycogen storage and survival upon macro- and micro-nutrient starvation

Nutrient limitation will for obvious reasons affect the gut microbiota. Nutrient availability in the GIT is affected by circadian oscillations, imposed by periods of fasting and feeding,^{29,43,165,166} and is in general characterized by the presence/absence of non-digestible complex carbohydrates as carbon sources.¹⁶⁷ Under such circumstances, metabolic flexibility and mixed-substrate utilization offer selective strategies for efficient gut colonization and survival.^29,168 These circumstances include: (i) availability of complex and non-digestible carbon sources, (ii) ability or lack thereof to utilize metabolites or sub-products released by other neighboring gut microbial inhabitants, and (iii) an ability to efficiently adapt metabolic activity to available nutrients where and when appropriate.

Several in vitro studies have investigated if the ability to accumulate glycogen affects survival of gut commensals under carbon starvation. For example, it was demonstrated that En. aerogenes cells containing glycogen survived better in an unfavorable environment in which carbon was limited.¹⁶⁹ In L. acidophilus, cells showed stable intracellular glycogen reserves during prolonged growth periods and for this reason it has been speculated that L. acidophilus maintains a higher level of intracellular glycogen to enhance its residence time when growing under conditions simulating the GIT environment.¹¹² Mutations that disrupt glycogen metabolism were also shown to negatively impact on E. coli fitness during nutrient starvation, demonstrating its role in maintenance of viability and alternative energy source acquisition during metabolic transitions.⁴²

These publications suggest that under carbon deprivation, glycogen is utilized to preserve cell viability, providing the energy and carbon source required by the bacteria for maintenance.⁴⁸ This bacterial durability has been linked with glycogen average chain length: glycogen with an average short chain length correlated with prolonged bacterial survival.¹³⁴ This effect was attributed to the slower degradation of this short chain length polymer, which can sustain bacterial viability for prolonged periods of time.¹²⁵

Regarding nutrient competition, the presence of specific alternative carbon sources to glucose, such as raffinose and the disaccharides trehalose and lactose, has been described to act as a signal promoting intracellular glycogen accumulation in L. acidophilus.⁸⁶ Raffinose is a non-digestible carbon source, known to be specifically fermented at intestinal level by several commensal species including representative strains of lactobacilli and bifidobacteria.¹⁷⁰ Specifically, growth on raffinose was associated with temporally increased expression of glg genes, which was shown to coincide with glycogen accumulation. Furthermore, glycogen accumulation was required to support L. acidophilus growth on raffinose as ΔglgA and ΔglgB mutants were shown to exhibit impaired growth on this carbohydrate.⁸⁶ These results suggest a close relationship between the metabolism of certain carbohydrates and glycogen metabolism, and although the specific molecular connections are currently not understood, both pathways are likely to be under the control of common central carbon regulators (see section about regulation in Box 2). Indeed, evidence in lactobacilli suggests that glg expression and glycogen accumulation are repressed by glucose as in other enteric bacteria, thus its expression might be enhanced in the presence of other carbon sources such as the ones typically encountered within the gut ecosystem. Thus, glycogen synthesis seems to be triggered by certain environmental conditions despite high energy and carbon availability, while glycogen degradation occurs during extended nutrient starvation.¹⁷¹

The limitation of micronutrients in the gut may also affect intracellular glycogen metabolism in bacteria. For example, the bacterial response to a shortage of phosphate in the gut, which can occur under physiological stress conditions of the host, includes activation of virulence in a broad range of pathogens, as well as of glycogen accumulation.¹⁷² Nitrogen is another limiting nutrient for gut microbial communities, that strongly determines gut microbial composition and their interactions with the host.^173,174 For example, E. coli MG1655 cells grown under nitrogen-limiting conditions were shown to exhibit the highest level of glycogen accumulation compared with carbohydrate-limited conditions during stationary phase.¹⁰⁸

Remarkably, a shortage of nitrogen has been demonstrated to activate the stringent response, evidenced by increased glycogen accumulation in E. coli and Vibrio cholerae cells grown in the presence of glucose excess.^121,175 In E. coli, limited availability of iron also connects the stringent response to glycogen metabolism as it promotes ppGpp accumulation which overall results in promotion of glycogen biosynthesis.¹⁷⁶ Accordingly, mutants defective in sensing and sequestering iron display altered glycogen accumulation phenotypes.¹⁴⁵ Consistent with this observation, it was found that the glgX gene of the commensal Bifidobacterium breve UCC2003 is important for survival under iron-limiting conditions.¹⁷⁷

Thus, glycogen accumulation as a means of intracellular carbon and energy storage may represent a common survival mechanism for commensals across different compartments of the GIT and an advantage in an environment with strong competition for available nutrients.

Glycogen storage and Survival upon acid and bile stress conditions

It is increasingly clear that many stress responses overlap and thus a stress response to one specific factor commonly results in cross-resistance to other, apparently unrelated stressors.¹⁷⁸ In this regard, it is conceivable that glycogen accumulation, when activated in response to decreased availability of nutrients and/or micronutrients, plays a role in the response and tolerance to other stress factors encountered in the GIT. As an example of this interconnection, nutrient starvation, which generally marks entry into the stationary growth phase, also represents the activation signal for glycogen accumulation.¹⁷⁹ Besides, it is well known that cells in the stationary phase are significantly more tolerant to acid stress than those in the log phase of growth.¹⁸⁰ It is also worth noting that some of the regulators controlling glycogen metabolism, control the expression of genes that contribute to the response to specific GIT stress factors, suggesting that intracellular glycogen metabolism may confer a selective advantage in order to better cope with specific GIT stress factors. The specific contribution of glycogen metabolism toward acid tolerance has not been investigated, although glycogen reserves act as intracellular energy sources, which are required for the maintenance of pH homeostasis under acidic conditions supported by the extrusion of protons via the F₀F₁ H⁺/ATPase.¹⁸¹ Thus, under acidic conditions, cells tend to adjust their metabolism, by increasing the synthesis of energy-rich intermediates (such as ATP and NADH) and/or glycogen.¹⁸² A connection between acid and bile stress responses has also been proposed as intracellular accumulation of BAs leads to proton release and thus cytoplasmic acidification. Early reports have described that bile salts promote higher intracellular accumulation of glycogen in several enterobacteria.⁹¹ For example, response to and survival under acidic conditions in E. coli is highly dependent on the alternative σ^S factor, RpoS, also known to control glycogen accumulation.¹⁵⁰ Furthermore, the AcrAB-TolC multidrug efflux system from enterobacteria, essential for bile resistance in Salmonella typhimurium, is activated as part of the RamA-controlled regulon, which is also known to regulate glg genes in other microorganisms.^183,184

More recently, glg gene inactivation in the commensal L. acidophilus did not appear to affect survival in simulated gastric juices, indicating the absence of a role in acid tolerance, although it was shown to negatively affect survival in simulated intestinal juices, suggesting a role in survival to bile. Indeed strains carrying a ΔglgB or ΔglgP mutation were shown to elicit growth defects, including longer lag phases during growth in the presence of bile extracts, further supporting a contribution of this metabolic pathway in the gut ecosystem.⁸⁶

The role of glycogen accumulation in biofilm formation

In nature bacteria are rarely encountered in planktonic lifestyles but are organized in matrix-enclosed communities known as biofilms. Recent findings suggest that bacteria within the gut environment are mainly encountered in the form of intricate biofilms that establish close association with the intestinal epithelia.¹⁸⁵ Besides, biofilm lifestyles confer resistance to harsh environments thereby rendering biofilm-associated bacteria more resilient to environmental challenges such as those encountered in the gut. In enteric bacteria, in vitro studies have demonstrated a correlation between glycogen accumulation and biofilm formation. Similarly, studies in E. coli have revealed that biofilm formation is enhanced in mutants defective for CsrA, a global carbon regulator that also represses glycogen storage during active phases of bacterial growth. Besides, it has been reported that biofilm formation phenotypes are affected in mutants with altered glycogen accumulation and subsequent catabolism, as illustrated by the finding that in E. coli glycogen accumulation and catabolism through GlgP is required to promote biofilm formation.¹⁸⁶ Consistent with this notion, in some bifidobacteria, in which biofilm production has been demonstrated to occur in response to bile exposure, a glgP insertional mutant (a mutation expected to prevent glycogen degradation) rendered the strain defective in biofilm formation when exposed to high bile concentrations.^187,188 However, it is worth noting that contradictory observations have been reported as a glycogen-deficient ΔglgC (glycogen synthesis) mutant was shown to accumulate high levels of biofilm in E. coli.¹⁸⁹ These observations clearly indicate that, glycogen metabolism and, particularly, degradation, affect biofilm formation. Further investigation is required so as to decipher the regulatory mechanisms connecting biofilm formation and glycogen metabolism although it has been speculated that glycogen catabolism can serve as energy and/or carbon source to promote biofilm formation.¹⁶² In agreement with this hypothesis, the ability to utilize glycogen was demonstrated to be important for the transition between planktonic and biofilm lifestyles and enabled increased glucose uptake during pulses of limited glucose availability in E. coli.¹⁶¹

Role of glycogen storage for colonization and fitness in the gut ecosystem

Deciphering the functions of gut microbiota members is important to understand the factors driving colonization, resilience or survival in the gut niche.²⁹ Since glycogen accumulation plays a role in survival under in vitro conditions, this bacterial metabolism is also thought to be a crucial mechanism involved in GIT colonization and fitness. Thus, this section focuses on several in vivo studies performed to determine the impact of bacterial glycogen storage on enhancing fitness in the gut ecosystem. It is possible that glycogen storage plays an important role in GIT colonization due to the necessity to support growth and/or to maintain viable in the intestinal environment when and where there is intense competition for (micro)nutrients.

The ability of L. acidophilus to synthesize and store glycogen was shown to support long-term colonization in the murine GIT.⁸⁶ In this in vivo study two bacterial strains, the parent L. acidophilus (able to synthesize and store glycogen) and its glgA mutant unable to synthesize glycogen were administered in a 1:1 ratio to germ-free (GF) mice. The results showed that colonization levels of the glgA mutant are significant lower compared to the parental strain for almost one month post-delivery. These findings represent compelling evidence that the ability of L. acidophilus to synthesize intracellular glycogen provides a competitive advantage that supports gut colonization. In the same study, the authors also showed that a glgA mutant is able to colonize the gut of GF mice in the absence of competition. However, when the parental strain was administered to the mice colonized with the glgA mutant the decline in the mutant abundance coincided with an increase of the parental population level. This supports the idea that intracellular glycogen storage plays a major role in the competitive fitness of L. acidophilus in the host environment. The importance of glycogen accumulation in murine GIT colonization has also been demonstrated using the commensal E. coli K-12. It has been shown that E. coli mutants which lack genes for glycogen accumulation are not able to colonize the murine GIT. However, by providing a constant supply of a readily metabolizable sugar in the animal’s drinking water, the competitive disadvantage of E. coli glycogen metabolism mutants was rescued, supporting the notion that glycogen storage may aid to maintain fitness when there is intense competition for resources and occasional nutrient deprivation.⁴³

The trait of glycogen storage in an ecological context has also been addressed by metagenomic and metatranscriptomic approaches. Metagenomic analysis can provide host-specific insights into diversity and functional potential of the gut microbiota. Studies such as the mouse intestinal bacterial collection (miBC) identified factors required for colonization in mice. This study described the presence of glycogen genes in the strains Blautia caecimuris, Cuneatibacter caecimuris, Extibacter muris, Irregularibacter muris, Longicatena caecimuris and Pasteurella caecimuris among others.¹⁹⁰ A metatranscriptomic study in Bacteroides defined in vivo fitness determinants for these gut members in a community context under different dietary conditions. Genes involved in glycogen storage were significantly upregulated in Ba. ovatus, Ba. cellulosyliticus and Ba. thetaiotaomicron, during intestinal colonization in mice. Thus, these genes appear to be important for colonization and survival in a given environmental context¹⁹¹. In another study involving the ruminal microbiome, the glycogen phosphorylase-encoding gene, among other functional features, was highly transcribed by 115 bacterial genera, mainly belonging to the phyla Pseudomonadota, Bacillota, and Actinomycetota.¹⁹²

Much information can be gained from the distribution of genes that are functionally important in complex microbial communities by targeting specific genes.^193,194 Using targeted metagenomic sequencing, the gene encoding the GlgB enzyme (which is crucial for glycogen storage and metabolism)¹⁹⁵ was found to be present in 69 gut microbiomes from human, pig, cow and chicken hosts.¹⁹⁶ For example, glgB sequences in human gut microbiota members included Clostridia sp., Faecalibacterium sp. and Lachnospiraceae. The activity of glgB in the gut, and their influence on microbial dynamics, are likely to be associated with diet, as carbohydrates are indeed the direct target of this enzymatic activity.¹⁹⁶ Remarkably, a metatranscriptomic analysis of the human gut microbiome revealed that specific genes involved in carbohydrate metabolism, including glgB (glucan branching enzyme), were highly expressed by Fusobacteria. These data indicate that important functional aspects of low abundance bacterial species are active contributors to the gut ecology and host physiology.¹⁹⁷ In this regard, the pathway for glycogen synthesis was shown to be active in the gut by members of Escherichia, Sutterela, Enterobacter and Enterococcus.¹⁹⁸

Another metatranscriptomic study on metabolic adaptation of the human gut microbiota during pregnancy and infancy revealed that glgC, involved in glycogen synthesis, was upregulated in the maternal microbiota prior to birth.¹⁹⁹ It has also been demonstrated that in many bacterial species synthesis of glycogen is stimulated when glucose is abundant in the medium¹²³. High glucose concentration in the gut environment can be found during the state of late pregnancy, where the ability of microbiota to transport and degrade a variety of carbohydrates is replaced by the overexpression of a single transporter capable of internalizing glucose, and of enzymes of the glycogen synthesis pathway.¹⁹⁹

All the above indicates that there is a correlation between the presence of glg genes and their expression among members of the gut ecosystem, supporting the notion that glycogen metabolism is an important fitness property for gut bacteria.

Glycogen and gut pathogenic bacteria

It has been suggested that glycogen storage increases bacterial virulence and survival at mucosal surfaces, although this view is still controversial. For example, it has been demonstrated that the ability of glycogen accumulation in Salmonella gallinarum and Salmonella pullorum does not confer a fitness increase but appeared to enhance long-term survival.²⁰⁰ On the other hand, it has been shown that glycogen accumulation in Salmonella enterica is important for biofilm formation to increase its virulence capacity.¹¹⁶ The pathogen V. cholerae is able to store glycogen in vivo and this mechanism plays an important role in the transition of V. cholerae between poor nutrient environment (such as an aquatic niche) and the host, because mutants that are either unable to store or degrade glycogen are attenuated for transmission.¹²¹ Genome analysis of Clostridioides difficile comparing in vitro versus in vivo conditions revealed that the genes glgA, glgC and glgD (glgCDA operon) are upregulated under in vivo conditions, suggesting that C. difficile accumulates glycogen under such circumstances.²⁰¹ The latter work also indicated that glycogen accumulation in C. difficile is one of the strategies used by this bacterium to cope with temporary starvation conditions typical of gut colonization.²⁰¹ To further explore the possible link between glycogen storage and C. difficile virulence a mutant of this bacterium lacking glgC was shown to produce significantly fewer spores compared to the parental strain, indicating that glycogen storage is required for efficient sporulation in C. difficile.²⁰² While more studies are needed to address the precise role of bacterial glycogen storage in vivo under various conditions of host environmental nutrient and metabolic perturbation are needed, currently available data indicate that this mechanism is used as a strategy to survive challenges encountered in the host environment. Sporulation is a mode of long-term survival that has been described as a widespread strategy used by gut bacteria to survive under nutritional limitations or other stressful conditions, and to transit from the environment to the host.²⁰³ Thus, in these bacteria which experience frequent oscillations of their environmental conditions, in particular when they transfer from a non-host environment to the gut of their host, glycogen accumulation is essential to survive outside the host.^121,204 In conclusion, commensal or pathogenic bacteria that are able to synthesize and store glycogen survive better and longer than their isogenic derivatives that don’t possess the ability to build up such energy and carbon reserves.

Concluding remarks

Determining the functional attributes of the microbiome is essential to understand its role in host physiology, metabolism and disease and it has also prompted a wide range of biotechnological applications to improve human health. This includes the production of novel probiotics and prebiotics, among others, to modulate the gut microbiota. However, these technological developments require an in-depth knowledge of the adaptive mechanisms of these microbial communities to the human GIT. In this regard, these microorganisms must have developed metabolic strategies to efficiently colonize and survive in such a harsh, dynamic and highly competitive environment. The genetic capability for glycogen metabolism as present in a large proportion of enteric bacteria, supports the notion that this metabolic pathway enhances adaptation and performance of gut commensals in their specific ecosystem. Not surprisingly, accumulation of this polymer is interconnected to central carbon metabolism and several fundamental biological processes as demonstrated by its intricate regulatory mechanisms. Available information suggests that the ability to synthesize and store intracellular glycogen by gut bacteria is linked to their long-term survival, adaptation to periods of starvation, protection against a range of stressors relevant to the gut ecosystem while also being important, and in some cases essential, for colonization of the GIT (Figure 5).

Figure 5. Role of glycogen metabolism in gut commensal bacteria.

Glycogen accumulation confers several potential advantages to bacteria enabling them to persist and survive into the gut environment. Among others, this metabolic process can: i) mediate sporulation, a strategy used by gut bacteria to survive under life-threatening conditions; ii) ensure long-term survival in periods of famine; iii) facilitate gut colonization; iv) efficiently response to stress conditions such as nutrient starvation, biofilm formation and promote acid tolerance and bile resistance.

This metabolic feature shapes functionality and composition of gut microbiome from a novel perspective. While most of the findings in this field are based on in vitro studies, the role of glycogen metabolism from an ecological perspective has been addressed in this review including metagenomics and metatranscriptomics of complex microbial communities and in vivo studies. Functional understanding and delineating the diversity, extent and regulation of this metabolic pathway in the gut microbiota may provide novel clues to improve their performance within the gut ecosystem.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Science Foundation of Ireland, through the Irish Government’s National Development Plan (SFI/12/RC/2273‐P1 and SFI/12/RC/2273‐P2) to DvS and KN, and by the Irish Research Council (grant GOIPD/2017/1302) and by the European union’s Horizon 2020 (MSCA-IF postdoctoral grant MicroMi-898088) to MET.