Insights into glycogen metabolism in Lactobacillus acidophilus: impact on carbohydrate metabolism, stress tolerance and gut retention

Abstract

In prokaryotic species equipped with glycogen metabolism machinery, the co-regulation of glycogen biosynthesis and degradation has been associated with the synthesis of energy storage compounds and various crucial physiological functions, including global cellular processes such as carbon and nitrogen metabolism, energy sensing and production, stress response and cell-cell communication. In addition, the glycogen metabolic pathway was proposed to serve as a carbon capacitor that regulates downstream carbon fluxes, and in some microorganisms the ability to synthesize intracellular glycogen has been implicated in host persistence. Among lactobacilli, complete glycogen metabolic pathway genes are present only in select species predominantly associated with mammalian hosts or natural environments. This observation highlights the potential involvement of glycogen biosynthesis in probiotic activities and persistence of intestinal lactobacilli in the human gastrointestinal tract. In this review, we summarize recent findings on (i) the presence and potential ecological distribution of glycogen metabolic pathways among lactobacilli, (ii) influence of carbon substrates and growth phases on glycogen metabolic gene expression and glycogen accumulation in L. acidophilus, and (iii) the involvement of glycogen metabolism on growth, sugar utilization and bile tolerance. Our present in vivo studies established the significance of glycogen biosynthesis on the competitive retention of L. acidophilus in the mouse intestinal tract, demonstrating for the first time that the ability to synthesize intracellular glycogen contributes to gut fitness and retention among probiotic microorganisms.

Figure 1

Classical pathway of glycogen metabolism in prokaryotes. Pgm converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by glucose-1-phosphate adenylyltransferase (GlgCD) encoded by glgC and glgD genes. Then, GlgA catalyzes the transfer of glucosyl moieties from ADP-glucose to the elongating chain of linear α-1,4-glucan; GlgB subsequently cleaves off a portion of the glucan and attaches it to existing chains via α-1,6 linkages to form the glycogen structure. For glycogen degradation, GlgP sequentially releases glucose moieties from the non-reducing ends to form glucose-1-phosphate and α-1,6-branched dextrins. GlgX or Amy hydrolyzes the α-1,6 branches of the phosphorylase-limit dextrins (typically 3–5 glucosyl residues in length) leading to the release of maltodextrins. Glycogen biosynthetic steps are indicated in red arrows, whereas glycogen degradation pathway is indicated in dashed purple arrows.

Figure 2

Genetic organization of glycogen metabolic ( glg ) operons in Lactobacillus species. (A) Organization of the glycogen metabolic genes encoded in L. acidophilus NCFM. Predicted rho-independent transcriptional terminators are indicated in hairpin loops with overall confidence score (ranges from 0 to 100) [31]. (B) Comparative gene mosaic arrangement of the glg operons among Lactobacillus and other representative microorganisms. All glg operons in Lactobacillus have the signature glgBCDAP core genes. In addition, species closely related to L. acidophilus have amy and pgm genes proceeding the core genes. Strain designations were included for specific Lactobacillus strains (indicated with asterisks, *) with intact glg operons but belong to species that are generally lacking the pathway based on the NCBI genome database to date. Putative glycogen biosynthesis genes are indicated in red arrows, whereas glycogen degradation genes are indicated in purple arrows. amyA or pulA, α-amylase/pullulanase/glycogen-debranching enzyme. Figure modified from [23].

Figure 3

Intracellular glycogen levels and glg expression during growth on raffinose versus trehalose. (A) Growth and glycogen accumulation profiles of L. acidophilus in semi-defined medium (SDM) [35] containing 2% of raffinose or trehalose as the sole carbon source. The glycogen levels at various growth phases (indicated by OD₆₀₀) from (A) were compiled and plotted against the transcript levels of the glg operon in (B). The data represent the mean ± standard deviation for two independent biological replicates. Growth phases are represented by OD₆₀₀: 0.3, early log; 0.6, mid-log; 1.0, early stationary; 1.5, stationary, and 2.2, late stationary. Intracellular glycogen content was quantified by hexokinase/glucose-6-phosphate dehydrogenase-based glucose assay and expressed as mg of glucose (released from glycogen by amyloglucosidase) per g of cell wet weight (mg glucose/g cww). Figure adapted from [23].

Figure 4

Role of glgA and glgB in glycogen biosynthesis. (A) Iodine staining of L. acidophilus NCK1909 parent strain and glg mutants grown on solid SDM containing 2% trehalose. Both ΔglgA and ΔglgB mutant cells appeared as yellow/colorless indicative of glycogen-deficient phenotype. Like the parent cells, the ΔglgP and Δamy mutants were stained brown, indicating that their ability to synthesize intracellular glycogen was unaffected. (B) Quantitative analysis of intracellular glycogen content in mid-log phase cells cultivated in SDM containing 2% trehalose, confirming the absence of intracellular glycogen in both ΔglgA and ΔglgB cultures. Data shown represent the mean ± standard deviation for two independent biological replicates. Figure adapted from [23].

Figure 5

Competitive co-colonization and colonization displacement experiments in vivo . (A) Competitive colonization study comparing GI retention of the glycogen-deficient ΔglgA mutant with the parent strain. Antibiotic-resistant derivatives of parent and ΔglgA mutant were generated for the studies as described in the text. Germ-free 129S6/SvEv mice (2 males, 2 females; 12–26 weeks old) were used in the co-colonization experiment carried out at the North Carolina State University Gnotobiotic Core Facility. Animal use protocols were approved by the Institutional Animal Care and Use Committee of North Carolina State University. Mice were maintained in cages in germ-free flexible film isolators housed in a room with cycles of 12 h of light and darkness, and were provided access to a standard diet (Prolab RMH 3500, LabDiet, St. Louis, MI) and water ad libitum. The mice were verified germ-free by culturing of fecal samples aerobically and anaerobically on plate count agar and MRS agar prior to experiments. On Day 0, both parent and ΔglgA mutant cells grown overnight in MRS broth were harvested, washed once with phosphate-buffered saline (PBS, pH 7.4; Life Technologies) and resuspended in fresh PBS. The cultures were each titered in PBS and combined in 1:1 ratio to obtain 1 x 10⁷ cells in total per 200 μL of gavage volume. Following gavage (200 μL/mouse), fecal samples were collected periodically, weighed, homogenized in PBS, diluted and plated onto antibiotic selective media for enumeration of the parent and mutant populations. (B) Germ-free mice (3 males, 27 weeks old) were first gavaged with the glgA mutant. A second gavage with the parent strain was subsequently performed when the glgA mutant population reached 6 x 10⁸ – 8 x 10⁸ CFU/g of fecal samples (indicated by a red arrow). Preparation of bacterial cells for gavage and sampling of fecal samples were carried out as described above.

Figure 6

Co-colonization and colonization displacement studies demonstrating the role of glycogen biosynthesis on competitive gut retention. (A) Co-colonization of both the glycogen-deficient ΔglgA mutant and the parent strain in germ-free 129S6/SvEv mice resulted in an overall 2 log reduction of the ΔglgA mutant population. Data shown represent the median cell counts values and mean cell counts ± standard deviation from all four mice. (B) Addition of the parent strain to gnotobiotic mice previously mono-colonized with the ΔglgA mutant resulted in a population shift with gradual displacement of the mutant population by the parent. Red arrow indicates the timepoint at which the parent strain was administered in a single gavage dose of 5 x 10⁸ CFU. Data shown represent the average cell counts values and mean cell counts ± standard deviation from all three mice. An asterisk (*) indicates a statistically significant difference between the mutant and parent populations (p-value < 0.05). Both in vivo studies established that a functional glycogen biosynthetic pathway contributes to the competitive advantage and retention of L. acidophilus in the GI tract.

Figure 7

Proposed mechanisms and functions of glycogen metabolic pathway in L. acidophilus . Based on proposed central functions of glycogen metabolism in other microorganisms [6,11,12] and findings from this study, carbohydrate substrates imported by the cells may be either metabolized through the glycolytic pathway, or shunted to glycogen biosynthetic pathway when a carbon source is not immediately required for glycolysis. The carbon pool may serve as an energy storage reserve and as a carbon capacitor that senses and modulates downstream carbon flow to maintain efficient carbon utilization and energetic homeostasis of the cells. The roles of glycogen metabolism in central carbon metabolism influence various physiological functions and consequently the retention and probiotic attributes of L. acidophilus. Our fundamental understanding of this pathway will inspire strategies to improve the stability and functionalities of probiotic and beneficial commensal microorganisms in the host.