Antiglycation effects of carnosine and other compounds on the long-term survival of Escherichia coli

Abstract

Glycation, or nonenzymatic glycosylation, is a chemical reaction between reactive carbonyl-containing compounds and biomolecules containing free amino groups. Carbonyl-containing compounds include reducing sugars such as glucose or fructose, carbohydrate-derived compounds such as methylglyoxal and glyoxal, and nonsugars such as polyunsaturated fatty acids. The latter group includes molecules such as proteins, DNA, and amino lipids. Glycation-induced damage to these biomolecules has been shown to be a contributing factor in human disorders such as Alzheimer's disease, atherosclerosis, and cataracts and in diabetic complications. Glycation also affects Escherichia coli under standard laboratory conditions, leading to a decline in bacterial population density and long-term survival. Here we have shown that as E. coli aged in batch culture, the amount of carboxymethyl lysine, an advanced glycation end product, accumulated over time and that this accumulation was affected by the addition of glucose to the culture medium. The addition of excess glucose or methylglyoxal to the culture medium resulted in a dose-dependent loss of cell viability. We have also demonstrated that glyoxylase enzyme GloA plays a role in cell survival during glycation stress. In addition, we have provided evidence that carnosine, folic acid, and aminoguanidine inhibit glycation in prokaryotes. These agents may also prove to be beneficial to eukaryotes since the chemical processes of glycation are similar in these two domains of life.

FIG. 1.

Glucose toxicity profile. Cells were inoculated with the indicated amounts of glucose added: 0.2% (open squares), 0.4% (open circles), 0.6% (open triangles), and 0.8% (open diamonds). The dashed line with filled squares represents LB medium without glucose. Asterisks indicate titers below the limit of detection of <1,000 CFU/ml; titering error was ±2- to 3-fold (28). Representative data are shown (n = 3).

FIG. 2.

ELISA using anti-CML antibody in cultures containing either LB medium (black bars) or LB medium supplemented with 0.4% glucose (GLU) (dark gray bars). Cell-free controls are indicated in the absence (light gray bars) or presence (white bars) of glucose. Cultures were initially inoculated with approximately 10⁶ CFU/ml and harvested after 1, 3, or 5 days of incubation. Data are the averages of 12 trials with standard deviation shown.

FIG. 3.

Carnosine protection against glucose toxicity. (A) Cells were incubated in LB medium with glucose (GLU; 0.6% [33.3 mM]) and the following concentrations of carnosine: 0 mM (filled squares), 1 mM (filled circles), 5 mM (filled triangles), 10 mM (filled diamonds), 20 mM (open squares), 50 mM (open circles), 100 mM (open triangles), and 200 mM (open diamonds). The dashed line with filled boxes represents an LB control culture with no additional glucose or carnosine. The asterisk indicates titers below the limit of detection. Representative data are shown (n = 3). (B) Addition of carnosine at different times after inoculation. Cells were inoculated simultaneously with glucose (0.6% [33.3 mM]), and then carnosine (50 mM) was added on different days for 5 days. Time of carnosine addition: day 0 (open circles), day 1 (open squares), day 2 (open triangles), day 3 (open diamonds), day 4 (filled circles), or day 5 (filled diamonds). Asterisks indicate titers below the limit of detection of <1,000 CFU/ml. Representative data are shown (n = 2).

FIG. 4.

ELISA using anti-CML antibody. Cells were harvested 1, 3, or 5 days after inoculation. Absorbance measurements were obtained to quantify the relative amount of CML per milligram of total protein in each culture (see Materials and Methods). Data are the averages of 12 measurements with standard deviation shown. GLU, glucose (0.4%); CAR, carnosine (50 mM).

FIG. 5.

Carnosine protection against methylglyoxal toxicity. (A) Cultures were inoculated at ∼10⁶ CFU/ml and then grown overnight (18 to 22 h) in the presence or absence of methylglyoxal (MG, 2.1 mM) and/or carnosine (Car) at the given concentrations. (B) Comparison of wild-type strains with glyoxylase mutants. Growth yield data are shown with the concentration of carnosine indicated from 0 to 20 mM. Asterisks indicate cell titers that were below the limit of detection (10³ CFU/ml).

FIG. 6.

Proposed model of carnosine protection against glycation. Carnosine most likely interacts with reactive carbonyl intermediate compounds (e.g., glyoxal and methylglyoxal) to inhibit the formation of advanced glycated end products (e.g., CML and carboxyethyl lysine). Other mechanisms of protection are also probable (see text for details). Abbreviations: MgsA, methylglyoxal synthase; CML, carboxymethyl lysine; CEL, carboxyethyl lysine. GloA and GloB are glyoxylase enzymes I and II, respectively.