Effect of β - hydroxy - γ -aminophosphonate (β - HPC) on the hydrolytic activity of Nocardia brasiliensis as determined by FT-IR spectrometry

Abstract

The use of immunomodulatory and metabolic modulating drugs has been considered a better strategy to improve the efficacy of conventional treatments against pathogens and metabolic diseases. L-carnitine is relevant in fatty acid metabolism and energy production by β-oxidation, but it also has a beneficial therapeutic immunomodulatory effect. The β-hydroxy-γ-aminophosphonate (β-HPC) was developed, synthesized and studied in different pathologies as a more soluble and stable analog than L-carnitine, which has been studied in bacterial physiology and metabolism; therefore, we set out to investigate the direct effect of β-HPC on the metabolism of N. brasiliensis, which causes actinomycetoma in Mexico and is underdiagnosed. To analyze the effect of β-HPC on the metabolic capacity of the bacterium for the hydrolysis of substrate casein, L-tyrosine, egg yolk, and tween 80, Fourier transform infrared spectroscopy (FT-IR) was employed. It was found that β-HPC increases the metabolic activity of N. brasiliensis associated with increased growth and increased hydrolysis of the substrates tested. By the effect of β-HPC, it was observed that, in the hydrolysis of L-tyrosine, the aromatic ring and functional groups were degraded. At 1515 cm^-1, any distinctive signal or peak for this amino acid was missing, almost disappearing at 839, 720, 647, and 550 cm^-1. In casein, hydrolysis is enhanced in the substrate, which is evident by the presence of NH, OH, amide, and CO. In casein, hydrolysis is enhanced in the substrate, which is evident by the presence of NH, OH, amide, COO, and P = O signals, characteristic of amino acids, in addition to the increase of the amide I and II bands. In Tween 80 the H-C = and C = C signals disappear and the ether signals are concentrated, it was distinguished by the intense band at 1100 cm^-1. Egg yolk showed a large accumulation of phosphate groups at 1071 cm^-1, where phosvitin is located. FT-IR has served to demonstrate that β-HPC is a hydrolysis enhancer. Furthermore, by obtaining the spectrum of N. brasiliensis, we intend to use it as a quick comparison tool with other spectra related to actinobacteria. Eventually, FT-IR may serve as a species identification option.

Keywords: FT-IR; L-carnitine analog; L-tyrosine; Nocardia brasiliensis spectrum; Tween 80; casein; egg yolk; hydrolysis.

FIGURE 1

Molecular comparison of L-carnitine and β-HPC. On the left, in the black line structure, L-carnitine is shown as an amino acid derivative with seven carbons, a carboxylate, and a quaternary amino group. On the right, the additions and changes to β-hydroxyphosphocarnitine or β-hydroxy-γ-aminophosphonate are shown in blue: five carbons, a phosphonate in carbon one and an isopropyl group in carbon 4.

FIGURE 2

Comparison of FT-IR spectra of the hydrolytic activity of N. brasiliensis on casein with and without β-HFC. Culture medium alone, control (blue line), culture medium with 7 days of growth of N. brasiliensis (orange line), and culture medium with 7 days of N. brasiliensis + β-HPC (red line); the increase in the signals of the alcohol functional group (O–H), alkanes (C–H), amides I, II, and III (C = O, N-H, C-N), the carboxylic acid (COO–), phosphate (P = O), and ethers (C–O–C) characteristic of amino acids, when N. brasiliensis was incubated in the presence of β-HPC indicating greater hydrolysis of casein.

FIGURE 3

Comparison of FT-IR spectra of the hydrolytic activity of N. brasiliensis on L-tyrosine with and without β-HFC. Culture medium alone, control (blue line), culture medium with 7 days of growth of N. brasiliensis (orange line), culture medium with 7 days of N. brasiliensis growth + β-HPC (red line). A decrease in the bands corresponding to the functional groups of L-tyrosine is observed with respect to the control when N. brasiliensis is incubated in the culture and the effect is more marked when N. brasiliensis + β-HPC is added since the characteristic signals of this amino acid that correspond to the functional group (OH), to the amino acid (NH), to alkenes (C = C), and to the aromatic signals (Ar d) notoriously decrease, these signals show a transmittance between 85 and 95 cm^–1 (red line).

FIGURE 4

Comparison of FT-IR spectra of the hydrolytic activity of N. brasiliensis on tween 80 with and without β-HFC. Culture medium (control blue line), hydrolyzate of 7 days of N. brasiliensis growth (orange line), hydrolyzate of growth of 7 days of N. brasiliensis + β-HPC (red line). A decrease in the alcohol group (O–H) is observed, as well as the signals of alkenes (= C-H), alkanes (C–H), and carboxylic acids (COO). In contrast, more intense signals are detected mainly from ethers (C–O–C), both groups of signals, when accumulating and intensifying the signal, also indicate an increase in the utilization of Tween 80 when N. brasiliensis + β-HPC is incubated.

FIGURE 5

Comparison of FT-IR spectra of the hydrolytic activity of N. brasiliensis on egg yolk with and without β-HFC. Culture medium alone, control (blue line), culture medium with 7 days of N. brasiliensis growth (orange line), culture medium with 7 days of N. brasiliensis growth + β-HPC (red line). A decrease in alcohol (O–H) signals is observed, as well as alkenes (= C-H, C = C), alkanes (CH₃, C-H), and signals of the COO ester. In contrast, C-C and C-O-C had strong signals. At 3280 cm^–1, O-H, and N-H stretches were detected. At 2923 cm^–1, C-H strain signals were detected with C sp³ hybridization (absorbance at all three: 0.55), and at 2850 cm^–1, C-H was observed (absorbance at 3 0.64). At 1750 cm^–1 COO^– signals characteristic of esters were detected (0.71, 0.65, 0.71); amide I carbonyl (C = O) signal was detected at 1627 cm^–1; amide II N-H, C-N at 1518 cm^–1; a signal characteristic of carboxylic acids (COO–) at 1400 cm^–1; and P = O at 1250 cm^–1. A strong signal (phosphate P = O) was identified at 1071 cm^–1. At 950 cm^–1, there was a signal of alcohol (C–OH); beyond the fingerprint zone, there was also a robust C-OH signal at 550 cm^–1.

FIGURE 6

Interpolation of the hydrolysis of N. brasiliensis on substrates within the calibration curve. In the presence and absence of β-HPC with different standard concentrations from the calibration curves (cc) of each substrate, using the SCiDAVis program. (A) Casein spectra (cc), a transmittance value tending to 100 indicates degradation of this substrate. The spectra of hydrolyzed casein (10 g) with N. brasiliensis alone (dashed fuchsia line) or N. brasiliensis + β-HPC (dotted red line) are similar to that of 1.25 g (black line), proving that hydrolysis took place. (B) The L-tyrosine spectra showed the functional groups previously described between 1500 and 400 cm^–1; even when the detected signals are shorter due to dilution. The spectrum of hydrolyzed L-tyrosine by N. brasiliensis (dashed fuchsia line) showed a broader OH signal at 3650 cm^–1 with a transmittance value of 75, and at 1550 cm^–1, with a transmittance of 60. On the contrary, the spectrum of hydrolyzate by N. brasiliensis + β-HPC is similar to that of 1.25 g (black line) with a transmittance greater than 90, indicating almost complete hydrolysis of the substrate. (C) In the Tween 80 cc, the hydrolyzate by N. brasiliensis corresponds to that of 0.2% concentration of this substrate. However, such results indicated a higher concentration of hydrolyzed compounds, since the concentration for the identification of this substrate is 0.1%; the spectrum hydrolyzed by N. brasiliensis + β-HPC (red dotted line) resembles that of 0.025% (green line). (D) Signals in egg yolk cc spectra show higher amplitude when this substrate is diluted, and in the 3000–3500 cm^–1 range, the hydrolyzed product by N, brasiliensis resembles that of 375 μL (green line) with a transmittance value of 86. The hydrolyzed lipid of N. brasiliensis + β-HPC correlates to that of 750 μL (deep blue line) with a transmittance value of 82. N. brasiliensis + β-HPC were closest to the highest concentrations. In the protein window (1800–1500 cm^–1), the substrate hydrolyzed by N. brasiliensis has a transmittance value of 60, and that hydrolyzed by N. brasiliensis + β-HPC presented a transmittance value of 54; such differences were similar throughout the mix and polysaccharide windows. However, broad signals were observed at 93, 75, and 1500 μL with transmittance values of 30 and 38 in the fingerprint area. In the presence of β-HPC, a signal with a transmittance value signal of 44 was detected.

FIGURE 7

Linear regression plots (x, y) of the interpolated data of N. brasiliensis on the calibration curve. (A) Casein did not show a linear relation even when the values were within the control limits (dotted lines in red). The r² value was 0.70. (B) The zigzagging behavior in L-tyrosine did not show linear regression, r² was 0.67. (C) The first plotted data prevented by Tween 80 showed a linear regression also affected by correlation, the r² was 0.69. (D) The curve relation in egg yolk exceeded the control limits, the r² was 0.73. The plots were generated in GraphPad Prism 5.0.

FIGURE 8

Least squares plots (x, y) of N. brasiliensis hydrolyzates interpolated on the calibration curve. The best fit was calculated. (A) Casein has linear regression and a pronounced slope with an r² of 1.0. (B) L-tyrosine showed a feeble slope in the linear regression. Therefore, the r² value was 0.67. (C) Tween 80 had linear regression, slope, and an r² value of 1.0. (D) The egg yolk showed a linear regression. The slope was not as pronounced as the casein, and the r² of Tween 80, was 1.0. The plots were generated in GraphPad Prism 5.0.

FIGURE 9

Grouping of data of the hydrolysis of N. brasiliensis within the calibration curve. (A) Hydrolysis of N. brasiliensis casein compared to calibration curve 1:16 dilution, corresponding to 1.25 g. (B) N. brasiliensis + β-HPC casein hydrolysis was close to the calibration curve 1:4 corresponding to 5 g. (C) The hydrolysis of N. brasiliensis on L-tyrosine approached the initial amount of the calibration curve (20 g). (D) N. brasiliensis + β-HPC hydrolysis approached a dilution of 1:2 of L-tyrosine from the calibration curve (10 g). (E) In egg yolk hydrolysis by N. brasiliensis, the dilution was 1: 8. (F) The hydrolysis of egg yolk from N. brasiliensis + β-HPC was greater because it approached the highest dilution. (G) N. brasiliensis and N. brasiliensis + β-HPC species over Tween 80 hydrolysis showed similarities to the 2% calibration curve; it represented the initial concentration to be diluted.

FIGURE 10

Principal component analysis (PCA) of the hydrolysis of N. brasiliensis within the calibration curve. Two main components were graphed that expressed the widest variation in the data set, a total of 1932 values given by FT-IR wavelength (X) and transmittance (Y). (A) Casein substrate at 1.25, 2.5, 5, 10, and 20 g, N. brasiliensis and N. brasiliensis + β-HPC. (B) L-Tyrosine substrate at 1.25, 2.5, 5, 10, and 20 g, N. brasiliensis and N. brasiliensis + β-HPC. (C) Tween 80 substrate at 0.1, 0.2, 0.025.0.01, and 0.5%, N. brasiliensis and N. brasiliensis + β-HPC. (D) Egg yolk substrate at 93.75, 187.5, 375, 750, 1500 mL, N. brasiliensis and N. brasiliensis + β-HPC. The PCA analysis was performed with OriginPro 2018 and R Commander 2.5–1.

FIGURE 11

Fourier transform infrared (FT-IR) spectrum of the FM-825 strain of N. brasiliensis isolated from a mycetoma patient. The absorbance spectra were collected between 4000 and 400 cm^–1 at a spectral resolution of 4 cm^–1 with 50 coadded and averaged scans and 50 background scans. The typical spectrum of bacteria is observed showing the assignments of the main signals within the spectral windows (W1–W5). (v) stretching vibrations, (d) bending vibrations, (s) symmetric vibrations, (as) asymmetric vibrations.

FIGURE 12

Comparison of the growth of control strain HUJEG-1 and FM-825 under the effect of β-HFC. (A) 24-well plate with Mueller-Hinton + β-HPC broth; 5 × 10⁵ CFU/mL N. brasiliensis HUJEG-1 (ATCC 700358) was seeded in nine wells from the first three columns: 1–3, and rows (A–C). In column 3 no β-HPC was added, and after 7 days of incubation there was no growth. A total of 5 × 10⁵ CFU/mL of N. brasiliensis FM-825 was seeded in columns 4–6 and rows (A–C). Column 6 was not added with β-HPC. Growth was observed in the seeded wells, and those without β-HPC had little growth. Row D was used as a sterility control of the procedure. (B) Chocolate agar plate, 5 × 10⁵ CFU/ml of N. brasiliensis HUJEG-1 (ATCC 700358) were seeded after thawing, grown in M-H with β-HPC and standardized with the McFarland Nephelometer; in all areas of striation, little growth is observed. (C) Chocolate agar plate, 5 × 10⁵ CFU/mL of N. brasiliensis FM-825 were seeded after thawing, grown on M-H agar with β-HPC and standardized with the McFarland Nephelometer; abundant growth is observed in all areas of striation.