Synergy of alanine and gentamicin to reduce nitric oxide for elevating killing efficacy to antibiotic-resistant Vibrio alginolyticus

Abstract

The present study explored the cooperative effect of both alanine (Ala) and gentamicin (Gent) on metabolic mechanisms by which exogenous Ala potentiates Gent to kill antibiotic-resistant Vibrio alginolyticus. To test this, GC-MS-based metabolomics was used to characterize Ala-, Gent- and both-induced metabolic profiles, identifying nitric oxide (NO) production pathway as the most key clue to understand metabolic mechanisms. Gent, Ala and both led to low, lower and lowest activity of total nitric oxide synthase (tNOS) and level of NO, respectively. NOS promoter L-arginine and inhibitor N^G-Monomethyl-L-arginine inhibited and promoted the killing, respectively, with the elevation and decrease of NOS activity and NO level. The present study further showed that CysJ is the enzyme-producing NO in V. alginolyticus. These results indicate that the cooperative effect of Ala and Gent causes the lowest NO, which plays a key role in Ala-potentiated Gent-mediated killing.

Keywords: Ala; Gent; NOS; V. alginolyticus; metabolomics; nitric oxide.

Figure 1.

Antibacterial efficiency by the synergistic use of Gent and Ala. A. Percent survival of ATCC33787 in the indicated concentrations of Ala and 40 μg/mL Gent. B. Percent survival of ATCC33787 in the indicated concentrations of Gent and 10 mM Ala. C. Percent survival of ATCC33787 in the indicated incubation time in the synergistic use of 40 μg/mL Gent and 10 mM Ala. D. Percent survival of ATCC33787 in the presence of other aminoglycosides. E. Percent survival of clinically isolated Vibrio spp. and lab-evolved antibiotic-resistant strains in the synergistic use of the indicated concentrations of Gent and 10 mM Ala. Results (a-e) are displayed as mean ± SEM, and significant differences are identified **p < 0.01) as determined by two-tailed Student’s t test

Figure 1.

Antibacterial efficiency by the synergistic use of Gent and Ala. A. Percent survival of ATCC33787 in the indicated concentrations of Ala and 40 μg/mL Gent. B. Percent survival of ATCC33787 in the indicated concentrations of Gent and 10 mM Ala. C. Percent survival of ATCC33787 in the indicated incubation time in the synergistic use of 40 μg/mL Gent and 10 mM Ala. D. Percent survival of ATCC33787 in the presence of other aminoglycosides. E. Percent survival of clinically isolated Vibrio spp. and lab-evolved antibiotic-resistant strains in the synergistic use of the indicated concentrations of Gent and 10 mM Ala. Results (a-e) are displayed as mean ± SEM, and significant differences are identified **p < 0.01) as determined by two-tailed Student’s t test

Figure 2.

Differential metabolomic profiling of Ala group, Gent group, and Ala + Gent group compared with untreated group (control). A. Heat map showing differential metabolites. Yellow color and blue color indicate increase and decrease of metabolites relative to the median metabolite level, respectively (see color scale). B. Category of identified metabolites of differential abundance. C. Number of differential abundance of metabolites. D. Venn diagram for comparison of differential metabolites among Ala (green color), Gent (blue color) and Ala + Gent group (red color). E. Z-score plot of differential metabolites based on control. The data of Ala (left), Gent (middle) and Ala + Gent (right) groups were separately scaled to the mean and standard deviation of control. Each point represents one metabolite in one technical repeat and colored by sample types

Figure 2.

Differential metabolomic profiling of Ala group, Gent group, and Ala + Gent group compared with untreated group (control). A. Heat map showing differential metabolites. Yellow color and blue color indicate increase and decrease of metabolites relative to the median metabolite level, respectively (see color scale). B. Category of identified metabolites of differential abundance. C. Number of differential abundance of metabolites. D. Venn diagram for comparison of differential metabolites among Ala (green color), Gent (blue color) and Ala + Gent group (red color). E. Z-score plot of differential metabolites based on control. The data of Ala (left), Gent (middle) and Ala + Gent (right) groups were separately scaled to the mean and standard deviation of control. Each point represents one metabolite in one technical repeat and colored by sample types

Figure 3.

Pathway enrichment. A. Pathway enrichment of varied metabolites in the Ala group, Gent group, and Ala + Gent group. Significant enriched pathways are selected to plot. P-value < 0.05. B. Venn diagram for significantly enriched pathways between the Ala (green color), Gent (blue color), and Ala + Gent group (red color). C. Integrative analysis of metabolites in significantly enriched pathways. Yellow color and blue color indicate increased and decreased metabolites, respectively

Figure 3.

Pathway enrichment. A. Pathway enrichment of varied metabolites in the Ala group, Gent group, and Ala + Gent group. Significant enriched pathways are selected to plot. P-value < 0.05. B. Venn diagram for significantly enriched pathways between the Ala (green color), Gent (blue color), and Ala + Gent group (red color). C. Integrative analysis of metabolites in significantly enriched pathways. Yellow color and blue color indicate increased and decreased metabolites, respectively

Figure 4.

Identification of crucial metabolites. A. The PCA analysis of control group, Ala group, Gent group, and Ala + Gent group. Each dot represents the technique replicates in the plot. t [1] and t [2] explain 98.7% of the total variance which allows confident interpretation of the variation. B. S-plot generated from OPLS-DA. Triangle represents individual metabolite, where potential biomarkers are highlighted with red, which is greater or equal to 0.05 and 0.5 for absolute value of covariance p and correlation p(corr), respectively. C. The scatter plot of glutamic acid, citrulline, and ornithine. Results are displayed as mean ± SEM, and significant differences are identified **p < 0.01

Figure 4.

Identification of crucial metabolites. A. The PCA analysis of control group, Ala group, Gent group, and Ala + Gent group. Each dot represents the technique replicates in the plot. t [1] and t [2] explain 98.7% of the total variance which allows confident interpretation of the variation. B. S-plot generated from OPLS-DA. Triangle represents individual metabolite, where potential biomarkers are highlighted with red, which is greater or equal to 0.05 and 0.5 for absolute value of covariance p and correlation p(corr), respectively. C. The scatter plot of glutamic acid, citrulline, and ornithine. Results are displayed as mean ± SEM, and significant differences are identified **p < 0.01

Figure 5.

iPath analysis showing comparison among Ala group, Gent group, and Ala + Gent group. The yellow and blue lines mean upregulation and downregulation of metabolic pathways, respectively. A. The TCA cycle. B. Amino acid metabolism. C. Urea cycle. D. Nucleotide metabolism. E. Activity of enzymes of the P cycle. Results (e) are displayed as mean ± SEM, and significant differences are identified (**p < 0.01) as determined by two-tailed Student’s t test

Figure 5.

iPath analysis showing comparison among Ala group, Gent group, and Ala + Gent group. The yellow and blue lines mean upregulation and downregulation of metabolic pathways, respectively. A. The TCA cycle. B. Amino acid metabolism. C. Urea cycle. D. Nucleotide metabolism. E. Activity of enzymes of the P cycle. Results (e) are displayed as mean ± SEM, and significant differences are identified (**p < 0.01) as determined by two-tailed Student’s t test

Figure 6.

NO generation and level mediated by Gent, Ala, and both. A. Metabolic diagram of NO generation. B. Activity of NO synthase (NOS) in V. alginolyticus ATCC33787 in response to Ala, Gent, and both. C. NO level in V. alginolyticus ATCC33787 in response to Ala, Gent, and both. D, Nitrite in V. alginolyticus ATCC33787 in response to Ala, Gent, and both. Results (b and c) are displayed as mean ± SEM, and significant differences are identified (*p < 0.05, **p < 0.01) as determined by two-tailed Student’s t test

Figure 6.

NO generation and level mediated by Gent, Ala, and both. A. Metabolic diagram of NO generation. B. Activity of NO synthase (NOS) in V. alginolyticus ATCC33787 in response to Ala, Gent, and both. C. NO level in V. alginolyticus ATCC33787 in response to Ala, Gent, and both. D, Nitrite in V. alginolyticus ATCC33787 in response to Ala, Gent, and both. Results (b and c) are displayed as mean ± SEM, and significant differences are identified (*p < 0.05, **p < 0.01) as determined by two-tailed Student’s t test

Figure 7.

Association of NO level with percent survival. A and B. NOS activity (a) and NO level (b) of V. alginolyticus ATCC33787 in the absence or presence of Ala, Gent or/and arginine. C. Percent survival of V. alginolyticus ATCC33787 in the presence of Ala + Gent plus the indicated concentration of arginine. D. Percent survival of V. alginolyticus ATCC33787 in the presence of Gent and L-NMMA. E. Percent survival of V. alginolyticus ATCC33787 in the presence of Gent plus the indicated concentration of L-NMMA. F. and G. NOS (f) and NO (g) of V. alginolyticus ATCC33787 in the presence of Ala + Gent plus L-NMMA. H. Percent survival of V. alginolyticus ATCC33787 in the presence of Ala and Gent, and the indicated concentration of H₂O₂ and time. Results (a-h) are displayed as mean ± SEM, and significant differences are identified (*p < 0.05, **p < 0.01) as determined by two-tailed Student’s t test

Figure 7.

Association of NO level with percent survival. A and B. NOS activity (a) and NO level (b) of V. alginolyticus ATCC33787 in the absence or presence of Ala, Gent or/and arginine. C. Percent survival of V. alginolyticus ATCC33787 in the presence of Ala + Gent plus the indicated concentration of arginine. D. Percent survival of V. alginolyticus ATCC33787 in the presence of Gent and L-NMMA. E. Percent survival of V. alginolyticus ATCC33787 in the presence of Gent plus the indicated concentration of L-NMMA. F. and G. NOS (f) and NO (g) of V. alginolyticus ATCC33787 in the presence of Ala + Gent plus L-NMMA. H. Percent survival of V. alginolyticus ATCC33787 in the presence of Ala and Gent, and the indicated concentration of H₂O₂ and time. Results (a-h) are displayed as mean ± SEM, and significant differences are identified (*p < 0.05, **p < 0.01) as determined by two-tailed Student’s t test

Figure 8.

Role of CysJ in NO generation in V. alginolyticus. A. Blast analysis of NOS-like protein CysJ in V. alginolyticus against NOS from human, mouse, fungi, and bacteria. B. Activity of NOS in recombinant CysJ in the absence or presence of SDS or L-NMMA. C. Activity of NOS and content of NO in cysJ-deleted mutant. D. Percent survival of cysJ-deleted mutant. E. qRT-PCR for expression of cysJ in the presence of Ala, Gent, L-NMMA or/and arginine

Figure 9.

Diagram for the synergistic use of Gent and Ala to kill antibiotic-resistant V. alginolyticus.

Figure 9.

Diagram for the synergistic use of Gent and Ala to kill antibiotic-resistant V. alginolyticus.