The Development of Bacteriophage Resistance in Vibrio alginolyticus Depends on a Complex Metabolic Adaptation Strategy

Abstract

Lytic bacteriophages have been well documented to play a pivotal role in microbial ecology due to their complex interactions with bacterial species, especially in aquatic habitats. Although the use of phages as antimicrobial agents, known as phage therapy, in the aquatic environment has been increasing, recent research has revealed drawbacks due to the development of phage-resistant strains among Gram-negative species. Acquired phage resistance in marine Vibrios has been proven to be a very complicated process utilizing biochemical, metabolic, and molecular adaptation strategies. The results of our multi-omics approach, incorporating transcriptome and metabolome analyses of Vibrio alginolyticus phage-resistant strains, corroborate this prospect. Our results provide insights into phage-tolerant strains diminishing the expression of phage receptors ompF, lamB, and btuB. The same pattern was observed for genes encoding natural nutrient channels, such as rbsA, ptsG, tryP, livH, lysE, and hisp, meaning that the cell needs to readjust its biochemistry to achieve phage resistance. The results showed reprogramming of bacterial metabolism by transcript regulations in key-metabolic pathways, such as the tricarboxylic acid cycle (TCA) and lysine biosynthesis, as well as the content of intracellular metabolites belonging to processes that could also significantly affect the cell physiology. Finally, SNP analysis in resistant strains revealed no evidence of amino acid alterations in the studied putative bacterial phage receptors, but several SNPs were detected in genes involved in transcriptional regulation. This phenomenon appears to be a phage-specific, fine-tuned metabolic engineering, imposed by the different phage genera the bacteria have interacted with, updating the role of lytic phages in microbial marine ecology.

Keywords: Vibrio alginolyticus; acquired phage resistance; bacteriophages; host metabolism; host–phage interaction; metabolic reprogramming; receptors; transporters.

Figure 1

Growth kinetics of phage-resistant and phage-susceptible strains. Growth (average, ±SE) of the Control (phage-susceptible strains; black circle; n = 3), VaAphrodite1 strains (phage strains resistant to bacteriophage Aphrodite1; white circle; n = 9), VaphiSt2 strains (phage strains resistant to bacteriophage phiSt2; black triangle; n = 9), and VaAres1 strains (phage strains resistant to bacteriophage Ares1; white triangle; n = 9).

Figure 2

Adsorption assay for bacteriophages Aphrodite1, phiSt2, and Ares1 against their corresponding resistant strains. Initial titer was 10⁴, and cultures upon infection were in their exponential phase (±SE; n = 3).

Figure 3

Relative expression levels of genes encoding for outer membrane and transmembrane proteins. Bars represent means ± SE of independent biological repeats (n = 3). Asterisks * indicate significantly different values based on Student’s t-test. Statistical significance was postulated for p ≤ 0.05.

Figure 4

Partial Least Squares Discriminant Analysis (PLS-DA) of metabolomics results. and representation of component 1 (46.4%) and component 2 (12.2%) (A) of the identified metabolites discussed in the results and discussion section comparing the wild type (Control, phage-susceptible strains; n = 4), VaAphrodite1 strains (phage strains resistant to bacteriophage Aphrodite1; n = 5), VaphiSt2 strains (phage strains resistant to bacteriophage phiSt2; n = 5), and VaAres1 strains (phage strains resistant to bacteriophage Ares1; n = 5). (B) Loadings of individually identified components showing the highest variability under the studied experimental conditions.

Figure 5

Schematic representation of nucleotide sequences of four CDSs and their orientation, involved in transcriptional regulation of V. alginolyticus, and their SNPs as presented in the text. Yellow represents CDS; orange, SNP positions; and blue, information of SNPs and protein effects in resistant bacteria VaAphoridte1 (A, B), VaphiSt2 (A, B), and VaAres1 (B, C). Orientation of the genome is also shown with blue numbers.

Figure 6

Schematic representation of metabolic reprogramming that takes place in phage-resistant strains. Relative transcript levels (heat maps) of sugar–polyol transporters (blue crescent moon) and amino acid transporters (brown crescent moon) of the inner bacterial membrane, accompanied by genes that are involved in important biochemical intracellular pathways of phage-susceptible (A) and phage-resistant (B, C, D) strains. Bars represent relative content levels of intracellular metabolites (±SE) in susceptible (white) and resistant strains in bacteriophages Aphrodite1 (light grey), phiSt2 (dark grey), and Ares1 (black). Blue arrows represent biochemical pathways in brief; the light blue round arrow represents part of the TCA cycle; yellow arrows represent putative extraction of metabolites through membrane proteins. White and black asterisks represent statistically significant differences between phage-susceptible strains and phage-resistant strains (ANOVA, post-hoc analysis Student’s t-test, p < 0.05).

Figure 7

Schematic representation of how metabolic reprogramming of bacterial cells could introduce phage resistance against lytic bacteriophages after phage–host interaction. Arrows represent genes (as seen in Table 3) involved in nutrient assimilation or exportation for which their relative transcript levels were significantly downregulated compared to the control (wild-type) strains in the current study. Gray: outer membrane receptors (lamb and ompF); Blue: inner bacterial membrane sugars and polyols (ptsG 2, crr, rbsA, celB, mtlA); Red: inner bacterial membrane amino acids (metQ, metL metN, livH, livB and hisP). OM: outer membrane; PG: peptidoglycan layer.