Screening and identification of BP100 peptide conjugates active against Xylella fastidiosa using a viability-qPCR method

Abstract

Background: Xylella fastidiosa is one of the most harmful bacterial plant pathogens worldwide, causing a variety of diseases, with huge economic impact to agriculture and environment. Although it has been extensively studied, there are no therapeutic solutions to suppress disease development in infected plants. In this context, antimicrobial peptides represent promising alternatives to traditional compounds due to their activity against a wide range of plant pathogens, their low cytotoxicity, their mode of action that make resistance more difficult and their availability for being expressed in plants.

Results: Peptide conjugates derived from the lead peptide BP100 and fragments of cecropin, magainin or melittin were selected and tested against the plant pathogenic bacteria X. fastidiosa. In order to screen the activity of these antimicrobials, and due to the fastidious nature of the pathogen, a methodology consisting of a contact test coupled with the viability-quantitative PCR (v-qPCR) method was developed. The nucleic acid-binding dye PEMAX was used to selectively quantify viable cells by v-qPCR. In addition, the primer set XF16S-3 amplifying a 279 bp fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed by comparing v-qPCR viable cells estimation with conventional qPCR and plate counting. When cells were treated with peptide conjugates derived from BP100, the observed differences between methods suggested that, in addition to cell death due to the lytic effect of the peptides, there was an induction of the viable but non-culturable state in cells. Notably, a contact test coupled to v-qPCR allowed fast and accurate screening of antimicrobial peptides, and led to the identification of new peptide conjugates active against X. fastidiosa.

Conclusions: Antimicrobial peptides active against X. fastidiosa have been identified using an optimized methodology that quantifies viable cells without a cultivation stage, avoiding underestimation or false negative detection of the pathogen due to the viable but non-culturable state, and overestimation of the viable population observed using qPCR. These findings provide new alternative compounds for being tested in planta for the control of X. fastidiosa, and a methodology that enables the fast screening of a large amount of antimicrobials against this plant pathogenic bacterium.

Keywords: Antimicrobial peptides; PEMAX; Plant pathogens; Viability-qPCR; Xylella fastidiosa.

Fig. 1

Relationship between C_T values and cell concentration in three strains of Xf using conventional qPCR (white symbols) and v-qPCR (black symbols), for viable cells, dead cells, and a mixture of viable cells with a fixed concentration of dead cells (1 × 10⁶ CFU/ml). TaqMan-based qPCR assay done with XF16S-3 primers. The thin line represents the detection limit at C_T = 37.5

Fig. 2

Effect of peptide BP178 on viability of Xff strain Temecula estimated by v-qPCR at different peptide concentrations (1.6, 12.5 and 50 μM). Two assays were performed at different initial Xff cell concentrations, 1 × 10⁷ CFU/ml (circles) and 1 × 10⁸ CFU/ml (squares). The exposure time to the peptide was 3 h. Xff concentration in non-treated cells was estimated after 3 h by v-qPCR. The detection limit of the v-qPCR is 3 log CFU/ml. Values are the means of three replicates, and error bars represent the standard deviation of the mean. Lowercase letters correspond to the means comparison of viable cells in 1 × 10⁷ CFU/ml. Capital letters correspond to the means comparison of viable cells in 1 × 10⁸ CFU/ml. Means sharing the same letters are not significantly different (P < 0.05), according to the Tukey’s test

Fig. 3

Effect of peptide BP178 on viability and culturability of Xff strain Temecula at different exposure times. Cell viability was estimated by v-qPCR (black symbols) and cell culturability by plate counting (grey symbols). Initial cell concentration was 1 × 10⁷ CFU/ml and the BP178 concentration used was 50 μM. Non-treated controls (NTC) were also performed by adding the corresponding volume of sterile distilled water. The dash line represents the detection limit of v-qPCR, whereas the normal line indicates the detection limit of the plate counting technic. Values are the means of three replicates, and error bars represent the standard deviation of the mean. Lowercase letters correspond to the means comparison of viable cells treated with BP178 (black triangles). Capital letters correspond to the means comparison of culturable cells treated with BP178 (grey triangles). Means sharing the same letters are not significantly different (P < 0.05), according to the Tukey’s test

Fig. 4

Effect of peptide BP178 on viability and culturability of Xff strain Temecula at different peptide concentrations. Total cell concentration was estimated by conventional qPCR (white symbols), cell viability was estimated by v-qPCR (black symbols), and cell culturability by plate counting (grey symbols). Exposure times of 3 h (triangles) and 24 h (circles) were used. Cell concentration was 1 × 10⁷ CFU/ml in both cases. The dash line represents the detection limit of v-qPCR, whereas the normal line indicates the detection limit of the plate counting technic. Values are the means of three replicates, and error bars represent the standard deviation of the mean. Letters correspond to the means comparison of viable cells treated with BP178 at exposure time of 3 h. Means sharing the same letters are not significantly different (P < 0.05), according to the Tukey’s test