Fig 1.
Identification of the paenilarvins in infected larvae and in P. larvae secretomes.
(A) Extracted ion chromatogram (EIC) for paenilarvins detected in experimentally infected bee larvae. Paenilarvins appear at retention times Rt = 9.06 and Rt = 9.69 minutes; the peak at 8.00 min does not correspond to the paenilarvins. The figure is representative for 4 independent experiments. (B) EICs for paenilarvins A/B/C of culture supernatants of P. larvae DSM25430 wt (red dotted line) producing paenilarvins and the knockout mutant P. larvae DSM25430 Δitu (solid black line) not able to produce paenilarvins. (C) Masses for paenilarvins A/B extracted from the corresponding peak (Rt = 5 min) in (B).
Table 1.
Measured and calculated nominal masses, corresponding retention times [min] and mass errors (Δppm) of the paenilarvins A, B, and C detected in infected larvae.
Fig 2.
Analysis of paenilarvin as putative virulence factor during pathogenesis.
(A) At the age of 12 hours after egg hatching, honey bee larvae were infected with P. larvae DSM25430 wt and DSM25430 Δitu. Daily mortality due to P. larvae infection was recorded and total mortality after 15 days was determined. Bars represent the percentage of exposed larvae that died from American Foulbrood after 15 days. Bars represent mean values ± SD of three independent exposure bioassays for each group and 30 larvae per group. No significant difference in total mortality (Student´s t-test; p-value = 0.89) was observed. (B) Daily mortality due to P. larvae infection was recorded and cumulative mortality per day was calculated. Curves represent mean cumulative mortality ± SD for each day of 3 replicates with 30 larvae each. Analysis of the data by two-way ANOVA did not reveal any significant difference between the two curves (p-value = 0.15).
Fig 3.
Toxicity of paenilarvins towards larvae and insect cells.
(A) Larvae were in vitro-reared on normal larval diet. At day 4 post grafting, the treatment group received larval diet supplemented with 65 μg/mL paenilarvin A/B. Of the two negative control groups, one received larval diet supplemented 65 μg/mL jellein IV and the other received un-supplemented larval diet. Larvae were reared for 14 days and total larval mortality until day 14 is shown. The results are shown as mean values ± SD of three replicates with 30 larvae each. Larval mortality stayed below 15% for all three groups and no statistically significant difference in larval mortality between the control groups and the paenilarvin treatment group could be established (one-way ANOVA, p-value = 0.44). (B) BTI-Tn5B1-4 cells were incubated for 48 h in cell culture medium (SF900 II) without supplement or supplemented with different concentrations of jellein IV as negative control and of paenilarvin A/B isolated from DSM25430 wt. Cell lysis buffer was used as positive control. Cell viability was measured quantitatively by MTT test and the results are shown as mean values ± SEM of three replicates. No statistically significant difference in cell viability after incubation with any paenilarvin A/B concentration compared to the negative controls was evident (one-way ANOVA, p-value = 0.40).
Fig 4.
Verification of the paenilarvin gene cluster inactivation mutant P. larvae DSM25430 Δitu.
(A) PCR analysis of the genomic region of P. larvae DSM25430 Δitu with primers flanking the insertion site in the malonyl CoA-ACP transacylase gene of the paenilarvin gene cluster revealed successful intron insertion. The amplicons of the wild-type strain and of the mutant strain carrying the insertion migrated at their expected sizes of 1005 bp and 1905 bp, respectively. (B) Growth curves of the wild-type strain P. larvae DSM25430 wt (closed circles) and the mutant strain DSM25430 Δitu (open circles) in liquid broth did not differ significantly (two-way-ANOVA, p-value = 0.15). Data points represent the mean ± SEM and consisted of three biological replicates with three technical replicates each.
Table 2.
Primers used for generating and verifying the paenilarvin gene cluster inactivation mutant.