Tyramine is not essential for European foulbrood pathogenesis in a virulent Melissococcus plutonius strain

Abstract

European foulbrood, caused by Melissococcus plutonius, is a major bacterial disease of honey bee brood. Although strains belonging to the genetically related group known as clonal complex 12 (CC12) are highly virulent, their virulence factors remain unclear. Since tyramine has been proposed as a putative virulence factor, we constructed a knockout mutant of the putative tyrosine decarboxylase gene (tdc) from a CC12 strain to assess its role in pathogenicity. The tdc knockout mutant lost the ability to produce tyramine, confirming that tdc is responsible for tyramine production. However, the mutant showed no attenuation in virulence toward honey bee brood. These results suggest that tyramine production is not essential for the pathogenicity of CC12 strains in European foulbrood.

European foulbrood (EFB), caused by the Gram-positive lanceolate coccus Melissococcus plutonius, is one of the globally distributed and economically important infectious diseases of honey bee brood [4]. M. plutonius strains are classified into 53 sequence types (STs) using the multilocus sequence typing (MLST) scheme (https://pubmlst.org/mplutonius/; accessed on June 3, 2026) [7]. These STs are further grouped into three clonal complexes (CC3, CC12, and CC13) [7, 18]. In M. plutonius, CC12 strains are more virulent to bee brood [14] and more resistant to chlorine-based disinfectants [15] than strains of the other CCs. All clonal complexes have been detected in Japan, and the isolation frequency of CC12 strains is higher in Japan than in European countries [18]. Furthermore, although both CC3 and CC12 strains have been isolated in North America, recent studies have reported an increasing frequency of CC12 strains among EFB cases [5, 6, 23, 24]. In particular, EFB caused by CC12 strains has also been detected in honey bee colonies used for blueberry pollination [6, 23, 24], suggesting that strains belonging to this group may pose a threat not only to apiculture but also to the agricultural sector. Therefore, there is a growing need to control the spread of CC12 strains not only in Japan but also globally.

To reduce the damage caused by CC12 strains, it is important to elucidate the virulence factors and pathogenic mechanisms underlying disease onset. Honey bee larvae are infected with M. plutonius through oral ingestion of larval food contaminated with the pathogen. Infected larvae typically die at 4–5 days old before being sealed in their cells. In honey bee larvae, the midgut occupies a large proportion of the body and plays an important role in larval development by digesting larval food and absorbing nutrients. The peritrophic matrix (PM), a chitin- and glycoprotein-rich layer lining the luminal side of the midgut epithelium, protects the epithelium from damage by food particles, digestive enzymes, and pathogens [8]. Takamatsu et al. [20] revealed that orally ingested bacterial cells of a CC12 strain, DAT561, were initially localized on the surface of the PM. The DAT561 cells then rapidly multiplied on the PM and eventually almost completely occupied the midgut lumen. The PM was degraded during DAT561 infection, and M. plutonius directly interacted with the midgut epithelium; concomitantly, the epithelial cells degenerated [20].

In CC3 strains, loss of a virulence plasmid pMP19 results in a loss of pathogenicity, rendering the strains nearly incapable of killing larvae [12]. In contrast, DAT561 retained its ability to kill larvae even in the absence of pMP19, strongly suggesting that other unidentified virulence factors are involved in the pathogenicity of CC12 strains. Djukic et al. [3] identified candidate genes involved in PM degradation as potential virulence factors through comparative genome analysis of M. plutonius strains. In our experiments, we found that deletion of these genes caused DAT561 to lose its ability to degrade the PM in the larval gut. However, in experimental infections using PM-degrading protein gene-deficient mutants, the mutants remained highly virulent to bee brood, comparable to the parent strain, suggesting that PM degradation is dispensable for the virulence of the highly virulent CC12 strain [13].

Djukic et al. [3] also identified a putative Enterococcus-type tyrosine decarboxylase gene (tdc), which is likely involved in tyramine production, as a potential virulence factor in the genomes of CC12 and CC3 strains. Building on this finding, our research group subsequently established a quantitative method for tyramine detection in culture supernatants and larval homogenates using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) [21]. Using this method, we demonstrated that DAT561 indeed produces tyramine in culture broth and in infected honey bee larvae [21]. According to Pérez-Ordóñez et al. [16], tyramine production in the medium varies among M. plutonius strains, with CC12 strains exhibiting the highest levels, even though CC3 strains also possess the tdc gene. Moreover, Kanbar et al. [9, 10] reported that tyramine has highly toxic effects on honey bee larvae, leading to EFB-like symptoms. Collectively, these findings suggest that tyramine may act as a virulence factor in CC12 strains. However, it has not yet been demonstrated whether the putative tdc gene has the capacity to produce tyramine, nor has the relationship between tyramine produced by M. plutonius and the development of EFB been elucidated. Therefore, in the present study, to clarify the function of the putative tdc gene and to investigate the role of tyramine as a virulence factor in the CC12 strain, we constructed a tdc-deficient mutant of the CC12 strain and analyzed its characteristics in exposure bioassays using in vitro-reared Apis mellifera brood.

Since pMP19 is unstable in bacterial cells and is lost during the gene knockout process [12], DAT561ΔpMP19, in which pMP19 had already been lost, was used as a parent strain in this study (Table 1). Construction of the tdc gene deletion mutant, DAT1705 (Table 1), was carried out according to a previous study [13] using the thermosensitive plasmid vector pSET6s [19]. Briefly, the upstream and downstream regions of the tdc gene were amplified from the genomic DNA of DAT561ΔpMP19 by PCR and fused by overlap-extension PCR [2, 22] using the primers and enzymes listed in Supplementary Table 1. The resulting PCR product was cloned into the PstI site of pSET6s, using Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA) as the cloning host, and the recombinant plasmid was subsequently introduced into DAT561ΔpMP19 by electroporation [2, 22]. Double-crossover gene deletion mutants were generated according to procedures described previously [2, 22]. Bacterial DNA was prepared using InstaGene Matrix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer’s instructions. Deletion of the target gene was confirmed by PCR using the primers and enzymes listed in Supplementary Table 1 and by sequencing analysis of the amplified products.

Table 1. Melissococcus plutonius strains used in this study and tyramine concentration in culture supernatants.

Strain	Tyrosine decarboxyrase gene (tdc)	Tyramine concentration in KSBHI broth (µg/mL)a	References
DAT561ΔpMP19 (parent strain)	+	190–201	[12]
DAT1705 (tdc deletion mutant derived from DAT561ΔpMP19)	−	41–44	This study

^aThe tyramine concentration of uninoculated KSBHI broth (negative control) was 31 µg/mL.

To measure tyramine concentration in M. plutonius culture supernatants, DAT561ΔpMP19 and DAT1705 were cultured in KSBHI broth according to a previous study [21]. Culture supernatants were obtained from three independent culture bottles and stored at −20°C until use. KSBHI broth incubated under the same conditions without M. plutonius inoculation was used as a negative control. Tyramine in the culture supernatants was measured using LC-MS/MS and deuterium-labeled tyramine (1,1,2,2-D4-tyramin HCl, Cambridge Isotope Laboratories, Inc., Tokyo, Japan; as an internal standard) as described in a previous study [21].

Exposure bioassays using A. mellifera brood were performed as described previously [13]. Briefly, inocula were prepared by culturing M. plutonius strains on KSBHI agar plates [1] for five days under anaerobic conditions. The bacteria suspended in sterile H₂O were mixed with an artificial diet B’ (Supplementary Table 2) at a ratio of 1 (bacterial suspension) to 9 (diet B’). Clinically healthy A. mellifera colonies were purchased from several different apiaries, and larvae less than 24 hr old were collected from three different queens for each experiment and randomly assigned to test groups. The larvae were infected with M. plutonius strains on day 2 post-grafting (pg) by feeding 20 μL of the inocula and were reared using 48-well cell culture plates until day 21 pg, as described previously [13]. Formulas and daily rations of the artificial diet are shown in Supplementary Tables 2 and 3, respectively. In this study, two exposure bioassays with different bacterial inoculum doses were conducted. In exposure bioassay I, larvae were infected with 3.5–8.3 × 10⁶ CFU/mL (7–16.6 × 10⁴ CFU/larva) of M. plutonius. In exposure bioassay II, larvae were infected with 4.2–4.4 × 10⁵ CFU/mL (8.4–8.8 × 10³ CFU/larva) of M. plutonius. In both assays, larvae were monitored daily until day 7 pg, and then at days 14 and 21 pg. In bioassay II, two groups of larvae were prepared. One group was used to assess the survival rate, and another was used to collect larval samples on day 4 post-infection (pi) (day 6 pg). The collected larvae were measured for their body weight, tyramine concentration, and bacterial load or analyzed histopathologically.

Bacterial loads in infected larvae were determined by plating serial dilutions of the larval homogenates onto KSBHI agar plates and counting colonies after incubation at 35°C for five or more days under anaerobic conditions. Tyramine concentration in larvae was measured using LC-MS/MS with deuterium-labeled tyramine, as described previously [21]. Some larvae were fixed in 10% phosphate-buffered formalin, dehydrated in a series of ethanol, embedded in paraffin, and sectioned for histopathological analysis. Sections were stained with the periodic acid–Schiff (PAS) reaction or subjected to immunohistochemical (IHC) staining using rabbit polyclonal antiserum against formalin-fixed M. plutonius cells, as described previously [13, 20]. The stained larval sections were examined using an EVOS M7000 Imaging System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and a Leica DM2000 microscope (Leica, Wetzlar, Germany).

Differences in larval survival throughout the experiments were analyzed using the log-rank test followed by a Bonferroni post hoc test. Larval weight and tyramine concentrations in infected and control larvae were analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Differences in logarithmically transformed bacterial loads were analyzed using Student’s t-test after assessing equality of variances by an F-test. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [11], a graphical user interface for R [17]. A P-value <0.05 was considered statistically significant.

PCR and sequencing analyses confirmed the deletion of the tdc gene region in the knockout mutant DAT1705 (Supplementary Fig. 1). Measurement of tyramine levels in culture supernatants showed that the negative control (i.e., KSBHI broth without bacteria inoculation) contained 31 µg/mL tyramine, whereas the supernatants from DAT561ΔpMP19 culture exhibited concentrations ranging from 190 to 201 µg/mL (Table 1), which were comparable to production levels previously reported for CC12 strains [16, 21]. In contrast, the tdc knockout mutant DAT1705 produced only 41–44 µg/mL tyramine, a level comparable to that of the negative control. This reduction indicated that DAT1705 had lost the ability to synthesize tyramine as a result of tdc deletion (Table 1). These findings demonstrate that the tdc gene is responsible for tyramine production in M. plutonius.

As shown in Fig. 1, in exposure bioassay I, 85% of larvae survived and developed into adults by day 21 pg under non-infected conditions. In contrast, the DAT561ΔpMP19- and DAT1705-infected groups exhibited low survival rates of 5% and 10%, respectively (Fig. 1). If the tdc gene were a virulence factor of the CC12 strain, a higher survival rate would be expected in the DAT1705-infected group. However, no significant difference was observed between the survival rates of the DAT561ΔpMP19- and DAT1705-infected groups. This absence of difference may be attributable to the high bacterial inoculum used in the first experiment (3.5–8.3 × 10⁶ CFU/mL), which may have been too strong to detect differences in virulence among M. plutonius-infected larvae. Therefore, a second exposure bioassay was conducted using a reduced bacterial inoculum (4.2–4.4 × 10⁵ CFU/mL) (Fig. 2A).

Fig. 1.

Survival of Apis mellifera brood experimentally infected with Melissococcus plutonius strains (DAT561ΔpMP19 or DAT1705) or left uninfected (Control) in exposure bioassay I. A total of 86 larvae were used in each experimental group. The bacterial concentration in inocula was 3.5 × 10⁶ to 8.3 × 10⁶ CFU/mL. Asterisks indicate significant differences (P<0.05). Day pg, day post-grafting; Day pi, day post-infection.

Fig. 2.

Minimal effect of the tdc gene deletion on the virulence of the CC12 strain DAT561ΔpMP19. Survival (A), tyramine concentration (B), larval weight (C), and bacterial load (D) of Apis mellifera brood experimentally infected with Melissococcus plutonius strains (DAT561ΔpMP19 or DAT1705) or left uninfected (Control) in exposure bioassay II. For the survival bioassay (A), a total of 42 larvae were used in each experimental group. The bacterial concentration in inocula was 4.2 × 10⁵ to 4.4 × 10⁵ CFU/mL. For measurements of tyramine concentration (B), larval weight (C), and bacterial load (D), larvae separate from those used in the survival bioassay were prepared, and surviving larvae were collected at day 4 post-infection (pi) (day 6 post-grafting [pg]). Tyramine concentration and larval weight are expressed as the mean ± standard error. Asterisks indicate significant differences (P<0.05).

In exposure bioassay II, the survival rate was 98% in the non-infected control group, whereas it was 17% in both the DAT561ΔpMP19- and the DAT1705-infected group. Again, no significant difference was observed between the two infected groups (Fig. 2A). The tyramine concentration in larvae infected with DAT1705 ranged from 0.2 to 2.8 ng/mg larva (average: 0.92 ng/mg larva), which was significantly lower than that in DAT561ΔpMP19-infected larvae (319–604 ng/mg larva [average: 455 ng/mg larva]) (Table 2 and Fig. 2B). Because tyramine levels in non-infected larvae ranged from 0.5 to 0.7 ng/mg larva (average: 0.6 ng/mg larva) and did not differ significantly from those in DAT1705-infected larvae, DAT1705 was confirmed not to produce tyramine even in larvae (Table 2 and Fig. 2B). In addition to the survival rates, no significant differences were observed in larval weight or bacterial loads between the DAT561ΔpMP19- and the DAT1705-infected groups (Table 2 and Fig. 2C and 2D). These findings indicate that DAT1705 remains highly virulent despite the loss of tyramine production, suggesting that deletion of the tdc gene does not affect the virulence of the CC12 strain of M. plutonius.

Table 2. Weight, Melissococcus plutonius load, and tyramine concentration in infected and non-infected honey bee larvae.

Group	Status	Sampling datea	Laval weight (mg)	Bacterial load (CFU/larva)b	Amount of tyramine detected
					(ng/larva)	(ng/mg larvaec)
Control (clinically healthy) group	Non-infected	Day 6 pg	112.7	0	56	0.5
		Day 6 pg	44.4	0	28	0.6
		Day 6 pg	107.1	0	69	0.6
		Day 6 pg	65.8	0	46	0.7
M. plutonius-infected group	DAT561ΔpMP19-infected	Day 6 pg	70.1	8.10 × 107	22349	319
		Day 6 pg	25.3	1.91 × 107	9833	389
		Day 6 pg	23.3	2.35 × 107	12564	539
		Day 6 pg	39.6	2.47 × 107	16813	425
		Day 6 pg	23.5	1.66 × 107	14199	604
	DAT1705-infected	Day 6 pg	18.4	1.25 × 107	51	2.8
		Day 6 pg	38.0	4.36 × 107	21	0.6
		Day 6 pg	26.7	1.01 × 107	16	0.6
		Day 6 pg	28.0	2.28 × 107	11	0.4
		Day 6 pg	22.1	1.76 × 107	5	0.2

^apg, post-grafting. ^bCFU, colony forming unit. ^cCalculated using unrounded raw tyramine detection data and larval weight, then rounded to the first (for DAT561ΔpMP19-infected larvae) or the second (for non-infected and DAT1705-infected larvae) decimal place.

In non-infected control larvae, the ingested food was surrounded by the PM, and M. plutonius was not detected (Fig. 3A and larvae nos. 1–5 in Supplementary Table 4). Degeneration of epithelial cells (i.e., cells containing numerous vacuoles or foam-like structures in the cytoplasm, or showing granular cytoplasm) was absent or only rarely observed in these larvae (Fig. 3B and larvae nos. 1–5 in Supplementary Table 4). In contrast, all DAT561ΔpMP19-infected larvae harbored M. plutonius in the midgut (Fig. 3C and larvae nos. 6–10 in Supplementary Table 4). Although the PM in most DAT561ΔpMP19-infected larvae remained partially intact, its thickness was relatively reduced compared with that in non-infected larvae. In addition, the midgut epithelium contained degenerated or disintegrated epithelial cells characterized by vacuoles or foam-like structures in the cytoplasm, granular or basophilic cytoplasm, and/or granular, swollen, shrunken nuclei (Fig. 3D and larvae nos. 6–10 in Supplementary Table 4). Many M. plutonius cells were also detected in the midgut of all the DAT1705-infected larvae, confirming the proliferation of ingested M. plutonius within larvae (Fig. 3E and larvae nos. 11–15 in Supplementary Table 4). Degenerated or disintegrated epithelial cells were also observed (Fig. 3F and larvae nos. 6–10 in Supplementary Table 4). Although most DAT1705-infected larvae retained a greater amount of the PM than the DAT561ΔpMP19-infected larvae (Supplementary Table 4), one larva exhibited almost complete loss of the PM, suggesting individual variation in infection progression (larva no. 14 in Supplementary Table 4). Despite deletion of the tdc gene, the pathological features in DAT1705-infected larvae were similar to those in larvae infected with DAT561ΔpMP19.

Fig. 3.

Light microscopic observations of representative non-infected control and Melissococcus plutonius-infected larvae. (A) A representative image of a larva from the non-infected control group. (B) Enlarged view of the boxed area in (A). (C–D) A larva infected with DAT561ΔpMP19. (E–F) A larva infected with DAT1705. Images were taken using an EVOS M7000 Imaging System (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for (A) and a Leica DM2000 microscope (Leica) for (B–F). PAS, Periodic acid-Schiff reaction; IHC, Immunohistochemical staining with rabbit polyclonal antiserum against M. plutonius cells [13, 20]; Fd, brood food in the midgut; ME, midgut epithelial cells; PM, peritrophic matrix; ML, midgut lumen; D-ME, degenerated midgut epithelial cells; B in ML, numerous bacterial cells in the midgut lumen.

In this study, we successfully constructed a tdc gene knockout mutant from the CC12 strain, DAT561ΔpMP19, and demonstrated that the tdc gene is responsible for tyramine production. However, the survival rate of bee brood infected with the tdc knockout mutant was comparable to that of the group infected with the parent strain. It should be noted that, in exposure bioassays, the survival rates appear to decrease dramatically at day 14 pg (day 12 pi) (Figs. 1 and 2A). However, the apparent sharp decline in survival is due to the cumulative counting of dead brood that died during the non-observation period (day 8 pg to 13 pg [day 6 pi to 11 pi]), since the bioassay conditions in this study did not monitor survival during this period in order to avoid potential adverse effects of frequent temperature fluctuations caused by daily observations on blood.

Although the inoculum dose in exposure bioassay II was deliberately reduced relative to that in exposure bioassay I, it is still possible that the dose remained sufficiently high to cause larval mortality in the tdc gene knockout mutant. However, when M. plutonius DAT606, a virulent CC3 strain, lacked the virulence determinant pMP19, larval mortality at day 21 pg was only 5%, even when larvae were administered a concentration of 7.0 × 10⁶ CFU/mL (1.4 × 10⁵ CFU/larva) [12]. Given that the inoculum used in exposure bioassay II was 4.2–4.4 × 10⁵ CFU/mL (8.4–8.8 × 10³ CFU/larva), these results suggest that the tdc gene is not an essential virulence determinant in M. plutonius DAT561ΔpMP19.

Pérez-Ordóñez et al. [16] reported that when 2- or 3-day-old larvae were fed 10 µL of a diet containing tyramine at a concentration of 0.5 mg/mL (5 µg/larva) on the first day of rearing, the survival rate on day 6 post-ingestion was around 25%. In the present study, larvae infected with the DAT561ΔpMP19 strain accumulated 9.8–22.3 µg of tyramine per larva at day 6 pg (day 4 pi), which exceeded the tyramine dose (5 µg/larva) reported by Pérez-Ordóñez et al. [16] to result in a 25% survival rate. Because Pérez-Ordóñez et al. [16] used larvae that were 2–3 days of age at the start of their experiment, their “day 6 post-ingestion” larvae likely correspond to approximately day 8 pg larvae in our experimental system. Although survival was not recorded at day 8 pg (day 6 pi) in our bioassays, the DAT561ΔpMP19-infected group still exhibited a survival rate of over 60% at day 7 pg (day 5 pi), suggesting that tyramine produced by M. plutonius within larvae exerts a weaker effect on larval survival than tyramine administered directly through the diet.

Kanbar et al. [9, 10] reported that brood were most sensitive to tyramine treatment when the larvae were 4–5 days old. In larvae infected with M. plutonius, it is expected that a certain amount of time is required for tyramine to accumulate to toxic levels after infection. Therefore, under the experimental conditions of this study (i.e., infection at day 2 pg), the peak of tyramine accumulation was unlikely to coincide with the period during which larvae are most sensitive to tyramine. This suggests that there remains scope to optimize bioassay conditions to better assess the effects of tyramine; for example, inoculating M. plutonius at day 0 pg may allow detection of differences in larval survival associated with tyramine production.

Kanbar et al. [9, 10] also reported that the lethal dose of tyramine for brood was approximately 0.3–0.5 mg/larva. However, this value was estimated based on experiments in which tyramine was artificially added to larval food and does not take into account the capacity of M. plutonius to produce tyramine within larvae. In this study and in our previous study [21], tyramine levels in larvae experimentally infected with M. plutonius reached at most 44.7 μg/larva [21], and no cases have so far been reported in which tyramine produced by M. plutonius accumulates to 0.3 mg/larva in larvae. Therefore, it remains unclear whether the amount of tyramine produced by M. plutonius in naturally infected larvae can reach the lethal dose reported by Kanbar et al. [9, 10]. Even if such levels could be achieved, the observation that larvae infected with the tyramine-non-producing strain DAT1705 still exhibited high mortality strongly suggests that the pathogenesis of EFB caused by CC12 strains involves additional virulence factors or mechanisms independent of tyramine. Nevertheless, even low levels of tyramine produced by M. plutonius may exert effects on larvae that are not directly associated with brood mortality, and thus the role of tyramine in the pathogenesis of EFB remains to be further elucidated.

Genome analysis of M. plutonius strains conducted by Djukic et al. [3] identified putative virulence factor genes in DAT561, including those encoding cell surface- and adhesion-associated proteins as well as capsule/cell envelope-forming proteins. In their report, genes encoding PM-degrading proteins were also proposed as potential virulence factors [3]; however, analyses using gene knockout strains have demonstrated that PM degradation does not affect larval mortality caused by the highly virulent CC12 strain [13]. In the present study, deletion of the tdc gene also did not reduce the virulence of DAT561ΔpMP19. Therefore, while it is possible that other putative virulence genes proposed by Djukic et al. [3] function as critical virulence factors, it also remains possible that genes examined thus far, including the PM-degrading protein genes and the tdc gene, may collectively contribute to virulence, such that simultaneous deletion of multiple genes could lead to a reduction in the virulence of the CC12 strain. Therefore, to elucidate the pathogenic mechanisms of the CC12 strain, it will be necessary to persistently investigate the roles of putative virulence genes through genetic manipulation, with particular attention to the potential combined effects of multiple factors.

CONFLICT OF INTEREST

The author declares no conflicts of interest associated with this study.

Funding Statement

This study was supported by JSPS KAKENHI Grant-in-Aid for Scientific Research (C) (Grant Number JP23K05546).