Transcriptional regulator SpxA1a controls the resistance of the honey bee pathogen Melissococcus plutonius to the antimicrobial activity of royal jelly

Abstract

Summary Royal jelly (RJ), a brood food of honey bees, has strong antimicrobial activity. Melissococcus plutonius, the causative agent of European foulbrood of honey bees, exhibits resistance to this antimicrobial activity and infects larvae orally. Among three genetically distinct groups (CC3, CC12 and CC13) of M. plutonius, CC3 strains exhibit the strongest RJ resistance. In this study, to identify genes involved in RJ resistance, we generated an RJ‐susceptible derivative from a highly RJ‐resistant CC3 strain by UV mutagenesis. Genome sequence analysis of the derivative revealed the presence of a frameshift mutation in the putative regulator gene spxA1a. The deletion of spxA1a from a CC3 strain resulted in increased susceptibility to RJ and its antimicrobial component 10‐hydroxy‐2‐decenoic acid. Moreover, the mutant became susceptible to low‐pH and oxidative stress, which may be encountered in brood foods. Differentially expressed gene analysis using wild‐type and spxA1a mutants revealed that 45 protein‐coding genes were commonly upregulated in spxA1a‐positive strains. Many upregulated genes were located in a prophage region, and some highly upregulated genes were annotated as universal/general stress proteins, oxidoreductase/reductase, chaperons and superoxide dismutase. These results suggest that SpxA1a is a key regulator to control the tolerance status of M. plutonius against stress in honey bee colonies.

Atypical <i>Melissococcus plutonius</i> strains: their characteristics, virulence, epidemiology, and mysteries

“…However, the isolation and characterization of causative agents by standard culture methods are both time-consuming and labor-intensive, and, thus, PCR, a rapid and easy method to detect and identify the causative agents of infectious diseases, has been widely utilized for these purposes in many diseases. Many PCR assays, including regular, hemi-nested, and real-time PCRs, have been developed and utilized for EFB; however, most were able to detect or identify M. plutonius , but not specifically distinguish atypical (CC12) strains from typical (CC3 and CC13) strains [ 12 , 15 , 20 , 27 , 32 , 47 , 56 , 62 , 71 ].…”

Influence of Honey bee Nutritive Jelly Type and Dilution on its Bactericidal Effect on Melissococcus plutonius, the Etiological Agent of European Foulbrood

“…The higher survival of the bacteria in the diluted jelly used as larval diet could lead to increased negative effects on the host compared to the natural situation and to an overestimation of its virulence. Furthermore, our model of bacterial survival showed a significant interaction between bacterial strain and jelly dilution, i.e., with the concentration of antibacterial compounds, indicating that the survival of the strains varied according to jelly dilution and pointing to differences in the resistance mechanisms between strains [ 19 ].…”

“…Vegetative cells of Paenibacillus larvae , the pathogenic agent of the American foulbrood disease, lose their viability after a few minutes in jelly, and this pathogen relies on spores to survive the bactericidal effect of jelly before they can replicate in the larval midgut lumen and later in their hemocoel [ 16 , 17 ]. Melissococcus plutonius , a non-spore forming bacterium causing European foulbrood disease [ 18 ], possesses vegetative cells endowed with spxA1a regulator gene–mediated stress resistance mechanisms to withstand the bactericidal effects of jelly until they reach the midgut lumen, where they multiply [ 18 , 19 ]. The bactericidal effects of jelly have been investigated using diluted queen-destined jelly or extracts of this jelly [ 7 ], most probably to alleviate the methodological constraints generated by the high viscosity of pure jelly.…”

A novel multiplex PCR assay to detect and distinguish between different types of <i>Paenibacillus larvae</i> and <i>Melissococcus plutonius</i>, and a survey of foulbrood pathogen contamination in Japanese honey

“…Colony-forming units (CFU) per milliliter of M. plutonius cell suspensions were calculated by plating serial dilutions of suspensions onto Basal agar plates [ 18 ] and counting colonies after the incubation of plates at 35°C for three days under anaerobic conditions. Since M. plutonius cells develop in chains [ 54 ], we also calculated the average number of cells in a single chain in the suspensions after staining M. plutonius cells using Gram’s method and counting cells in 100 chains under a microscope (CME, Leica, Wetzlar, Germany). A single colony on an agar plate originates from a single chain of cells; therefore, M. plutonius cell concentrations (cells/ml) in suspensions were measured using the following formula: cells/ml=CFU/ml × the average number of cells in a single chain.…”