Paenibacillus larvae enolase as a virulence factor in honeybee larvae infection

Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, C.P. 11600, Montevideo, Uruguay.
Veterinary Microbiology (Impact Factor: 2.73). 01/2011; 147(1-2):83-9. DOI: 10.1016/j.vetmic.2010.06.004
Source: PubMed

ABSTRACT Paenibacillus larvae is a gram-positive spore-forming bacteria, causative agent of American Foulbrood (AFB), a severe disease affecting larvae of the honeybee Apis mellifera. In an attempt to detect potential virulence factors secreted by P. larvae, we identified an enolase among different secreted proteins. Although this protein is a cytosolic enzyme involved in glycolytic pathways, it has been related to virulence. The aim of the present work was to evaluate its role during the infection of honeybee larvae. Toxicity assays showed that enolase was highly toxic and immunogenic to honeybee larvae. Its production was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. P. larvae enolase production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyze milk proteins as described for P. larvae, suggesting that could be involved in larval degradation, maybe through the plasmin(ogen) system. These results suggest that P. larvae enolase may have a role in virulence and could contribute to a general insight about insect-pathogen interaction mechanisms.

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    • "Dancer and Chantawannakul [21] associated the pathogenicity of P. larvae with the secretion of metalloproteases. Antúnez et al. [22] reported the production by P. larvae of an enolase that could have a role in the virulence of the pathogen. Recently, P. larvae virulence has been associated with an S layer protein [23] whose presence determined the difference in the virulence between ERIC I and ERIC II genotypes [24] with the former showing a weaker virulence due to the absence of the specific S-layer [23]. "
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    ABSTRACT: Paenibacillus larvae is the causative agent of American foulbrood (AFB), a virulent disease of honeybee (Apis mellifera) larvae. In Tunisia, AFB has been detected in many beekeeping areas, where it causes important economic losses, but nothing is known about the diversity of the causing agent. Seventy-five isolates of P. larvae, identified by biochemical tests and 16S rRNA gene sequencing, were obtained from fifteen contaminated broods showing typical AFB symptoms, collected in different locations in the northern part of the country. Using BOX-PCR, a distinct profile of P. larvae with respect to related Paenibacillus species was detected which may be useful for its identification. Some P. larvae-specific bands represented novel potential molecular markers for the species. BOX-PCR fingerprints indicated a relatively high intraspecific diversity among the isolates not described previously with several molecular polymorphisms identifying six genotypes on polyacrylamide gel. Polymorphisms were also detected in several biochemical characters (indol production, nitrate reduction, and methyl red and oxidase tests). Contrary to the relatively high intraspecies molecular and phenotypic diversity, the in vivo virulence of three selected P. larvae genotypes did not differ significantly, suggesting that the genotypic/phenotypic differences are neutral or related to ecological aspects other than virulence.
    09/2013; 2013:479893. DOI:10.1155/2013/479893
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    • "Finally, enolase is an enzyme widely described as secreted or surface exposed in several microorganisms of the genera Paenibacillus, Bacillus, Lactobacillus, and Streptococcus [17, 27–30], showing immunogenic properties [15] [21] [29] and ability to bind to fibronectin [30] [31]. Enolase is also shown to be a virulence factor in P. larvae [32] and is found on the surface of L. crispatus in a pH-dependent way, being released to the medium at pH close to its isoelectric point or more alkaline [28]. In Mycoplasmas it has been detected on the surface of M. gallisepticum and M. fermentans, in both cases able to bind to plasminogen [33] [34]. "
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    ABSTRACT: Mycoplasma synoviae is a Gram positive bacteria lacking of cell wall that affects chickens and turkeys causing infection in the upper respiratory tract and in some cases arthritis, with economical impact to broiler breeders. Treatment and prevention of avian synovitis depend on knowledge of the infectious process. Secreted or surface-exposed proteins play a critical role in disease because they often mediate interactions between host and pathogen. In the present work, we sought to identify possible M. synoviae secreted proteins by cultivating the bacteria in a modified protein-free Frey medium. Using this approach, we were able to detect in the cell-free fraction a number of proteins that have been shown in other organisms to be secreted, suggesting that they may also be secreted by M. synoviae.
    09/2012; 2012:802308. DOI:10.5402/2012/802308
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    • "Microbiology (2011), 157, 1474–1480 DOI 10.1099/mic.0.044321-0 "
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    ABSTRACT: American foulbrood is a bacterial disease of worldwide distribution that affects larvae of the honeybee Apis mellifera. The causative agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Several authors have proposed that P. larvae secretes metalloproteases that are involved in the larval degradation that occurs after infection. The aim of the present work was to evaluate the production of a metalloprotease by P. larvae during larval infection. First, the complete gene encoding a metalloprotease was identified in the P. larvae genome and its distribution was evaluated by PCR in a collection of P. larvae isolates from different geographical regions. Then, the complete gene was amplified, cloned and overexpressed, and the recombinant metalloprotease was purified and used to generate anti-metalloprotease antibodies. Metalloprotease production was evaluated by immunofluorescence and fluorescence in situ hybridization. The gene encoding a P. larvae metalloprotease was widely distributed in isolates from different geographical origins in Uruguay and Argentina. Metalloprotease was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. Its production was also confirmed in vivo, during the infection of honeybee larvae. This protein was able to hydrolyse milk proteins as described for P. larvae, suggesting that could be involved in larval degradation. This work contributes to the knowledge of the pathogenicity mechanisms of a bacterium of great economic significance and is one step in the characterization of potential P. larvae virulence factors.
    Microbiology 02/2011; 157(Pt 5):1474-80. DOI:10.1099/mic.0.044321-0 · 2.84 Impact Factor
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