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: 2.51). 01/2011; 147(1-2):83-9. DOI: 10.1016/j.vetmic.2010.06.004
Source: PubMed


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.
    Full-text · Article · Sep 2013
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    • "Contig 355 encodes an enolase which is usually present on the cell surface or even secreted and is a potential virulence factor. In bacterial systems enolase has been demonstrated to contribute to pathogenicity by binding plasminogen in the infected host, potentially allowing the bacteria to acquire surface-associated proteolytic activity [33-35]. The basic leucine zipper transcription factor, encoded by contig 395, is a member of the bZIP family, one bZIP family member (Moatf1) from the rice fungus Magnaporthe oryzae mediates oxidative stress responses and is necessary for full virulence [36]. "
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    ABSTRACT: Background White mold, caused by Sclerotinia sclerotiorum, is one of the most important diseases of pea (Pisum sativum L.), however, little is known about the genetics and biochemistry of this interaction. Identification of genes underlying resistance in the host or pathogenicity and virulence factors in the pathogen will increase our knowledge of the pea-S. sclerotiorum interaction and facilitate the introgression of new resistance genes into commercial pea varieties. Although the S. sclerotiorum genome sequence is available, no pea genome is available, due in part to its large genome size (~3500 Mb) and extensive repeated motifs. Here we present an EST data set specific to the interaction between S. sclerotiorum and pea, and a method to distinguish pathogen and host sequences without a species-specific reference genome. Results 10,158 contigs were obtained by de novo assembly of 128,720 high-quality reads generated by 454 pyrosequencing of the pea-S. sclerotiorum interactome. A method based on the tBLASTx program was modified to distinguish pea and S. sclerotiorum ESTs. To test this strategy, a mixture of known ESTs (18,490 pea and 17,198 S. sclerotiorum ESTs) from public databases were pooled and parsed; the tBLASTx method successfully separated 90.1% of the artificial EST mix with 99.9% accuracy. The tBLASTx method successfully parsed 89.4% of the 454-derived EST contigs, as validated by PCR, into pea (6,299 contigs) and S. sclerotiorum (2,780 contigs) categories. Two thousand eight hundred and forty pea ESTs and 996 S. sclerotiorum ESTs were predicted to be expressed specifically during the pea-S. sclerotiorum interaction as determined by homology search against 81,449 pea ESTs (from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings) and 57,751 S. sclerotiorum ESTs (from mycelia at neutral pH, developing apothecia and developing sclerotia). Among those ESTs specifically expressed, 277 (9.8%) pea ESTs were predicted to be involved in plant defense and response to biotic or abiotic stress, and 93 (9.3%) S. sclerotiorum ESTs were predicted to be involved in pathogenicity/virulence. Additionally, 142 S. sclerotiorum ESTs were identified as secretory/signal peptides of which only 21 were previously reported. Conclusions We present and characterize an EST resource specific to the pea-S. sclerotiorum interaction. Additionally, the tBLASTx method used to parse S. sclerotiorum and pea ESTs was demonstrated to be a reliable and accurate method to distinguish ESTs without a reference genome.
    Full-text · Article · Nov 2012 · BMC Genomics
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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.
    Full-text · Article · Sep 2012
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