Research Article: In vivo and in vitro expression of five genes involved in Corynebacterium pseudotuberculosis virulence

Date Published: May 30, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Jefferson Ivan Corrêa, Andreas Stocker, Soraya Castro Trindade, Vera Vale, Thais Brito, Bruno Bastos, José Tadeu Raynal, Patrícia Mares de Miranda, Adriano Costa de Alcantara, Songeli Menezes Freire, Lília Moura Costa, Roberto Meyer.

http://doi.org/10.1186/s13568-018-0598-z

Abstract

Caseous lymphadenitis (LC) is a chronic contagious disease caused by Corynebacterium pseudotuberculosis, which mainly affects goats and sheep. Vaccination is an effective but not yet well-established method, partly due to a lack of knowledge surrounding the most effective immunoprotective components. The present study aimed to quantify and compare the in vivo expression of genes pld (phospholipase D), cpp (CP40), nanH (neuraminidase H), sodC (superoxide dismutase C) and spaC (adhesin) using qRT-PCR, with the respective expression in vitro. Caseous material of abscesses removed from five animals was cultured, with colonies suggestive of C. pseudotuberculosis identified. RNA extraction was performed on these samples, as well as on the respective pellets derived from liquid cultures brain heart infusion. After evaluating RNA integrity, complementary DNA was synthesized, followed by the relative quantification each of the genes of interest. Mean mRNA expression of the five genes found in abscesses and in cultures differed significantly, with respective values of: nanH 811.50 ± 198.27 and 359.35 ± 75.45 (p = 0.009); cpp 856.31 ± 385.11 and 154.54 ± 94.34 (p = 0.0039); plD 922.70 ± 450.73 and 212.41 ± 153.10 (p = 0.016); sodC 1,293.53 ± 564.75 and 223.63 ± 145.58 (p = 0.016); spaC 1,157.10 ± 525.13 and 214.26 ± 125.70 (p = 0,016). Expression was observed to be 6–8 times higher in abscesses than in cultures, Indicative that is a genetic expression of the in vitro bacterium exists, yet in vivo has a greater magnitude corroborating to one of these virulence factors in the pathogenesis of LC.

Partial Text

Caseous lymphadenitis (CL), a chronic contagious disease that affects small ruminants, is caused by Corynebacterium pseudotuberculosis, a gram-positive, pleomorphic, non-sporulated, facultatively anaerobic bacillus belonging to the Corynebacteriaceae family (Actinomycetales order).

mRNA detection of the nanH, cpp, pld, sodC, and spaC genes in bacterial cells obtained from culture indicated that transcription of these genes occurred, in addition to the possible synthesis of the respective proteins. Although it is possible to transcribe genes without the corresponding translation (Houben et al. 2009), in the context of our research, the probability of this occurring in five genes simultaneously is extremely low. Additionally, it is known that the synthesis and secretion of phospholipase D and CP40 proteins in cultures of C. pseudotuberculosis occurs, since these substances are detected in culture supernatants (Walker et al. 1994; Wilson et al. 1995). In addition, the interference of phospholipase D in the attenuation of hemolysis caused by β-hemolysin from Staphylococcus aureus has been documented in vitro, which serves as an auxiliary test in the identification of C. pseudotuberculosis.

 

Source:

http://doi.org/10.1186/s13568-018-0598-z

 

Leave a Reply

Your email address will not be published.