Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis

Authors: Boggiatto, Paola; Vrentas, Catherine; Fitzsimmons, Daniel; Bayles, Darrell; Alt, David; Olsen, Steven

Submitted to: BioMed Central (BMC) Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2018
Publication Date: 8/1/2018
Citation: Boggiatto, P.M., Vrentas, C.E., Fitzsimmons, D.J., Bayles, D.O., Alt, D.P., Olsen, S.C. 2018. Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis. BioMed Central (BMC) Molecular Biology. 19:10. https://doi.org/10.1186/s12867-018-0111-x.
DOI: https://doi.org/10.1186/s12867-018-0111-x

Interpretive Summary: Brucella species are intracellular bacteria that cause persistent infections in the mammalian host. Study of the bacterial transcriptome is hindered by the intracellular nature of the bacteria, yet this is critical in the understanding of host-pathogen interactions and in the development of therapeutic approaches to treat these infections. Coincidence cloning is a technique that allows for the enrichment and amplification of bacterial RNA extracted from infected tissues. Using this methodology, we demonstrated the utility of coincidence cloning to study host-Brucella interactions and provide the first characterization of Brucella gene expression within the tissues of an infected mammalian host. This work will be of interest to scientists, regulatory personnel, and producers to not only understand how Brucella adapts to infection in natural hosts, but also what genes are utilized that might be targets for vaccination or therapeutic interventions.

Technical Abstract: Background: Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed shortly after invasion of mammalian cells in culture. Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Results: Using the coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph node sections of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. The RNA levels were sufficient for analysis of gene expression patterns by RNA-sequencing, which provides evidence of metabolic activity in both the short-term and the long-term samples. Indel (insertion/deletion) analysis indicated recovery of the inoculation strain in the sequencing reads, and clustering analysis demonstrated a distinct transcriptional profile present in samples recovered in long-term infection. We demonstrate the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, such as observed in in vivo brucellosis, and alternatively characterize intermediate products of the coincidence cloning reaction. Conclusions: Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species.