An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

Abstract

Bacterial transcriptome analyses during host colonization are essential to decipher the complexity of the relationship between the bacterium and its host. RNA sequencing (RNA-seq) is a promising approach providing valuable information about bacterial adaptation, the host response and, in some cases, mutual tolerance underlying crosstalk, as recently observed in the context of Mycobacterium ulcerans infection. Buruli ulcer is caused by M. ulcerans. This neglected disease is the third most common mycobacterial disease worldwide. Without treatment, M. ulcerans provokes massive skin ulcers. A healing process may be observed in 5% of Buruli ulcer patients several months after the initiation of disease. This spontaneous healing process suggests that some hosts can counteract the development of the lesions caused by M. ulcerans. Deciphering the mechanisms involved in this process should open up new treatment possibilities. To this end, we recently developed the first mouse model for studies of the spontaneous healing process. We have shown that the healing process is based on mutual tolerance between the bacterium and its host. In this context, RNA-seq seems to be the most appropriate method for deciphering bacterial adaptation. However, due to the low bacterial load in host tissues, the isolation of mycobacterial RNA from skin tissue for RNA-seq analysis remains challenging. We developed a method for extracting and purifying mycobacterial RNA whilst minimizing the amount of host RNA in the sample. This approach was based on the extraction of bacterial RNA by a differential lysis method. The challenge in the development of this method was the choice of a lysis system favoring the removal of host RNA without damage to the bacterial cells. We made use of the thick, resistant cell wall of M. ulcerans to achieve this end.

Combined Transcriptome Sequencing of Mycoplasma hyopneumoniae and Infected Pig Lung Tissue Reveals Up-Regulation of Bacterial F1-Like ATPase and Down-Regulation of the P102 Cilium Adhesin in vivo

“…To overcome this hurdle, we developed alternative methods to isolate bacterial RNA from lungs infected with M. hyopneumoniae (Figure 2A). Application of differential lysis techniques to isolate bacterial RNA (Rienksma et al, 2015;Robbe-Saule et al, 2017), was not an option for Mycoplasma hyopneumoniae since this bacterium does not contain a cell wall. As previously reported, we identified with immunohistochemistry that M. hyopneumoniae is found specifically on the surface of the respiratory epithelium (Figure 2A).…”

Environmental Variations in Mycobacterium ulcerans Transcriptome: Absence of Mycolactone Expression in Suboptimal Environments

“…Total RNA extraction was carried out using a trizol/chloroform extraction followed by a passage in Qiagen column using RNeasy Mini Kit (Qiagen, Hilden, Germany) [31].…”

Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus

“…The protocol for differential lysis was adapted from Robbe-Saule et al (2017), Two living larvae were randomly selected, externally disinfected with 70% ethanol, dried, then placed into Petri dishes. Using sterile forceps holding the head of the larvae, the larval contents were extracted by applying pressure using a 10 ml syringe plunger.…”