Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

Abstract

Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

Keywords: bacteria; intracellular infection; non-coding RNA; pathogen; regulation.

Figure 1

Approaches leading to the identification of novel non-coding RNAs in intracellular bacterial pathogens. (A) Comparative genomics of two closely and phylogenetically-related bacterial species, one pathogenic, and the other not, allows the identification of pathogen-specific genome regions predicted to generate non-coding RNA. These candidates can be further validated experimentally and tested for putative function in virulence with the corresponding defective mutants constructed ad-hoc; (B) analyses of transcriptomic data obtained with classical microarray technology or, alternatively by RNA-seq, allow the identification of non-coding RNAs in (i) the pathogen when growing in laboratory media mimicking infection conditions; or, (ii) in bacteria isolated from eukaryotic cells infected in in vitro using tissue cultured cells. These new non-coding RNAs are further validated experimentally and tested for putative role in virulence using defective mutants; (C) Transcriptomic data including information about non-coding RNA can also be obtained from the pathogen grown in tissues or organs of the animal. Although this approach does not provide a direct proof of production of the non-coding RNA in bacteria located inside the host cell, this possibility can be further tested in tissue culture cells. Experimental validation of the non-coding RNA and testing of its putative role in virulence are logical subsequent steps.

Figure 2

Examples of sRNAs produced by intracellular bacterial pathogens inside the host eukaryotic cell. All sRNAs shown here were validated experimentally in bacteria isolated from eukaryotic cells. Note the existence of negative and positive regulation, indicated with (−) and (+) signs respectively. In those sRNAs indicated with an asterisk, the regulation over the indicated target has been demonstrated in bacteria residing within the infected eukaryotic cell. For the case of RyhB-2 in S. Typhimurium, only the regulation over yeaQ has been proved to occur in intracellular bacteria. Intracellular bacteria are shown in phagosomal and cytosolic locations, covering the different lifestyles of the distinct pathogens shown. See also Table 1 for details.