Engineering of obligate intracellular bacteria: progress, challenges and paradigms

Key Points Extracellular bacteria are free-living organisms, whereas facultative intracellular bacteria replicate either inside eukaryotic host cells or in an environmental niche. Obligate intracellular bacteria, which include Chlamydia spp., Anaplasma spp., Ehrlichia spp., Rickettsia spp., Orientia spp. and Coxiella spp., replicate exclusively inside of eukaryotic host cells. Genetic tools for the manipulation of obligate intracellular bacteria have historically been limited; however, there has been considerable recent progress in refining these methods. Such tools include transformation strategies, shuttle vectors, random and targeted mutagenesis through allelic exchange, and mobile group II introns. Novel bacterial molecules that shed light on both microbial pathogenesis mechanisms and host cell biology have been characterized by applying genetic tools to study Chlamydia trachomatis serovar L2 and Rickettsia parkeri . Vaccines against obligate intracellular bacterial infections are lacking. Refining genetic tools would enable the characterization of virulence factors and the development of vaccine candidates. Key questions in bacterial pathogenesis and physiology are primed for investigation once all obligate intracellular bacteria can be genetically manipulated on a routine basis. In this Review, Pedra and colleagues describe the advances and challenges in the genetic engineering of obligate intracellular bacteria, and highlight examples of how the use of genetically manipulated pathogens has improved our understanding of microbial pathogenesis and host–pathogen interactions. It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp. , Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria co