LAUSR

As their designation implies, obligate intracellular bacteria are microbes that have developed lifestyles so closely entwined with the cells of the hosts they infect that they can reproduce only within the confines of these cells. Among this group of bacteria are several pathogenic species with a significant impact on human health. For instance, Chlamydia spp. are responsible for millions of cases of urogenital, ocular, and respiratory infections every year [1]. Moreover, Coxiella burnetii is the agent of Q fever [2], whereas members of the order Rickettsiales (specifically the genera Rickettsia, Ehrlichia, and Anaplasma) cause life-threatening vector-borne diseases, such as spotted fever and typhus [3]. While these pathogens are all restricted to life in an intracellular niche, they differ in the ways they reproduce and engage with their host cells. Chlamydia spp., Ehrlichia spp., Anaplasma spp., and C. burnetii have biphasic life cycles, in which the bacteria alternate between infectious (environmentally stable) and replicative (metabolically highly active) forms inside specialized membrane-enclosed vacuoles [1–3]. In contrast, Rickettsia spp. escape their vacuoles after uptake and replicate in the host cytosol without undergoing developmental transitions [3]. The various obligate intracellular lifestyles are enabled by distinct sets of virulence factors, which, for instance, engage in the subversion of host defenses and the hijacking of host resources and machineries. Studying the molecular basis of these intimate relationships between host and bacteria can uncover unknown aspects of host–pathogen interactions and new targets for pharmacological intervention to help relieve the significant disease burden of intracellular infections. However, historically, the study of obligate intracellular bacteria was neglected due to the impeding lack of tools enabling their molecular genetic manipulation. While whole-genome sequencing provided the opportunity to predict their virulence traits, in the absence of genetic tools, direct links to specific bacterial genes could not be formally demonstrated. Fortunately, recent years have seen a significant expansion of our genetic toolbox for these important pathogens. Here, we summarize these developments, with a focus on Chlamydia spp., along with a brief overview for other obligate intracellular bacteria. Furthermore, we provide examples of insights into chlamydial biology enabled by the expanded toolbox, and we conclude with a discussion on future perspectives for the molecular genetic manipulation of this group of bacteria. PLOS PATHOGENS