Advances in Genetic Manipulation of Obligate Intracellular Bacterial Pathogens

Abstract

Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia, and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic) growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long-term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection

“…Successful colonization of mammalian cells by C. burnetii requires the formation of a single CCV that is actively controlled by the bacterial T4SS and its secreted factors (46)(47)(48). Consequently, the T4SS and its secreted factors have been described as a novel virulence factor of Coxiella in mammals (16,17,49). We used Drosophila S2 cells and adult flies to investigate the role of the T4SS during C. burnetii infection.…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…However, Coxiella's obligate intracellular lifestyle made development of genetic methods technically challenging. Reliance on a eukaryotic host cell for growth dramatically complicates selection, scoring, cloning and expansion of genetic transformants [100]. Moreover, mutants with lowered intracellular growth fitness may not be recovered.…”

“…Multiple methods for gene inactivation are now available including random mutagenesis using Himar1 [80] and targeted mutagenesis using a 'loop in/loop out' strategy coupled with sacB counterselection or a Cre-lox approach that allows deletion of large chromosomal regions [104]. For single and multiple copy complementation, a Tn7-based system and RSF1010 ori-based shuttle vectors, respectively, are available [80,88,91,100]. Additional tools include inducible gene expression using the anhydrotetracycline-inducible promoter tetA [80] and a luciferase-based gene reporter system [105].…”

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Isolating clonal populations by plaque assay has not been reported for environmental Chlamydiae strains that can only be propagated in amoebal hosts. In these situations, alternative approaches, such as limiting dilution, flow cytometry (59,60), laser microdissection, or use of micromanipulators, have been employed to isolate bacterial cells directly from infected cells in a monolayer, as reported for Chlamydia and other intracellular pathogens (61,62).…”