Engineering of obligate intracellular bacteria: progress, challenges and paradigms

Abstract

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 could enable the discovery of novel signalling circuits in host-pathogen interactions.

Figure 1. Advances in the genetic manipulation of obligate intracellular bacteria

EMS, ethyl methanesulfonate; FRAEM, fluorescence-reporteda allelic exchange mutagenesis; Pld, phospholipase D.

Figure 2. Transformation methods, expression vectors and random mutagenesis

a Chemical transformation with calcium chloride is used to transform Chlamydia trachomatis serovar L2. Electroporation is primarily used to transform members of the order Rickettsiales^,,. Plasmids complexed with polyamidoamine (PAMAM) dendrimers provide an alternative method to transform obligate intracellular bacteria. b | Shuttle vectors have been developed for C. trachomatis and Rickettsia spp. In both cases, portions of endogenous plasmids (pSW2 and pL2 for C. trachomatis, and pRAM18 for Rickettsia spp.) are fused to Escherichia coli plasmid backbones to enable replication. The shuttle vectors include E. coli origins of replication (oriV), fluorescent markers (GFP and GFP optimized for excitation by ultraviolet light (GFPuv) or red fluorescent protein (mCherry)), antibiotic selection genes (β-lactamase (bla), chloramphenicol (cat), spectinomycin (aadA) and rifampicin resistance (rif)) and multiple cloning sites (MCS)^,,. Two chlamydial shuttle vectors, pBOMB4 and pASK-GFP/mKate2-L2, can be modified to include a tetracycline-inducible promoter for conditional gene expression^,. The shuttle vector pL2dest enables the expression of proteins that are fused with a β-lactamase reporter to study protein secretion. The fluorescence-reported allelic exchange mutagenesis (FRAEM) vector pSU6 enables allelic exchange in C. trachomatis by behaving as a suicide vector in the absence of tetracycline. c | The Himar1 transposase randomly inserts the transposon into the bacterial genome. Successful transformants are selected with antibiotics and the expression of a fluorescent protein. The mutants are selected and sequenced to determine insertion sites. In chemical mutagenesis, mutations are caused by DNA-alkylating agents (for example, ethyl methanesulfonate (EMS)). Mutants are selected, pooled and subjected to forward or reverse genetics screensA. phagocytophilum, Anaplasma phagocytophilum; R. amblyommatis, Rickettsia amblyommatis; R. bellii, Rickettsia bellii; SFG, spotted fever group; tetO, tetracycline operator; tetR, tetracycline repressor.

Figure 3. Targeted mutagenesis and selection

a Targeted mutagenesis enables the alteration of specific bacterial genes. Allelic exchange can be used to introduce point mutations, and to insert and delete specific genes. Group II intron technology (TargeTron) enables introns to be specifically inserted into bacterial genes to disrupt gene function through LtrA, a multifunctional protein derived from Lactococcus lactis, which reverse transcribes and splices the intron and cleaves the recipient DNA for intron insertion. TargeTrons have been successfully applied to generate transient mutants of Ehrlichia chaffeensis and stable mutants of Chlamydia trachomatis and Rickettsia rickettsii^,. b | Two methods are currently used to distinguish mutants from wild-type bacteria: antibiotic selection and physical selection. Mutants can be physically separated from wild-type bacteria by fluorescence-activated cell sorting (FACS) and laser microdissection. One final step is obtaining clonal mutants, which can be isolated by limiting dilution, or, in some cases, plaque purification. BSL3, biological safety level 3.

Figure 4. Discoveries in microbial pathogenesis facilitated by genetic tools

a Chemical mutagenesis of Chlamydia trachomatis led to the identification of inclusion membrane protein for actin assembly (InaC), which recruits f ilamntous actin (F-actin) and induces Golgi redistribution around the inclusion in manner that is dependent on ADP ribosylation factor 1 (ARF1), ARF4 and ARF5 (REF. 8). b | Rickettsia parkeri disseminates intercellularly through a mechanism that is not used by facultative intracellular bacteria. Screening of a Himar1 transposon mutagenesis library of R. parkeri identified a mutant deficient in intercellular spread named surface cell antigen 4 (sca4)Himar1 (REF. 10). The sca4, Himar1 mutant was complemented by pRAM18dRGA[Sca4], which restored intercellular spread. Sca4 was found to inhibit vinculin binding to α-catenin (not shown), thereby reducing intercellular force transduction and enabling the intercellular spread of R. parkeri. c | Himar1 transposon mutagenesis was used to generate an Anaplasma phagocytophilum mutant library. One mutant exhibited a single transposon insertion in the dihydrolipoamide dehydrogenase 1 (lpda1) gene. Compared with wild-type infection, the lpda1, Himar1 A. phagocytophilum mutant infected neutrophils less well, and activated nuclear factor-κB (NF-κB) poorly (as measured by decreased phosphorylated inhibitory subunit of NF-κB (pIκB)), and elicited the production of less tumour necrosis factor (TNF) and macrophage inflammatory protein 2 (MIP2; also known as CXCL2) in neutrophils. Transient infection of macrophages by the lpda1, Himar1 A. phagocytophilum strain correlated with enhanced nuclear NF-κB activity, which led to the increased production of reactive oxygen species (ROS) and the release of proinflammatory cytokines. Mice that were infected with the lpda1, Himar1 mutant or wild-type A. phagocytophilum HZ exhibited differences in immunopathology and cell-specific outcomes, which suggests that LPDA1 may have a role in inhibiting excessive host immune activation. Arrow size correlates with relative quantity of cytokines secreted. IL-12p40, interleukin-12 subunit p40.