Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria

Abstract

The ability to genetically manipulate a pathogen is fundamental to discovering factors governing host-pathogen interactions at the molecular level and is critical for devising treatment and prevention strategies. While the genetic "toolbox" for many important bacterial pathogens is extensive, approaches for modifying obligate intracellular bacterial pathogens were classically limited due in part to the uniqueness of their obligatory lifestyles. Many researchers have confronted these challenges over the past two and a half decades leading to the development of multiple approaches to construct plasmid-bearing recombinant strains and chromosomal gene inactivation and deletion mutants, along with gene-silencing methods enabling the study of essential genes. This review will highlight seminal genetic achievements and recent developments (past 5 years) for Anaplasma spp., Rickettsia spp., Chlamydia spp., and Coxiella burnetii including progress being made for the still intractable Orientia tsutsugamushi. Alongside commentary of the strengths and weaknesses of the various approaches, future research directions will be discussed to include methods for C. burnetii that should have utility in the other obligate intracellular bacteria. Collectively, the future appears bright for unraveling the molecular pathogenic mechanisms of these significant pathogens.

Keywords: Anaplasma; Chlamydia; Coxiella; Ehrlichia; Orientia; Rickettsia; genetics; obligate.

Figure 1

Infection and replication lifestyles of select obligate intracellular bacteria in the animal host. (A) Chlamydia spp. undergo a biphasic development cycle with the infectious elementary body (EB) internalized into the host cell (primarily epithelial cells) where it resides in a host-membrane-derived vacuole termed the inclusion (Abdelrahman and Belland, 2005; Elwell et al., 2016). The EB converts into the replicating reticulate body (RB), which divides and eventually differentiates back into the EB form. EBs exit the host cell through cell lysis or inclusion extrusion [not pictured, (Hybiske and Stephens, 2007)] ~36–72 h postinfection. (B) C. burnetii infects macrophages where it resides within an acidified phagolysosome-like vacuole known as the Coxiella-containing vacuole (CCV) (Minnick and Raghavan, 2012). Both forms of C. burnetii, the small-cell and large-cell variants (SCV/LCV), are infectious with the SCV being more environmentally stable and having a lower metabolic rate than the LCV. C. burnetii exits the cell through cell lysis, although the timing of natural release is uncertain. For experimental usage, cells are artificially lysed once the bacteria reach the stationary phase after ~5–6 days. C. burnetii may also be grown axenically in acidified citrate cysteine medium [ACCM, (Omsland et al., 2009; Vallejo Esquerra et al., 2017)]. (C) Both Anaplasma spp. and Ehrlichia spp. have an infectious dense-core cell form (DC) and a replicative reticulate cell form (RC) (Salje, 2021). Cell types infected vary by species and include neutrophils, monocytes/macrophages, erythrocytes, or endothelial cells. Following internalization in a membrane-bound vacuole, the DC form converts into the RC form which then replicate as a bacterial microcolony known as a morulae. RCs eventually convert back to the DC form and are released from the cell ~3 days postinfection by either lysis (pictured), exocytosis, or filopodia in an actin-dependent mechanism. The exit mechanisms can vary by species and the stage of infection. (D) Rickettsia spp. replicate within the cytoplasm and most commonly infect endothelial cells, although some species will also infect macrophages. Members of the typhus group (TG) exit the cell via cell lysis (~4–5 days postinfection), whereas the spotted fever group (SPG) members can enter into adjacent cells via actin-based motility (depicted) or be released following lysis of the host cell (~48 h postinfection). (E) Orientia spp. infect a wide variety of cells including endothelial cells, dendritic cells, monocytes, and macrophages (Salje, 2021). Bacteria replicate within the cytoplasm in close proximity to the nucleus and are released from the cell in a budding process ~4 days postinfection. Note that for all of the bacteria above, the cell types listed reflect natural infection reservoirs and that multiple permissive cell types may be used in the lab for bacterial growth and propagation. In addition to the listed differences in time for bacterial entry, replication, and release, the infectious burdens per host cell vary across species from as little as ~30–50 for the SPG Rickettsia to a few hundred for Chlamydia spp. Images created with BioRender.com.

Figure 2

Updates to the chlamydial genetics toolbox. (A) Allelic exchange (FLAEM) utilizes the pSUmC-4.0 vector to obtain recombinant gene deletion mutants (Keb and Fields, 2020). 5′- and 3′- gene sequences from the targeted gene are cloned flanking the resistance gene (aadA) and reporter gene (gfp) which are expressed from their own promoters. Upon transformation, the strain exhibits red and green fluorescence and resistance to spectinomycin. Removal of aTc to halt pgp6 expression creates a suicide plasmid scenario promoting homologous recombination under spectinomycin selection. A double-recombination event will result in a strain with green fluorescence and spectinomycin resistance. A single-recombination event is noted by a red and green fluorescent bacterium. Mini-shuttle vectors would only possess sequences defined by the red dashed lines and would include the native plasmid origin of replication (Fields et al., 2022). (B) The cre-bearing vector, pSU-CRE, can then be transformed into the strain and selected via ampicillin yielding a red and green fluorescent strain resistant to spectinomycin and ampicillin. Expression of Cre will mediate excision of the loxP-flanked aadA-GFP cassette generating a spectinomycin sensitive, red fluorescent bacterium. Removal of aTc can then be performed to cure the plasmid allowing for complementation of the mutant or construction of additional mutants. The cured strain will be sensitive to ampicillin and not fluorescent. (C) A base CRISPRi vector is shown with the guide RNA location shown in light blue (Ouellette et al., 2021). Guide expression is constitutive owing to a dnaK ^P, and expression is isolated from downstream elements via an rrnB1 terminator sequence. Either dcas9 or dcas12 can be used for gene silencing, and expression of the dCas9/12 encoding an SsrA VAA degradation tag is controlled by the tet promoter. Accessory genes may be inserted 3′ of the dcas gene to make transcriptional fusions for complementation studies. (D) For transposon mutagenesis, a pUC-based suicide vector is used carrying the himar1 c9 transposase and either bla [used for C. trachomatis (LaBrie et al., 2019)] or gfp and cat [for C. muridarum (Wang et al., 2019)] in the transposon region defined by the inverted repeats (IR, shown in blue). (E) A generic riboswitch is shown to represent the synthetic riboswitch E, which is responsive to theophylline (Grieshaber et al., 2022). In the absence of theophylline, the 5′ UTR folds to block the RBS. Binding of theophylline changes the folding of the 5′ UTR revealing the RBS and allowing for binding of the ribosome and translation. To create a “tighter” expression system, the riboswitch can be inserted downstream of the tet promoter (tet systems are shown in A–C) to allow for gene regulation via aTc induction and translational regulation via theophylline (not pictured). Vector maps were drawn with SnapGene, and sizes are approximate. Vectors are not drawn to scale. Images were drawn with BioRender.com.

Figure 3

Complementation of C. burnetii proline and tyrosine auxotrophies for transformant selection. (A) Pathway of proline biosynthesis from glutamate. C. burnetii has lost the proA and proB genes that catalyze the first two steps of proline biosynthesis from glutamate while retaining proC, which catalyzes the final step in the proline biosynthesis pathway. (B) Schematic of the proBA operon cloned from Legionella and placed under the control of the CBU1169 promoter. The 1169^P -proBA operon was cloned into the pJB-CAT shuttle vector and the plasmid transformed into C. burnetii. (C) Complementation of the proline auxotrophy was tested using ACCM-D plus or minus proline. Expression of proBA in NMII (NMII proBA) complemented growth of the bacterium in the absence of proline, whereas the wild-type bacteria (NMII) could only grow in the presence of added proline. (D) Pathway of tyrosine biosynthesis from chorismate. C. burnetii has lost both genes (tyrA and tyrB) required for biosynthesis of tyrosine from chorismate. (E) Schematic of the tyrB gene cloned from E. coli and placed under the control of the CBU1169 promoter. This fragment was then cloned into pJB-CAT and the plasmid (pJB-CAT-tyrB) transformed into C. burnetii. (F) Complementation of the tyrosine auxotrophy was tested using ACCM-D with the addition of the TyrB substrate 4-hydroxyphenylpyruvic acid (4-HPP) and plus or minus tyrosine. Expression of tyrB in NMII (NMII tyrB) complemented growth of the bacterium in ACCM-D containing 4-HPP but minus tyrosine, whereas the wild-type bacteria (NMII) could only grow in the presence of added tyrosine and not in the presence of the TyrB substrate 4-HPP alone.

Figure 4

MinTn7 transposition in bacteria. (A) Schematic of site-specific miniTn7 transposition into the region downstream of the glutamine-fructose-6-phosphate gene (glmS). The glmS gene has a 30-bp attTn7 site in the end of it that is recognized by the miniTn7 transposase (consisting of the TnsA, TnsB, TnsC, and TnsD proteins). Subsequent miniTn7 insertion occurs 36 bp downstream of the attTn7 site. This transposition system utilizes two plasmids, one containing the miniTn7 transposon with a selectable marker and the second containing the miniTn7 transposase genes. (B) Alignment of predicted miniTn7 attTn7 sites in different obligate intracellular bacteria compared with Pseudomonas aeruginosa. No predicted attTn7 sites were identified in Rickettsia, Ehrlichia, or Orientia species and even though other Anaplasma species have an attTn7 site, these were not found in A. phagocytophilum. Gray boxes indicate conserved sequences. (C) Schematic of the C. burnetii glmS region. Due to a duplication event between the end of cbu01787 and cbu01788, C. burnetii has two different attTn7 sites allowing for integration into either site.

Figure 5

Creation of new IPTG and arabinose-inducible expression systems in C. burnetii. (A) Schematics of two different IPTG-induction systems allowing inducible expression of mCherry and their respective immunoblots detecting mCherry production. The upper schematic shows the original IPTG-inducible system contained on the pJB-CAT and pKM230 C. burnetii shuttle vectors, which consists of a truncated lacI gene (missing the tetramerization domain), a tac promoter, and a single LacI binding site upstream of mCherry. Production of mCherry was seen in the minus IPTG induction by immunoblot (upper right) indicating leaky expression of mCherry. The lower schematic shows the new IPTG-inducible system consisting of a full-length lacI gene and lacO promoter containing dual LacI binding sites upstream of mCherry. No background mCherry production was seen in the uninduced control, whereas good production of mCherry was evident in the IPTG-induced sample. (B) Schematic of the arabinose-induction system allowing inducible expression of mCherry. The mCherry gene was cloned downstream of the araB promoter. The sequence of the native araB promoter is shown. This sequence was modified so that the -35 and -10 regions had a smaller 16-bp spacer region and their sequences were closer to the consensus sigma 70 promoter sequence (TTGACA and TATAAT, respectively). The native and modified arabinose induction systems were cloned into the pJB-CAT shuttle vector and then transformed into C. burnetii. Arabinose-based gene expression was examined by immunoblot of 0-, 4-, or 7-h post-arabinose induction of 5-day C. burnetii cultures. Production of mCherry from the native araB promoter was very low at 7 h, whereas in the modified araB system, high levels of protein were detected at both 4 and 7 h post induction. No background mCherry production was seen in the uninduced control (0 h).

Figure 6

Creation of synthetic promoters for predictable gene expression in C. burnetii. (A) Schematic of the variable strength ProSeries synthetic promoters fused upstream of the mCherry gene. The synthetic promoters are flanked by insulation regions that help prevent unwanted gene transcription. (B) Sequence of the four different ProSeries promoters (proA, proB, proC, and proD) is shown. The only difference between each promoter lies in the -10 box. (C) The four different promoter-mCherry fusions were cloned into pJB-CAT, and the resulting vectors were transformed into C. burnetii. The level of promoter activity was examined by looking at mCherry production by immunoblot. An increasing level of mCherry production was seen across the four promoter constructs.