Transformation of Chlamydia: Current approaches and impact on our understanding of chlamydial infection biology

Abstract

The intonation “The king is dead, long live the king” aptly describes the state of Chlamydia research. Genetic-based approaches are rapidly replacing correlative strategies to provide new insights. We describe how current transformation technologies are enhancing progress in understanding Chlamydia infection biology and present key opportunities for further development.

1. Introduction

The Chlamydiales are a group of obligate intracellular parasites of eukaryotic cells. Susceptible hosts range from single-cell protists to humans and includes a broad array of animals [1]. Elucidating the pathogenic mechanisms employed by Chlamydia spp. represents a common research goal, particularly due to their significance in human disease. Chlamydia trachomatis represents the prototypical species pathogenic for humans and is the agent of blinding trachoma and sexually transmitted disease. Additional species capable of commonly infecting humans include the respiratory pathogen C. pneumoniae and the zoonotic agent C. psittaci [2]. Members of the C. trachomatis trachoma biovar mediate ocular (serovars A–C) or genital infections (serovars D–K), whereas members of the lymphogranuloma venereum (LGV) biovar (serovars L1–L3) are agents of disseminating genital infection. C. trachomatis genomes are composed of a ca.1Mb circular chromosome and a 7.5 kb plasmid [3]. Despite differences in tissue tropism and clinical presentation, C. trachomatis genomes are remarkably similar, displaying >98% genomic conservation [4]. Compared to LGV strains, serovars A–K are more fastidious and grow more slowly. For this reason, C. trachomatis L2 represents a common laboratory agent employed to model chlamydial infection in cell culture. Although debate has arisen regarding the most appropriate agent to model human disease in animals, C. muridarum and C. caviae are routinely used to study infection parameters using mice and guinea pigs, respectively [5].

To fully understand the challenges, limitations, and promise of genetics in Chlamydia, one must appreciate key biological complexities exemplified among this group of unique Gram-negative bacteria. Long-term (>700 mya) co-evolution with eukaryotic hosts has resulted in intricate physiology and infection biology [4]. A biphasic developmental cycle is a hallmark of chlamydial biology [6] and is initiated when infectious elementary bodies (EBs) invade a eukaryotic cell. EBs are minimally active metabolically and are considered spore-like given their condensed nucleoid and highly disulfide crosslinked envelope. EBs differentiate into vegetative, yet non-infectious, reticulate bodies (RBs) that divide until an unknown signal(s) induces asynchronous conversion back into EBs. Development occurs entirely within in a parasitophorous intracellular vesicle termed an inclusion and is concluded when chlamydiae exit cells via lysis or extrusion [7]. The timing for completion of development among Chlamydia spp can vary from 48–96 hrs [6]. Temporal gene expression programs correlate with development [8], yet the details for how development is controlled remain largely unknown. Chlamydia are regarded as master manipulators of host cell biology and possess virulence determinants that include metabolic capabilities, adhesins, and secreted effectors. These factors culminate to establish and maintain a protected intracellular niche by influencing host cell cytoskeleton, vesicular trafficking, and immune surveillance mechanisms (reviewed in [9]). The current understanding of Chlamydia infection was achieved over a >40 yr time-span without the benefit of the direct genetic manipulation that has driven progress in tractable microbial pathogens.

Past progress relied heavily on in vitro correlative studies and the use of surrogate systems. The “Molecular Koch’s postulates” were introduced to establish a set of experimental criteria used to define pathogenicity genes or virulence factors [10]. The standards can also be applied more broadly to describe the experimental requirements to definitively assign molecular function to a respective gene product. Experimental criteria include i) targeted inactivation of a gene(s) should cause a measurable loss of a given phenotype/function and ii) complementation, reintroducing the wild-type gene, should restore the phenotype [10]. Addressing these postulates via rapid and definitive elucidation of the molecular mechanisms governing the host-pathogen interactions impacting C. trachomatis survival and pathogenesis is now possible due to advances in genetic manipulation. Broad aspects of Chlamydia genetics have been the topic of several excellent reviews [11–14]. This review will focus on transformation of Chlamydia, how the capability has revolutionized the study of Chlamydia infection biology, and what remains to be accomplished to fully exploit the power of genetics. An overview of chlamydial genes that have studied using transformation-based approaches is provided in Table 1.

Table 1.

Summary of genes investigated using transformation-based approaches

Genea (Protein)	Transformation-based approach	Selection markerb	Importance	Ref
incA (Inclusion membrane protein A)	Mutagenesis (TargeTron)	bla, cat	First application of TargeTron in Chlamydia	[31]
	Mutagenesis (TargeTron)	aadA, cat	Demonstrated the use of different selection and the ability to generate double mutants	[37]
	Ectopic expression of epitope (FLAG) tagged protein/Complementation	bla	Subdomain expression used in complementation studies to address domain-specific protein functions.	[52] [71]
incD (Inclusion membrane protein D)	Epitope (Flag) or reporter (CyaA, Gsk) tagged protein	bla	Shuttle vector systems for study of protein function, localization, and secretion into host cytosol.	[5, 23, 3]
incF, incA	Reporter (Apex2)-tagged expression	bla	First application of proximity labeling using transformed chlamydiae	[53]
ct005, ct179, ct224, ct229, ct288, ct383, ct449, ct813, ct850, and incC (Inclusion membrane proteins)	- Mutagenesis (TargeTron) - Ectopic (Flag)-tagged expression -Complementation	bla	Functional study of Inc proteins revealed a requirement of CT229, IncC, and CT383 in inclusion membrane integrity and host survival	[70]
inaC/ct813 (Chlamydial inclusion membrane protein)	- Complementation of EMS mutant - Epitope (Flag)-tagged expression - Mutagenesis (TargeTron)	bla	Parallel studies demonstrating effects of InaC/CT813 on ARF GTPases	[34, 72]
ct006, ct134, ct135, ct179, ct192, ct195, ct224, ct227, ct244, ct345, ct365, ct383, ct449, ct483, ct484, ct529, ct565, ct616, ct788, ct789, ct846	- Epitope (Flag)-tagged expression	bla	Expression of putative Incs in C. trachomatis to test for localization to the inclusion membrane.	[69]
rsbV1 (Anti-anti- sigma factor RsbV1)	- Mutagenesis (TargeTron)	aadA, cat	Capability of TargeTron for using different selection & ability to generate double mutants	[37, 64]
CpoS (Chlamydia promoter of survival)	- Mutagenesis (TargeTron) - Complementation	bla, cat bla	Functional studies revealing an importance of CpoS in host cell survival	[56]
tepP (Translocated early phosphoprotein)	- Complementation of a EMS mutant - Mutagenesis (TargeTron) - Epitope (Flag)-tagged expression	bla	Functional studies of a new type III effector	[12, 11]
ChgroEL2 and ChgroEL3 (chlamydial chaperonin)	- Mutagenesis (TargeTron)	aadA	Functional studies of potentially redundant chlamydial chaperones	[28]
Cdu1 (Chlamydial deubiquitinating enzyme 1)	- Transposon mutagenesis - Epitope (Flag)-tagged expression	bla	First application of transposon mutagenesis in Chlamydia	[20]
trpA (Tryptophan synthase alpha subunit) & Ctl0063, Ctl0064 and Ctl0065	- Mutagenesis (FRAEM) - Complementation	bla aadA	First application of allelic exchange for targeted gene deletion in Chlamydia	[41]
ct694, ct695, ct696 and euo	- Reporter (BlaM)-tagged expression	bla	TEM-1 β-lactamase reporter to assay secretion	[39]
tarp (Translocated actin-recruiting phosphoprotein)	- Reporter (BlaM)-tagged expression - Epitope (c-myc)-tagged expression	bla	TEM-1 β-lactamase reporter to assay secretion Subdomain epitope tagging expression used to address domain-specific functions	[39, 50]
cpaf (Chlamydia protease-like activity factor)	- Complementation of a EMS mutant - Epitope (Flag) tagged expression	bla	Studies demonstrating the importance of CPAF during murine infection and evidence of secretion in tissue culture	[5, 76, 77]
pgp1–8 (Plasmid- encoded proteins)	- Deletion mutagenesis	bla	Studies addressing the function of plasmid- encoded genes within Chlamydia	[35, 60, 66]
pgp3, pgp5	- Deletion mutagenesis	bla	Application of C. muridarum mutants for virulence studies in mice.	[26, 36]

^aGene designations are given as indicated in respective papers.

^bAntibiotic resistances for penicillin G, spectinomycin, and chloramphenicol are provided by the bla, aadA, or cat genes, respectively

2. Transformation

Fulfillment of the Koch’s molecular postulates requires the ability to transform bacteria with exogenous DNA. This has been challenging for obligate intracellular bacteria since host cell and bacterial membranes represent barriers to reagents—necessitating protocols using purified bacteria—and all growth occurs under the selective pressure of host cells. In addition, the biphasic developmental cycle manifested by Chlamydia, Anaplasma, Coxiella, and Ehrlichia where infectious particles are likely not competent adds a further level of complication [15]. These barriers have been overcome for Coxiella burnetii by development of an axenic medium capable of supporting the complete developmental process [16, 17]. Although initial attempts at defining growth requirements of Chlamydia have emerged [18], successful host-free cultivation awaits further progress. Despite these challenges, significant progress has been made for Chlamydia, and C. trachomatis L2 is now regarded as somewhat genetically tractable.

Interestingly, lateral gene transfer (LGT) via natural transformation occurs in Chlamydia spp. Evidence from C. trachomatis clinical isolates revealed genome-wide mosaics [19–21], raising the possibility of genetic transfer and homologous recombination. Experimentally, recombinant strains can be generated with a frequency of 10⁻³–10⁻⁴ by co-cultivation [22–24] of strains. Both intra- and interspecies LGT is possible and likely occurs intracellularly since cohabitation of a single inclusion appears to be a requirement [23]. Unlike zoonotic Chlamydia spp., C. trachomatis lacks a prophage necessary for transduction (reviewed in [25]). In addition, chlamydial genomes lack obvious conjugation machinery. LGT in the absence of these factors implies some degree of natural competence. Limited amino acid similarity between C. trachomatis serovar D CT339 and Bacillus ComEC has been noted [11], yet direct functional evidence is currently lacking. Regardless of mechanism, LGT represents an important tool and has already been employed for gene linkage studies [26–28] and could be leveraged to more efficiently shuttle vectors among strains and engineer multi-gene mutants in the future. Selection and isolation of recombinants requires selectable markers and generation of spontaneous chromosomal mutants in gyrA, ropB, folA, and 23s rrna have been employed to provide resistance to ofloxacin, rifampin, trimethoprim, and lincomycin, respectively.

The first success with artificial transformation of Chlamydia with recombinant plasmids employed electroporation. Tam et al. [29] electroporated a shuttle vector (pPBW100) into purified C. trachomatis serovar E EBs. pPBW100 was generated from an E. coli backbone plasmid (pUC6 version) ligated to a C. trachomatis E endogenous plasmid (pCTE1) and employed chloramphenicol resistance as a selectable marker. In 2009, a similar electroporation method was used to mobilize an engineered vector into C. psittaci 6BC EBs [30]. In both cases, maintenance of the episomal element was transient. This feature was leveraged to transfer kasugamycin and spectinomycin resistance into the chromosome via allelic exchange [30]. These important, proof-of-principle achievements indicated the possibility of artificial transformation and laid the ground work for further progress. Similar to the situation in Rickettsia [31], engineering a vector containing endogenous plasmid (termed pL2 in C. trachomatis L2) sequences facilitated stable plasmid maintenance in Chlamydia. Wang, et al. [32] developed a CaCl₂-based method to transform C. trachomatis with a chimeric shuttle vector (pGFP::SW2) expressing pL2-encoded genes fused with an element containing an E. coli origin, blaM, and gfp. The chlamydial persistence phenotype [33] induced by β-lactam antibiotics enabled efficient selection with PenG, and long-term GFP-fluorescence indicated stable maintenance of the plasmid. The CaCl₂-method has become the preferred technique for chlamydial transformation, and multiple shuttle vectors leveraging diverse antibiotic selections have been generated (Suppl Table 1 in [11]).

Routine transformation has brought a degree of genetic tractability to C. trachomatis L2. This capability needs to be extended to other Chlamydia spp. to take full advantage of genetics. The LGV serovars are not the most relevant clinically, and animal model studies commonly employ C. muridarum. While, transformation of other serovars and species with basic shuttle vectors has been achieved [34–37], capabilities and reagents developed for L2 are not widespread. While there are no obvious confounding barriers, plasmid incompatibility issues will need to be addressed since some transformations appear to require strains lacking endogenous plasmid for greatest efficiency [38, 39]. Although the function of plasmid-encoded genes is incompletely understood, shuttle vectors for use in other Chlamydia will also likely require backbones derived from respective endogenous plasmids due to plasmid tropism [35, 36]. Finally, it should be noted that the rare clinical application of β-lactam antibiotics previously prevented use of PenG in transformation of biovar trachoma C. trachomatis [39]. However, the NIH Office of Science Policy recently issued an opinion stating that the use of β-lactam antibiotics for serovars D–K does not constitute a Major Action experiment under NIH guidelines.

3. Impact of Transformation

Mutagenesis is required to definitively elucidate protein function. Forward genetics, enabled by random, chemical-mediated mutagenesis coupled with whole-genome sequencing, represents a powerful approach that is currently making significant progress in understanding chlamydial biology (reviewed in [11]). While mutant strain production by this method is transformation-independent, these approaches rely on natural transformation and LGT for gene linkage studies and artificial transformation for mutant complementation (discussed below) since genomes often contain multiple mutations. Pregenetic-era progress revealed a plethora of factors implicated in Chlamydia pathogenesis. For example, all Chlamydia spp. express a virulence-associated type III secretion system (T3SS) that contributes to pathogenesis at multiple levels [9, 40]. Transformation has been applied most directly in reverse genetic approaches aimed at characterizing these factors. The greatest success has been in elucidating the role(s) of plasmid-encoded proteins since plasmid ORFs can be easily deleted during manipulations in E. coli then returned to plasmid deficient Chlamydia for phenotypic studies [41, 42]. These studies have delineated proteins required for plasmid replication and maintenance as well as proteins that directly impact pathogenesis as regulators of chromosomal genes or as secreted effectors (reviewed in [43]).

Engineered pL2-based plasmids have also been developed to enable mutagenesis of chromosomal loci. TargeTron technology has been adapted for Chlamydia to enable site-specific disruption of genes via insertion of a Group II intron and an antibiotic resistance gene [44]. Initial studies targeted the T3 secreted inclusion membrane protein A (IncA) to study requirements for homotypic fusion of inclusions [44, 45]. This method has been adopted by investigators as a comparatively rapid method to generate targeted mutations in suspected virulence genes. The TargeTron approach has been extended to deduce a role of Incs in inclusion integrity and host cell survival [46, 47], investigate the function of effectors [48], and deduce the role(s) of physiological proteins such as chaperonins [49]. More recently, Himar1-based transposition has been added to the molecular toolbox. Fischer, et al. disrupted C. trachomatis cdu1 to show that this secreted deubiquitinase targets host Mcl-1 [50]. Although experimental details were not provided, this represents the first application of transposon mutagenesis in Chlamydia. Chemical and insertion-based gene disruption leave behind gene sequences, raising the possibility of partially functional protein fragments. A fluorescence-reported allelic exchange mutagenesis (FRAEM) technique was developed to overcome this barrier [51]. Key features include a conditionally-replicating suicide vector backbone, differential fluorescence indicators of transformation and allelic exchange events, and the ability to delete entire coding sequences [52]. This method is recent and thus far only proof-of-principle mutations in trpA, ctl0063, ctl0064, and ctl0065 have been reported [51]. Like insertion mutagenesis, FRAEM leaves a selection marker (gfp and bla) integrated at the targeted locus. Marker-less gene inactivation would be desirable since insertions have the possibility of exerting polar effects on downstream genes. Indeed, deletion of ctl0063 and replacement with a gfp-bla selection marker resulted in decreased expression of ctl0064 [51]. Theoretically, the impact of insertions could be mitigated using site-specific recombinases [53], such as CRE-loxP or FLP-frt, to excise DNA elements to leave behind only 34 bp scar sequences.

Transformation of Chlamydia was actually first applied for ectopic expression of reporter proteins conferring bioluminescence [54] or fluorescence [32, 55, 56] so that bacteria could be tracked during live infection. Expression has now been broadly applied for trans production of epitope-tagged chlamydial proteins to reliably examine protein localization during infection [46, 48, 50, 57–60]. This important feature bypasses the need to generate antibodies and avoids specificity questions that traditionally plagued immunolocalization studies in Chlamydia. While native promoters have been useful, non-native promoters such as that for Neisseria meningitidis porA [32, 61] have been employed for constitutive expression while, tet-responsive promoters have been developed to enable conditional gene expression [59, 62]. Verification of chlamydial effector protein section into the eukaryotic cytosol via immunofluorescence was previously complicated by limits of detection. Evidence of extra-inclusion localization can now be assessed by expressing fusion proteins containing the target chlamydial proteins fused to reporters active only in the eukaryotic cytosol [59, 61]. Finally, ectopic expression has also been fruitful to gain functional insights for chlamydial proteins. For example, the power of proximity labeling to elucidate live-cell protein associations has been made possible by expressing secreted chlamydial proteins fused with promiscuous biotin ligases [63]. Subdomain expression has also been leveraged to address domain-specific functions [45, 64] to gain more refined mechanistic insights.

Importantly, transformation has enabled complementation studies as a follow-up to mutagenesis. Allelic replacement is necessary to fulfill the second half of the molecular Koch’s postulates and is essential to definitively assign function to a respective gene product. This is true even when whole genome sequencing indicates that two strains differ only at the targeted locus. Failure to complement ignores the possibility that a given mutation—especially insertion mutations—could have cis-acting affects such as changes in DNA topology or accessibility that alter expression of other genes. Direct restoration of the chromosomal gene is most ideal since expression levels and topology would be consistent. Efficacious replacement would require the development of a counter selectable marker usable in Chlamydia to manipulate plasmids. Although the use of temperature-sensitive (TS) alleles has been suggested [65], this approach is not currently available. Reversion is most commonly achieved by ectopic expression of the mutated gene via trans complementation. However, this has proved challenging in Chlamydia and is sometimes inexplicably ignored. Several studies have reported partial complementation or complete lack of phenotypic reversion. This could be due to non-target-gene associated contributions to a particular phenotype, particularly when other mutations are present in the genome. Expression levels are a prime candidate since non-physiologic amounts of a protein can skew the host-pathogen balance. Endogenous promoters are likely the best choice when relying on trans complementation. Finally, all chlamydial expression plasmids contain the pL2 backbone, and plasmid-dependent effects could complicate complementation experiments. Given contributions of plasmid-encoded factors to pathogenesis [43], there is a need to employ vector-only controls in complementation studies.

4. Future Opportunities

While significant progress has been achieved, additional advances will be necessary to render Chlamydia significantly more genetically tractable. Improved transformation efficiency and the ability to inactivate genes encoding essential proteins represent two prominent examples. Ideally, development of a system for host-free cultivation [66] would address both issues. An axenic medium has certainly facilitated work with Coxiella [16]. However, host-free cultivation of Chlamydia may not be achievable in the near term. Improved electroporation parameters may be one avenue, yet decreased chlamydial viability may limit the efficacy of this approach [30]. Dendrimer-based approaches represent an alternative since they are non-toxic and have been reported to increase transformation efficiency in C. trachomatis [67]. These findings, however, have not yet been confirmed or widely adopted by other labs. Given the observed high rates of DNA exchange via LGT [22], physical introduction of exogenous DNA to a niche containing competent chlamydiae likely represents a major rate-limiting step. Hence, new insights regarding chlamydial competence biology represents one area that could lead to more effective transformation methods. Increased efficiency will certainly be required to facilitate alternative random and targeted mutagenesis techniques such as transposition or CRISPR/Cas, respectively.

Genes necessary for fundamental physiological processes such as replication, development, or establishment and maintenance of the replication-competent niche are designated as essential. In their absence, chlamydiae fail to replicate and/or generate de novo infectious particles. Proteins encoded by these genes represent the most important and interesting targets of investigation, yet their essential nature precludes direct inactivation. To date, chemical mutagenesis approaches yielding conditional, hypo- or hypermorphic phenotypes represent the only avenue to investigate these gene products. For example, temperature-sensitive alleles [65] and a hypomorphic allele of gspE [68] have provided functional information regarding chlamydial physiology and type II secretion, respectively. The fluorescence-reporting feature of FRAEM can also indicate essential genes. Single-recombinant merodiploids fail to resolve to a deletion mutant after repeated passages when the event would be lethal (Mueller and Fields, Unpublished). In these instances, transformation could be used to introduce a plasmid encoding a conditionally-expressed wild-type copy of the target gene. This would enable deletion of the chromosomal copy when the trans gene is expressed and subsequent phenotypic characterization in the absence of expression.

Chlamydia genetics will likely never reach the tractability level of free-living bacteria, yet current capabilities have revolutionized the approach to investigating infection biology. The ability to transform Chlamydia has played a large role in those advances. Overall, more rapid and definitive progress can be expected for this important and interesting intracellular parasite.