Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation.
In Sinorhizobium meliloti, the production of exopolysaccharides such as succinoglycan and exopolysaccharide II (EPS II) enables the bacterium to invade root nodules on Medicago sativa and establish a nitrogen-fixing symbiosis. While extensive research has focused on succinoglycan, less is known concerning the regulation of EPS II or the mechanism by which it mediates entrance into the host plant. Previously, we reported that the ExpR/Sin quorum-sensing system is required to produce the symbiotically active low-molecular-weight fraction of this exopolysaccharide. Here, we show that this system induces EPS II production by increasing expression of the expG-expC operon, encoding both a transcriptional regulator (ExpG) and a glycosyl transferase (ExpC). ExpG derepresses EPS II production at the transcriptional level from MucR, a RosR homolog, while concurrently elevating expression of expC, resulting in the synthesis of the low-molecular-weight form. While the ExpR/Sin system abolishes the role of MucR on EPS II production, it preserves a multitude of other quorumsensing-independent regulatory functions which promote the establishment of symbiosis. In planktonic S. meliloti, MucR properly coordinates a diverse set of bacterial behaviors by repressing a variety of genes intended for expression during symbiosis and enhancing the bacterial ability to induce root nodule formation. Quorum sensing precisely modulates the functions of MucR to take advantage of both the production of symbiotically active EPS II as well as the proper coordination of bacterial behavior required to promote symbiosis.
Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia.
Members of the order Chlamydiales comprise a group of exquisitely evolved parasites of eukaryotic hosts that extends from single-celled amoeba to mammals. The most notable are human pathogens and include the agent of oculogenital disease Chlamydia trachomatis, the respiratory pathogen C. pneumoniae, and the zoonotic agent C. psittaci. All of these species are obligate intracellular bacteria that develop within parasitophorous vesicles termed inclusions. This demanding lifestyle necessitates orchestrated entry into nonphagocytic cells, creation of a privileged intracellular niche, and subversion of potent host defenses. All chlamydial genomes contain the coding capacity for a nonflagellar type III secretion system, and this mechanism has arisen as an essential contributor to chlamydial virulence. The emergence of tractable approaches to the genetic manipulation of chlamydiae raises the possibility of explosive progress in understanding this important contributor to chlamydial pathogenesis. This minireview considers challenges and recent advances that have revealed how chlamydiae have maintained conserved aspects of T3S while exploiting diversification to yield a system that exerts a fundamental role in the unique biology of Chlamydia species.
Development of approaches to genetically manipulate is fostering important advances in understanding pathogenesis.luorescence-eported llelicxchange utagenesis (FRAEM) now enables the complete deletion of specific genes in L2. We have leveraged this technology to delete the coding sequences for a known type III effector. The evidence provided here indicates that CT694/CTL0063 is a virulence protein involved in chlamydial invasion. Based on our findings, we designate the gene product corresponding to ranslocatedembrane-associated ffector A (TmeA). Deletion of did not impact development of intracellular chlamydiae. However, the absence of TmeA manifested as a decrease in infectivity in both tissue culture and murine infection models. The defect was reflected by impaired invasion of host cells. TmeA binds human AHNAK, and we demonstrate here that AHNAK is transiently recruited by invading chlamydiae. TmeA, however, is not required for endogenous AHNAK recruitment. TmeA also impairs AHNAK-dependent actin bundling activity. This TmeA-mediated effect likely does not explain impaired invasion displayed by the strain of , since AHNAK-deficient cells revealed no invasion phenotype. Overall, our data indicate the efficacy of FRAEM and reveal a role of TmeA during chlamydial invasion that manifests independently of effects on AHNAK.
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