Chlamydia trachomatis TmeB antagonizes actin polymerization via direct interference with Arp2/3 activity.

Chlamydia trachomatis is an obligate intracellular pathogen that actively promotes invasion of epithelial cells. A virulence-associated type III secretion system contributes to chlamydial entry and at least four effectors have been described that are deployed during this time. Two of these invasion-related effectors, the translocated membrane-associated effectors A and B (TmeA and TmeB), are encoded in a bi-cistronic operon. TmeA directly activates host N-WASP to stimulate Arp2/3-dependent actin polymerization. According to current working models, TmeA-mediated N-WASP activation contributes to invasion. TmeB has not been functionally characterized. Unlike a tmeA null strain, loss of tmeB does not impact invasion efficiency of C. trachomatis. Using strains deficient for multiple genes, we provide evidence that TmeA is dispensable for invasion in the absence of TmeB. Our data indicate that overabundance of TmeB interferes with invasion and that this activity requires active Arp2/3 complex. We further show that TmeB is capable of interfering with Arp2/3-mediated actin polymerization. In aggregate, these data point to opposing functions for TmeA and TmeB that manifest during the invasion process. These studies raise intriguing questions regarding the dynamic interplay between TmeA, TmeB, and branched actin polymerization during chlamydial entry.

Transformation and Mutagenesis of Chlamydia trachomatis and C. muridarum Utilizing pKW Vector.

A gene deletion by allelic exchange via homologous recombination from a bacterial genome represents a valuable genetic tool for studying a role(s) of determinants involved in various aspects of pathogenesis. Due to chlamydial obligate intracellular lifestyle and comparatively low transformation rate, the mutagenesis of Chlamydia utilizes types of suicide vectors that have to be maintained and propagated by the bacteria throughout several rounds of their intracellular developmental cycle. These deletion constructs must be lost by chlamydiae once null mutant formation is achieved. pKW is a small, 5.45 bp, pUC19-derived vector, which has been recently successfully employed for the generation of deletion mutants in C. trachomatis, serovariant D, and C. muridarum. This vector contains both, E. coli as well as chlamydial species-specific plasmid origins of replication, allowing for its propagation by both bacterial genera under a selective pressure. However, once the selective antibiotic is removed from culture, chlamydiae rapidly lose pKW, and the subsequent reintroduction of the selective antibiotic back to chlamydiae-infected cells results efficiently in the selection of generated deletion mutants. Protocols provided here describe in detail the preparation of pKW deletion constructs for C. trachomatis and C. muridarum applicable for chlamydial transformation and the production of null mutant in non-essential genes. Protocols provided here, are describing in detail methods for assembly of the pKW shuttle vector and generation of deletion mutants in C. trachomatis and C. muridarum. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of pKW shuttle vector Basic Protocol 2: Generation of a deletion mutant in C. trachomatis, serovars D and L2 and, Chlamydia muridarum Support Protocol: Transformation of C. trachomatis, serovars B.

A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum.

The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.

Genetic Manipulation of Chlamydia trachomatis: Chromosomal Deletions.

Progress in understanding molecular mechanisms contributing to chlamydial pathogenesis has been greatly facilitated by recent advances in genetic manipulation of C. trachomatis. Valuable approaches such as random, chemically induced mutagenesis or targeted, insertion-based gene disruption have led to significant discoveries. We describe herein a technique for generating definitive null strains via complete deletion of chromosomal genes in C. trachomatis. Fluorescence-reported allelic exchange mutagenesis (FRAEM), using the suicide vector pSUmC, enables targeted deletion of desired chromosomal DNA. The protocol provided here describes steps required to produce transformation competent chlamydiae, generate a specific allelic exchange plasmid construct, carry out mutagenesis, and isolate clonal populations of resulting mutant strains.

Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis.

Gene inactivation is essential for forward and reverse genetic approaches to establish protein function. Techniques such as insertion or chemical mutagenesis have been developed to mutagenize chlamydiae via targeted or random mutagenesis, respectively. Both of these approaches require transformation of chlamydiae to either introduce insertion elements or complement mutants. We have recently developed a targeted mutagenesis strategy, fluorescence-reported allelic exchange mutagenesis (FRAEM), to delete Chlamydia trachomatis L2 genes. This approach overcomes several barriers for genetically manipulating intracellular bacteria. Perhaps most significantly, FRAEM employs fluorescence reporting to indicate successful transformation and subsequent recombination events. Three protocols are provided that detail methods to construct gene-specific suicide vectors, transform C. trachomatis L2 to select for recombinants, and isolate clonal populations via limiting dilution. In aggregate, these protocols will allow investigators to engineer C. trachomatis L2 strains carrying complete deletions of desired gene(s). © 2017 by John Wiley & Sons, Inc.

Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.

UNLABELLED: 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 ß-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. IMPORTANCE: The obligate intracellular nature of a variety of infectious bacteria presents a significant obstacle to the development of molecular genetic tools for dissecting pathogenicity. Although progress in chlamydial 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. Here, 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 L2. Furthermore, this approach permits monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms.

C. pneumoniae disrupts eNOS trafficking and impairs NO production in human aortic endothelial cells.

Endothelial nitric oxide synthase (eNOS) generated NO plays a crucial physiological role in the regulation of vascular tone. eNOS is a constitutively expressed synthase whose enzymatic function is regulated by dual acylation, phosphorylation, protein-protein interaction and subcellular localization. In endothelial cells, the enzyme is primarily localized to the Golgi apparatus (GA) and the plasma membrane where it binds to caveolin-1. Upon stimulation, the enzyme is translocated from the plasma membrane to the cytoplasm where it generates NO. When activation of eNOS ceases, the majority of the enzyme is recycled back to the membrane fraction. An inability of eNOS to cycle between the cytosol and the membrane leads to impaired NO production and vascular dysfunction. Chlamydia pneumoniae is a Gram-negative obligate intracellular bacterium that primarily infects epithelial cells of the human respiratory tract, but unlike any other chlamydial species, C. pneumoniae displays tropism toward atherosclerotic tissues. In this study, we demonstrate that C. pneumoniae inclusions colocalize with eNOS, and the microorganism interferes with trafficking of the enzyme from the GA to the plasma membrane in primary human aortic endothelial cells. This mislocation of eNOS results in significant inhibition of NO release by C. pneumoniae-infected cells. Furthermore, we show that the distribution of eNOS in C. pneumoniae-infected cells is altered due to an intimate association of the Golgi complex with chlamydial inclusions rather than by direct interaction of the enzyme with the chlamydial inclusion membrane.

Chlamydia pneumoniae impairs the innate immune response in infected epithelial cells by targeting TRAF3.

Type I IFNs are induced during microbial infections and have well-characterized antiviral activities. TRAF3 is a signaling molecule crucial for type I IFN production and, therefore, represents a potential target for disarming immune responses. Chlamydia pneumoniae is a human pathogen that primarily infects respiratory epithelial cells; the onset of symptoms takes several weeks, and the course of infection is protracted. C. pneumoniae has also been associated with a variety of chronic inflammatory conditions. Thus, typical C. pneumoniae infections of humans are consistent with an impairment in inflammatory responses to the microorganism. We demonstrate that infection of epithelial cells with C. pneumoniae does not lead to IFN-ß production. Instead, infected cells are prevented from activating IFN regulatory factor 3. This effect is mediated by C. pneumoniae-dependent degradation of TRAF3, which is independent of a functional proteasome. Hence, it is likely that C. pneumoniae expresses a unique protease targeting TRAF3-dependent immune effector mechanisms.

Scc1 (CP0432) and Scc4 (CP0033) function as a type III secretion chaperone for CopN of Chlamydia pneumoniae.

The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secreted proteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effector proteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demonstrated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. The Scc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3 chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.

Effector protein modulation of host cells: examples in the Chlamydia spp. arsenal.

As obligate intracellular parasites, Chlamydia spp. must create and maintain a specialized intracellular niche while simultaneously contending with potent host defenses. Discoveries that chlamydiae deploy an array of anti-host proteins have placed new emphasis on deciphering the impact of host cell biology on chlamydial development and virulence. Recent advances in the understanding of chlamydial pathogenesis are exemplified by work describing potential roles of (i) chlamydial Tarp in invasion, (ii) Inc proteins in modulation of vesicular interactions, and (iii) chlamydial proteins in disregulation of NF-kappaB signal transduction. Characterization of these chlamydial effector proteins promises to answer old questions and reveals previously unappreciated biology. The challenge will be to determine how these molecular mechanisms mesh together and collectively contribute to Chlamydia-mediated disease.

A protein secreted by the respiratory pathogen Chlamydia pneumoniae impairs IL-17 signalling via interaction with human Act1.

Chlamydia pneumoniae is a common respiratory pathogen that has been associated with a variety of chronic diseases including asthma and atherosclerosis. Chlamydiae are obligate intracellular parasites that primarily infect epithelial cells where they develop within a membrane-bound vacuole, termed an inclusion. Interactions between the microorganism and eukaryotic cell can be mediated by chlamydial proteins inserted into the inclusion membrane. We describe here a novel C. pneumoniae-specific inclusion membrane protein (Inc) CP0236, which contains domains exposed to the host cytoplasm. We demonstrate that, in a yeast two-hybrid screen, CP0236 interacts with the NFκB activator 1 (Act1) and this interaction was confirmed in HeLa 229 cells where ectopically expressed CP0236 was co-immunoprecipitated with endogenous Act1. Furthermore, we demonstrate that Act1 displays an altered distribution in the cytoplasm of HeLa cells infected with C. pneumoniae where it associates with the chlamydial inclusion membrane. This sequestration of Act1 by chlamydiae inhibited recruitment of the protein to the interleukin-17 (IL-17) receptor upon stimulation of C. pneumoniae-infected cells with IL-17A. Such inhibition of the IL-17 signalling pathway led to protection of Chlamydia-infected cells from NFκB activation in IL-17-stimulated cells. We describe here a unique strategy employed by C. pneumoniae to achieve inhibition of NFκB activation via interaction of CP0236 with mammalian Act1.

Degradation of Chlamydia pneumoniae by peripheral blood monocytic cells.

Chlamydia pneumoniae is a common human respiratory pathogen that has been associated with a variety of chronic diseases, including atherosclerosis. The role of this organism in the pathogenesis of atherosclerosis remains unknown. A key question is how C. pneumoniae is transferred from the site of primary infection to a developing atherosclerotic plaque. It has been suggested that circulating monocytes could be vehicles for dissemination of C. pneumoniae since the organism has been detected in peripheral blood monocytic cells (PBMCs). In this study we focused on survival of C. pneumoniae within PBMCs isolated from the blood of healthy human donors. We found that C. pneumoniae does not grow and multiply in cultured primary monocytes. In C. pneumoniae-infected monocyte-derived macrophages, growth of the organism was very limited, and the majority of the bacteria were eradicated. We also found that the destruction of C. pneumoniae within infected macrophages resulted in a gradual diminution of chlamydial antigens, although some of these antigens could be detected for days after the initial infection. The detected antigens present in infected monocytes and monocyte-derived macrophages represented neither chlamydial inclusions nor intact organisms. The use of {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]}-6-aminocaproyl-d-erythro-sphingosine as a vital stain for chlamydiae proved to be a sensitive method for identifying rare C. pneumoniae inclusions and was useful in the detection of even aberrant developmental forms.