The Chlamydia trachomatis type III-secreted effector protein CteG induces centrosome amplification through interactions with centrin-2.

The centrosome is the main microtubule organizing center of the cell and is crucial for mitotic spindle assembly, chromosome segregation, and cell division. Centrosome duplication is tightly controlled, yet several pathogens, most notably oncogenic viruses, perturb this process leading to increased centrosome numbers. Infection by the obligate intracellular bacterium Chlamydia trachomatis (C.t.) correlates with blocked cytokinesis, supernumerary centrosomes, and multipolar spindles; however, the mechanisms behind how C.t. induces these cellular abnormalities remain largely unknown. Here we show that the secreted effector protein, CteG, binds to centrin-2 (CETN2), a key structural component of centrosomes and regulator of centriole duplication. Our data indicate that both CteG and CETN2 are necessary for infection-induced centrosome amplification, in a manner that requires the C-terminus of CteG. Strikingly, CteG is important for in vivo infection and growth in primary cervical cells but is dispensable for growth in immortalized cells, highlighting the importance of this effector protein to chlamydial infection. These findings begin to provide mechanistic insight into how C.t. induces cellular abnormalities during infection, but also indicate that obligate intracellular bacteria may contribute to cellular transformation events. Centrosome amplification mediated by CteG-CETN2 interactions may explain why chlamydial infection leads to an increased risk of cervical or ovarian cancer.

In Search of a Mechanistic Link between Chlamydia trachomatis-Induced Cellular Pathophysiology and Oncogenesis.

Centrosome duplication and cell cycle progression are essential cellular processes that must be tightly controlled to ensure cellular integrity. Despite their complex regulatory mechanisms, microbial pathogens have evolved sophisticated strategies to co-opt these processes to promote infection. While misregulation of these processes can greatly benefit the pathogen, the consequences to the host cell can be devastating. During infection, the obligate intracellular pathogen Chlamydia trachomatis induces gross cellular abnormalities, including supernumerary centrosomes, multipolar spindles, and defects in cytokinesis. While these observations were made over 15 years ago, identification of the bacterial factors responsible has been elusive due to the genetic intractability of Chlamydia. Recent advances in techniques of genetic manipulation now allows for the direct linking of bacterial virulence factors to manipulation of centrosome duplication and cell cycle progression. In this review, we discuss the impact, both immediate and downstream, of C. trachomatis infection on the host cell cycle regulatory apparatus and centrosome replication. We highlight links between C. trachomatis infection and cervical and ovarian cancers and speculate whether perturbations of the cell cycle and centrosome are sufficient to initiate cellular transformation. We also explore the biological mechanisms employed by Inc proteins and other secreted effector proteins implicated in the perturbation of these host cell pathways. Future work is needed to better understand the nuances of each effector's mechanism and their collective impact on Chlamydia's ability to induce host cellular abnormalities.

Identification and Preliminary Characterization of Novel Type III Secreted Effector Proteins in Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a host-derived vacuole termed the inclusion. Central to pathogenesis is a type III secretion system that translocates effector proteins into the host cell, which are predicted to play major roles in host cell invasion, nutrient acquisition, and immune evasion. However, until recently, the genetic intractability of C. trachomatis hindered identification and characterization of these important virulence factors. Here, we sought to expand the repertoire of identified effector proteins and confirm they are secreted during C. trachomatis infection. Utilizing bioinformatics, we identified 18 candidate substrates that had not been previously assessed for secretion, of which we show four to be secreted, using Yersinia pseudotuberculosis as a surrogate host. Using adenylate cyclase (CyaA), BlaM, and glycogen synthase kinase (GSK) secretion assays, we identified nine novel substrates that were secreted in at least one assay. Interestingly, only three of the substrates, shown to be translocated by C. trachomatis, were similarly secreted by Y. pseudotuberculosis. Using large-scale screens to determine subcellular localization and identify effectors that perturb crucial host cell processes, we identified one novel substrate, CT392, that is toxic when heterologously expressed in Saccharomyces cerevisiae. Toxicity required both the N- and C-terminal regions of the protein. Additionally, we show that these newly described substrates traffic to distinct host cell compartments, including vesicles and the cytoplasm. Collectively, our study expands the known repertoire of C. trachomatis secreted factors and highlights the importance of testing for secretion in the native host using multiple secretion assays when possible.

Functional Characterization of Non-Ankyrin Repeat Domains of Orientia tsutsugamushi Ank Effectors Reveals Their Importance for Molecular Pathogenesis.

Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium, causes scrub typhus, and has one of the largest known armamentariums of ankyrin repeat-containing effectors (Anks). Most have a C-terminal F-box presumed to interact with the SCF ubiquitin ligase complex primarily based on their ability to bind overexpressed Skp1. Whether all F-box-containing Anks bind endogenous SCF components and the F-box residues essential for such interactions has gone unexplored. Many O. tsutsugamushi Ank F-boxes occur as part of a PRANC (pox protein repeats of ankyrin-C-terminal) domain. Roles of the non-F-box portion of the PRANC and intervening sequence region (ISR) that links the ankyrin repeat and F-box/PRANC domains are unknown. The functional relevance of these effectors' non-ankyrin repeat domains was investigated. The F-box was necessary for Flag-tagged versions of most F-box-containing Anks to precipitate endogenous Skp1, Cul1, and/or Rbx1, while the ISR and PRANC were dispensable. Ank toxicity in yeast was predominantly F-box dependent. Interrogations of Ank1, Ank5, and Ank6 established that L1, P2, E4, I9, and D17 of the F-box consensus are key for binding native SCF components and for Ank1 and Ank6 to inhibit NF-κB. The ISR is also essential for Ank1 and Ank6 to impair NF-κB. Ectopically expressed Ank1 and Ank6 lacking the ISR or having a mutagenized F-box incapable of binding SCF components performed as dominant-negative inhibitors to block O. tsutsugamushi NF-κB modulation. This study advances knowledge of O. tsutsugamushi Ank functional domains and offers an approach for validating their roles in infection.

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion.

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.

A Functional Core of IncA Is Required for Chlamydia trachomatis Inclusion Fusion.

UNLABELLED: Chlamydia trachomatis is an obligate intracellular pathogen that is the etiological agent of a variety of human diseases, including blinding trachoma and sexually transmitted infections. Chlamydiae replicate within a membrane-bound compartment, termed an inclusion, which they extensively modify by the insertion of type III secreted proteins called Inc proteins. IncA is an inclusion membrane protein that encodes two coiled-coil domains that are homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) motifs. Recent biochemical evidence suggests that a functional core, composed of SNARE-like domain 1 (SLD-1) and part of SNARE-like domain 2 (SLD-2), is required for the characteristic homotypic fusion of C. trachomatis inclusions in multiply infected cells. To verify the importance of IncA in homotypic fusion in Chlamydia, we generated an incA::bla mutant. Insertional inactivation of incA resulted in the formation of nonfusogenic inclusions, a phenotype that was completely rescued by complementation with full-length IncA. Rescue of homotypic inclusion fusion was dependent on the presence of the functional core consisting of SLD-1 and part of SLD-2. Collectively, these results confirm in vitro membrane fusion assays identifying functional domains of IncA and expand the genetic tools available for identification of chlamydia with a method for complementation of site-specific mutants. IMPORTANCE: Chlamydia trachomatis replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusions are nonfusogenic with vesicles in the endocytic pathway but, in multiply infected cells, fuse with each other to form a single large inclusion. This homotypic fusion is dependent upon the presence of a chlamydial inclusion membrane-localized protein, IncA. Specificity of membrane fusion in eukaryotic cells is regulated by SNARE (soluble N-ethylmaleimide sensitive factor attachment receptor) proteins on the cytosolic face of vesicles and target membranes. IncA contains two SNARE-like domains. Newly developed genetic tools for the complementation of targeted mutants in C. trachomatis are used to confirm the minimal requirement of SNARE-like motifs necessary to promote the homotypic fusion of inclusions.

The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

Expression and localization of predicted inclusion membrane proteins in Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a membrane-bound vacuole termed the inclusion. Early in the infection cycle, the pathogen extensively modifies the inclusion membrane through incorporation of numerous type III secreted effector proteins, called inclusion membrane proteins (Incs). These proteins are characterized by a bilobed hydrophobic domain of 40 amino acids. The presence of this domain has been used to predict up to 59 putative Incs for C. trachomatis; however, localization to the inclusion membrane with specific antibodies has been demonstrated for only about half of them. Here, we employed recently developed genetic tools to verify the localization of predicted Incs that had not been previously localized to the inclusion membrane. Expression of epitope-tagged putative Incs identified 10 that were previously unverified as inclusion membrane localized and thus authentic Incs. One novel Inc and 3 previously described Incs were localized to inclusion membrane microdomains, as evidenced by colocalization with phosphorylated Src (p-Src). Several predicted Incs did not localize to the inclusion membrane but instead remained associated with the bacteria. Using Yersinia as a surrogate host, we demonstrated that many of these are not secreted via type III secretion, further suggesting they may not be true Incs. Collectively, our results highlight the utility of genetic tools for demonstrating secretion from chlamydia. Further mechanistic studies aimed at elucidating effector function will advance our understanding of how the pathogen maintains its unique intracellular niche and mediates interactions with the host.

Global mapping of the Chlamydia trachomatis conventional secreted effector - host interactome reveals CebN interacts with nucleoporins and Rae1 to impede STAT1 nuclear translocation.

Chlamydia trachomatis (C.t.), the leading cause of bacterial sexually transmitted infections, employs a type III secretion system (T3SS) to translocate two classes of effectors, inclusion membrane proteins and conventional T3SS (cT3SS) effectors, into the host cell to counter host defense mechanisms. Here we employed three assays to directly evaluate secretion during infection, validating secretion for 23 cT3SS effectors. As bioinformatic analyses have been largely unrevealing, we conducted affinity purification-mass spectrometry to identify host targets and gain insights into the functions of these effectors, identifying high confidence interacting partners for 21 cT3SS effectors. We demonstrate that CebN localizes to the nuclear envelope in infected and bystander cells where it interacts with multiple nucleoporins and Rae1, blocking STAT1 nuclear import following IFN-γ stimulation. By building a cT3SS effector-host interactome, we have identified novel pathways that are targeted during bacterial infection and have begun to address how C.t. effectors combat cell autonomous immunity.

Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation.

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 ß-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii.

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based ß-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.

Indole production promotes Escherichia coli mixed-culture growth with Pseudomonas aeruginosa by inhibiting quorum signaling.

Indole production by Escherichia coli, discovered in the early 20th century, has been used as a diagnostic marker for distinguishing E. coli from other enteric bacteria. By using transcriptional profiling and competition studies with defined mutants, we show that cyclic AMP (cAMP)-regulated indole formation is a major factor that enables E. coli growth in mixed biofilm and planktonic populations with Pseudomonas aeruginosa. Mutants deficient in cAMP production (cyaA) or the cAMP receptor gene (crp), as well as indole production (tnaA), were not competitive in coculture with P. aeruginosa but could be restored to wild-type competitiveness by supplementation with a physiologically relevant indole concentration. E. coli sdiA mutants, which lacked the receptor for both indole and N-acyl-homoserine lactones (AHLs), showed no change in competitive fitness, suggesting that indole acted directly on P. aeruginosa. An E. coli tnaA mutant strain regained wild-type competiveness if grown with P. aeruginosa AHL synthase (rhlI and rhlI lasI) mutants. In contrast to the wild type, P. aeruginosa AHL synthase mutants were unable to degrade indole. Indole produced during mixed-culture growth inhibited pyocyanin production and other AHL-regulated virulence factors in P. aeruginosa. Mixed-culture growth with P. aeruginosa stimulated indole formation in E. coli cpdA, which is unable to regulate cAMP levels, suggesting the potential for mixed-culture gene activation via cAMP. These findings illustrate how indole, an early described feature of E. coli central metabolism, can play a significant role in mixed-culture survival by inhibiting quorum-regulated competition factors in P. aeruginosa.

Got mutants? How advances in chlamydial genetics have furthered the study of effector proteins.

Chlamydia trachomatis is the leading cause of infectious blindness and a sexually transmitted infection. All chlamydiae are obligate intracellular bacteria that replicate within a membrane-bound vacuole termed the inclusion. From the confines of the inclusion, the bacteria must interact with many host organelles to acquire key nutrients necessary for replication, all while promoting host cell viability and subverting host defense mechanisms. To achieve these feats, C. trachomatis delivers an arsenal of virulence factors into the eukaryotic cell via a type 3 secretion system (T3SS) that facilitates invasion, manipulation of host vesicular trafficking, subversion of host defense mechanisms and promotes bacteria egress at the conclusion of the developmental cycle. A subset of these proteins intercalate into the inclusion and are thus referred to as inclusion membrane proteins. Whereas others, referred to as conventional T3SS effectors, are released into the host cell where they localize to various eukaryotic organelles or remain in the cytosol. Here, we discuss the functions of T3SS effector proteins with a focus on how advances in chlamydial genetics have facilitated the identification and molecular characterization of these important factors.

Orientia tsutsugamushi Nucleomodulin Ank13 Exploits the RaDAR Nuclear Import Pathway To Modulate Host Cell Transcription.

Orientia tsutsugamushi is the etiologic agent of scrub typhus, the deadliest of all diseases caused by obligate intracellular bacteria. Nucleomodulins, bacterial effectors that dysregulate eukaryotic transcription, are being increasingly recognized as key virulence factors. How they translocate into the nucleus and their functionally essential domains are poorly defined. We demonstrate that Ank13, an O. tsutsugamushi effector conserved among clinical isolates and expressed during infection, localizes to the nucleus in an importin ß1-independent manner. Rather, Ank13 nucleotropism requires an isoleucine at the thirteenth position of its fourth ankyrin repeat, consistent with utilization of eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import. RNA-seq analyses of cells expressing green fluorescent protein (GFP)-tagged Ank13, nucleotropism-deficient Ank13I127R, or Ank13ΔF-box, which lacks the F-box domain essential for interacting with SCF ubiquitin ligase, revealed Ank13 to be a nucleomodulin that predominantly downregulates transcription of more than 2,000 genes. Its ability to do so involves its nucleotropism and F-box in synergistic and mutually exclusive manners. Ank13 also acts in the cytoplasm to dysregulate smaller cohorts of genes. The effector's toxicity in yeast heavily depends on its F-box and less so on its nucleotropism. Genes negatively regulated by Ank13 include those involved in the inflammatory response, transcriptional control, and epigenetics. Importantly, the majority of genes that GFP-Ank13 most strongly downregulates are quiescent or repressed in O. tsutsugamushi-infected cells when Ank13 expression is strongest. Ank13 is the first nucleomodulin identified to coopt RaDAR and a multifaceted effector that functions in the nucleus and cytoplasm via F-box-dependent and -independent mechanisms to globally reprogram host cell transcription. IMPORTANCE Nucleomodulins are recently defined effectors used by diverse intracellular bacteria to manipulate eukaryotic gene expression and convert host cells into hospitable niches. How nucleomodulins enter the nucleus, their functional domains, and the genes that they modulate are incompletely characterized. Orientia tsutsugamushi is an intracellular bacterial pathogen that causes scrub typhus, which can be fatal. O. tsutsugamushi Ank13 is the first example of a microbial protein that coopts eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import. It dysregulates expression of a multitude of host genes with those involved in transcriptional control and the inflammatory response being among the most prominent. Ank13 does so via mechanisms that are dependent and independent of both its nucleotropism and eukaryotic-like F-box domain that interfaces with ubiquitin ligase machinery. Nearly all the genes most strongly downregulated by ectopically expressed Ank13 are repressed in O. tsutsugamushi-infected cells, implicating its importance for intracellular colonization and scrub typhus molecular pathogenesis.

A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response.

Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm(-2)) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3 %) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility).

Quorum sensing: implications on rhamnolipid biosurfactant production.

Quorum sensing (QS) has received significant attention in the past few decades. QS describes population density dependent cell to cell communication in bacteria using diffusible signal molecules. These signal molecules produced by bacterial cells, regulate various physiological processes important for social behavior and pathogenesis. One such process regulated by quorum sensing molecules is the production of a biosurfactant, rhamnolipid. Rhamnolipids are important microbially derived surface active agents produced by Pseudomonas spp. under the control of two interrelated quorum sensing systems; namely las and rhl. Rhamnolipids possess antibacterial, antifungal and antiviral properties. They are important in motility, cell to cell interactions, cellular differentiation and formation of water channels that are characteristics of Pseudomonas biofilms. Rhamnolipids have biotechnological applications in the uptake of hydrophobic substrates, bioremediation of contaminated soils and polluted waters. Rhamnolipid biosurfactants are biodegradable as compared to chemical surfactants and hence are more preferred in environmental applications. In this review, we examine the biochemical and genetic mechanism of rhamnolipid production by P. aeruginosa and propose the application of QS signal molecules in enhancing the rhamnolipid production.

Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci.

The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions.IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.

Mutagenesis of Chlamydia trachomatis Using TargeTron.

Chlamydia trachomatis is an important human pathogen that prior to 2011 was largely intractable to genetic manipulation. Here we describe the application of a group II intron, referred to as TargeTron, for site-specific insertional inactivation of target genetic loci in the obligate, intracellular bacteria C. trachomatis. In this chapter, we outline the methods for intron retargeting, chlamydia transformation, and mutant verification. We also outline a method for complementation of TargeTron mutants. Furthermore, we discuss potential pitfalls and alternative strategies for generating mutants with TargeTron technology.

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Propagation and Purification of Chlamydia trachomatis Serovar L2 Transformants and Mutants.

Chlamydia trachomatis (C.t.) is an obligate intracellular pathogen that cannot be cultured axenically and must be propagated within eukaryotic host cells. There are at least 15 distinct chlamydial serovariants that belong to 2 major biovars commonly referred to as trachoma and lymphogranuloma venereum (LGV). The invasive chlamydia LGV serovar L2 is the most widely used experimental model for studying C.t. biology and infection and is the only strain with reliable genetic tools available. New techniques to genetically manipulate C.t. L2 have provided opportunities to make mutants using TargeTron and allelic exchange as well as strains overexpressing epitope-tagged proteins, in turn necessitating the regular purification of transformant and mutant clones. Purification of C.t. is a labor-intensive exercise and one of the most common reagents classically used in the purification process, Renografin, is no longer commercially available. A similar formulation of diatrizoate meglumine called Gastrografin is readily available and we as well as others have had great success using this in place of Renografin for chlamydial purifications. Here, we provide a detailed general protocol for infection, propagation, purification, and titering of Chlamydia trachomatis serovar L2 with additional notes specifically pertaining to mutants or recombinant DNA carrying clones.