Chlamydial Inclusions Research Articles

Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Chlamydia trachomatis is an obligate intracellular human pathogen responsible for the most prevalent sexually-transmitted infection in the world. For decades C. trachomatis has been considered an "energy parasite" that relies entirely on the uptake of ATP from the host cell. The genomic data suggest that C. trachomatis respiratory chain could produce a sodium gradient that may sustain the energetic demands required for its rapid multiplication. However, this mechanism awaits experimental confirmation. Moreover, the relationship of chlamydiae with the host cell, in particular its energy dependence, is not well understood. In this work, we are showing that C. trachomatis has an active respiratory metabolism that seems to be coupled to the sodium-dependent synthesis of ATP. Moreover, our results show that the inhibition of mitochondrial ATP synthesis at an early stage decreases the rate of infection and the chlamydial inclusion size. In contrast, the inhibition of the chlamydial respiratory chain at mid-stage of the infection cycle decreases the inclusion size but has no effect on infection rate. Remarkably, the addition of monensin, a Na+/H+ exchanger, completely halts the infection. Altogether, our data indicate that chlamydial development has a dynamic relationship with the mitochondrial metabolism of the host, in which the bacterium mostly depends on host ATP synthesis at an early stage, and at later stages it can sustain its own energy needs through the formation of a sodium gradient.

Making Contact: VAP Targeting by Intracellular Pathogens.

In naïve cells, the endoplasmic reticulum (ER) and the ER-resident VAP proteins are common components of sites of membrane contacts that mediate the non-vesicular transfer of lipids between organelles. There is increasing recognition that the hijacking of VAP by intracellular pathogens is a novel mechanism of host-pathogen interaction. Here, we summarize our recent findings showing that the Chlamydia inclusion membrane protein IncV tethers the ER to the inclusion membrane by binding to VAP via the molecular mimicry of two eukaryotic FFAT motifs. We extend the discussion to other microorganisms that have evolved similar mechanisms.

Inhibition of Wnt Signaling Pathways Impairs Chlamydia trachomatis Infection in Endometrial Epithelial Cells.

Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis.

Chlamydia pneumoniae inclusion membrane protein Cpn0147 interacts with host protein CREB3.

Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impacts. Like other chlamydial species, Chlamydia pneumoniae possesses a unique developmental cycle, the infectious elementary body gains access to the susceptible host cell, where it transforms into the replicative reticulate body. The cytoplasmic vacuole where Chlamydia pneumoniae replicates is called an inclusion, which is extensively modified by the insertion of chlamydial effectors known as inclusion membrane proteins (Incs). The C. pneumoniae-specific inclusion membrane protein (Inc) Cpn0147 contains domains that are predicted to be exposed to the host cytoplasm. To map host cell binding partners of Cpn0147, a yeast two-hybrid system was used to screen Cpn0147 against a HeLa cell cDNA library, which led to the finding that Cpn0147 interacted with the host cell protein cyclic adenosine monophosphate (cAMP)-responsive element (CRE)-binding protein (CREB3). The interaction was validated by co-immunoprecipitation of Cpn0147 with CREB3 from HeLa cells ectopically expressing both. Furthermore, Cpn0147 and CREB3 were co-localised in HeLa cells under confocal fluorescence microscopy. The above observations suggest that CREB3 may directly bind to the cytoplasmic domain of Cpn0147 to mediate the interactions of chlamydial inclusions with host cell endoplasmic reticulum.

Broad recruitment of mGBP family members to Chlamydia trachomatis inclusions.

Chlamydia, the most common sexually transmitted pathogen, is an exquisitely adapted Gram-negative obligate intracellular bacterium. Intracellular Chlamydia trachomatis replicate in a specialized vacuole, termed inclusion, which shields the bacterium from antimicrobial immunity of the host cells and acts as a signalling interface. Previously it was shown that members of the interferon induced guanylate binding protein (mGBP) family, in particular murine GBP1 and mGBP2, were found to accumulate at the bacterial inclusions, similar to previously published recruitment of GBPs to the parasitophorous vacuole of Toxoplasma gondii. Here, we provide a wide comparison of mGBPs roles within the host cell in the context of Chlamydia and Toxoplasma infection. By confocal microscopy on fixed and living infected cells we show localization of mGBP3, mGBP6, mGBP7, mGBP9, and mGBP10, in addition to mGBP1 and mGBP2, at chlamydia inclusions. In time lapse videos using GFP expressing Chlamydia we show rapid and transient dynamics of mGBP9 accumulation onto chlamydia inclusions. Taken together this study reveals a broad activation of mGBP recruitment towards Chlamydia trachomatis inclusions after infection and provides evidence for time limited action of mGBP9 at the chlamydia inclusion.

Acquisition of Rab11 and Rab11-Fip2—A novel strategy for Chlamydia pneumoniae early survival

The initial steps in chlamydial infection involve adhesion and internalization into host cells and, most importantly, modification of the nascent inclusion to establish the intracellular niche. Here, we show that Chlamydia pneumoniae enters host cells via EGFR-dependent endocytosis into an early endosome with a phosphatidylinositol 3-phosphate (PI3P) membrane identity. Immediately after entry, the early chlamydial inclusion acquires early endosomal Rab GTPases including Rab4, Rab5, Rab7, as well as the two recycling-specific Rabs Rab11 and Rab14. While Rab5, Rab11 and Rab14 are retained in the vesicular membrane, Rab4 and Rab7 soon disappear. Loss of Rab7 enables the C. pneumoniae inclusion to escape delivery to, and degradation in lysosomes. Loss of Rab4 and retention of Rab11/ Rab14 designates the inclusion as a slowly recycling endosome—that is protected from degradation. Furthermore, we show that the Rab11/ Rab14 adaptor protein Rab11-Fip2 (Fip2) is recruited to the nascent inclusion upon internalization and retained in the membrane throughout infection. siRNA knockdown of Fip2 demonstrated that the protein is essential for internalization and infection, and expression of various deletion variants revealed that Fip2 regulates the intracellular positioning of the inclusion. Additionally, we show that binding to Rab11 and Fip2 recruits the unconventional actin motor protein myosin Vb to the early inclusion and that together they regulate the relocation of the nascent inclusion from the cell periphery to the perinuclear region, its final destination. Here, we characterize for the first time inclusion identity and inclusion-associated proteins to delineate how C. pneumoniae establishes the intracellular niche essential for its survival.

Chlamydia Hijacks ARF GTPases To Coordinate Microtubule Posttranslational Modifications and Golgi Complex Positioning.

ABSTRACTThe intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion.

A novel role for Connexin 43 in IFN beta expression during Chlamydia trachomatis infection and cytosolic DNA-sensing

Abstract Background The DNA sensor cGAS is essential for IFNβ expression in cells infected with Chlamydia. cGAMP generated during DNA sensing can also transfer via intercellular gap-junction proteins, connexins CX43/45 leading to IFNβ expression in adjacent uninfected cells. Present study highlights a novel role for CX43 in IFNβ expression during DNA-sensing and during Chlamydia infection, independent of its gap junction function. Methods Total IFNβ transcripts were measured by qRT-PCR following CX43 or CX45 siRNA mediated knockdown (KD). Single cell RNA-ISH method view RNA™ was used to visualize IFNβ mRNA in infected HeLa cells. CX43 localization was studied using IF and confocal microscopy. Results CX43-KD in HeLa cells reduced IFNβ expression by &amp;gt;90% (p&amp;lt;0.001) compared to control or CX45 KD, as measured by qRT-PCR. CX43-KD also reduced IFNβ mRNA in dsDNA transfected cells (p&amp;lt;0.001) but not with poly IC RNA. RNA-ISH showed IFNβ mRNA transcripts in infected cells (&amp;gt;90%) and in adjacent uninfected (~10%) cells, but not in distal uninfected cells, suggesting cell contact is required for adjacent-cell effect. Surprisingly, RNA-ISH of CX43-KD cells showed &amp;gt;85% reduction in IFNβ transcripts (p&amp;lt;0.0001) in infected cells, in addition to the loss of transcripts in cells adjacent to infected cells. CX43 localized proximal to the chlamydial inclusion membrane. Co-localization of CX43 was also observed with transfected dsDNA, but not dsRNA. Conclusion CX43 depletion has been shown to increase autophagy in cells. Here we show that CX43 depletion decreases IFNβ induction during DNA sensing. Our results suggest that like STING, CX43 could be a key player in both DNA sensing and autophagy, but with opposing roles. (Funded by NIAID R01-AI067678 to UN).

Ca. Similichlamydia in Epitheliocystis Co-infection of Gilthead Seabream Gills: Unique Morphological Features of a Deep Branching Chlamydial Family.

The Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum constitutes a broad range of organisms with an intriguing array of ultrastructural morphologies, including intracellular membranes and compartments and their corresponding complex genomes encoding these forms. The phylum Chlamydiae are all obligate intracellular bacteria and, although much is already known of their genomes from various families and how these regulate the various morphological forms, we know remarkably little about what is likely the deepest rooting clade of this phylum, which has only been found to contain pathogens of marine and fresh water vertebrates. The disease they are associated with is called epitheliocystis; however, analyses of the causative agents is hindered by an inability to cultivate them for refined in vitro experimentation. For this reason, we have developed tools to analyse both the genomes and the ultrastructures of bacteria causing this disease, directly from infected tissues. Here we present structural data for a member of the family Ca. Similichlamydiaceae from this deep-rooted clade, which we have identified using molecular tools, in epitheliocystis lesions of gilthead seabream (Sparus aurata) in Greece. We present evidence that the chlamydial inclusions appear to develop in a perinuclear location, similar to other members of the phylum and that a chlamydial developmental cycle is present, with chlamydial forms similar to reticular bodies (RBs) and elementary bodies (EBs) detected. Division of the RBs appeared to follow a budding process, and larger RBs with multiple condensed nucleoids were detected using both transmission electron microscopy (TEM) and by focused-ion beam, scanning electron microscopy (FIB-SEM). As model hosts, fish offer many advantages for investigation, and we hope by these efforts to encourage others to explore the biology of fish pathogens from the PVC.

Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense.

Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.

A Co-infection Model System and the Use of Chimeric Proteins to Study Chlamydia Inclusion Proteins Interaction.

Chlamydia trachomatis is an obligate intracellular bacterium associated with trachoma and sexually transmitted diseases. During its intracellular developmental cycle, Chlamydia resides in a membrane bound compartment called the inclusion. A subset of Type III secreted effectors, the inclusion membrane proteins (Inc), are inserted into the inclusion membrane. Inc proteins are strategically positioned to promote inclusion interaction with host factors and organelles, a process required for bacterial replication, but little is known about Inc proteins function or host interacting partners. Moreover, it is unclear whether each Inc protein has a distinct function or if a subset of Inc proteins interacts with one another to perform their function. Here, we used IncD as a model to investigate Inc/Inc interaction in the context of Inc protein expression in C. trachomatis. We developed a co-infection model system to display different tagged Inc proteins on the surface of the same inclusion. We also designed chimeric Inc proteins to delineate domains important for interaction. We showed that IncD can self-interact and that the full-length protein is required for dimerization and/or oligomerization. Altogether our approach can be generalized to any Inc protein and will help to characterize the molecular mechanisms by which Chlamydia Inc proteins interact with themselves and/or host factors, eventually leading to a better understanding of C. trachomatis interaction with the mammalian host.

Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis

Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.

Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane.

Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H2O2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncATM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncATM-APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.

In vitro activity of a partially purified and characterized bark extract of Castanea sativa Mill. (ENC®) against Chlamydia spp

ABSTRACTCastanea sativa Mill (ENC®), containing tannins against 33 Chlamydia strains, was compared to SMAP-29 with inhibitory effect against C. trachomatis and C. pneumoniae. The ENC® activity against Chlamydia spp. was evaluated determining the lowest concentration to achieve more than half reduction of intact chlamydial inclusions versus controls. ENC® reduced all Chlamydia strains tested at 1 µg/mL, while SMAP-29 induced reductions of C. trachomatis and C. pneumoniae infectivity at 10 µg/mL. A great reduction of C. trachomatis, C. pneumoniae, and C. abortus infectivity was achieved with a 10 µg/mL ENC® concentration, whereas their infectivity was almost inhibited at 100 µg/mL ENC® concentration.

STUDIES ON CAUSES OF ABORTION IN MAGHRABIAN CAMELS

History of abortion, stillbirths and death of camel calves within 6 months of birth were appeared in the year of 2013 in Maghribian camels in a farm in Mersa Matrouh governorate. Whole blood and serum samples were collected from 34 camels ≥ 5 years and from 19 camels < 5 years. Blood films prepared for detection of blood parasites by Giemsa stain. Serum samples used for detection of Toxoplasma antibodies by slide Toxo-Latex agglutination test (LAT) and Chlamydophilla spp. antibodies were detected by complement fixation test (CFT). Fifteen internal organs (liver, heart, lung and spleen) from aborted calves and twenty vaginal swabs from she camels that aborted were collected for isolation of chlamydiae via inoculation in embryonated chicken eggs (ECE). Toxoplasma antibodies, Theileria, Anaplasma and mixed infection (Theileria and Anaplasma) were detected in 70.6%, 26.5%, 17.6% and 8.8% of camels ≥ 5 years, while they were detected in 42.1%, 26.3%, 10.5% and 5.3% of camels < 5 years respectively. Examined serum samples by CFT showed 27% were positive for C. psittaci antibodies. Chlamydial inclusion bodies were detected in 45% and 20% of vaginal swabs and internal organs respectively by chicken embryo inoculation. Camels were treated with coliprim, butalex, imizol and alamycin which repeated routinely every 6 months. Number of aborted fetuses and stillbirths were decreased in years 2014 and 2015, while the number of still alive camel calves were increased in the same 2 years. No camel calves were dead within 6 months of birth in year 2015.

Isolation of Chlamydia spp. from Ewes and Does in Iran.

Enzootic ovine abortion is caused by Chlamydia abortus and may result in abortion among small ruminants during the last 2-3 weeks of pregnancy. Enzootic abortion is diagnosed by isolation of the agent or detection of its nucleic acid in the products of abortion or vaginal excretions of freshly aborted females. Isolation of chlamydial agents in cell culture is the gold standard, so in the present study this method was employed. Twenty-eight vaginal and conjunctival swab samples were selected from ewes and does that had recently aborted. The samples were inoculated to McCoy cells. The inoculated cells were fixed, stained by Giemsa staining, and mounted on slides. Finally, the slides were observed by an optical microscope for the presence chlamydial inclusion bodies. Chlamydia was isolated from four conjunctival and three vaginal samples. All the negative cultures were passaged a further two times. Cell culture was identified as the most convenient method for the isolation of Chlamydia and remains essential to document the viability of the organism. Isolation of Chlamydia in the present study, highlights the importance of paying more attention to the bacterium as one of the main abortifacient pathogens along with other infectious causes of abortion.

Host Nectin-1 Promotes Chlamydial Infection in the Female Mouse Genital Tract, but Is Not Required for Infection in a Novel Male Murine Rectal Infection Model.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection.

Fast implementation of the Niblack binarization algorithm for microscope image segmentation

A fast way to implement the Niblack binarization algorithm is described. It uses not only the integral image for the local mean values calculation, but also the second order integral image for the local variance calculation. Following the proposed approach the time of segmentation has been significantly reduced providing the possibility of its use in practice. The generalization of integral image representation, called `k-order integral image' could be used for fast calculation of higher order local statistics. An example of algorithm for the segmentation of cells and Chlamydial inclusions on microscope images, containing the steps for color deconvolution and fast adaptive local binarization is presented.

Amphipathic β2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae

We demonstrate in the current work that small cationic antimicrobial β2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 μM, i.e. < 3.4 μg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the β2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these β2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic β2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen.

Inhibitory effects of the Chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia psittaci strain guinea pig inclusion conjunctivitis and Chlamydia trachomatis serovar E

Objective To evaluate inhibitory effects of the Chlamydiaphage phiCPG1 capsid protein Vp1 on Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC)and Chlamydia trachomatis (Ct)serovar E, and to provide new ideas for the treatment of Ct infection. Methods The Chlamydiaphage phiCPG1 capsid protein Vp1 was expressed in Escherichia coli BL21 transfected with the recombinant plasmid Vp1-pET30a (+), identified by Western blot analysis and purified by using dialysis bags. Bicinchonininc acid (BCA)assay was performed to determine the concentration of Vp1 protein. GPIC and Ct serovar E strains were both classified into 4 groups to be firstly incubated with Vp1 protein (Vp1 group), Tris-glycine solution (Tris group), S protein (S group)or Dulbecco's Modified Eagle Medium (DMEM, DMEM group)at room temperature for 3 hours, then were used to infect Hela cells followed by 72-hour (GPIC) or 48-hour (Ct serovar E)culture with the presence of Vp1 protein (Vp1 group), Tris-glycine solution (Tris group), S protein (S group)or DMEM (DMEM group). Subsequently, immunofluorescence staining was conducted to observe and count chlamydial inclusions. Results The number of GPIC inclusions was significantly different between the 4 groups after 72-hour culture (F= 476.632, P 0.05). Compared with the DMEM group, the Vp1 group showed a significant decrease of 80.2%±3.99% and 77.2%±1.79% in the number of GPIC and Ct serovar E inclusions respectively, with no significant difference in the inhibitory effect of Vp1 on GPIC versus Ct serovar E (t = 2.057, P > 0.05). Conclusion The phiCPG1 capsid protein Vp1 can obviously inhibit GPIC and Ct serovar E infections to a similar degree. Key words: Chlamydia trachomatis; Chlamydia muridarum; Capsid proteins; Phages; Protein Vp1