Type III Secretion Research Articles

Role of enteroaggregative E.coli induced activation of epidermal growth factor receptor in cultured human intestinal epithelial cell lines

Enteric pathogens exploit the versatility of cytoskeletal system for internalization into non-phagocytic cells, as a crucial step in their pathogenic life cycle. Enteropathogenic Escherichia coli(EPEC) andEnterohemorrhagic Escherichia coli (EHEC) were shown to form actin pedestals in host cells using type-III secretion system. Earlier, we reported that EAEC induced increase in intracellular calcium ions might have crucial role in F-actin rearrangements in INT-407 cells leading to its invasion. It was suggested that EGFR might contribute in Rck-mediated adherence and invasion of Salmonella in host cells. In the present study, we assessed the role of EAEC induced activated EGFR in human intestinal epithelial cell lines (INT-407 & HCT-15). The presence of activated EGFR was detected in membrane fractions ofeach cell line, infected with two different strains of EAEC (EAEC-T8 & EAEC-O42) separatelyfor 3h in presence and absence of Tyrphostin AG1478 (EGFR-inhibitor), by western immunoblotting using p-EGFR (Y1068) antibody. Adherence and invasion of EAEC-T8 to each cell line were checked in presence of Tyrphostin AG1478. Further, the effect of Tyrphostin AG1478 on cytoskeletal F-actin rearrangement of EAEC-T8 infected cells was assessed under confocal microscope following staining with TRITC-phalloidin. EAEC-T8 induced maximum increase in EGFR autophosphorylation at Y1068. Adherence and invasion of EAEC-T8 as well as this organism induced cytoskeletal F-actin polymerization were found to be inhibited in presence of Tyrphostin AG1478. Our study revealed that EAEC induced activated EGFR might play a major role in host cell adherence and cytoskeletal rearrangements leading to invasion of the organism in these cells.

Scaffolding Protein GspB/OutB Facilitates Assembly of the Dickeya dadantii Type 2 Secretion System by Anchoring the Outer Membrane Secretin Pore to the Inner Membrane and to the Peptidoglycan Cell Wall.

ABSTRACTThe phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal, and human bacterial pathogens. This multiprotein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in D. dadantii, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic homology region domain that interacts directly with the N-terminal secretin domain. Site-specific photo-cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB provides a scaffold for the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or the pilotin GspS.

Detection of proper Type 3 Secretion System Translocon Assembly using a split Luciferase Assay

Type 3 secretion system (T3SS) is used by many Gram‐negative bacteria to colonize the host and inject effector proteins into target cells. Inhibitors targeting T3SS are important to combat the appearance of multidrug‐resistant pathogens. In Pseudomonas aeruginosa, the T3SS secreted proteins PopB (SctE) and PopD (SctB) are required to inject effector proteins into the host cells. PopB and PopD associate with the target membrane and they are proposed to form a translocon pore by which effectors are translocated. This translocon is a potential target for inhibitors because these molecules do not need to overcome the intrinsic problems associated with cell membrane permeability and the presence of efflux pumps.In previous studies, we have reported that the PopD N‐terminus is exposed to host cell cytosol (J. Biol. Chem. 2018 293 8982–8993). We reasoned that the PopD N‐terminal exposure could be used as a reporter for properly assembled translocons. Using a split form of the small NanoLuc (NLuc) luciferase, we have developed an assay to detect the exposure of this N‐terminus to the host cytosol. HeLa cells producing the large truncated version of NLuc are used as targets. These cells are incubated with a P. aeruginosa ΔpopD PAK strain that produces a PopD variant fused with the missing portion of NLuc at the N‐terminus (Nluc10‐PopD). Assembly of a functional translocon is detected by the maturation of NLuc enzyme in the HeLa cell cytosol and the production of luminescence. Failure to assemble a functional translocon did not produce luminescence. The assay has a typical signal‐to‐background ratio (S/B) of 6 ‐ 10 and a Z factor above 0.5, constituting an excellent tool for the screening and detection of small molecule inhibitors of T3SS translocon assembly.

Role of ConE’s ATPase Motifs in Protein‐Protein Interactions within the Conjugation Machinery of Bacillus subtilis

Conjugation is the transfer of DNA from one bacterial cell to another. Conjugation is a major form of horizontal gene transfer, resulting in the spread of genes that play roles in antibiotic resistance, virulence, symbiosis and metabolism. ICEBs1 is an integrative and conjugative element of Bacillus subtilis. The conjugation machinery that transfers DNA is a Type 4 Secretion System (T4SS). The ICEBs1 T4SS includes the proteins ConB, ConC, ConD, ConE, ConG, ConQ, and CwlT. The focus of our work is the ConE peripheral membrane protein, which belongs to the VirB4 family of ATPases. We analyzed whether ConE interacts with any of the other ICEBs1 T4SS proteins using bacterial two hybrid (BACTH). We found that ConE interacts with itself, ConB, and ConQ. We used site‐directed mutagenesis of the conE gene to make alanine substitutions within five conserved residues within ATPase motifs of the ConE protein that have previously been shown to be critical for mating. We used BACTH to determine if these mutations affect ConE’s protein interactions. We discovered that interaction of ConE with itself or ConB is unaffected by mutations in any of the ATPase residues tested, but that the ConE‐ConQ interaction depends on two conserved residues within the ATPase motifs. Our research sheds new light on the role of conserved residues within the ATPase motifs of ConE.

Retrieving the in vivo Scopoletin Fluorescence Excitation Band Allows the Non-invasive Investigation of the Plant-Pathogen Early Events in Tobacco Leaves.

In this study, we developed and applied a new spectroscopic fluorescence method for the in vivo detection of the early events in the interaction between tobacco (Nicotiana tabacum L.) plants and pathogenic bacteria. The leaf disks were infiltrated with a bacterial suspension in sterile physiological solution (SPS), or with SPS alone as control. The virulent Pseudomonas syringae pv. tabaci strain ATCC 11528, its non-pathogenic ΔhrpA mutant, and the avirulent P. syringae pv. tomato strain DC3000 were used. At different post-infiltration time–points, the in vivo fluorescence spectra on leaf disks were acquired by a fiber bundle-spectrofluorimeter. The excitation spectra of the leaf blue emission at 460 nm, which is mainly due to the accumulation of coumarins following a bacterial infiltration, were processed by using a two-bands Gaussian fitting that enabled us to isolate the scopoletin (SCT) contribution. The pH-dependent fluorescence of SCT and scopolin (SCL), as determined by in vitro data and their intracellular localization, as determined by confocal microscopy, suggested the use of the longer wavelength excitation band at 385 nm of 460 nm emission (F385_460) to follow the metabolic evolution of SCT during the plant–bacteria interaction. It was found to be directly correlated (R2 = 0.84) to the leaf SCT content, but not to that of SCL, determined by HPLC analysis. The technique applied to the time-course monitoring of the bacteria–plant interaction clearly showed that the amount and the timing of SCT accumulation, estimated by F385_460, was correlated with the resistance to the pathogen. As expected, this host defense response was delayed after P. syringae pv. tabaci ATCC 11528 infiltration, in comparison to P. syringae pv. tomato DC3000. Furthermore, no significant increase of F385_460 (SCT) was observed when using the non-pathogenic ΔhrpA mutant of P. syringae pv. tabaci ATCC 11528, which lacks a functional Type Three Secretion System (TTSS). Our study showed the reliability of the developed fluorimetric method for a rapid and non-invasive monitoring of bacteria-induced first events related to the metabolite-based defense response in tobacco leaves. This technique could allow a fast selection of pathogen-resistant cultivars, as well as the on-site early diagnosis of tobacco plant diseases by using suitable fluorescence sensors.

Chromosome-encoded IpaH ubiquitin ligases indicate non-human enteroinvasive Escherichia

Until recently, Shigella and enteroinvasive Escherichia coli were thought to be primate-restricted pathogens. The base of their pathogenicity is the type 3 secretion system (T3SS) encoded by the pINV virulence plasmid, which facilitates host cell invasion and subsequent proliferation. A large family of T3SS effectors, E3 ubiquitin-ligases encoded by the ipaH genes, have a key role in the Shigella pathogenicity through the modulation of cellular ubiquitination that degrades host proteins. However, recent genomic studies identified ipaH genes in the genomes of Escherichia marmotae, a potential marmot pathogen, and an E. coli extracted from fecal samples of bovine calves, suggesting that non-human hosts may also be infected by these strains, potentially pathogenic to humans. We performed a comparative genomic study of the functional repertoires in the ipaH gene family in Shigella and enteroinvasive Escherichia from human and predicted non-human hosts. We found that fewer than half of Shigella genomes had a complete set of ipaH genes, with frequent gene losses and duplications that were not consistent with the species tree and nomenclature. Non-human host IpaH proteins had a diverse set of substrate-binding domains and, in contrast to the Shigella proteins, two variants of the NEL C-terminal domain. Inconsistencies between strains phylogeny and composition of effectors indicate horizontal gene transfer between E. coli adapted to different hosts. These results provide a framework for understanding of ipaH-mediated host-pathogens interactions and suggest a need for a genomic study of fecal samples from diseased animals.

Small Things Matter: The 11.6-kDa TraB Protein is Crucial for Antibiotic Resistance Transfer Among Enterococci.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci. In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely.

Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family

Multidrug-resistant bacteria pose serious problems in hospital-acquired infections (HAIs). Most antibiotic resistance genes are acquired via conjugative gene transfer, mediated by type 4 secretion systems (T4SS). Although most multidrug-resistant bacteria responsible for HAIs are of Gram-positive origin, with enterococci being major contributors, mostly Gram-negative T4SSs have been characterized. Here, we describe the structure and organization of PrgL, a core protein of the T4SS channel, encoded by the pCF10 plasmid from Enterococcus faecalis. The structure of PrgL displays similarity to VirB8 proteins of Gram-negative T4SSs. Invitro experiments show that the soluble domain alone is enough to drive both dimerization and dodecamerization, with a dimerization interface that differs from all other known VirB8-like proteins. Invivo experiments verify the importance of PrgL dimerization. Our findings provide insight into the molecular building blocks of Gram-positive T4SS, highlighting similarities but also unique features in PrgL compared to other VirB8-like proteins.

Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system.

Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for Dickeya virulence. Previous studies showed that the D. dadantii T2SS secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins possess a signal peptide and are first addressed to the periplasm before their recruitment by Out. T2SS-specific secretion signals remain unknown which prevents in silico identification of T2SS substrates. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced and encoding proteins with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

The propensity of selected Indian plant extracts for polyphenolics, antioxidant, and inhibition of Pseudomonas aeruginosa biofilms through type-3 secretion system

Detection of virulence genes and investigation of the immunogenicity of outer membrane proteins of Salmonella entericaMohamed A. El-Tayeb , Abdelnasser S. S. Ibrahim, Dina A. Maany , Yahya B. Elbadawi, Ali A. Al-Salamah

Enteroinvasive Escherichia coli O96:H19 is an Emergent Biofilm-Forming Pathogen.

Enteroinvasive Escherichia coli (EIEC) is a diarrheagenic E. coli pathotype carrying a virulence plasmid that encodes a type III secretion system (TTSS) directly implicated in bacterial cell invasion. Since 2012, EIEC serotype O96:H19 has been recognized in Europe, Colombia, and most recently Uruguay. In addition to the invasion phenotype, the strains isolated from Colombian children with moderate-to-severe gastroenteritis had a strong biofilm formation phenotype, and as a result, they are referred to as biofilm-forming enteroinvasive E. coli (BF-EIEC). The objective of this study was to characterize the biofilm formation phenotype of the BF-EIEC O96:H19 strain 52.1 isolated from a child with moderate-to-severe gastroenteritis in Colombia. Random mutagenesis using Tn5 transposons identified 100 mutants unable to form biofilm; 20 of those had mutations within the pgaABCD operon. Site-directed mutagenesis of pgaB and pgaC confirmed the importance of these genes in N-acetylglucosamine-mediated biofilm formation. Both biofilm formation and TTSS-mediated host cell invasion were associated with host cell damage on the basis of cytotoxic assays comparing the wild type, invasion gene mutants, and biofilm formation mutants. Multilocus sequence typing-based phylogenetic analysis showed that BF-EIEC strain 52.1 does not cluster with classic EIEC serotype strains. Instead, BF-EIEC strain 52.1 clusters with EIEC serotype O96:H19 strains described in Europe and Uruguay. In conclusion, BF-EIEC O96:H19, an emerging pathogen associated with moderate-to-severe acute gastroenteritis in children under 5 years of age in Colombia, invades cells and has a strong biofilm formation capability. Both phenotypes are independently associated with in vitro cell cytotoxicity, and they may explain, at least in part, the higher disease severity reported in Europe and Latin America. IMPORTANCE Enteroinvasive Escherichia coli (EIEC), a close relative of Shigella, is implicated in dysenteric diarrhea. EIEC pathogenicity involves cell invasion mediated by effector proteins delivered by a type III secretion system (TTSS) that disrupt the cell cytoskeleton. These proteins and the VirF global regulator are encoded by a large (>200 kb) invasion plasmid (pINV). This study reports an emergent EIEC possessing a cell invasion phenotype and a strong polysaccharide matrix-mediated biofilm formation phenotype. Both phenotypes contribute to host cell cytotoxicity in vitro and may contribute to the severe disease reported among children and adults in Europe and Latin America.

CrkII/Abl phosphorylation cascade is critical for NLRC4 inflammasome activity and is blocked by Pseudomonas aeruginosa ExoT

Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection.

Mechanisms Underlying Contact Lens-Related Keratitis Caused by Pseudomonas aeruginosa.

Infectious keratitis is a severe complication associated with contact lens (CL) wear, and can progress rapidly with suppurative infiltration, resulting in the loss of vision. Contact lens wearers with poor and improper care are susceptible to develop infectious keratitis. Gram-negative bacilli such as Pseudomonas aeruginosa, have an ability to form biofilms on CL cases and CLs. Moreover, P. aeruginosa has various virulence factors such as type III secretion system (TTSS) which is an important factor for pathogenicity in keratitis. The effector proteins of TTSS have been identified, namely ExoU, ExoS, ExoT, and ExoY. Pseudomonas aeruginosa strains with ExoU show resistance to disinfection. The strains isolated from CL-related keratitis have higher ExoU gene positivity. Expression of elastase and swarming motility of P. aeruginosa isolates significantly correlates with focus size of keratitis. In addition to education of lens care for the CL wearer, development of CL cleaning solutions targeting suppression of virulence factors are needed for prevention of CL-related keratitis in the future.

Evidence for common ancestry and microevolution of passerine-adapted Salmonella enterica serovar Typhimurium in the UK and USA.

The evolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) within passerines has resulted in pathoadaptation of this serovar to the avian host in Europe. Recently, we identified an S. Typhimurium lineage from passerines in North America. The emergence of passerine-adapted S. Typhimurium in Europe and North America raises questions regarding its evolutionary origin. Here, we demonstrated that the UK and US passerine-adapted S. Typhimurium shared a common ancestor from ca. 1838, and larids played a key role in the clonal expansion by disseminating the common ancestor between North America and Europe. Further, we identified virulence gene signatures common in the passerine- and larid-adapted S. Typhimurium, including conserved pseudogenes in fimbrial gene lpfD and Type 3 Secretion System (T3SS) effector gene steC. However, the UK and US passerine-adapted S. Typhimurium also possessed unique virulence gene signatures (i.e. pseudogenes in fimbrial gene fimC and T3SS effector genes sspH2, gogB, sseJ and sseK2), and the majority of them (38/47) lost a virulence plasmid pSLT that was present in the larid-adapted S. Typhimurium. These results provide evidence that passerine-adapted S. Typhimurium share a common ancestor with those from larids, and the divergence of passerine- and larid-adapted S. Typhimurium might be due to pseudogenization or loss of specific virulence genes.

Multi-parameter photon-by-photon hidden Markov modeling

Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may cause artifacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species, all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.

Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria

Contractile injection systems (CISs) mediate cell–cell interactions by phage tail-like structures, using two distinct modes of action: extracellular CISs are released into the medium, while type 6 secretion systems (T6SSs) are attached to the cytoplasmic membrane and function upon cell–cell contact. Here, we characterized a CIS in the multicellular cyanobacterium Anabaena, with features distinct from extracellular CISs and T6SSs. Cryo-electron tomography of focused ion beam-milled cells revealed that CISs were anchored in thylakoid membrane stacks, facing the cell periphery. Single particle cryo-electron microscopy showed that this unique in situ localization was mediated by extensions of tail fibre and baseplate components. On stress, cyanobacteria induced the formation of ghost cells, presenting thylakoid-anchored CISs to the environment. Functional assays suggest that these CISs may mediate ghost cell formation and/or interactions of ghost cells with other organisms. Collectively, these data provide a framework for understanding the evolutionary re-engineering of CISs and potential roles of these CISs in cyanobacterial programmed cell death.

Effectidor: an automated machine-learning-based web server for the prediction of type-III secretion system effectors.

Type-III secretion systems are utilized by many Gram-negative bacteria to inject type-3 effectors (T3Es) to eukaryotic cells. These effectors manipulate host processes for the benefit of the bacteria and thus promote disease. They can also function as host-specificity determinants through their recognition as avirulence proteins that elicit immune response. Identifying the full effector repertoire within a set of bacterial genomes is of great importance to develop appropriate treatments against the associated pathogens. We present Effectidor, a user-friendly web server that harnesses several machine-learning techniques to predict T3Es within bacterial genomes. We compared the performance of Effectidor to other available tools for the same task on three pathogenic bacteria. Effectidor outperformed these tools in terms of classification accuracy (area under the precision-recall curve above 0.98 in all cases). Effectidor is available at: https://effectidor.tau.ac.il, and the source code is available at: https://github.com/naamawagner/Effectidor. Supplementary data are available at Bioinformatics online.

The type 3 secretion effector IpgD promotes S. flexneri dissemination.

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.

Recent Advancements in Tracking Bacterial Effector Protein Translocation.

Bacteria-host interactions are characterized by the delivery of bacterial virulence factors, i.e., effectors, into host cells where they counteract host immunity and exploit host responses allowing bacterial survival and spreading. These effectors are translocated into host cells by means of dedicated secretion systems such as the type 3 secretion system (T3SS). A comprehensive understanding of effector translocation in a spatio-temporal manner is of critical importance to gain insights into an effector’s mode of action. Various approaches have been developed to understand timing and order of effector translocation, quantities of translocated effectors and their subcellular localization upon translocation into host cells. Recently, the existing toolset has been expanded by newly developed state-of-the art methods to monitor bacterial effector translocation and dynamics. In this review, we elaborate on reported methods and discuss recent advances and shortcomings in this area of tracking bacterial effector translocation.

Immunogenicity and Protective Capacity of a Virus-like Particle Vaccine against Chlamydia trachomatis Type 3 Secretion System Tip Protein, CT584.

An effective vaccine against Chlamydia trachomatis is urgently needed as infection rates continue to rise and C. trachomatis causes reproductive morbidity. An obligate intracellular pathogen, C. trachomatis employs a type 3 secretion system (T3SS) for host cell entry. The tip of the injectosome is composed of the protein CT584, which represents a potential target for neutralization with vaccine-induced antibody. Here, we investigate the immunogenicity and efficacy of a vaccine made of CT584 epitopes coupled to a bacteriophage virus-like particle (VLP), a novel platform for Chlamydia vaccines modeled on the success of HPV vaccines. Female mice were immunized intramuscularly, challenged transcervically with C. trachomatis, and assessed for systemic and local antibody responses and bacterial burden in the upper genital tract. Immunization resulted in a 3-log increase in epitope-specific IgG in serum and uterine homogenates and in the detection of epitope-specific IgG in uterine lavage at low levels. By contrast, sera from women infected with C. trachomatis and virgin controls had similarly low titers to CT584 epitopes, suggesting these epitopes are not systemically immunogenic during natural infection but can be rendered immunogenic by the VLP platform. C. trachomatis burden in the upper genital tract of mice varied after active immunization, yet passive protection was achieved when immune sera were pre-incubated with C. trachomatis prior to inoculation into the genital tract. These data demonstrate the potential for antibody against the T3SS to contribute to protection against C. trachomatis and the value of VLPs as a novel platform for C. trachomatis vaccines.