Hijacking host cell vesicular transport: New insights into the nutrient acquisition mechanism of Chlamydia.

Chlamydia infection is an important cause of public health diseases, and no effective vaccine is currently available. Owing to its unique intracellular lifestyle, Chlamydia requires a variety of nutrients and substrates from host cells, particularly sphingomyelin, cholesterol, iron, amino acids, and the mannose-6-phosphate receptor, which are essential for inclusion development. Here, we summarize the recent advances in Chlamydia nutrient acquisition mechanism by hijacking host cell vesicular transport, which plays an important role in chlamydial growth and development. Chlamydia obtains the components necessary to complete its intracellular developmental cycle by recruiting Rab proteins (major vesicular trafficking regulators) and Rab effector proteins to the inclusion, interfering with Rab-mediated multivesicular trafficking, reorienting the nutrition of host cells, and reconstructing the intracellular niche environment. Consequently, exploring the role of vesicular transport in nutrient acquisition offers a novel perspective on new approaches for preventing and treating Chlamydia infection.

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

A single chlamydial protein reshapes the plasma membrane and serves as recruiting platform for central endocytic effector proteins.

Uptake of obligate intracellular bacterial pathogens into mammalian epithelial cells is critically dependent on modulation of the host's endocytic machinery. It is an open question how the invading pathogens generate a membrane-bound vesicle appropriate to their size. This requires extensive deformation of the host plasma membrane itself by pathogen-derived membrane-binding proteins, accompanied by substantial F-actin-based forces to further expand and finally pinch off the vesicle. Here we show that upon adhesion to the host cell, the human pathogenic bacterium Chlamydia pneumoniae secretes the scaffolding effector protein CPn0677, which binds to the inner leaflet of the invaginating host's PM, induces inwardly directed, negative membrane curvature, and forms a recruiting platform for the membrane-deforming BAR-domain containing proteins Pacsin and SNX9. In addition, while bound to the membrane, CPn0677 recruits monomeric G-actin, and its C-terminal region binds and activates N-WASP, which initiates branching actin polymerization via the Arp2/3 complex. Together, these membrane-bound processes enable the developing endocytic vesicle to engulf the infectious elementary body, while the associated actin network generates the forces required to reshape and detach the nascent vesicle from the PM. Thus, Cpn0677 (now renamed SemD) acts as recruiting platform for central components of the endocytic machinery during uptake of chlamydia.

Identification and Structural Modeling of the RNA Polymerase Omega Subunits in Chlamydiae and Other Obligate Intracellular Bacteria.

Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing σ factor. The core enzyme comprises two α subunits, one ß subunit, one ß' subunit, and one ω subunit. The ω subunit plays critical roles in the assembly of the core enzyme and other cellular functions, including the regulation of bacterial growth, the stress response, and biofilm formation. However, the identity of an ω subunit for the obligate intracellular bacterium Chlamydia has not previously been determined. Here, we report the identification of the hypothetical protein CTL0286 as the probable chlamydial ω subunit based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. Our findings indicate that CTL0286 functions as the missing ω subunit of chlamydial RNAP. Our extended analysis also indicates that all obligate intracellular bacteria have ω orthologs. IMPORTANCE Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase ω subunit, and it has been hypothesized that the chlamydial RNA polymerase ω subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle. Here, we report the identification of the chlamydial RNA polymerase ω subunit, based on conserved sequence, conserved synteny, AlphaFold-predicted conserved three-dimensional structure, and AlfaFold-Multimer-predicted conserved interactions. Our identification of the previously elusive chlamydial RNA polymerase ω subunit sets the stage for investigation of its roles in regulation of gene expression during chlamydial growth, development, and stress responses, and sets the stage for preparation and study of the intact chlamydial RNA polymerase and its interactions with inhibitors.

Phosphoregulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion membrane contact sites.

Membrane contact sites (MCS) are crucial for nonvesicular trafficking-based interorganelle communication. Endoplasmic reticulum (ER)-organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT motif-containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV-VAP tethering complex and the formation of ER-Inclusion MCS. Via a C-terminal region containing three CK2 phosphorylation motifs, IncV recruits CK2 to the inclusion leading to IncV hyperphosphorylation of the noncanonical FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.

Differential Effects of Small Molecule Inhibitors on the Intracellular Chlamydia Infection.

Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis. We compared published and novel inhibitors and showed that they can differentially alter inclusion size, chlamydial number and infectious EB production, and that these effects can vary over the course of the intracellular infection. Our results provide the justification for analysis with multiple assays performed either at the end of the infection or over a time course. We also show that this approach has the potential to identify the particular step in the developmental cycle that is impacted by the inhibitor. We furthermore propose that the magnitude of inhibitor-induced progeny defects are best quantified and compared by using a new value called maximal progeny production (Progenymax). As a demonstration of the validity of this systematic approach, we applied it to inhibitors of Akt and AMPK, which are host kinases involved in lipid synthesis and cholesterol trafficking pathways. Both inhibitors reduced EB production, but Akt disruption primarily decreased RB-to-EB conversion while AMPK inhibition paradoxically enhanced RB replication. IMPORTANCE Chlamydia is the most reported cause of bacterial, sexually transmitted infection in the United States. This bacterium infects human cells and reproduces within a cytoplasmic inclusion via an unusual developmental cycle involving two specialized chlamydial forms. Small molecule compounds have been reported to negatively affect the inclusion as well as chlamydial replication and infectious progeny production, but we showed that these effects can be discordant and vary over the course of the 48- to 72-hour long intracellular infection. We propose approaches to analyze these nonuniform effects, including measurements at the end of the intracellular infection, and more detailed analysis with multiple assays performed over the course of the developmental cycle. We then applied this approach to investigate and compare the anti-chlamydial effects of two inhibitors that alter host lipid synthesis and cholesterol trafficking.

[Interleukins in Chlamydia infection: An update].

Chlamydia infection remains a problem for the world. Hundreds of millions of people suffer from Chlamydia-related diseases, but the specific infection mechanism is still unclear. Studies have shown that interleukins is involved in the innate immune process after Chlamydia infection. In the early stage of infection, Chlamydia, through receptor-mediated multiple signal transduction pathways, such as mitogen-activated protein kinase (MAPK), signal transducers and activators of transcription 3 (STAT3), myeloid differentiation factor 88 (MyD88) pathways, promotes the body to release a variety of pro-inflammatory interleukins, such as interleukin 1ß (IL-1ß), IL-6, IL-8 and IL-17, which inhibits Chlamydia replication and accelerates the clearance of Chlamydia. With the continuous secretion of pro-inflammatory interleukins, the body regulates immune cells to secrete anti-inflammatory interleukins, such as IL-4, IL-10 and IL-22, to reduce inflammatory reaction and tissue damage. We summarized the role of interleukins in Chlamydia infection in order to provide reference for clinical treatment.

Inhibition of Chlamydial Infection by CRISPR/Cas9-SAM Mediated Enhancement of Human Peptidoglycan Recognition Proteins Gene Expression in HeLa Cells.

The global problem of emerging resistance of microorganisms to antibiotics makes the search for new natural substances with antibacterial properties relevant. Such substances include peptidoglycan recognition proteins (PGLYRP), which are the components of the innate immunity of many organisms, including humans. These proteins have a unique mechanism of action that allows them to evade the resistance of bacteria to them, as well as to be active against both Gram-positive and Gram-negative bacteria. However, the use of antimicrobial recombinant proteins is not always advisable due to the complexity of local delivery of the proteins and their stability; in this regard it seems appropriate to activate the components of the innate immunity. The aim of this study was to increase the expression level of native peptidoglycan recognition protein genes in HeLa cells using genome-editing technology with synergistic activation mediators (CRISPR/Cas9-SAM) and evaluate antichlamydial effect of PGLYRP. We demonstrated activation of the chlamydial two-component gene system (ctcB-ctcC), which played a key role in the mechanism of action of the peptidoglycan recognition proteins. We generated the HeLa cell line transduced with lentiviruses encoding CRISPR/Cas9-SAM activation system with increased PGLYRP gene expression. It was shown that activation of the own peptidoglycan recognition proteins gene expression in the cell line caused inhibition of the chlamydial infection development. The proposed approach makes it possible to use the capabilities of innate immunity to combat infectious diseases caused by Gram-positive and Gram-negative bacteria.

Molecular causes of an evolutionary shift along the parasitism-mutualism continuum in a bacterial symbiont.

Symbiosis with microbes is a ubiquitous phenomenon with a massive impact on all living organisms, shaping the world around us today. Theoretical and experimental studies show that vertical transmission of symbionts leads to the evolution of mutualistic traits, whereas horizontal transmission facilitates the emergence of parasitic features. However, these studies focused on phenotypic data, and we know little about underlying molecular changes at the genomic level. Here, we combined an experimental evolution approach with infection assays, genome resequencing, and global gene expression analysis to study the effect of transmission mode on an obligate intracellular bacterial symbiont. We show that a dramatic shift in the frequency of genetic variants, coupled with major changes in gene expression, allow the symbiont to alter its position in the parasitism-mutualism continuum depending on the mode of between-host transmission. We found that increased parasitism in horizontally transmitted chlamydiae residing in amoebae was a result of processes occurring at the infectious stage of the symbiont's developmental cycle. Specifically, genes involved in energy production required for extracellular survival and the type III secretion system-the symbiont's primary virulence mechanism-were significantly up-regulated. Our results identify the genomic and transcriptional dynamics sufficient to favor parasitic or mutualistic strategies.

Chlamydial MreB Directs Cell Division and Peptidoglycan Synthesis in Escherichia coli in the Absence of FtsZ Activity.

Cell division is the ultimate process for the propagation of bacteria, and FtsZ is an essential protein used by nearly all bacteria for this function. Chlamydiae belong to a small group of bacteria that lack the universal cell division protein FtsZ but still divide by binary fission. Chlamydial MreB is a member of the shape-determining MreB/Mbl family of proteins responsible for rod shape morphology in Escherichia coliChlamydia also encodes a homolog of RodZ, an MreB assembly cytoskeletal protein that links MreB to cell wall synthesis proteins. We hypothesized that MreB directs cell division in Chlamydia and that chlamydial MreB could replace FtsZ function for cell division in E. coli Overexpression of chlamydial mreB-rodZ in E. coli induced prominent morphological changes with production of large swollen or oval bacteria, eventually resulting in bacterial lysis. Low-level expression of chlamydial mreB-rodZ restored viability of a lethal ΔmreB mutation in E. coli, although the bacteria lost their typical rod shape and grew as rounded cells. When FtsZ activity was inhibited by overexpression of SulA in the ΔmreB mutant of E. coli complemented with chlamydial mreB-rodZ, spherical E. coli grew and divided. Localization studies using a fluorescent fusion chlamydial MreB protein indicated that chlamydial RodZ directs chlamydial MreB to the E. coli division septum. These results demonstrate that chlamydial MreB, in partnership with chlamydial RodZ, acts as a cell division protein. Our findings suggest that an mreB-rodZ-based mechanism allows Chlamydia to divide without the universal division protein FtsZ.IMPORTANCE The study of Chlamydia growth and cell division is complicated by its obligate intracellular nature and biphasic lifestyle. Chlamydia also lacks the universal division protein FtsZ. We employed the cell division system of Escherichia coli as a surrogate to identify chlamydial cell division proteins. We demonstrate that chlamydial MreB, together with chlamydial RodZ, forms a cell division and growth complex that can replace FtsZ activity and support cell division in E. coli Chlamydial RodZ plays a major role in directing chlamydial MreB localization to the cell division site. It is likely that the evolution of chlamydial MreB and RodZ to form a functional cell division complex allowed Chlamydia to dispense with its FtsZ-based cell division machinery during genome reduction. Thus, MreB-RodZ represents a possible mechanism for cell division in other bacteria lacking FtsZ.

Chlamydia: what is on the outside does matter.

This review summarises major highlights on the structural biology of the chlamydial envelope. Chlamydiae are obligate intracellular bacteria, characterised by a unique biphasic developmental cycle. Depending on the stage of their lifecycle, they appear in the form of elementary or reticulate bodies. Since these particles have distinctive functions, it is not surprising that their envelope differs in lipid as well as in protein content. Vice versa, by identifying surface proteins, specific characteristics of the particles such as rigidity or immunogenicity may be deduced. Detailed information on the bacterial membranes will increase our understanding on the host-pathogen interactions chlamydiae employ to survive and grow and might lead to new strategies to battle chlamydial infections.

Proteomic characterisation of the Chlamydia abortus outer membrane complex (COMC) using combined rapid monolithic column liquid chromatography and fast MS/MS scanning.

Data are presented on the identification and partial characterisation of proteins comprising the chlamydial outer membrane complex (COMC) fraction of Chlamydia abortus (C. abortus)-the aetiological agent of ovine enzootic abortion. Inoculation with the COMC fraction is known to be highly effective in protecting sheep against experimental challenge and its constituent proteins are therefore of interest as potential vaccine candidates. Sodium N-lauroylsarcosine (sarkosyl) insoluble COMC proteins resolved by SDS-PAGE were interrogated by mass spectrometry using combined rapid monolithic column liquid chromatography and fast MS/MS scanning. Downstream database mining of processed tandem MS data revealed the presence of 67 proteins in total, including putative membrane associated proteins (n = 36), such as porins, polymorphic membrane proteins (Pmps), chaperonins and hypothetical membrane proteins, in addition to others (n = 22) that appear more likely to have originated from other subcellular compartments. Electrophoretic mobility data combined with detailed amino acid sequence information derived from secondary fragmentation spectra for 8 Pmps enabled peptides originating from protein cleavage fragments to be mapped to corresponding regions of parent precursor molecules yielding preliminary evidence in support of endogenous post-translational processing of outer membrane proteins in C. abortus. The data presented here will facilitate a deeper understanding of the pathogenesis of C. abortus infection and represent an important step towards the elucidation of the mechanisms of immunoprotection against C. abortus infection and the identification of potential target vaccine candidate antigens.

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription.

A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two σ factors, namely σ66 and σ28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Antibiotic treatment of Chlamydia-induced cystitis in the koala is linked to expression of key inflammatory genes in reactive oxygen pathways.

Chlamydial-induced cystitis in the koala (Phascolarctos cinereus) is currently treated by antibiotics. However, while reducing the chlamydial load, this treatment can also lead to gastrointestinal complications and death. Development of alternative treatments, such as a therapeutic chlamydial vaccine, are hindered by the lack of detailed understanding of the innate immune response to chlamydial clearance and disease regression during antibiotic treatment. Through clinical, microbiological and transcriptomic approaches, disease regression, bacterial clearance and innate immune responses were mapped in koalas with signs of chlamydial-induced cystitis while receiving anti-chlamydial antibiotics. Significant reduction in the signs of cystitis were observed during and post antibiotic treatment. This was observed as a thinning of the bladder wall and complete reversal of urinary incontinence. Transcriptomic analysis before treatment, at the end of treatment and prior to release identified significant down-regulation of specific genes involved in 21 biological pathways. Of these, the chemokine receptor signalling and NOD-like receptor signalling pathways where identified as important markers of inflammation. Specific genes within these pathways (NCF1 and NOX2) were significantly down-regulated, suggesting a decrease in reactive oxygen species production. Through the monitoring of specific clinical and transcriptomic markers, these findings allow detailed profiling of the clinical response to therapeutic vaccination in koalas with current signs of disease. This also adds to our understanding of innate immune responses to chlamydial infections and indicates that chlamydial-induced cystitis in the koala is linked to the regulation of reactive oxygen pathways.

A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum.

Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target.IMPORTANCEChlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

ChlamBase: a curated model organism database for the Chlamydia research community.

The accelerating growth of genomic and proteomic information for Chlamydia species, coupled with unique biological aspects of these pathogens, necessitates bioinformatic tools and features that are not provided by major public databases. To meet these growing needs, we developed ChlamBase, a model organism database for Chlamydia that is built upon the WikiGenomes application framework, and Wikidata, a community-curated database. ChlamBase was designed to serve as a central access point for genomic and proteomic information for the Chlamydia research community. ChlamBase integrates information from numerous external databases, as well as important data extracted from the literature that are otherwise not available in structured formats that are easy to use. In addition, a key feature of ChlamBase is that it empowers users in the field to contribute new annotations and data as the field advances with continued discoveries. ChlamBase is freely and publicly available at chlambase.org.

Life inside and out: making and breaking protein disulfide bonds in Chlamydia.

Disulphide bonds are widely used among all domains of life to provide structural stability to proteins and to regulate enzyme activity. Chlamydia spp. are obligate intracellular bacteria that are especially dependent on the formation and degradation of protein disulphide bonds. Members of the genus Chlamydia have a unique biphasic developmental cycle alternating between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body. The proteins in the envelope of the EB are heavily cross-linked with disulphides and this is known to be critical for this infectious phase. In this review, we provide a comprehensive summary of what is known about the redox state of chlamydial envelope proteins throughout the developmental cycle. We focus especially on the factors responsible for degradation and formation of disulphide bonds in Chlamydia and how this system compares with redox regulation in other organisms. Focussing on the unique biology of Chlamydia enables us to provide important insights into how specialized suites of disulphide bond (Dsb) proteins cater for specific bacterial environments and lifecycles.

Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events.

The emergence of semiautonomous organelles, such as the mitochondrion, the chloroplast, and more recently, the chromatophore, are critical steps in the evolution of eukaryotes. They resulted from primary endosymbiotic events that seem to share general features, i.e., an acquisition of a bacterium/cyanobacteria likely via a phagocytic membrane, a genome reduction coinciding with an escape of genes from the organelle to the nucleus, and finally the appearance of an active system translocating nuclear-encoded proteins back to the organelles. An intense mobilization of foreign genes of bacterial origin, via horizontal gene transfers, plays a critical role. Some third partners, like Chlamydia, might have facilitated the transition from cyanobacteria to the early chloroplast. This chapter describes our current understanding of primary endosymbiosis, with a specific focus on primary chloroplasts considered to have emerged more than one billion years ago, and on the chromatophore, having emerged about one hundred million years ago.

Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

Feasibility of a Conditional Knockout System for Chlamydia Based on CRISPR Interference.

Chlamydia is an obligate intracellular bacterium and, as such, has significantly reduced its genome size and content. Although recent advances have allowed for transformation of C. trachomatis with an exogenous plasmid, genetic manipulation of Chlamydia remains challenging. In particular, the ability to create conditional knockouts has not been developed. This is particularly important given the likelihood that most genes within the small genome of Chlamydia may be essential. Here, I describe the feasibility of using CRISPR interference (CRISPRi) based on the catalytically inactive Cas9 variant (dCas9) of Staphylococcus aureus to inducibly, and reversibly, repress gene expression in C. trachomatis. CRISPRi has been developed and used successfully in a variety of bacterial organisms including E. coli and Mycobacterium tuberculosis. I first describe the creation of a single plasmid system for CRISPRi in Chlamydia, targeted to a non-essential gene, incA, that expresses a dispensable inclusion membrane protein. Control transformations of C. trachomatis serovar L2 with plasmids encoding only the dCas9, under the control of an inducible promoter, or only the guide RNA (gRNA) targeted to the 5' UTR of incA, expressed constitutively, failed to prevent expression of IncA. Importantly, expression of dCas9 alone did not have a deleterious effect on chlamydiae. Transformation of C. trachomatis with a plasmid combining the dCas9 and a gRNA targeting incA and induction of expression of the dCas9 resulted in the reversible inhibition of IncA expression. Consequently, conditional knockout mediated by CRISPRi is feasible in Chlamydia. Potential improvements and experimental concerns in using the system are also discussed.