scholarly journals New Frontiers in Type III Secretion Biology: the Chlamydia Perspective

2013 ◽  
Vol 82 (1) ◽  
pp. 2-9 ◽  
Author(s):  
K. E. Mueller ◽  
G. V. Plano ◽  
K. A. Fields
Keyword(s):  
Type Iii Secretion ◽  
Genetic Manipulation ◽  
Respiratory Pathogen ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Type Iii ◽  
Obligate Intracellular Bacteria ◽  
Coding Capacity

ABSTRACTMembers of the orderChlamydialescomprise a group of exquisitely evolved parasites of eukaryotic hosts that extends from single-celled amoeba to mammals. The most notable are human pathogens and include the agent of oculogenital diseaseChlamydia trachomatis, the respiratory pathogenC. pneumoniae, and the zoonotic agentC. psittaci. All of these species are obligate intracellular bacteria that develop within parasitophorous vesicles termed inclusions. This demanding lifestyle necessitates orchestrated entry into nonphagocytic cells, creation of a privileged intracellular niche, and subversion of potent host defenses. All chlamydial genomes contain the coding capacity for a nonflagellar type III secretion system, and this mechanism has arisen as an essential contributor to chlamydial virulence. The emergence of tractable approaches to the genetic manipulation of chlamydiae raises the possibility of explosive progress in understanding this important contributor to chlamydial pathogenesis. This minireview considers challenges and recent advances that have revealed how chlamydiae have maintained conserved aspects of T3S while exploiting diversification to yield a system that exerts a fundamental role in the unique biology ofChlamydiaspecies.

scholarly journals A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicolas Jacquier ◽  
Akhilesh K. Yadav ◽  
Trestan Pillonel ◽  
Patrick H. Viollier ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Peptidoglycan Biosynthesis ◽  
Peptidoglycan Synthesis ◽  
Lytic Transglycosylase ◽  
Obligate Intracellular Bacteria

ABSTRACT 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. IMPORTANCE Chlamydiales 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.

scholarly journals The growing repertoire of genetic tools for dissecting chlamydial pathogenesis

2021 ◽  
Author(s):  
Arkaprabha Banerjee ◽  
David E Nelson
Keyword(s):  
Genetic Engineering ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Pathogenic Potential ◽  
Host Preferences ◽  
Genetic Tools ◽  
Obligate Intracellular ◽  
Obligate Intracellular Bacteria ◽  
Multiple Species

Abstract Multiple species of obligate intracellular bacteria in the genus Chlamydia are important veterinary and/or human pathogens. These pathogens all share similar biphasic developmental cycles and transition between intracellular vegetative reticulate bodies and infectious elementary forms, but vary substantially in their host preferences and pathogenic potential. A lack of tools for genetic engineering of these organisms has long been an impediment to the study of their biology and pathogenesis. However, the refinement of approaches developed in C. trachomatis over the last ten years, and adaptation of some of these approaches to other Chlamydia spp. in just the last few years, has opened exciting new possibilities for studying this ubiquitous group of important pathogens.

scholarly journals Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Nicholas A. Wood ◽  
Krystal Y. Chung ◽  
Amanda M. Blocker ◽  
Nathalia Rodrigues de Almeida ◽  
Martin Conda-Sheridan ◽  
...  
Keyword(s):  
Host Cells ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Clp Protease ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Developmental Form ◽  
Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

scholarly journals Piericidin A1 Blocks Yersinia Ysc Type III Secretion System Needle Assembly

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Jessica M. Morgan ◽  
Miles C. Duncan ◽  
Kevin S. Johnson ◽  
Andreas Diepold ◽  
Hanh Lam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Human Pathogens ◽  
Content Type ◽  
Bacterial Surface ◽  
Type Iii ◽  
Animal Pathogens ◽  
Bacterial Type

ABSTRACT The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS. The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS.

scholarly journals Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Timothy P. Driscoll ◽  
Victoria I. Verhoeve ◽  
Mark L. Guillotte ◽  
Stephanie S. Lehman ◽  
Sherri A. Rennoll ◽  
...  
Keyword(s):  
Metabolic Network ◽  
Genetic Manipulation ◽  
Eukaryotic Cell ◽  
Eukaryotic Cells ◽  
Intracellular Bacteria ◽  
Axenic Medium ◽  
Obligate Intracellular ◽  
Metabolic Genes ◽  
Obligate Intracellular Bacteria ◽  
Host Metabolism

ABSTRACTReductive genome evolution has purged many metabolic pathways from obligate intracellularRickettsia(Alphaproteobacteria;Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenicRickettsiaculture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed theRickettsiametabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell.IMPORTANCEA hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species ofRickettsia, arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed theRickettsiametabolic network and identified 51 host metabolites required to compensate patchworkRickettsiabiosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what aRickettsiaaxenic medium might entail.IMPORTANCEA hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species ofRickettsia, arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed theRickettsiametabolic network and identified 51 host metabolites required to compensate patchworkRickettsiabiosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what aRickettsiaaxenic medium might entail.

scholarly journals Apoptosis Functions in Defense against Infection of Mammalian Cells with Environmental Chlamydiae

2020 ◽  
Vol 88 (6) ◽  
Author(s):  
Dominik Brokatzky ◽  
Oliver Kretz ◽  
Georg Häcker
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Intracellular Bacteria ◽  
Mitochondrial Apoptosis ◽  
Content Type ◽  
Obligate Intracellular ◽  
Human Epithelial Cells ◽  
Antiapoptotic Activity ◽  
Obligate Intracellular Bacteria ◽  
Induced Apoptosis

ABSTRACT Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All Chlamydiales are obligate intracellular bacteria, and different species infect vastly different hosts. Chlamydia trachomatis infects human epithelial cells; Parachlamydia acanthamoebae replicates in amoebae. We here report that apoptosis impedes growth of P. acanthamoebae in mammalian cells. In HeLa human epithelial cells, P. acanthamoebae infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-XL or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of P. acanthamoebae in HeLa cells, as measured by fluorescence in situ hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with C. trachomatis inhibited P. acanthamoebae-induced apoptosis, suggesting that the known antiapoptotic activity of C. trachomatis can also block P. acanthamoebae-induced apoptosis. C. trachomatis coinfection could not rescue P. acanthamoebae growth in HeLa; in coinfected cells, C. trachomatis even suppressed the growth of P. acanthamoebae independently of apoptosis, while P. acanthamoebae surprisingly enhanced the growth of C. trachomatis. Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.

scholarly journals Reductive Evolution in Bacteria:Buchnerasp.,Rickettsia prowazekiiandMycobacterium leprae

2001 ◽  
Vol 2 (1) ◽  
pp. 44-48 ◽  
Author(s):  
Jo Wixon
Keyword(s):  
Acyrthosiphon Pisum ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Orthologous Genes ◽  
Reductive Evolution ◽  
Obligate Intracellular ◽  
Growth Requirements ◽  
Obligate Intracellular Bacteria ◽  
The Relationship ◽  
Host Genes

Obligate intracellular bacteria commonly have much reduced genome sizes compared to their nearest free-living relatives. One reason for this is reductive evolution: the loss of genes rendered non-essential due to the intracellular habitat. This can occur because of the presence of orthologous genes in the host, combined with the ability of the bacteria to import the protein or metabolite products of the host genes. In this article we take a look at three such bacteria whose genomes have been fully sequenced.Buchnerais an endosymbiont of the pea aphid,Acyrthosiphon pisum, the relationship between these two organisms being so essential that neither can reproduce in the absence of the other.Rickettsia prowazekiiis the causative agent of louse-borne typhus in humans andMycobacterium lepraeinfection of humans leads to leprosy. Both of these human pathogens have fastidious growth requirements, which has made them very difficult to study.

scholarly journals A Coming of Age Story: Chlamydia in the Post-Genetic Era

2015 ◽  
Vol 84 (3) ◽  
pp. 612-621 ◽  
Author(s):  
Anna J. Hooppaw ◽  
Derek J. Fisher
Keyword(s):  
Global Economy ◽  
Financial Burden ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Reticulate Body ◽  
Technological Advances ◽  
Obligate Intracellular Bacteria

Chlamydiaspp. are ubiquitous, obligate, intracellular Gram-negative bacterial pathogens that undergo a unique biphasic developmental cycle transitioning between the infectious, extracellular elementary body and the replicative, intracellular reticulate body. The primaryChlamydiaspecies associated with human disease areC. trachomatis, which is the leading cause of both reportable bacterial sexually transmitted infections and preventable blindness, andC. pneumoniae, which infects the respiratory tract and is associated with cardiovascular disease. Collectively, these pathogens are a significant source of morbidity and pose a substantial financial burden on the global economy. Past efforts to elucidate virulence mechanisms of these unique and important pathogens were largely hindered by an absence of genetic methods. Watershed studies in 2011 and 2012 demonstrated that forward and reverse genetic approaches were feasible withChlamydiaand that shuttle vectors could be selected and maintained within the bacterium. While these breakthroughs have led to a steady expansion of the chlamydial genetic tool kit, there are still roads left to be traveled. This minireview provides a synopsis of the currently available genetic methods forChlamydiaalong with a comparison to the methods used in other obligate intracellular bacteria. Limitations and advantages of these techniques will be discussed with an eye toward the methods still needed, and how the current state of the art for genetics in obligate intracellular bacteria could direct future technological advances forChlamydia.

scholarly journals A Salmonella Virulence Factor Activates the NOD1/NOD2 Signaling Pathway

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
A. Marijke Keestra ◽  
Maria G. Winter ◽  
Daisy Klein-Douwel ◽  
Mariana N. Xavier ◽  
Sebastian E. Winter ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Intestinal Inflammation ◽  
Inflammatory Responses ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Content Type ◽  
Type Iii

ABSTRACTThe invasion-associated type III secretion system (T3SS-1) ofSalmonella entericaserotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasionin vitrobut required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responsesin vitroandin vivo.IMPORTANCESalmonella entericaserotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol.

scholarly journals Chlamydial Lytic Exit from Host Cells Is Plasmid Regulated

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Chunfu Yang ◽  
Tregei Starr ◽  
Lihua Song ◽  
John H. Carlson ◽  
Gail L. Sturdevant ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Actin Polymerization ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Cytoskeletal Proteins ◽  
Host Cells ◽  
Genetic Mechanism ◽  
Developmental Cycle ◽  
Obligate Intracellular ◽  
Type Iii

ABSTRACTChlamydia trachomatisis an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknownpgp4-regulated chromosomal T3S effector gene.IMPORTANCEChlamydia's obligate intracellular life style requires both entry into and exit from host cells. Virulence factors that function in exiting are unknown. The chlamydial inclusion is stabilized late in the infection cycle by F-actin. A prerequisite of chlamydial exit is its ability to disassemble actin from the inclusion. We show that chlamydial plasmid-free organisms, and also a plasmid gene protein 4 (pgp4) null mutant, do not disassociate actin from the inclusion and fail to exit cells. We further provide evidence that Pgp4-regulated exit is dependent on the chlamydial type III secretion system. This study is the first to define a genetic mechanism that functions in chlamydial lytic exit from host cells. The findings also have practical implications for understanding why plasmid-free chlamydiae are highly attenuated and have the ability to elicit robust protective immune responses.

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