Cell Type Development in Chlamydia trachomatis Follows a Program Intrinsic to the Reticulate Body

AbstractThe obligate intracellular bacterial pathogen Chlamydia trachomatis (Ctr) is reliant on an unusual developmental cycle consisting of two cell forms termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but does not replicate. The RB replicates in the host cell but is non-infectious. This developmental cycle is central to chlamydial pathogenesis. In this study we developed mathematical models of the chlamydial developmental cycle that account for potential factors influencing the timing of RB to EB cell type switching during infection. Our models predicted that two broad categories of regulatory signals for RB to EB development could be differentiated experimentally; an “intrinsic” cell autonomous program inherent to each RB or an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between these hypotheses, we tracked the expression of Ctr developmental specific promoters using fluorescent reporters and live cell imaging. These experiments indicated that EB production was not influenced by increased MOI or by superinfection, suggesting the cycle follows an intrinsic program that is not influenced by environmental factors. Additionally, live cell imaging of these promoter constructs revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a cell type gene expression progression with the promoters for euo and ihtA active in RBs, while the promoter for hctA was active in early EBs/intermediate cells and finally the promoters for the true late genes, hctB, scc2, and tarp active in the maturing EB.ImportanceChlamydia trachomatis is an obligate intracellular bacteria that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells Chlamydia must complete a multi cell type developmental cycle. The developmental cycle consists of two specialized cells; the EB which mediates infection of new cells and the RB which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB to EB cell type switching. We demonstrate that RB to EB development follows a cell autonomous program that does not respond to environmental cues. Additionally, we show that RB to EB development is a function of cell growth and cell division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

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Single-Inclusion Kinetics of Chlamydia trachomatis Development

mSystems ◽

10.1128/msystems.00689-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Travis J. Chiarelli ◽

Nicole A. Grieshaber ◽

Anders Omsland ◽

Christopher H. Remien ◽

Scott S. Grieshaber

Keyword(s):

Chlamydia Trachomatis ◽

Mathematical Models ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Host Cells ◽

Developmental Cycle ◽

Cell Type ◽

Content Type ◽

Obligate Intracellular

ABSTRACT The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an “intrinsic” cell-autonomous program inherent to each RB and an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

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Chlamydial infection and disease in the koala

Microbiology Australia ◽

10.1071/ma05065 ◽

2005 ◽

Vol 26 (2) ◽

pp. 65 ◽

Cited By ~ 3

Author(s):

Peter Timms

Keyword(s):

Chlamydia Trachomatis ◽

Chlamydial Infection ◽

Developmental Cycle ◽

Elementary Body ◽

Obligate Intracellular ◽

Molecular Approaches ◽

Reticulate Body ◽

Wide Range ◽

The Family ◽

Serious Disease

Chlamydiae are obligate intracellular bacterial pathogens able to infect and cause serious disease in humans, birds and a remarkably wide range of warm and cold-blooded animals. The family Chlamydiaciae have traditionally been defined by their unique biphasic developmental cycle, involving the interconversion between an extracellular survival form, the elementary body and an intracellular replicative form, the reticulate body. However, as with many other bacteria, molecular approaches including 16SrRNA sequence are becoming the standard of choice. As a consequence, the chlamydiae are in a taxonomic state of flux. Prior to 1999, the family Chlamydiaceae consisted of one genus, Chlamydia, and four species, Chlamydia trachomatis, C. psittaci, C. pecorum and C. pneumoniae. In 1999, Everett et al proposed a reclassification of Chlamydia into two genera (Chlamydia and Chlamydophila) and nine species (Chlamydia trachomatis, C. suis, and C. muridarum and Chlamydophila psittaci, C. pneumoniae, C. felis, C. pecorum, C. abortus, and C. caviae). While some of these species are thought to be host specific (C. suis ? pigs, C. muridarum ? mice, C. felis ? cats, C. caviae ? guinea pigs) many are known to infect and cause disease in a wide range of hosts.

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A Dynamic, Ring-Forming Bactofilin Critical for Maintaining Cell Size in the Obligate Intracellular Bacterium Chlamydia trachomatis

Infection and Immunity ◽

10.1128/iai.00203-21 ◽

2021 ◽

Author(s):

Mary R. Brockett ◽

Junghoon Lee ◽

John V. Cox ◽

George W. Liechti ◽

Scot P. Ouellette

Keyword(s):

Cell Division ◽

Chlamydia Trachomatis ◽

Cell Size ◽

Cytoskeletal Proteins ◽

Intracellular Bacterium ◽

Developmental Cycle ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular ◽

Peptidoglycan Biosynthesis ◽

Obligate Intracellular Pathogen

Bactofilins are polymer-forming cytoskeletal proteins that are widely conserved in bacteria. Members of this protein family have diverse functional roles such as orienting subcellular molecular processes, establishing cell polarity, and aiding in cell shape maintenance. Using sequence alignment to the conserved bactofilin domain, we identified a bactofilin ortholog, BacACT, in the obligate intracellular pathogen Chlamydia trachomatis. Chlamydia species are obligate intracellular bacteria that undergo a developmental cycle alternating between infectious, non-dividing EBs (elementary bodies) and non-infectious, dividing RBs (reticulate bodies). As Chlamydia divides by a polarized division process, we hypothesized that BacACT may function to establish polarity in these unique bacteria. Utilizing a combination of fusion constructs and high-resolution fluorescence microscopy, we determined that BacACT forms dynamic, membrane-associated filament- and ring-like structures in Chlamydia’s replicative RB form. Contrary to our hypothesis, these structures are distinct from the microbe’s cell division machinery and do not colocalize with septal peptidoglycan or MreB, the major organizer of the bacterium’s division complex. Bacterial two-hybrid assays demonstrated BacACT interacts homotypically but does not directly interact with proteins involved in cell division or peptidoglycan biosynthesis. To investigate the function of BacACT in chlamydial development, we constructed a conditional knockdown strain using a newly developed CRISPR interference system. We observed that reducing bacACT expression significantly increased chlamydial cell size. Normal RB morphology was restored when an additional copy of bacACT was expressed in trans during knockdown. These data reveal a novel function for chlamydial bactofilin in maintaining cell size in this obligate intracellular bacterium.

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Chlamydia trachomatis Encodes a Dynamic, Ring-Forming Bactofilin Critical for Maintaining Cell Size and Shape

10.1101/2020.10.19.346502 ◽

2020 ◽

Author(s):

Mary R. Brockett ◽

Junghoon Lee ◽

John V. Cox ◽

George W. Liechti ◽

Scot P. Ouellette

Keyword(s):

Cell Division ◽

Chlamydia Trachomatis ◽

Cell Size ◽

Cell Shape ◽

Ring Structure ◽

Intracellular Bacterium ◽

Developmental Cycle ◽

Obligate Intracellular Bacterium ◽

Obligate Intracellular ◽

Crispr Interference

ABSTRACTBactofilins are polymer-forming cytoskeletal proteins that are widely conserved in bacteria. Members of this protein family have diverse functional roles such as orienting subcellular molecular processes, establishing cell polarity, and aiding in cell shape maintenance. Chlamydia species are obligate intracellular bacteria that undergo a developmental cycle alternating between an infectious, non-dividing EB and a non-infectious, dividing RB. As Chlamydia divides by a polarized division process, we hypothesized that BacACT may function to establish polarity in these unique bacteria. Using sequence alignment to the conserved bactofilin domain, we identified a bactofilin ortholog, BacACT, in the obligate intracellular pathogen Chlamydia trachomatis. Utilizing a combination of fusion constructs and high-resolution fluorescence microscopy, we determined that BacACT forms a dynamic, membrane-associated, ring-like structure in Chlamydia’s replicative RB form. Contrary to our hypothesis, this filamentous ring structure is distinct from the microbe’s cell division machinery and does not colocalize with septal peptidoglycan or MreB, the major organizer of the bacterium’s division complex. Bacterial two-hybrid assays demonstrated BacACT interacts homotypically but does not directly interact with proteins involved in cell division or peptidoglycan biosynthesis. To investigate the function of BacACT in chlamydial development, we constructed a conditional knockdown strain using a newly developed CRISPR interference system. We observed that reducing bacACT expression significantly impacted chlamydial cell size and morphology. Normal RB morphology was restored when an additional copy of BacACT was expressed in trans during knockdown. These data reveal a novel function for chlamydial bactofilin in maintaining cell shape in this obligate intracellular bacterium.IMPORTANCEChlamydia is an ancient, obligate intracellular bacterium with a unique biphasic developmental cycle. As a result of its evolution within the osmotically stable environment of a host cell, Chlamydia has lost its dependence on side-wall peptidoglycan, and maintains only a fraction of the components thought to be required for regulating bacterial cell size and division. As such, very little is known about how Chlamydia species carry out these critical processes in the absence of a stabilizing peptidoglycan layer. In the current study, we identify a novel cytoskeletal element, termed a bactofilin, that is critical for maintaining the morphology of the bacteria. Using state-of-the-art genetic techniques for this organism, we demonstrate that chlamydial bactofilin forms a dynamic ring structure independent of the microbe’s division machinery and that abrogating its expression level using CRISPR interference results in abnormal morphologic forms. These findings enhance our understanding of chlamydial biology and bactofilins more generally.

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Effects ofMentha suaveolensEssential Oil onChlamydia trachomatis

BioMed Research International ◽

10.1155/2015/508071 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 9

Author(s):

Rosa Sessa ◽

Marisa Di Pietro ◽

Fiorenzo De Santis ◽

Simone Filardo ◽

Rino Ragno ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Substantial Reduction ◽

Developmental Cycle ◽

Elementary Body ◽

Promising Candidate ◽

Common Cause ◽

Sexually Transmitted ◽

Reticulate Body ◽

Preventative Strategy

Chlamydia trachomatis, the most common cause of sexually transmitted bacterial infection worldwide, has a unique biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body.C. trachomatisis responsible for severe reproductive complications including pelvic inflammatory disease, ectopic pregnancy, and obstructive infertility. The aim of our study was to evaluate whetherMentha suaveolensessential oil (EOMS) can be considered as a promising candidate for preventingC. trachomatisinfection. Specifically, we investigated thein vitroeffects of EOMS towardsC. trachomatisanalysing the different phases of chlamydial developmental cycle. Our results demonstrated that EOMS was effective towardsC. trachomatis, whereby it not only inactivated infectious elementary bodies but also inhibited chlamydial replication. Our study also revealed the effectiveness of EOMS, in combination with erythromycin, towardsC. trachomatiswith a substantial reduction in the minimum effect dose of antibiotic. In conclusion, EOMS treatment may represent a preventative strategy since it may reduceC. trachomatistransmission in the population and, thereby, reduce the number of new chlamydial infections and risk of developing of severe sequelae.

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Red Fluorescent Chlamydia trachomatis Applied to Live Cell Imaging and Screening for Antibacterial Agents

Frontiers in Microbiology ◽

10.3389/fmicb.2018.03151 ◽

2018 ◽

Vol 9 ◽

Author(s):

Sergio A. Mojica ◽

Anna U. Eriksson ◽

Rohan A. Davis ◽

Wael Bahnan ◽

Mikael Elofsson ◽

...

Keyword(s):

Chlamydia Trachomatis ◽

Live Cell Imaging ◽

Antibacterial Agents ◽

Cell Imaging ◽

Live Cell

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Role for GrgA in Regulation of σ28-Dependent Transcription in the Obligate Intracellular Bacterial PathogenChlamydia trachomatis

Journal of Bacteriology ◽

10.1128/jb.00298-18 ◽

2018 ◽

Vol 200 (20) ◽

Cited By ~ 3

Author(s):

Malhar Desai ◽

Wurihan Wurihan ◽

Rong Di ◽

Joseph D. Fondell ◽

Bryce E. Nickels ◽

...

Keyword(s):

Transcription Factor ◽

Chlamydia Trachomatis ◽

Sigma Factor ◽

Bacterial Pathogen ◽

Direct Interaction ◽

Developmental Cycle ◽

Content Type ◽

Sexually Transmitted ◽

Obligate Intracellular ◽

Specific Transcription Factor

ABSTRACTThe obligate intracellular bacterial pathogenChlamydia trachomatishas a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primaryChlamydiasigma factor, σ66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor, σ28. In prior work, we identified GrgA as aChlamydia-specific transcription factor that activates σ66-dependent transcription by binding DNA and interacting with a nonconserved region (NCR) of σ66. Here, we extend these findings by showing GrgA can also activate σ28-dependent transcription through direct interaction with σ28. We measure the binding affinity of GrgA for both σ66and σ28, and we identify regions of GrgA important for σ28-dependent transcription. Similar to results obtained with σ66, we find that GrgA's interaction with σ28involves an NCR located upstream of conserved region 2 of σ28. Our findings suggest that GrgA is an important regulator of both σ66- and σ28-dependent transcription inC. trachomatisand further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.IMPORTANCEChlamydia trachomatisis the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion ofC. trachomatis-infected women develop infertility, pelvic inflammatory syndrome, and other serious complications.C. trachomatisis also a leading infectious cause of blindness in underdeveloped countries. The pathogen has a unique developmental cycle that is transcriptionally regulated. The discovery of an expanded role for theChlamydia-specific transcription factor GrgA helps us understand the progression of the chlamydial developmental cycle.

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