Single-Inclusion Kinetics of Chlamydia trachomatis Development

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.

Regulation of the Mitochondrion-Fatty Acid Axis for the Metabolic Reprogramming of Chlamydia trachomatis during Treatment with β-Lactam Antimicrobials

“…While we morphologically observed a large number of RBs in productive infection at 24 hpi ( Fig. S2A ), expression of late genes that function in RB-to-EB differentiation was observed at 24 hpi ( 9 , 59 ). Therefore, we are aware that the interpretation of the observed changes in gene expression could be influenced by the stage of chlamydial development.…”

In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

“…C . trachomatis , an obligate intracellular bacterium, has an intriguing and unique biphasic developmental cycle alternating between the extracellular, infectious elementary body (EB) and the intracellular, noninfectious, reticulate body (RB) [ 3 , 7 , 8 ]. The developmental cycle begins when EBs attach and enter the host cell by endocytosis.…”

“…The developmental cycle begins when EBs attach and enter the host cell by endocytosis. Once inside the host cell, EBs are internalized and confined to a vacuole termed inclusion, through a process requiring the secretion of several proteins including Type-III secretion system effector proteins [ 7 , 9 ]. Within the inclusion, EBs then differentiate to RBs, which replicate by binary fission within 24 hours post-infection and, as the inclusion expands, RBs begin to transition back to EBs in an asynchronous process [ 7 , 9 ].…”

“…Once inside the host cell, EBs are internalized and confined to a vacuole termed inclusion, through a process requiring the secretion of several proteins including Type-III secretion system effector proteins [ 7 , 9 ]. Within the inclusion, EBs then differentiate to RBs, which replicate by binary fission within 24 hours post-infection and, as the inclusion expands, RBs begin to transition back to EBs in an asynchronous process [ 7 , 9 ]. At the end of the developmental cycle, the inclusion occupies most of the host cell’s cytoplasm and, after approximately 48 hours, the EBs are finally released from the host cell mainly by lysis [ 7 , 10 ].…”

“…Within the inclusion, EBs then differentiate to RBs, which replicate by binary fission within 24 hours post-infection and, as the inclusion expands, RBs begin to transition back to EBs in an asynchronous process [ 7 , 9 ]. At the end of the developmental cycle, the inclusion occupies most of the host cell’s cytoplasm and, after approximately 48 hours, the EBs are finally released from the host cell mainly by lysis [ 7 , 10 ].…”