Image: 3D reconstruction of the Chlamydia inclusion within an infected host cell, with four developmental forms shown in different colors; reproduced from the paper in Nature Communications: http://rdcu.be/DYCQ
How do you solve a problem like Chlamydia?
Let’s start at the very beginning with how this bacterium multiplies – chlamydial reproduction is complicated compared to its prokaryotic cousins because it only occurs inside an infected host cell, and involves conversion between two developmental forms of the organism. The reticulate body (RB), which is the vegetative form, divides repeatedly before converting into the infectious, environmentally-stable elementary body (EB). Conventional depiction of this developmental cycle shows a Chlamydia-infected cell at different steps in the intracellular infection:
Thinking about Chlamydia development as a cell-fate decision
But this host-cell-centric view hides the relationship between RB replication and RB-to-EB conversion: they are mutually exclusive cell-fate decisions.
Here’s how the developmental cycle looks as a series of cell-fate decisions:
This RB-centric developmental cycle emphasizes the delayed and asynchronous nature of conversion, which are its defining features. But what signals and mechanisms control the timing of conversion? The task is made harder by the paucity of information about the changing proportions of RBs and EBs in the chlamydial inclusion. 2D electron micrographs provide a snapshot but have limitations for quantitative analysis, especially at late times when the inclusion contains up to 1,000 chlamydiae and fills the host cell cytoplasm.
3D EM analysis of the chlamydial inclusion
This problem seemed tailor-made for serial block-face scanning EM (SBEM), which has been used to visualize the 3D architecture of cells and subcellular structures. The approach is similar to a CT scan and allowed us to generate 3D reconstructions of the chlamydial inclusion within an infected host cell at different times of the intracellular infection.
From this 3D EM analysis, we quantified the RBs, EBs, and intermediates of replication (dividing RB) and conversion (intermediate body, IB), in infected cells over time. A special shout-out goes to UC Irvine undergrads Chris Chander and Sean Pairawan, and then-high school students Tracy Lou and Melody Guo, who spent countless hours at the computer tracing tens of thousands of chlamydiae.
This comprehensive analysis generated growth curves showing expansion of the RB population followed by the onset of conversion, which leads to EB accumulation and RB depletion.
Chlamydiae get smaller with replication
Unexpectedly, we found that RBs progressively decrease in size by 6-fold and that the timing of conversion correlates with small RB size. We proposed a size control model in which RBs get smaller through replication and can only convert into EBs at a threshold size. This allows the RB population to expand, while delaying conversion. We also proposed that conversion is asynchronous because the observed heterogeneity in RB size varies the number of replication cycles required to reach the conversion size.
Size control model of chlamydial development
A modified, RB-centric developmental cycle illustrates how size control could produce delayed and asynchronous conversion. This model is supported by mathematical modeling, which showed that size control is sufficient to reproduce the dynamic balance that we measured between replication and conversion over the course of the intracellular infection, without the need for an external signal.
In noting the asynchrony of RB-to-EB conversion, the revered chlamydiologist Jim Moulder wrote, “It seems that the signal must originate at different times in individual RBs” (Moulder, J. W. Interaction of chlamydiae and host cells in vitro. Microbiol. Rev. 55, 143-190, 1991). Our study reveals that this elusive signal does not have to be external because conversion could be intrinsically controlled by RB size.