We have previously proposed a hypothesis for the development of intracellular chlamydiae based on a combination of electron microscopic and other observations (26
) and further developed the hypothesis using biomathematical modeling (35
). Tenets of the so-called contact-dependent hypothesis of chlamydial development are the following: (i) as RBs, chlamydiae grow strictly in contact with the plasma membrane-derived CIM, (ii) contact with the CIM is mediated by surface projections hypothesized to correspond to T3S injectisomes, and (iii) disruption of T3S activity through physical detachment from the CIM is associated with the onset of late differentiation. The implied biological significance of the hypothesis is that maintaining contact with the CIM permits continued delivery of chlamydial T3S effectors into the host cell cytosol and subsequent subversion of cellular processes to benefit chlamydial growth; moreover, disruption of contact through physical detachment interrupts T3S effector translocation, thus rendering the host cell less hospitable for chlamydial growth. Because contact of chlamydial particles with the CIM, or loss thereof, has direct implications on the ability of chlamydiae to move inside the inclusion, we sought to quantify the movement (distance traveled and velocity) of individual chlamydial particles at different times along development. For this analysis, we used a Richardson RTM3 light microscope (28
) that is optimized for high-resolution white-light microscopy and live recording (30 frames per s) of Chlamydia
-infected cells in real time. There are some limitations to the quantification of movement. (i) The movies are a cross-sectional, two-dimensional slice of the infected cells, and so particles that move in and out of focus are difficult to track. We have made allowances for this in our image-processing computer algorithms, but if fast-moving particles move far when out of focus, then identification and matching of particles are prevented. (ii) During late stages of development, the inclusion lumen is densely packed with EBs, and spatial constraints are likely to prevent unhindered movement. The high density at late stages also makes it difficult to distinguish some particles between frames. To alleviate this problem we included only particles for which we could clearly identify and match particles between frames. (iii) We could not record the same cell for each stage of the developmental cycle included in our analysis. We recorded representative cells in the culture at each time point. We acknowledge that there would be some asynchrony of infection or development and that there may be some differences at times where the same phenotypes are seen.
The results of our analysis reveal a close relationship between key developmental stages and motion properties of the chlamydiae. While movement of chlamydial bodies has been noted previously (22
), this is the first detailed study using advanced microscopic techniques to (i) confirm that chlamydiae definitely do undergo movement and (ii) link this movement to specific stages of development. The magnitude and speed of RB wobbling from early to middle stages of development (Fig. , 8 to 20 hpi) steadily increase with time, suggesting that individual RBs gradually lose contact with the CIM, allowing for increased movement and reciprocally supporting their hypothesized tethering to the CIM via T3S injectisomes. This is consistent with the observation that the number of surface projections decreases during development, as observed by Matsumoto in C. psittaci
). Between 20 and 24 hpi, a remarkable gain in movement is observed for approximately half of the particles in an inclusion such that average velocities of individual particles are approximately four times greater at 24 hpi than at 20 hpi (Table ). Coincidentally, the distances traveled by individual particles are substantially increased. These results are consistent with the contact-dependent hypothesis whereby untethering of individual particles is predicted to be asynchronous during late differentiation.
Naturally, larger-sized particles will move at a slower rate than smaller particles when acted upon by the same force. It is, therefore, predictable that the decreasing size of the chlamydial particle (from 0.8 to 1.0 μm for the RB to 0.25 μm for the EB) will affect the degree of movement during late differentiation. However, we found that the fastest particles in the developmental cycle (at 24 hpi) were of the size of RB particles (Fig. ). Given the very marked change observed from essentially no movement or slight in-place wobbling of RBs to very fast motion of newly detached RBs, IBs, or EBs, we conclude that the large gain of velocity observed between 20 and 24 hpi is not accounted for significantly by the change in the size of the chlamydial particle as it undergoes late differentiation. A steady decrease in speed was also observed between 24 and 49 hpi, i.e., during late differentiation. We speculate that particles gradually lose velocity as they bounce off each other and off the CIM in the increasingly crowded inclusion lumen. Biomathematical simulations of the contact-dependent hypothesis predict that both the multiplicity of inclusions within a single infected cell and the size of the chlamydial particle relative to that of the inclusion are determining factors in the outcome of an infection (17
). In multiple, smaller inclusions within an infected cell, the size differential between the inclusion and that of a replicating RB it contains may become small enough such that loss of T3S-mediated contact between the RB and the CIM becomes improbable. Following the same logic, the likelihood of an extremely large RB's becoming untethered from the CIM diminishes with increasing RB size. We explored this part of the hypothesis using C. trachomatis
grown in the presence of penicillin. Penicillin-exposed chlamydial cultures are known to produce aberrantly enlarged mRBs and provide a model for chlamydial persistence, a hallmark of chlamydial chronic infection and disease in humans (1
). In our experiments, cultures were supplemented with penicillin G at 24 hpi and observed at 48 hpi, allowing for inclusions to contain a mixture of persistent mRBs as well as normal RBs, IBs, and EBs. We found that EBs in inclusions exposed to penicillin moved at maximal speeds similar to those observed at 49 hpi in normal cultures (Fig. and Table ). Particles the size of RBs were observed to wobble in place, while mRBs were either completely static or wobbled slightly in place. This is consistent with the idea that these particles are more extensively tethered to the underlying CIM and suggests that mRBs may not become untethered from the CIM, with the consequence that they do not enter late differentiation and that they persist de facto. Hence, the motion properties of chlamydial particles within persistent inclusions are consistent with the results of biomathematical simulations that predict that mRBs persist in vitro owing to their continued tethering to the CIM. Although this hypothesis ultimately requires experimental verification, it is consistent with gene expression studies that have shown at both the transcriptional and protein levels that expression of the T3S injectisome genes is not significantly affected during persistent growth (5
The T3S system is thought to be central to the virulence of many bacterial pathogens including Chlamydia
). However, there is no consensus as to whether a functional T3S system exists during the intracellular growth phase of chlamydiae and, if so, whether tethering of each single RB to the inclusion membrane via T3S injectisomes is necessary for development. A role of T3S in chlamydial pathogenesis is supported by virulence-related properties of several chlamydial T3S-translocated effectors (7
). Other studies have coupled T3S expression or T3S activity with development (12
). An attractive hypothesis emerging from these converging results is that T3S-mediated translocation of early mid-cycle effector(s) to the infected cell cytosol maintains the host in a state optimized for chlamydial exponential growth and that disruption of this state through interruption of T3S translocation may “alert” chlamydiae within the inclusion to initiate late differentiation and subsequent progress of the infection. Stress-induced inhibition of this process would then represent a survival mechanism of the chlamydiae whereby a sustained level of T3S translocation activity maintains viability of fewer chlamydiae but for extended periods of time. Although our study does not directly address T3S activity and its potential role in development, our findings are consistent with the contact-dependent hypothesis. Further experiments beyond the scope of this study would be required to investigate the potential role of the T3S system on chlamydial development. Our novel approach of using real-time light microscopy and kinetics analysis with Chlamydia
has described chlamydial movement in a way that has never been done previously. It has elucidated properties in the regulation of the unique developmental cycle of this medically important pathogen.