It is hypothesized that chlamydial particles have T3S injectisomes and that the Chlamydia
T3S system functions as a molecular nanosyringe involved in injecting effector proteins from the intracellular inclusion into the host cell cytosol which, in turn, subvert various host cell pathways [12
]. RBs are observed to replicate in close proximity to the surface of the host inclusion membrane [16
] and as an inclusion expands (to accommodate the growth in the number of chlamydiae), the surface area of contact between the inclusion membrane and RBs on the inclusion membrane surface decreases. Consequently, the number of injectisomes per RB in contact with the inclusion membrane decreases. We have hypothesized that when the decreasing number of injectisomes on the RB surface that are in contact with the inclusion membrane falls below a threshold number, detachment of the RB from the surface occurs, providing a signal for differentiation of RB into EB.
A wide range of Gram negative bacteria have the T3S system including Yersinia
, a variety of plant pathogens, and of course Chlamydia
]. Possible T3S-mediated functions include the intracellular survival of Chlamydia
in the acquisition of nutrients, inhibiting fusion with host cell lysosomes, and diverting lipids. T3S activity as regulated by contact between the RB and the inclusion membrane supports the hypothesis that T3S inactivation is a possible trigger for late differentiation and it would explain why chlamydial development, initially almost synchronous, becomes asynchronous in the mature inclusion as reticulate bodies differentiate into elementary bodies [19
]. Furthermore, our T3S hypothesis for chlamydial development is not only consistent with, but predicts, persistent infection under various conditions.
There is a large portion of the Chlamydia
literature that describes chlamydial persistence (a topic that had seemed mutually exclusive to T3S). Persistent maxi-RBs express unique RNA and protein profiles, including reduced amounts of the chlamydial MOMP, a potential protective antigen [20
], and increased levels of the chlamydial heat shock proteins [4
], which correlates with the pathological serious disease sequelae associated with chlamydial infections [22
]. We have shown quantitatively how our hypothesis is consistent with the atypical persistent model. According to our hypothesis, chlamydiae can persist, that is, remain in RB form for long periods of time, by maintaining a number of injectisomes above a detachment threshold. There are two conditions by which this will occur: (i) if the inclusion becomes very small (e.g., small, dividing inclusions) or (ii) if the RBs become relatively very large (e.g, under stress RBs do not divide). This is precisely what happens in the persistence models. In persistent models in vitro
, the maxi-RBs are larger and the inclusions are actually much smaller (especially in C. pneumoniae
models). Here, a larger RB in a smaller inclusion would necessarily make more contact with the membrane, hence would maintain a sufficient number of T3S needles and never fall off, hence never differentiate. In continuous growth models, there are often many inclusions per host cell and they are of different sizes. Some inclusions are relatively small and would include only a few chlamydiae (in a persistent state). Infection by certain Chlamydia
species, such as C. pneumoniae
or many veterinary isolates, consistently results in multiple inclusions that do not fuse [19
]. Further, in cases where infected host cells may still have the ability to divide such that the inclusions are distributed between the daughter cells, then small persistent inclusions would potentially have the ability to persevere for an extended period of time. We must make special note that we are not suggesting any causality in the mode for inducing persistence in terms of the external conditions that lead to spatial constraints. But we do hypothesize that if external or infection conditions, or even potentially stochasticity, contribute to larger sizes of RBs (including their inability to divide), to multiple chlamydial inclusions, or to other spatial constraints, that these geometric effects are then what inhibits detachment of RBs and triggering for differentiation into EBs. Our simulation analysis suggests that the relationships we established between the various geometrical attributes could be expected to be equivalent across various models of persistence. Because different persistent models interrupt development in various ways, the influence of the stress on all geometric factors could differ and must be considered, but our threshold relationships should be robust across models. Further, the size of RBs under in vitro
stressed conditions, such as the addition of penicillin, may depend on the number of RBs present when the stressor is added. Our results are not influenced by the observed physical relationship between the number of RBs and their size, but our results are essentially based on the initial condition of a certain number of RBs, their size, and other geometric properties of the system in predicting whether or not RB detachment and differentiation will occur.
In this study we have analyzed chlamydial infection and predicted the conditions that will lead to persistence or normal development, according to our T3S contact-dependent hypothesis. Our investigation was carried out through the development of a novel mathematical model that extensively advanced previous similar analyses. Modeling can predict specific relationships between pathogen characteristics and infection parameters and it can determine threshold levels critical for development. We have included considerable detail of geometrical features and investigated a deterministic ordinary differential equation model and also a detailed stochastic analog of these equations. This level of detail was useful in accurately modeling persistent infection. As far as we are aware this is the first time that bacterial development and replication have been modeled in such detail. This model is useful not only to verify the plausibility of the T3S contact dependent hypothesis of development, but the quantification also produces experimentally testable predictions regarding the threshold between normal development and persistence.
The study of chlamydial biology is reputedly difficult because of the superimposed complexity of the obligate intracellular and developmental life cycle, and the genetic intractability of the organisms. In practical terms, this means that genetic characterization is limited to sequence analysis, that a molecular Koch postulate-type analysis for virulence genes is not possible, and that any attempt at molecular characterization suffers from the constant threat of contamination by host cell components. Alternative strategies such as biomathematical modeling therefore provide avenues for developing testable hypotheses that could not be obtained otherwise. Experimental verification of the predicted model results can form the basis for further modeling which may in turn generate motivation for further experimentation. Several lines of experimentation should be undertaken based on the results of the biomathematical model. For example, a prediction is that the length of the T3S needle directly impacts detachment of the RB from the inclusion membrane. Since in other pathogens the length of the T3S needle is tightly controlled and length is directly related to T3S activity [25
], this concept begs for experimental verification in Chlamydia
, for example using newly developed cryo-transmission electron microscopy or tomography methodologies. Indeed a tantalizing possibility is that the needle length varies during development and/or even between Chlamydia
species. Another prediction from this study is that the number of inclusions per cell is directly related to the productive versus persistent growth outcome. Spontaneously arising mutations of the chlamydial gene incA
, encoding an inclusion membrane protein, have been described that are altered in inclusion fusogenicity within the infected cell [24
]. Although these do not represent true isogenic mutants, a comparison of the normal versus persistent growth outcome using standard methodologies in these variants compared with their "wild-type" counterparts would be very worthwhile. The fact that the incA
gene product is itself a T3S effector protein makes the case for this phenotypic comparative analysis even more compelling.
Our modeling has shown that increasing the RB radius, or the length of needles mediating contact between RBs and the CIM, or the number of inclusions, will contribute to the establishment of the persistent mode of growth. The RB radius is the most important determinant for deciding if persistent infection will ensue and we predict that it will always occur if the average RB radius is greater than ~1.3 μm. The RB radius is also the main determinant of the EB yield and there is an optimal RB radius for producing the greatest EB yield. However, the EB yield is also dependent on the number of inclusions. Together, there is a relationship between the RB radius and the number of inclusions that predicts whether persistent growth or normal development will occur within a host cell. Within an inclusion, the RB radius and the length of the T3S needles determine the level of established contact between the RB and CIM and the level of T3S activity. We calculated an analytical curve for the threshold between infection outcomes and also predicted the maximum RB numbers and EB yield based on these variables.
We were initially surprised that the length of the T3S needles was as sensitive as it was towards the outcome of infection. The physical structure, size, and density of the needles on the chlamydial surface are crucial and we strongly recommend that experiments be performed to obtain measurements of these quantities over the developmental cycle for several Chlamydia
species. In the early 1980's Matsumoto published a series of papers providing such quantities for surface projections of Chlamydia psittaci
strain CAL-10 [10
] that have since been proposed to correspond to the T3S injectisomes and their outermost needle structures. This work was carried out long before T3S was discovered and the results were not utilized for the following two decades. However, the experimental measurements have not been repeated and although surface projections have been observed on other chlamydial species [34
] quantitative measurements have not yet been obtained. Greater understanding of chlamydial T3S and the contact relationship between the RB and the CIM is required. Experiments should also be designed to test all the outcomes predicted in this study. In vitro
experiments can be used to match against the threshold relationships presented in this study. These threshold curves are easily understandable and the relationships can be tested. If the hypothesis and model outcomes are verified experimentally, then the data obtained can be used to further fine-tune the model and greater complexity can also be introduced, leading to further predictions.