Experimental results presented here demonstrate that for a number of chlamydial strains and species, the intracellular inclusions indeed acquire caveolin-2 during the developmental cycle. These strains include C. trachomatis serovars A, B, C, E, F, and K, C. pneumoniae (A39), and GPIC (Table 1). C. trachomatis serovars E, F, and K, and the C. pneumoniae and C. caviae species of Chlamydophila previously were shown to enter host cells via lipid rafts. Their vacuoles/inclusions were demonstrated to acquire host cell caveolin-1 at entry and to accumulate it during later stages of infection [18, 19].
Consequently, in host cells that express both caveolin proteins, inclusions of these strains display caveolin-2, as well as caveolin-1. However, in this same cell type, inclusions of serovars A, B, and C, are marked only by caveolin-2, a result implying that for chlamydial inclusions this acquisition can occur independently of caveolin-1. This implication is somewhat surprising since there is experimental evidence demonstrating that caveolin-2 usually can not exit from the Golgi and traffic to the plasma membrane in the absence of caveolin-1[25, 26]. However, if recombinant caveolin-1 DNA is expressed by caveolin-1 negative cells, then caveolin-2 can transit from the Golgi and be delivered to the plasma membrane, as a hetero-oligomeric complex with caveolin-1[25, 26, 30, 31].
To confirm that transport of caveolin-2 to chlamydial inclusions indeed might occur independently of caveolin-1, we asked whether chlamydial inclusions might similarly acquire caveolin-2 in FRT cells, a cell line that does not express caveolin-1 [27–29]. In our earlier report [19], we confirmed that FRT cells in fact do not express caveolin-1. Also, we demonstrated that whereas for some chlamydial strains, entry into those cells was dependent on intact lipid rafts (Table 1), in no case was entry dependent on caveolin-1 and caveolae. We show here that in the caveolin-1 negative FRT cells, inclusions of each of the above chlamydial strains in fact acquired caveolin-2. Moreover, since FRT cells do not contain caveolin-2 at the plasma membrane, pathogen-associated caveolin-2 could not have been acquired at entry. Rather, it had to derive from an intracellular source.
Chlamydial inclusions thus acquired caveolin-2 despite the fact that in the absence of caveolin-1 this protein usually does not traffic from the Golgi. We therefore suggested that the pathogen might influence the host cell and actually induce a transport and sorting pathway that normally may not actively function in the uninfected cell. Consistent with this suggestion is the fact that for all strains and species tested in FRT cells, if the pathogens were inactivated by prior EB treatment with UV irradiation, none of the EB containing vesicles acquired caveolin-2. In contrast, in HeLa cells, there were low levels of caveolin-2 associated with vesicles containing UV-treated EBs. The source of these low levels of caveolin-2 on vesicles/inclusions in HeLa cells is not yet clear. Perhaps the EBs are acquiring minimal amounts of caveolin-2 from the membranes of these cells upon entry. This suggestion is supported by the fact that the vesicles of these UV irradiated EBs contain both caveolin-1 and -2 (data not shown); suggesting that the proteins might be co-localizing in hetero-oligomeric complexes.
It is well known that Chlamydia-containing vacuoles and later, the developing inclusions, are able to intercept and fuse with exocytic vesicles of the biosynthetic pathway that originate from the Golgi [6, 7]. These Golgi-derived vesicles provide the inclusions with sphingolipids and perhaps other key metabolites [7, 32]. In addition, the ability of at least some chlamydial strains to intercept these Golgi-derived vesicles is dependent on pathogen viability and therefore likely on gene expression. Considering these points, and the fact that caveolin-1/2 hetero-oligomers traffic to the plasma membrane from the Golgi [25, 26, 30, 31], it is reasonable to suggest that the previously described pathogen induced interception of Golgi-derived vesicles might account for the acquisition of caveolin-2 by inclusions, as seen in the current study. However, although studies by others indicate that all chlamydial strains appear to intercept vesicles of the biosynthetic pathway, not all strains acquire caveolin-2 and this fact provides a strong counter argument. Thus for example, in the current study LGV (L2) inclusions did not acquire caveolin-2 (Table 1), although they have been reported to intercept Golgi-derived vesicles of the biosynthetic pathway [7]. Furthermore, in the current study, acquisition of caveolin-2 by inclusions of some strains was independent of caveolin-1, while among other strains, inclusions acquired both caveolin proteins. Thus there may be more than one pathway by which inclusions acquire caveolin-2. Importantly, acquisition of caveolin-2 by inclusions of any particular strain did not correlate with entry by a raft-mediated pathway. In contrast, acquisition of caveolin-1 at early as well as late stages in pathogen development did correlate with a route of entry involving lipid raft microdomains. Together, these facts are consistent with the conclusion that acquisition of caveoloin-1 and -2 can represent independent and distinct processes. As expected, similar incubations with anti-caveolin-3 demonstrated this component was not present in any of these cells whether infected with Chlamydiae or not.
To date, no chlamydial protein has been identified that might be secreted into the host cell cytosol to influence host intracellular trafficking. However, several chlamydial proteins, termed Incs, have been identified in the inclusion membrane [33–35]. Several of these Inc proteins have cytoplasmic domains, making them potential mediators for interactions with the host that might influence trafficking [36]. One of these proteins, IncG, interacts with host protein, 14-3-3β [37]. This particular interaction can not underlie the trafficking of caveolin-2 to the inclusions since GPIC (C. caviae) and C. pneumoniae (AR39) do not express IncG [37], although, as shown here, they do acquire caveolin-2. Furthermore, the reverse also is true. Thus, as demonstrated above, mouse pneumonitis strain, MoPn and LGV (L2) do not acquire caveolin-2, but they do express IncG [37]. There are, however, other Inc proteins and they may underlie the phenomena we have presented here.
Despite their presence on chlamydial inclusions, it remains unclear what role, if any, caveolin proteins may play in the developmental cycle of Chlamydia. As noted above, all ten of the chlamydial strains and species we examined were able to enter caveolin-1 negative FRT cells [19]. Since these caveolin-1 negative cells do not express caveolin-2 at the plasma membrane [25, 26], it would appear that for any of these strains neither of the caveolin proteins is necessary for entry per se. Although following internalization all ten strains and species remained viable in the FRT cells [19], the only strains able to generate large inclusions in the FRT cells were GPIC which acquires both caveolin-1 and -2 in caveolin-positive cells, and MoPn which acquires neither caveolin protein. Despite these latter two instances, our findings remain consistent with the possibility that caveolin-1 may yet play a post entry role in the development of at least some strains. The fact that successful MoPn development does occur in inclusions that acquire neither caveolin-1 nor -2 [19] (and current study) could mean that neither caveolin-1 nor -2 has a necessary role in the development of MoPn. The ability of GPIC to develop mature inclusions in FRT cells where it acquires only caveolin-2, despite acquiring both caveolin proteins in caveolin-1 positive cells, may imply that for GPIC caveolin-2 rather than caveolin-1 is a key for development. Likewise, inclusions of C. trachomatis serovars A, B, and C, whether developing in HeLa or FRT cells, display only caveolin-2, suggesting a potentially broader chlamydial requirement for the caveolin-2 protein during development.
The series of merged optical sections demonstrating the association of caveolin-2 with the inclusions (Figure 2) implies that caveolin-2 is in apposition to pathogen cells that are located at the inclusion membrane. Nevertheless, the caveolin-2 is not in direct contact with the pathogens. Caveolin is not a transmembrane protein, a fact that can be deduced from the experimental finding that cell-surface biotinylation does not label caveolin proteins [38]. Thus, these membrane proteins are not accessible from the extracellular milieu, and this originally extracellular membrane surface, is topologically equivalent to the lumen of the chlamydial inclusion. Hence, whereas our findings imply a specific association between the pathogens and caveolin-2, this association appears to be indirect. The paucity of information concerning caveolin-2 does not enable us to suggest an identity for the linking molecule. Moreover, how this indirect association between caveolin-2 and the pathogen might relate to the protein's function in the chlamydial developmental cycle, or in its acquisition by the pathogen, is also not yet clear.