The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood. HSPGs are thought to play a role in the initial binding interactions. Since cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report the novel finding that FGF2 is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. Unexpectedly, we found that FGF2 binds directly to EBs, where it may function as a bridging molecule to facilitate interactions of EBs with FGFR on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of Erk1/2 signaling but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the HMW isoforms of FGF2 and increased amounts and release of the LMW isoforms. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for FGF2 and that this accounts for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread ().
Working model for how C. trachomatis co-opts the FGF2 signaling pathway to enhance infection.
By several criteria, we found that the binding of FGF2 to EBs appears to be quite specific. We postulate that FGF2 functions as a bridging molecule, by binding simultaneously to EB surface proteins and to HSPGs and/or FGFR on the host cell (). Co-localization of FGF2 with purified EBs was not diminished by pre-treatment of EBs with heparinase, suggesting that FGF2 binding to EBs was not mediated by HSPGs. However, EB-FGF2 binding may involve synergistic interactions with OmcB, a cysteine rich outer membrane protein found in most chlamydial species that contains a HS binding domain and mediates attachment to HSPGs 
. We further show that a consequence of FGF2 binding to EBs is that activated FGFR and FRS2α are recruited to the site of bacterial binding, facilitating uptake. Activation of FGFR, however, is not required for the Chlamydia-induced upregulation of fgf2
transcription, production, processing, and release.
In previous work, we have shown that phospho-PDGFR co-localizes with bound EBs, but other growth factor receptors, such as EGFR, are not recruited 
, suggesting selectivity and specificity in growth factor receptor recruitment. Although PDGFR signaling has been shown to stimulate FGFR under some conditions, we did not find evidence for cross-talk in the setting of C. trachomatis
-induced activation of FGFR in the absence of serum. However, using informative pharmacologic inhibitors, we found evidence that the PDGFR and FGFR pathways may function redundantly in C. trachomatis
entry. Growth factor signaling may also be important at steps downstream of entry, for example by providing pro-survival signals for the host cell 
We found that C. trachomatis
infection upregulates FGF2 transcription, production, and secretion. FGF2 transcription and production were upregulated within the first 12 hpi and continued for at least 24 hpi. This process was independent of FGFR activation, but involved biphasic activation of Erk1/2 kinases. Early Erk1/2 activation was independent of de novo bacterial protein synthesis. We speculate that the first wave of Erk1/2 activation may involve TARP, a chlamydial type III secreted protein that is present in EBs and then injected into the host cell cytoplasm upon bacterial binding. TARP has recently been shown to bind to SHC1 
, a Src homology-2 domain containing protein that is recruited to and phosphorylated by FGFR (and EGFR) upon its activation and that subsequently mediates Erk1/2 activation 
. Thus, recruitment and activation of FGFR may facilitate or synergize with TARP and other chlamydial factors to activate Erk1/2 as well as to enhance bacterial internalization. Indeed, the failure of the FGFR inhibitor PD173074 to completely block C. trachomatis
-induced Erk1/2 activation may result from the redundant involvement of chlamydial factors, such as TARP, together with the activation of FGFR. The second peak of Erk1/2 activation required active bacterial protein synthesis. This finding suggests either that a de novo synthesized chlamydial protein (as opposed to an immediate early protein such as TARP) is secreted from the vacuole to activate the Erk pathway, or that Erk is activated in response to vacuolar and/or bacterial intracellular growth. In any case, we conclude that the two waves of Erk1/2 activation, which occur through separate pathways, contribute to upregulation of FGF2 expression.
Our work also reveals that midway through the chlamydial intracellular life cycle, there is a loss of the HMW FGF2 isoforms and a concurrent increase in the LMW isoforms (16/18 kDa). The change in the spectrum of FGF2 proteins was independent of Erk1/2 activation but required bacterial intracellular growth. We favor the idea that the HMW forms are degraded rather than processed into the 16 and/or 18 kDa form. The molecular identity of the 16 kDa isoform is currently under investigation, but may represent a previously reported pepstatin-sensitive acid proteinase cleavage product 
We considered several possible mechanisms for the change in FGF2 isoforms. First, the change in isoforms could result from a shift in the translation initiation sites. However, a modified pulse-chase experiment, in which we followed the isoform distribution after inhibiting host protein synthesis at 6–12 hpi with cycloheximide, demonstrated that the change in FGF2 isoforms still occurred, eliminating this possibility. Second, we tested whether the Chlamydia
protease CPAF might be responsible for degrading or processing the FGF2 isoforms, but in vitro experiments using recombinant CPAF ruled out this notion. Third, and most likely, the change in FGF2 isoforms may be a consequence of C. trachomatis
-induced activation of a host protease, as pretreatment with lactacystin or MG132 prevented the isoform change. In hematopoietic cells, thrombin has been reported to process the HMW FGF2 isoforms into an 18 kDa species 
, though this process seems less likely in epithelial cells that lack thrombin. However, it is possible that an as yet-identified bacterial-encoded protease could account for the processing.
Finally, we demonstrate that by enhancing secondary rounds of infection, C. trachomatis-induced up-regulation of FGF2 is physiologically important. Conditioned media from C. trachomatis-infected cells (CT-CM) stimulated EB binding. Two pieces of evidence provide support that FGF2 contributed to the activity of the CT-CM. First, there was an increase in FGF2 levels in CT-CM compared to CM isolated from mock-infected cells. Second, immunodepletion of FGF2 from the CT-CM decreased its ability to stimulate EB binding, whereas depletion with a control antibody or an irrelevant antibody was without effect. In addition to stimulating EB binding, we speculate that FGF2 production enhances secondary rounds of infection by its prosurvival activity.
We found both similarities and differences in the HSPG-dependence and modulation of FGF2 signaling of serovar E compared to serovar L2. As observed with L2, serovar E binding to HeLa cells was stimulated by FGF2 in an HSPG-dependent manner but was not affected by depletion of host cell FGF2. Serovar E bound to FGF2 in vitro, though perhaps less avidly. It is intriguing to speculate that the absence of a functional heparan sulfate binding domain in the OmcB surface protein of serovar E 
may explain in part the decreased FGF2 binding (). Nonetheless, serovar E stimulated transcription, production, and processing of FGF2. Together these results suggest that serovar E activates the Erk pathway and FGF2 production similarly to serovar L2 and that it may utilize FGF2/HSPG-dependent pathway for binding. In the future, it will be interesting to determine whether FGFR signaling is activated upon serovar E binding.
Modulation of growth factor expression or distribution is an emerging theme in bacterial infections. Neisseria gonorrhoeae
infection induces expression, processing and release of amphiregulin, an epidermal growth factor (EGF) family member that is anti-apoptotic 
. H. pylori
infection stimulates HB-EGF production, which may contribute to cancer progression 
. C. pneumoniae
infection of cultured endothelial cells has been reported to increase FGF2 and PDGF production, which may be responsible for smooth muscle cell proliferation and intimal thickening in aortic tissues, and could account for its potential association with atherosclerosis 
In summary, our results demonstrate that C. trachomatis
co-opts FGF2 to enhance infection and bacterial spread (). Activation of the Erk1/2 pathway, either at the time of binding and entry or during subsequent intracellular growth, leads to increased fgf2
transcription and production. In addition, intracellular growth activates host protease(s), resulting in alterations in the distribution of FGF2 isoforms and enhanced release of the secreted forms during host cell lysis. The released FGF2 serves as a bridging molecule to facilitate subsequent rounds of binding, entry, and intracellular development. This positive feedback loop amplifies secondary infection as well as promoting efficient bacterial spread. FGF2 may play additional roles in the pathogenesis of chlamydial infection, by potentiating the inflammatory response, by inhibiting apoptosis, or by modulating gene expression 
. In the future, it will be interesting to determine whether FGF2 contributes to pelvic inflammatory disease and whether other human adapted chlamdyial species, such as C. pneumoniae,
utilize FGF2 to enhance infection.