Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA

Chlamydia invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors – TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1–23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.


Introduc.on
Chlamydia trachoma.s is an obligate intracellular bacterium which infects ocular and genital epithelial cells, causing pelvic inflammatory disease, tubal factor inferRlity, ectopic pregnancy, and preventable blindness 1 .Chlamydia features a biphasic developmental cycle divided between metabolically quiescent elementary bodies (EBs) which invade host cells and vegetaRve reRculate bodies (RBs) which replicate inside membrane vacuoles termed inclusions 2 .Given its obligate intracellular nature, entry into host cells is essenRal for pathogen survival; consequently, Chlamydia possesses a robust suite of resources that regulate its uptake.Invasion also underpins pathogenicity, as it promotes access to the intracellular niche where it hijacks several host cell processes.IniRal interacRon with host epithelial cells is mediated by a reversible electrostaRc interacRon between a Chlamydia adhesin and host heparin sulfate proteoglycans 3 .Subsequently, Chlamydia engages mulRple host receptors and delivers a variety of protein effectors via a type III secreRon system [4][5][6] .Signaling from the effectors TarP and TmeA establishes a robust acRn modulatory network that induces the assembly of acRn-rich structures that engulf invading bacteria [7][8][9] .
The resultant acRn recruitment is characterisRcally highly localized to invading EBs and exhibits rapid kineRcs of acRn recruitment and turnover, such that acRn network assembly and disassembly occurs within 200 seconds 7,10 .The majority of studies regarding chlamydial invasion focus on the mechanism of acRn recruitment, while the process of disassembly at the end of invasion remains understudied, despite evidence poinRng to its importance to elementary body uptake.We recently reported that altering the dynamics of acRn turnover correlated with decreased invasion efficiency 7 .
Although mulRple uptake mechanisms have been implicated as potenRal pathways for C. trachoma.sinvasion [11][12][13] , the role of host dynamins during this process has been controversial.Dynamins are large GTPases that form oligomeric structures in a helical configuraRon around membrane lipids during clathrin-and caveolin-mediated endocytosis, mediaRng scission of vesiculated cargoes following GTP hydrolysis 14 .They are comprised of a catalyRc G domain, a lipid-binding pleckstrin homology (PH) domain, and a proline-rich domain (PRD) that interacts with Src homology 3 (SH3) domain-containing proteins 15 .
Absent acRvaRon, dynamins possess low intrinsic GTPase acRvity and assemble into dimers or tetramers 16 .These are uRlized to generate higher-order oligomers such as half-rings, rings, and helices, the la'er forming at the collar of invaginaRng vesicles 17 .GTP hydrolysis induces a conformaRonal change along the oligomer that promotes constricRon followed by vesicle scission, prompRng rapid turnover of the dynamin superstructure 18 .Dynamin oligomerizaRon is promoted by several effectors, including SH3 domaincontaining proteins 19 , acRn filaments 20 , and membrane lipids 21 .Many known effectors of dynamin oligomerizaRon are present at C. trachoma.sinvasion sites, raising the possibility that that dynamindependent scission is uRlized during terminal stages of this process.Several host proteins present during invasion are also directly or indirectly targeted by chlamydial effectors 4,[22][23][24] , highlighRng the level of control the pathogen exerts on the invasion process.
RNA interference of dynamin 2 (Dyn2) restricted C. trachoma.suptake 12 , bolstering support for a dynamin-dependent uptake mechanism.In contrast, pretreatment with the dynamin inhibitor MiTMAB, which targets the PH domain of dynamin, did not alter C. trachoma.sinvasion efficiency 11 .However, this study also idenRfied that SNX9, a BAR domain protein which promotes dynamin oligomerizaRon, is recruited during invasion, and that its depleRon a'enuated Chlamydia entry.Furthermore, overexpression of dominant negaRve GTPase-inacRve Dyn1 K44A did not prevent C. trachoma.sinfecRon of HeLa cells 25 .
Notably, this study did not invesRgate C. trachoma.suptake frequency and did not target Dyn2, the predominant dynamin species expressed in epithelial cells.In this study, we aim to reconcile the controversial involvement of host dynamins during C. trachoma.sentry, monitoring its involvement using a series of high-resoluRon tools previously employed to characterize the regulaRon of acRn remodeling during invasion 7 .
Given that dynamin interacts both with acRn itself and with several proteins that regulate acRn polymerizaRon 20,[26][27][28][29] , it has become increasingly apparent that the dynamin GTPase cycle and acRn polymerizaRon are co-regulated.On this basis, the secreted effectors TarP and TmeA, which are themselves regulators of acRn dynamics, likely also regulate host Dyn2 during invasion.Once secreted, TmeA associates with the plasma membrane and acRvates N-WASP, followed by Arp2/3 complex acRvaRon and nucleaRon of acRn polymerizaRon 9,30 .Likewise, TarP signaling acRvates host signaling proteins such as Rac1, PI3K, and the WAVE2 complex, in addiRon to recruiRng the acRn effectors formin and Arp2/3 7,31 .
Many host proteins associated with TarP and TmeA signaling are known to regulate Dyn2 oligomerizaRon, such as cortacRn 32 , EPS8 33 , profilin 32,34 and the Arp2/3 complex 35 .Thus, in addiRon to the previously established role of TarP and TmeA signaling as synergisRc effectors of rapid acRn kineRcs 7,8 , it is likely that they have a role in Dyn2 localizaRon dynamics during Chlamydia entry.
Here, we demonstrate that Dyn2 is co-recruited alongside acRn during Chlamydia invasion and coordinates efficient engulfment of the pathogen.This phenomenon is conRngent upon signaling from both TarP and TmeA, such that TarP signaling is necessary for local recruitment of Dyn2, whereas TmeA signaling acRvates Dyn2 by promoRng oligomerizaRon.The applicaRon of the Dyn2 acRvator Ryngo 1-23, which promotes oligomerizaRon and stabilizes Dyn2 polymers rescues invasion defects associated with TmeA deleRon, enhancing its entry efficiency, and restoring kineRcs of Dyn2 and acRn recruitment and turnover to near wild-type levels.Further, we discovered that acRn disassembly is dependent on Dyn2 funcRon, thus ensuring the compleRon of invasion.Altogether, these findings resolve the long-standing controversy within the field, providing a novel regulatory funcRon which accounts for both rapid assembly and disassembly of Chlamydia engulfment machinery in addiRon to a comprehensive model for the uRlizaRon and regulaRon of host Dyn2 during C. trachoma.sinvasion.They also highlight cooperaRon between TarP and TmeA and illustrate the broader impact of establishing their respecRve acRn networks beyond the formaRon of engulfment structures.

Dynamin 2 and ac.n are co-recruited during Chlamydia entry
Since conflicRng reports persist regarding host dynamin-2 (Dyn2) involvement during C. trachoma.sinvasion, we revisited the quesRon and evaluated its recruitment in greater detail using quanRtaRve imaging approaches.We first determined whether Dyn2 was present within entry sites by co-transfecRng Cos7 cells with GFP-Dyn2 and iRFP670-LifeAct prior to infecRon with wild-type C. trachoma.s(MOI=20) stained with the red fluorescent dye CMTPX.Using live-cell confocal microscopy, we monitored Dyn2 and acRn recruitment during entry, acquiring images at 20 second intervals (Fig. 1A).As previously reported 7 , we observed rapid acRn recruitment, which was concomitant with arrival of Dyn2 and resulted in rapid uptake of Chlamydia, characterized by loss of CMTPX-CTL2 signal within 200-300 sec.In contrast, expression of mutant Dyn2 K44A (Dyn2 DN), which is defecRve in GTPase binding and hydrolysis and cannot mediate vesicle scission (Fig. 1B), prolonged internalizaRon to 400-700 sec.Delayed pathogen uptake following Dyn2 DN expression could arise from several potenRal sources, such as inefficient Dyn2 recruitment, impaired acRn dynamics, or disrupRons within the Dyn2 GTPase cycle that prevent vesicle scission.To address each of these possibiliRes, we employed a previously established protocol for quanRtaRvely assessing host protein recruitment dynamics during Chlamydia invasion, starRng by characterizing Dyn2 WT and Dyn2 DN recruitment dynamics (Fig. 1C).While both Dyn2 WT and Dyn2 DN were recruited during entry, we noted that Dyn2 DN achieved peak mean fluorescence intensity (MFI) roughly 80 seconds later than Dyn2 WT and persisted within entry sites for a longer duraRon, indicaRng that rapid recruitment of Dyn2 and subsequent rapid entry of Chlamydia is conRngent upon Dyn2 GTPase acRvity.To further substanRate this claim, we converted Rme-lapse images of acRn, Dyn2, and Chlamydia into kymographs, upon which we indicated the start (i.e.iniRaRon of acRn/Dyn2 recruitment) and end (i.e.loss of EB fluorescence) of invasion (Fig. 1D, S1).The duraRon between iniRaRon of acRn/Dyn2 recruitment and pathogen entry was prolonged by expression of Dyn2 DN (Fig. 1D,E), such that Chlamydia uptake in cells expressing Dyn2 WT occurred within 180 sec, which was delayed by over two-fold (380 sec) when Dyn2 DN was expressed.Moreover, slow pathogen uptake following Dyn2 DN expression coincided with slower Dyn2 recruitment and turnover (Fig. 1F,G), reducing the rate of Dyn2 recruitment by 40 percent and turnover by 60 percent compared to Dyn2 WT.Altogether, these data indicate that Dyn2 is corecruited alongside acRn during Chlamydia entry, and that Dyn2 GTPase acRvity is necessary for efficient recruitment dynamics and rapid pathogen entry.

Dynamin 2 inhibi.on restricts Chlamydia entry and ac.n turnover
The recruitment of Dyn2 alongside its role in facilitaRng rapid pathogen entry suggests that dynamindependent uptake is an important component of Chlamydia invasion.Previous reports indicate that Dyn2 self-assembly and acRn polymerizaRon are co-regulated 26,29,35 , such that delayed Chlamydia entry following Dyn2 disrupRon may be due to defecRve acRn polymerizaRon.To test this, we disrupted Dyn2 acRvity via pharmacological inhibiRon or by RNA interference prior to monitoring acRn recruitment during Chlamydia invasion.Since co-overexpression of Dyn2 DN and acRn may arRficially influence acRn dynamics, we instead inhibited endogenous Dyn2 using the dynamin inhibitor Dynasore, which mimics Dyn2 DN by restricRng Dyn2 GTPase acRvity and subsequent scission (Fig. 2A).Furthermore, we were limited to ~50% Dyn2 knockdown via RNA interference (Fig. 2B), as excessive Dyn2 depleRon prevented cell adherence and cell proliferaRon, rendering these cells unsuitable for further analysis.Nonetheless, we noted that both 25 µM Dynasore treatment and parRal siRNA depleRon of Dyn2 a'enuated acRn dynamics during CTL2 WT invasion (Fig 2B ), resulRng in prolonged acRn retenRon within entry sites.InteresRngly, acRn recruitment kineRcs were largely unchanged by Dyn2 disrupRon, yielding comparable rates across all condiRons (Fig. 2C).In contrast, acRn turnover was significantly a'enuated by both Dynasore treatment and Dyn2 siRNA knockdown, with Dynasore treatment halving the acRn turnover rate, while Dyn2 siRNA treatment slowed acRn turnover by 25 percent (Fig. 2D).Given the importance of rapid acRn turnover kineRcs toward efficient invasion 7 , it is possible that Dyn2 inhibiRon (or absence) prolongs Chlamydia entry through defects in acRn turnover.In support of this noRon, we observed that both inhibiRon and depleRon of Dyn2 delayed Chlamydia entry by roughly two-fold (Fig. 2E,F), comparable to the delay observed following Dyn2 DN overexpression (Fig. 1E), indicaRng that acRve Dyn2 is required for efficient acRn turnover and rapid Chlamydia entry.Moreover, we observed a comparable a'enuaRon in wild-type Chlamydia entry efficiency following Dyn2 inhibiRon (Fig. 2G) or siRNA depleRon (Fig. 2H), reducing Chlamydia uptake by roughly 20 percent.Therefore, Dyn2 acRvity regulates acRn turnover during invasion such that disrupRon of Dyn2 impedes acRn depolymerizaRon within entry sites.

Signaling from both TarP and TmeA is required for dynamin-dependent entry
Several studies have indicated that mutant Chlamydia strains harboring TarP and TmeA deleRon or lossof-funcRon mutaRons exhibit substanRally dysregulated pathogen entry 7,36,37 .As such, we monitored invasion of Chlamydia mutant strains lacking either TarP or TmeA (ΔTmeA, ΔTarP) or both (DKO) to determine if their respecRve routes of entry were affected by Dyn2 inhibiRon or depleRon.Loss of either TarP or TmeA rendered their respecRve invasion processes resistant to Dyn2 inhibiRon (Fig. 2G), likely indicaRng the uRlizaRon of an alternaRve entry mechanism, i.e. fluid-phase uptake, which is dynaminindependent (Fig. S2).Entry efficiency of these strains were similarly insensiRve to Dyn2 depleRon via RNA interference (Fig. 2H), confirming that Dyn2 does not contribute to pathogen invasion following TarP or TmeA deleRon.Finally, we noted that cis-complementaRon of the DTarP and DTmeA mutants (cis-TmeA, cis-TarP) restored Dynasore sensiRvity (Fig. 2G).
For the ΔTarP mutant, Dyn2 dispensability was unsurprising given the spaRal profiles of acRn exhibited by this mutant, which assembles structures typically associated with fluid-phase uptake, such as large blooms and mini-ruffles 38 (Fig. S2A,E).Indeed, ΔTarP EBs frequently colocalized with the fluid-phase marker Dextran-Alexa Fluor 647; 40 percent of EBs were dextran posiRve within 20 minutes post-entry (Fig. S2F,G).In contrast, the DTmeA mutant retained punctate recruitment of acRn characterisRc of wildtype EBs (Video S2,3) and exhibited lower incidence of dextran colocalizaRon (Fig. S2F).Thus, invasion of DTmeA EBs is mechanisRcally disRnct from ΔTarP, adopRng a spaRal configuraRon that may benefit from Dyn2 acRvity.As such, the apparent insensiRvity of DTmeA EBs toward Dyn2 inhibiRon might reflect that Dyn2 is required for entry, but present in a non-funcRonal state that rendered inhibiRon by Dynasore moot, which will be addressed in detail later in this study.Altogether, our data unequivocally reveal that dynamin-dependent uptake is an important component of C. trachoma.sinvasion which is conRngent upon both TarP and TmeA signaling, wherein each effector likely regulates different invasion-associated aspects of Dyn2.
We next tested the role of PI3K/Vav2 signaling, which is one of the Rac-acRvaRng pathways linked to TarP, the other being Abi1/Eps8/Sos1 signaling 31 (Fig. 4D).To determine the funcRonal outcome of PI3K signaling toward Dyn2 regulaRon, we monitored the invasion of wild-type and ΔTmeA EBs in the presence of the PI3K inhibitor Wortmannin (100 nM).Pretreatment with Wortmannin yielded intense and long-lasRng Dyn2 localizaRon relaRve to mock at wild-type entry sites (Fig. 4A,B) and a'enuated the rate of Dyn2 turnover (Fig. 4H), consistent with PI3K signaling through Rac (Fig. 3C,H).InteresRngly, PI3K inhibiRon did not alter Dyn2 recruitment during ΔTmeA invasion (Fig. 4A,B), indicaRng that in absence of TmeA, Dyn2 is not in its proper context to be affected further by wortmannin treatment.Moreover, wortmannin pretreatment did not alter the invasion efficiency of any strain tested (Fig. 4C) yet induced a significant delay in CTL2 WT uptake (Mock = 180 sec, Wort = 320 sec) (Fig. 4E,F).This disparity may arise due to the enhanced sensiRvity of our kymograph-based internalizaRon assay (Fig. 4E,F), which employs quanRtaRve fluorescence-based live-cell imaging to idenRfy invasion defects.The former internalizaRon assay (Fig. 4C) relies on anRbody accessibility to measure invasion efficiency, a low-resoluRon approach with elevated likelihood of missing regulatory interacRons between host and pathogen.In summary, our data indicate that TarP signaling is essenRal for dynamin-dependent entry of Chlamydia and is required for local recruitment of Dyn2 within entry sites, while also regulaRng its retenRon as a consequence of the acRn network generated via the PI3K/Rac1 signaling axis.
TmeA and Dyn2 func.on.Although ΔTmeA EBs recruit Dyn2 in a highly localized and punctate manner, similar to CTL2 WT (Fig 3A , 4A), inhibiRon of funcRon via ectopic expression of dominant negaRve Dyn2 or 25 µM Dynasore treatment did not alter uptake duraRon or Dyn2 dynamics associated with ΔTmeA (Fig. S3).The apparent insensiRvity toward Dyn2 disrupRon following TmeA deleRon may reflect a lack of Dyn2 involvement during ΔTmeA entry, or that TmeA deleRon induces Dyn2 loss of funcRon.To disRnguish between these two possibiliRes, we employed the Dyn2 acRvator Ryngo 1-23, a small molecule compound that sRmulates Dyn2 oligomerizaRon in a manner comparable to short acRn filaments 39 .As such, we quanRfied Chlamydia entry arer 30 minute preincubaRon with 40 µM Ryngo 1-23, wherein DTmeA invasion efficiency was improved to near wild-type levels (Mock CTL2 WT = 79.8%,Ryngo ΔTmeA = 71.0%)(Fig. 5A).Moreover, this compound restored normal Dyn2 recruitment dynamics during ΔTmeA entry (Fig. 5B), generating a Dyn2 recruitment profile comparable to mock-treated CTL2 WT (Fig. 5D,G,H).Likewise, both mock CTL2 WT and Ryngo ΔTmeA were internalized within 180 seconds on average, which was prolonged to 240 seconds for ΔTmeA in absence of Ryngo (Fig. 5E,F), and that compound-assisted entry reduced the incidence of fluid-phase uptake (Fig. S2F).Taken together, these data suggest that Dyn2 oligomerization is defective when TmeA signaling is absent, and that Ryngo bypasses the requirement for TmeA signaling, enabling dynamin-dependent entry of ΔTmeA EBs.In contrast, invasion efficiency, Dyn2 localization dynamics, and duration of internalization associated with wild type CTL2 were all negatively affected by Ryngo (Fig. 5A-F).A possible explanation may be that joint activation of Dyn2 by both TmeA signaling and Ryngo administration results in Dyn2 hyperactivation that prevents normal completion of the Dyn2 GTPase cycle.Indeed, Gu et.al found that Ryngo 1-23 abrogated Dyn1 helical collar assembly, instead promoting stacked ring assembly (Fig. 5C), exhibiting reduced GTPase activity and attenuated vesicle scission compared to helices 39 .Additionally, CTL2 WT entry was comparably attenuated by either Dynasore-mediated inhibition of Dyn2 (Fig. 2G) or Ryngo-mediated Dyn2 activation (Fig. 5A), implying that dynamin-dependent entry of Chlamydia is sensitive to both hypo-and hyperacRvaRon of Dyn2.Finally, whereas Ryngo administraRon prior to infecRon with ΔTarP EBs restored localized recruitment of Dyn2 (Figs.S2C, S4B, Video S1), its recruitment was vastly dysregulated relaRve to wild-type (Fig. S4C,F,G) and failed to elicit rapid internalizaRon of the pathogen (Fig. S4E).Together, this implies that Dyn2 is not organized in a proper context within entry sites when TarP is absent despite restoraRon of recruitment by Ryngo.In contrast, Dyn2 dynamics and funcRon were restored by Ryngo treatment in ΔTmeA EB invasion because Dyn2 proteins were in a context that favors oligomerizaRon.In summary, these data indicate that TmeA signaling acRvates Dyn2, promoRng its oligomerizaRon in support of rapid dynamin-dependent entry of Chlamydia.In addiRon, the ordered roles of TarP and TmeA regarding Dyn2 funcRon highlights the previously reported collaboraRon between these two effectors.

Ac.n turnover is correlated with Dynamin 2 ac.va.on status and Chlamydia uptake
Previous studies have identified that TmeA deletion dysregulates the actin network generated by Chlamydia during invasion, causing poor actin retention and abnormally fast actin turnover [7][8][9] .Moreover, in this study, we have noted a functional link between Dyn2 activity and actin turnover, wherein actin recruitment was abnormally persistent upon pharmacological inhibition of Dyn2 or upon expression of Dyn2 K44A (Figs. 2B, S1F), resulting in delayed pathogen uptake.In light of these observations, we opted to evaluate the influence of the dynamin activator Ryngo 1-23 on actin kinetics to determine whether compound-mediated restoration of Dyn2 activity within ΔTmeA entry sites also restores normal actin dynamics.While administration of Ryngo prior to infection strongly increased the persistence of actin recruitment at entry sites of both wild-type and ΔTmeA EBs (Fig. 6A), relative to the respective mocktreated controls, the slowed turnover associated with the ΔTmeA mutant was indistinguishable from mock-treated wild type control (Fig. 6A-C, Video S2, S3).As expected, we observed that Ryngo treatment restored the duration of internalization of ΔTmeA mutants to levels of mock-treated CTL2 WT (Fig. 6E,F).However, when invasion signaling was intact, i.e. when TarP and TmeA are both present, the additional Dyn2 activation by Ryngo had a negative effect on actin turnover and pathogen uptake (Fig. 6A,F, Video S3).This paralleled the effects of Ryngo on Dyn2 recruitment (Fig. 5), underscoring a possible relationship between actin disassembly and Dyn2 turnover (Fig. 6D).Indeed, either insufficient Dyn2 acRvity (i.e., Dynasore treatment, Dyn2 DN, Mock/ΔTmeA; Fig. 2B-D) or Dyn2 hyperacRvaRon (i.e., Ryngo/CTL2 WT; Fig. 6A-F) results in similar dysregulated acRn turnover and delayed pathogen uptake.CollecRvely, our data is consistent with a model whereby acRn remodeling by TarP and TmeA, in addiRon to forming engulfment structures, also ensures Dyn2 recruitment and acRvaRon.With Dyn2 regulaRng acRn turnover, this selfcontained invasion mechanism ensures that disassembly of the invasion structures is properly coordinated with a successful scission event indicated by Dyn2 turnover.

Discussion
In this study, we conclusively demonstrated that C. trachoma.suRlizes host Dyn2 to complete invasion.Dyn2 funcRon is modulated by the effectors TarP and TmeA, which respecRvely mediate recruitment to invasion sites and acRvaRon by promoRng oligomerizaRon.Neither TarP nor TmeA possesses domains that mediate direct interacRon with Dyn2 to facilitate recruitment and oligomerizaRon; instead, TarP and TmeA modulate Dyn2 via their respecRve acRn networks.InteresRngly, Dyn2 influences acRn turnover, wherein perturbaRon of Dyn2 funcRon induces persistent acRn retenRon.This funcRonal interdependence consRtutes a self-regulaRng system, such that Dyn2 funcRon and pathogen engulfment are regulated by the acRn network assembled via TarP and TmeA signaling.Reciprocally, Dyn2 funcRon and subsequent membrane fission promotes acRn disassembly and mediates resoluRon of engulfment structures.
Moreover, TarP and TmeA signaling are sequenRally coordinated such that the essenRal steps of invasion are iniRated and completed.Specifically, we found that TarP signaling via PI3K/Rac1 coordinated iniRal recruitment and retenRon of Dyn2 within entry sites.Once recruited, Dyn2 is acRvated by TmeA signaling on the basis that defects associated with TmeA deleRon were rescued by administraRon of the small molecular acRvator Ryngo 1-23, which promotes Dyn2 oligomerizaRon.Moreover, these data are consistent with previous observaRons suggesRng that TmeA regulates la'er stages of invasion.Finally, our study provides several high-resoluRon methods for tracking pathogen uptake, enabling detailed analysis of host-pathogen interacRons underpinning Chlamydia entry, exceeding the limitaRons of previously employed techniques.In summary, we report that Dyn2 acRvaRon is an important component of Chlamydia invasion, which is regulated synergisRcally by TarP and TmeA to mediate scission of Chlamydiacontaining vesicles and iniRate turnover of host proteins following invasion.Altogether, findings underscore the high degree of control Chlamydia has over its invasion process.
TarP-deficient strains were incapable of localized and punctate Dyn2 recruitment, indicaRng that TarP signaling is required to prompt Dyn2 recruitment into a scission-competent configuraRon.Given that Dyn2 directly interacts with several TarP-associated acRn regulators, including cortacRn 32 , EPS8 33 , and profilin 32,34 , we propose that the acRn network generated by TarP signaling regulates Dyn2 funcRon.
Whether this interacRon is mediated by direct interacRon with acRn, which has been reported previously 20 , or by various signaling molecules recruited by TarP is not known.One possibility is that TarPmediated acRn remodeling induces changes to the local environment that enrich and retain Dyn2 at sufficient quanRRes to achieve funcRonality.For example, robust acRn polymerizaRon can promote membrane curvature to support binding of Bin/amphiphysin/Rvs (BAR) domain proteins, some of which (e.g., SNX9) are known Dyn2 interactors 40 .This would also account for temporal regulaRon of Dyn2, wherein the Rming of host protein recruitment influences both the concentraRon and orientaRon of Dyn2.
Although our study demonstrates that Dyn2 and acRn dynamics are funcRonally linked, a comprehensive model of Dyn2 involvement during invasion will require further characterizaRon of its recruitment and acRvaRon.
Recently, we reported that TarP signaling uniquely recruited host formins 7 , which uRlize profilin/acRn complexes to acquire monomeric acRn 41 , and are important regulators of acRn polymerizaRon during Chlamydia entry.Moreover, the Arp2/3 complex is extensively associated with Dyn2 acRvity [42][43][44] and collaborates with host formins to enhance acRn remodeling during invasion 7 .Robust acRn remodeling provides a mechanism to ensure Dyn2 recruitment at sufficient levels; consequently, the pathways employed by Chlamydia to mediate acRn nucleator acRvaRon are highly relevant points of Dyn2 regulaRon.For instance, we observed that TarP signaling via the PI3K/Rac1 axis, which regulates acRn polymerizaRon during invasion 31 , also governed Dyn2 retenRon within entry sites.There is also precedence for Dyn2 modulaRon of acRn remodeling, specifically insofar as disrupRon of Dyn2 dysregulates Rac localizaRon and impairs acRn dynamics within lamellipodia 45 , highlighRng that regulaRon of Dyn2 and Rac1 are funcRonally linked.Furthermore, acRn stability and Dyn2 oligomerizaRon are coregulated 28,29 , such that inhibiRon of Arp2/3 was sufficient to shir the balance of acRn dynamics toward net disassembly, prevenRng scission of phagocyRzed parRcles and increasing Dyn2 persistence 46 .Thus, destabilizaRon of invasion-associated acRn networks following Rac inhibiRon likely interferes with Dyn2 scission and subsequent turnover, yielding abnormally persistent signal.Conversely, Rac acRvaRon would promote Dyn2 funcRon, a role demonstrably fulfilled by TarP.
We also found that TmeA signaling promoted Dyn2 acRvaRon, wherein strains lacking TmeA exhibited defecRve uptake that could be rescued by Ryngo 1-23 administraRon.Several lines of evidence suggest that TmeA regulates Dyn2 via its previously established role in acRn remodeling 30,47,48 .In-vitro assays idenRfied that short acRn filaments sRmulate Dyn2 ring assembly 49 , and that Ryngo 1-23 promotes Dyn2 ring formaRon via a comparable mechanism 50 .Thus, one possibility is that membrane localized TmeA generates acRn filaments which scaffold the iniRal acRvaRon of Dyn2 at the plasma membrane.Signaling via the TmeA/N-WASP axis drives Arp2/3 acRvaRon, which synergizes with TarP signaling to promote acRn polymerizaRon and pathogen engulfment 30,47 .CollaboraRon between TarP and TmeA may addiRonally regulate Dyn2, wherein TmeA-mediated acRn polymerizaRon funcRons arer Dyn2 recruitment to promote oligomerizaRon.Indeed, both TarP and TmeA were necessary for dynamin-dependent entry, as strains lacking either effector were insensiRve to Dyn2 disrupRon, although the basis for their insensiRvity differed.How might the regulatory contribuRons of TarP and TmeA be disRnguished, given the shared importance of their respecRve acRn remodeling funcRons?For TmeA, the involvement of N-WASP might offer some clues.This nucleaRon promoRng factor harbors a proline-rich domain (PRD) that binds proteins with Src-homology 3 (SH3) domains.The SH3 domain-containing protein SNX9 interacts with dynamin and sRmulates Dyn2 oligomerizaRon 40 and is important for C. trachoma.sinvasion 51 .As such, interacRon between N-WASP and SNX9 might account for Dyn2 dependence toward TmeA signaling.Intriguingly, TmeA also bears similarity with the C. pneumonaie secreted effector SemD 52,53 , which recruits the BARdomain proteins PACSIN and SNX9 to induce membrane curvature and promote pathogen engulfment.On this basis, TmeA-mediated Dyn2 regulaRon could manifest via the formaRon of SNX9/Dyn2 heterodimers, providing a mechanism of Dyn2 modulaRon disRnct from its acRn remodeling funcRon.Therefore, there are at least two molecular interacRons that uniquely link TmeA signaling with Dyn2 funcRon.
While the precise nature of how TmeA signaling modulates the Dyn2 GTPase cycle remains unknown, analysis of Dyn2 mutants may provide insight toward TmeA/Dyn2 regulaRon, and perhaps the mechanism of Ryngo-mediated rescue.Studies regarding the formaRon of progressive higher-order dynamin oligomers have benefited from various mutaRons that affect protein-protein interacRons, GTPase acRvity, conformaRonal changes during constricRon, etc. Determining the exact mechanism of compoundmediated rescue following TmeA deleRon will require elucidaRng which oligomeric species of Dyn2 is induced by either Ryngo or TmeA signaling.MutaRons which prevent dynamin self-assembly (i.e.Dyn1 I670K 54 ) or those which ablate membrane associaRon (i.e.Dyn2 K562E 55 ) could be informaRve toward this end, as these mutants are membrane scission-deficient and are not rescued by Ryngo 50,56 .Our working model predicts that these mutants should likewise be unaffected by TmeA signaling.Dyn1 K/E exhibits reduced affinity for acRn filaments and is parRally rescued by Ryngo in-vitro 50 , whereas Dyn2ΔPRD cannot bind SH3 domain-containing proteins and is dominant-negaRve for endocytosis 57 .Studies incorporaRng these mutants could disambiguate whether TmeA signaling operates by mediaRng Dyn2/acRn interacRons, or by promoRng interacRon with SH3 domain-containing proteins like SNX9.Using this report as a foundaRon, future studies could interrogate the effects of each Dyn2 mutant during Chlamydia invasion and determine the precise nature of effector signaling toward dynamin-dependent entry.
InteresRngly, unlike ΔTmeA, Ryngo treatment impaired wild-type Chlamydia invasion, restricRng pathogen entry and yielding obvious defects in Dyn2 and acRn recruitment.One explanaRon may be that in certain contexts, Ryngo sRmulates Dyn2 oligomerizaRon into a scission-incompetent configuraRon.FRET analysis of dynamin oligomerizaRon found that Ryngo prompted the assembly of stacked Dyn2 rings around membrane tubules 50 , represenRng a lower-order oligomerizaRon state that achieved insufficient GTPase acRvity to induce membrane scission.As such, co-sRmulaRon of Dyn2 acRvaRon by both Ryngo and Chlamydia/TmeA signaling may interfere with the relaRve abundance of Dyn2 oligomeric species.Specifically, sRmulaRon with Ryngo is expected to generate a disproporRonate quanRty of Dyn2 rings which interfere with further oligomerizaRon steps.EliminaRon of Chlamydia-specific Dyn2 acRvaRon (i.e., ΔTmeA) may prevent oversRmulaRon, encouraging proper assembly of higher-order, scission-competent Dyn2 oligomers.Meanwhile, whereas Ryngo pretreatment restored local Dyn2 recruitment at ΔTarP entry sites, it failed to prompt rapid engulfment of ΔTarP EBs and had no rescuing effect on its entry efficiency, suggesRng that post-recruitment, Dyn2 needs to be primed for acRvaRon by Ryngo.
Finally, our study idenRfied that both Dyn2 and acRn turnover were co-regulated.MechanisRcally, Dyn2 turnover is intuiRve, occurring either during or shortly arer membrane scission as a funcRon of GTP hydrolysis 58,59 .As such, Dyn2-mediated scission of Chlamydia-containing vacuoles may intrinsically prompt Dyn2 turnover while also providing a signal to iniRate acRn turnover.IntervenRons which prevent dynamin-mediated membrane fission also accumulate F-acRn around tubulated membranes 60,61 , whereas scission is consistently associated with acRn turnover and sensiRzes acRn filaments toward cofilinmediated severing 29,62,63 .Furthermore, given that dynamin extensively interacts with acRn-associated proteins 28,[63][64][65] , post-scission turnover of acRn regulatory machinery alongside Dyn2 may shir acRn regulaRon toward turnover.Importantly, acRn polymerizaRon during Chlamydia invasion is both intricately regulated and pathogen-directed 66 ; consequently, turnover of acRn and other invasion-associated host proteins could be regulated disRnctly from turnover associated with rouRne engulfment of cellular cargoes (i.e., growth factors, transferrin).This could require addiRonal factors that fine-tune their funcRon and/or dynamics to accommodate pathogen-mediated uptake mechanisms.As such, further study is required to gain a more comprehensive perspecRve on host protein turnover post-invasion.
Overall, our findings of Dyn2 modulaRon by TarP and TmeA fit well with the proposed pathogendirected invasion model proposed by Byrne and Moulder 67 .While the majority of molecular studies of chlamydial invasion focus on acRn recruitment, we demonstrate here that la'er stages are also targeted by TarP and TmeA, highlighRng their central funcRon in invasion, comprising a self-contained signaling module capable of mediaRng the iniRal, middle, and end stages of invasion.

Cell and Bacterial Culture
Green monkey kidney fibroblast-like (Cos7) cells and cervical adenocarcinoma epithelial (HeLa) cells were cultured at 37˚ C with 5% atmospheric CO2 in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Thermo Fisher ScienRfic, Waltham, MA, USA) supplemented with 10 µg/mL gentamicin, 2 mM L-glutamine, and 10% (v/v) filter-sterilized fetal bovine serum (FBS).HeLa and Cos7 cells were cultured for a maximum of 15 passages for all experiments.McCoy B mouse fibroblasts (originally from Dr. Harlan Caldwell, NIH/NIAID) were cultured under comparable condiRons.Chlamydia trachoma.sserovar L2 (434/Bu) was propagated in McCoy cells and EBs were purified using a Gastrografin density gradient as described previously 68 .

Invasion Assay
C. trachoma.sinternalizaRon efficiency was conducted using HeLa cells and was performed as described previously 10 .Briefly, HeLa cells were seeded in 24-well plates containing acid-etched glass coverslips and allowed to adhere overnight.Cells were pretreated with Wortmannin (40nM), Dynasore (25µM), EHop-016 (10µM), or Ryngo (40µM) for 30 minutes prior to infecRon.Dyn2 siRNA or scramble RNA were transfected and allowed to incubate 24 hours prior to infecRon.Following inhibitor treatment or RNA interference, cells were infected with EBs derived from wild-type C. trachoma.sL2 (434/Bu), C. trachoma.s in which TarP, TmeA, or both were deleted by FRAEM (ΔTarP, ΔTmeA, ΔTmeA/ΔTarP), or C. trachoma.s in which TarP or TmeA expression was restored by cis-complementaRon (cis-TarP, cis-TmeA) at MOI=50.EBs were allowed to a'ach onto HeLa cells for 30 min at 4°C before rinsing coverslips with cold HBSS, followed by addiRon of supplemented DMEM prewarmed to 37°C, before incubaRng cells at 37°C for 10 min.Arer incubaRon, cells were stringently washed with cold HBSS containing 100 μg/mL heparin to remove any transiently adherent EBs before fixaRon in 4% paraformaldehyde at room temperature for 15 min.Fixed cells were labeled with a mouse monoclonal anR-MOMP anRbody (Novus Biologicals, Centennial, CO, USA #NB10066403), rinsed with 1x PBS, and fixed once more in 4% paraformaldehyde for 10 min.Next, cells were permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with rabbit polyclonal anR-Chlamydia trachoma.sanRbody (Abcam ab252762).Cells were then rinsed in 1x PBS and labeled with Alexa Fluor 594 anR-mouse (ThermoFisher #A11032, Waltham, MA, USA) and Alexa Fluor 488 anR-rabbit (ThermoFisher #A11034) IgG secondary anRbodies.Coverslips were mounted and observed on a Nikon CSU-W1 confocal microscope (Nikon, Melville, NY, USA), obtaining Z-stacks using a 0.3 micron step size across the height of the cell monolayer.
Monolayer Z-stacks were transformed via Z-projecRon according to maximal fluorescence intensity in ImageJ prior to quanRfying percent invasion efficiency as follows: total EBs (green) -extracellular EBs (red)/total EBs (green) x 100%.

Quan.ta.ve live cell imaging of Chlamydia invasion
Cos7 cells were seeded onto Ibidi µ-Slide 8-well glass-bo'omed chambers (Ibidi, Fitchburg, WI, USA) and allowed to adhere overnight prior to transfecRon.Cells were transfected with fluorescent proteins as indicated, using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA) according to manufacturer direcRons.TransfecRon was allowed to proceed overnight before replacing media with fresh DMEM + 10% FBS/2 mM L-glutamine and allowing protein expression to conRnue for a total of 24 hours post-transfecRon.TransfecRon efficiency was verified on a Nikon CSU-W1 spinning disk confocal microscope prior to applicaRon of DMEM containing Wortmannin (40nM), Dynasore (25µM), EHop-016 (10µM), or Ryngo (40µM).For RNA interference, Dyn2 siRNA or scramble RNA was co-transfected alongside GFP-acRn or mRuby-LifeAct and allowed to incubate for 24 hours prior to imaging.Wells were individually infected with CMTPX-labeled wild-type C. trachoma.sL2 (434/Bu), unless otherwise indicated, at MOI=20 and promptly imaged using a 60x objecRve (NA 1.40) in a heated and humidified enclosure.Images were collected once every 20 seconds for 30 minutes, with focal plane maintained using an infrared autofocusing system.Upon compleRon of the imaging protocol, the next well was infected and imaging repeated; mock-treated wells were imaged first to allow inhibitor treatment sufficient Rme to achieve inhibiRon.Images were compiled into videos using NIH ImageJ and analyzed to idenRfy protein recruitment events.The mean fluorescence intensity (MFI) of recruitment events was measured for each Rmepoint alongside the local background MFI of a concentric region immediately outside the recruitment event.Background MFI was subtracted from recruitment MFI for each Rmepoint and normalized as percent maximal fluorescence intensity for each Rmepoint, repeaRng this normalizaRon process for each recruitment event.

RNA interference
Cos-7 or HeLa cells were seeded onto Ibidi µ-Slide 8-well glass-bo'omed chambers (live-cell imaging) or in 24-well plates containing acid-etched glass coverslips (invasion assay) and allowed to adhere overnight.
Mission esiRNAs were custom-ordered to target Cos7 Dyn2 mRNA, ensuring that the resultant esiRNA targeted a shared sequence found in all recorded mRNA transcript variants.Cells were transfected with either 100 nM Mission anR-Dyn2 esiRNA (Eupheria Biotech, Dresden, Germany) or 100 nM Trilencer-27 Universal scrambled negaRve control (Origene SR30004, Rockville, MD, USA) using Lipofectamine RNAiMAX reagent (Thermo Fisher) according to manufacturer direcRons.IncubaRon was allowed to proceed for 24 hours before conducRng live-cell imaging or invasion assays using methods described earlier.Lysates for Western blo~ng were obtained from Cos7 cells by applying 2x Laemmli buffer to cells arer the compleRon of live-cell imaging.

Western BloKng
Lysates generated as described above were resolved via SDS-PAGE in 10% polyacrylamide gels at 120 volts for 1.5 hours or unRl the dye front has begun to evacuate the bo'om of the gel casse'e.Gels were transferred onto 0.45µM pore size nitrocellulose in 1x Towbin buffer + 10% methanol at 90 mA for 16 hours.Western blots were blocked in 5% bovine serum albumin for 1 hour, briefly rinsed in Tris buffered saline + 0.1% Tween-20 (TBST) and incubated with appropriate primary anRbody for 1 hour.Blots were then washed three Rmes for 5 minutes in TBST and probed with appropriate HRP-conjugated secondary anRbodies for 1 hour.Protein bands were resolved by chemiluminescence using Immobilon Western HRP Substrate (Millipore Sigma, St Louis, MO, USA).Dynamin 2 knockdown efficiency was calculated by densitometry analysis, comparing the raRo of Dyn2 anRbody signal (Thermo PA1-661) against β-acRn loading control (Abcam ab49900).

Dextran Uptake Assay
Cos7 cells were seeded onto 24-well plates containing acid-etched glass coverslips and allowed to adhere overnight.Cells were treated with 40 µM Ryngo 1-23 or DMSO in media containing 100 µg/mL Dextran Alexa Fluor 647; 10,000 MW (Thermo D22914) and incubated at 37°C for 30 minutes.Cells were infected with wild-type or mutant Chlamydia strains at MOI=50, synchronizing infecRon by sedimentaRon at 4°C on a rocking incubator for 30 minutes.InfecRon was iniRated by addiRon of prewarmed media, followed by incubaRon in a 37°C incubator for 20 minutes prior to fixaRon in 4% paraformaldehyde for 10 minutes at room temperature.Fluorescent dextran and inhibitor were maintained in media for each indicated stage.Cells were then permeabilized using 0.1% (w/v) Triton X-100 for 10 minutes at room temperature, rinsed with HBSS and labeled with a mouse monoclonal anR-MOMP anRbody (Novus #NB10066403).Cells were rinsed in 1x PBS and labeled with Alexa Fluor 488 anR-mouse (ThermoFisher # A-11001) IgG secondary anRbody.Coverslips were mounted and observed on a Nikon CSU-W1 confocal microscope (Nikon), obtaining Z-stacks using a 0.3 micron step size across the height of the cell monolayer.Monolayer Z-stacks were transformed via Z-projecRon according to maximal fluorescence intensity in ImageJ prior to quanRfying the percentage of elementary bodies which colocalize with fluorescent dextran, according to the following equaRon: [ Dextran + EBs (magenta/green) / Total EBs (green) ] x 100%.
Plasmids and DNA prepara.onpEGFP-AcRn-C1 69 was a gir from Dr Sco' Grieshaber (University of Idaho), and mRuby-LifeAct-7 (Addgene plasmid #54560) was a gir from Michael Davidson.Dyn2-pmCherryN1 was a gir from ChrisRen Merrifield (Addgene plasmid #27689), RFP Dynamin2 K44A was a gir from Jennifer Lippinco'-Schwartz (Addgene plasmid #128153), WT Dyn2 pEGFP was a gir from Sandra Schmid (Addgene plasmid #34686), and GFP-Dynamin 2 K44A was a gir from Pietro De Camilli (Addgene plasmid #22301).Upon receipt, bacterial stab cultures were streak-plated onto LB agar containing appropriate anRbioRc (Kanamycin, carbenicillin) for each plasmid.Resultant anRbioRc-resistant colonies were selected and propagated in LB broth + anRbioRc for plasmid isolaRon prior to sequence verificaRon.All plasmids were isolated using MiniPrep DNA isolaRon kits (Qiagen, Valencia, CA, USA) following a variant protocol for DNA isolaRon termed MiraPrep 70 .Following plasmid isolaRon, the eluate was precipitated by addiRon of 3M sodium acetate (Invitrogen, Waltham, MA, USA) at 10% (v/v) of eluate volume followed by addiRon of 250% (v/v) absolute ethanol calculated arer addiRon of sodium acetate.The mixture was incubated at 4°C overnight and centrifuged at 14,000×g for 15 minutes at 4°C.Supernatant was removed and 70% ethanol was added, followed by centrifugaRon at 14,000×g for 10 minutes at 4°C.Supernatant was removed once more, and precipitated  obtaining images every 20 seconds for 30 minutes to idenRfy sites of acRn recruitment proximal to invading bacteria.AcRn recruitment at pathogen entry sites was quanRfied as described earlier (Fig. 1C) and plo'ed as %max acRn MFI for each Rmepoint +/-SEM compiled from a minimum N=36 recruitment events.Upon compleRon of imaging, cells which received either scramble RNA or Dyn2 siRNA were lysed in 2x Laemmli buffer, resolving protein expression via Western blot to determine the knockdown efficiency of Dyn2 siRNA compared to acRn loading control.KineRcs of (C) acRn recruitment and (D) acRn turnover, and (F) internalizaRon duraRon were obtained using the same methodology described in Fig. 1E-G 1C) and plo'ed as %max Dyn2 MFI for each Rmepoint +/-SEM compiled from a minimum N=18 recruitment events.(C) HeLa cells were treated with 40nM Wortmannin for 30 minutes before infecRon with the indicated Chlamydia strains at MOI=50 and stained using the "in-and-out" method described earlier to quanRfy pathogen entry

Figure 1 :
Figure 1: Dynamin 2 and ac.n are co-recruited during Chlamydia entry

Figure 2 :
Figure 2: Disrup.on of Dynamin 2 restricts ac.n turnover and Chlamydia entry . Violin plots contain a minimum N=34 individual events, reporRng the median value +/-SD.StaRsRcal significance was determined by Wilcoxon Rank-sum.(E) Kymographs depicRng RFP-Dyn2 and GFP-Chlamydia fluorescence over a 30 minute Rmelapse.Top arrow indicates iniRaRon of protein recruitment and bo'om arrow indicates compleRon of pathogen entry.(G,H) HeLa cells were infected with the indicated Chlamydia strain at MOI=50 and stained using the "in-and-out" method which disRnguishes non-internalized EBs from total cell-associated EBs, as described in Materials and Methods.(G) Cells were pre-treated with 25 µM Dynasore for 30 minutes prior to infecRon, or (H) transfected with either scramble or Dyn2-specific siRNA for 24 hours prior to infecRon.Invasion efficiency of each Chlamydia strain was plo'ed as mean +/-SEM.Data was collected from 15 fields, with each field containing an average of 50 Chlamydiae.StaRsRcal significance was determined by pairwise T-test with Bonferroni post-correcRon.All data are representaRve of at least 3 independent experiments, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Figure 6 :
Figure 6: Ac.n turnover is correlated with Dynamin 2 ac.va.on status and Chlamydia uptake