Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In this interesting manuscript the authors present experiments examining the relationship between the obligate intracellular bacterium Chlamydia pneumoniae (Cpn) and the microtubule (MT) cytoskeleton of the host eukaryotic cell using both mammalian cells and yeast as a model. They demonstrate that host microtubule stability contributes to the rate of entry of the bacteria into the cells. Interphase MT architecture is therefore important and correspondingly they show that mitotic cells are less permissive to bacterial entry. Other experiments show that Cpn entry is accompanied by changes to MT stability (measured indirectly via post-translational modifications). Finally they investigate the effects of overexpressing a Cpn virulence factor Cpn0572 in mammalian cells and yeast that they have previously shown to interact with both the actin and MT cytoskeletal networks. Cpn0572 expression induces MT acetylation (stability) which correlates with their previous observations and when expressed ectopically in yeast Cpn0572 suppresses force-dependent MT catastrophe. They propose that chlamydial effectors like Cpn0572 influence MT architecture and stability during Cpn entry, revealing a previously unappreciated role for MT in the bacterial entry process.
Our Response to Summary ____Reviewer #1
We thank the reviewer for the summary of our study. We would like to clarify one point. The mammalian-cell experiments demonstrating that CPn0572 interacts with and alters the MT cytoskeleton, including increased MT acetylation, were reported in our previous work (Höhler et al., 2024; doi: 10.1242/jcs.263450) and are not experiments performed in the present study. In the current manuscript, we use controlled ectopic expression of CPn0572 in S. pombe to analyse its effects on MT dynamics by live-cell imaging and show that CPn0572 reduces catastrophe and depolymerization and suppresses the normal catastrophe response at the cell cortex.
Experimental points
*
__Comment 1*__
Fig 1A - to assist interpretation whole cell images similar to those in the supplementary file should be included. It is difficult to relate the images in the small immunofluorescence panels to the phenotypes depicted.* *
Answer to comment 1
We have provided whole cell images of the enlarged images shown in Fig 1A. These are shown in Supp. Fig. S1A. We have added the following statement in the revised results section (line 174): Whole cell images of the zoom images in Fig 1A are shown in Fig S1A.
Comment 2
Cold recovery assay: one wonders what happens to the actin and intermediate filament networks under these conditions and when treated with MT-targetted agents. Some data should be included to rule out that there are additional effects on these systems as off target effects on actin might also influence the data. This is an essential control to support the conclusions drawn.
Answer to comment 2
Cold treatment efficiently perturbs the MT cytoskeleton without causing a comparable gross disruption of the actin cytoskeleton. These images are now included in the revised version of the manuscript (new Supp. Fig. S2). We have added the following sentence to our revised manuscript:(lines 129-130) Cold-treatment did not lead to gross alteration of F-actin organization (Fig. S2A).
Comment 3a
*Is MT repolymerisation synchronous following cold recovery? Its difficult to assess from the included images and sample size. *
Answer to comment 3a
Cold-induced MT depolymerization followed by rewarming results in rapid and highly synchronized MT regrowth in U2OS cells. This has been quantitatively demonstrated by Didier et al. (2008), who showed that MT asters were detectable within 30 s after rewarming and that a centrosome-radiating MT network had reformed in 95% of control U2OS cells within 60 s (Didier et al., 2008; doi: 10.1091/mbc.E06-12-1140). The images shown in our study are representative examples and were not intended to independently quantify the synchrony of MT regrowth.
We have clarified this point in the revised results section as follows: (lines 120–122)
“Cold-induced MT depolymerization followed by rewarming results in rapid and highly synchronized re-polymerization of the MT cytoskeleton in U2OS cells [32].”
Comment 3b
Why was this method selected in preference to nocodazole treatment and washout, where synchrony is easier to establish.
Answer to comment 3b
We selected cold-induced MT depolymerization because this approach provides both rapid and synchronized MT regrowth upon rewarming. We do not consider synchrony of MT repolymerization to be inherently easier to establish following nocodazole treatment and washout. In U2OS cells, cold-induced MT depolymerization followed by rewarming is a well-established MT-regrowth assay, and rapid, highly coordinated repolymerization has been demonstrated previously (Didier et al., 2008; doi: 10.1091/mbc.E06-12-1140).
Importantly, the cold-recovery approach is particularly suitable for our experimental question because transfer of the cells from ice to 37°C provides a precisely defined starting point for MT repolymerization and, at the same time, initiates the early infection period. This allows us to analyse C. pneumoniae entry during the first minutes of MT recovery. Nocodazole washout can likewise be used to induce synchronized MT regrowth, but requires drug removal and repeated washing before recovery can be initiated. We therefore chose cold-induced depolymerization as the more appropriate approach for coupling synchronized MT regrowth directly to the very early stages of infection.
Comment 4
Although EB are scored in Fig 1A, they are not shown alongside the microtubules and modified microtubules as described.
Answer to comment 4
We apologize for the misleading representation in Fig 1A. The upper part of the figure is a schematic illustration of the experimental setup, whereas the lower part shows representative microscopic images of the tubulin and acetylated MT phenotype at the indicated time points. The schematic depicts the experimental workflow subsequently used for the infection experiments and was not intended to indicate that EBs were visualized in the images shown in Fig 1A.
We have modified the schematic in the revised manuscript to make this distinction clear and to avoid further confusion.
Comment 5a (please note- we have divided this into several sub comments)
What happens to actin/intermediate filaments following the treatments with Taxol?
Answer to comment 5a
Because actin has a central role in chlamydial entry, we examined whether the Taxol treatment used in our experiments causes a major reorganization of the actin cytoskeleton. U2OS cells treated with DMSO or 10 µM Taxol for 2 h showed prominent F-actin fibres under both conditions, with no obvious gross disruption of the actin cytoskeleton following Taxol treatment (included now as new Fig. S2B). We have added the following sentence to our revised manuscript:(lines 202-204): Under the conditions used, Taxol treated cells showed prominent F-actin fibres with no obvious gross disruption of the actin cytoskeleton (Fig S2B).
We did not analyse intermediate filaments. To our knowledge, there are no data implicating intermediate filaments in chlamydial entry. In C. trachomatis, their reorganization has been described during later inclusion development (Kumar and Valdivia, 2008, doi:10.1016/j.chom.2008.05.018), while in C. pneumoniae-infected cells alterations of vimentin and keratins 8/18 were detected at 48-72 h post-infection (Savijoki et al., 2008, doi:10.1111/j.1574-695X.2008.00488.x).
Comment 5b
Is this specifically targeting the MT under these conditions?
Answer to comment 5b
Taxol directly binds β-tubulin within polymerized MTs and stabilizes the MT lattice (Xiao et al., 2006, doi:10.1073/pnas.0603704103). Nevertheless, because actin and MTs are functionally interconnected, we experimentally assessed the actin cytoskeleton under the exact Taxol conditions used in our infection assay and did not observe a major alteration of F-actin organization.
The purpose of the Taxol/Tubacin comparison was specifically to distinguish MT stabilization from increased tubulin acetylation per se. Taxol stabilizes MTs and consequently increases their acetylation, whereas Tubacin inhibits HDAC6-mediated tubulin deacetylation and increases MT acetylation without stabilizing MTs (Haggarty et al., 2003, doi:10.1073/pnas.0430973100). Under our experimental conditions, both treatments produced a comparable increase in acetylated MTs, whereas only Taxol generated MTs resistant to cold-induced depolymerization. These data are already shown in Fig 2 and Fig S3 and described in the Results (lines 195-220).
Thus, both Taxol and Tubacin increase MT acetylation, but only Taxol stabilizes the MT network and only Taxol increases EB internalization. This is the basis for our conclusion that the long-lived MT state, rather than acetylation alone, is associated with enhanced C. pneumoniae entry.
Comment 5c
How toxic are the treatments and how were they titrated - this does not seem to be included.
Answer to comment 5c
The concentrations and treatment times are already given in both the Fig 2 legend and the Materials and Methods. U2OS cells were treated with 10 µM Taxol or 10 µM Tubacin for 2 h at 37°C before infection (Fig 2, lines 222-232; Materials and Methods, lines 675-686).
These conditions were not established by a de novo dose-response titration in the present study but were selected on the basis of established short-term treatments for manipulating MT stability and acetylation in U2OS cells. Importantly, Jansen et al. used the same conditions - 10 µM Taxol for 2 h and 10 µM Tubacin for 2 h in U2OS cells - to experimentally distinguish stable from acetylated MT populations (Jansen et al., 2023, doi:10.1083/jcb.202106105). Tubacin as an inhibitor of HDAC6-dependent tubulin deacetylation was originally characterized by Haggarty et al. (2003, doi:10.1073/pnas.0430973100).
We additionally verified the intended differential effects of these treatments in our own U2OS cells: both Taxol and Tubacin increased MT acetylation, whereas only Taxol protected MTs against cold-induced depolymerization (Fig 2 and Fig S3; lines 199-209).
We did not perform a separate quantitative cytotoxicity assay. However, treatment was limited to 2 h, we observed no obvious signs of acute cellular deterioration or major changes in cell morphology, and the new F-actin analysis shows no gross disruption of the actin cytoskeleton under the Taxol conditions used.
Comment 6
line 156 - it is unclear what 'microtubule subsets' are referred to here and how the authors arrive at the fact that ~9% of MT are acetylated.
Answer to comment 6
We apologize that the term “microtubule subsets” was not sufficiently defined. By this term, we referred to microtubules distinguished by post-translational modification, in this case acetylated versus non-acetylated MTs. U2OS cells vary considerably in the abundance of acetylated MTs, ranging from cells containing few to cells containing many acetylated MTs.
We have changed the wording in the results section accordingly.
Comment 7
Can the effects of taxol be modulated by changing the bacterial load (MOI)? The dose dependency of taxol is considered but not the reciprocal i.e. whether the effect can be suppressed by increasing the number of bacteria.
Answer to comment 7
We understand the proposed experiment to mean varying the bacterial load (MOI) at a constant taxol concentration to determine whether the taxol-dependent increase in entry becomes less apparent at higher MOIs.
While such an experiment could test how the magnitude of the taxol effect depends on bacterial input, increasing the MOI is not mechanistically reciprocal to the taxol treatment. Taxol alters a host-cell property before addition of C. pneumoniae EBs by stabilizing MTs and increasing the population of long-lived/acetylated MTs. Our experiment therefore addresses whether this pre-existing MT state influences bacterial entry. Increasing the number of bacteria does not reverse or otherwise alter this host-cell state.
Moreover, at high MOIs, a reduced relative difference between control and taxol-treated cells could result simply from saturation of available entry sites or cellular uptake capacity rather than from suppression of the taxol effect. We therefore consider an MOI titration in taxol-treated cells difficult to interpret with respect to the specific question of whether a pre-existing stabilized MT state promotes C. pneumoniae entry.
Comment 8
Figure 1 shows limited co-localisation between EB and MT. Are the authors certain that this is not stochastic? How many EB align with F-actin stress fibres on intermediate filaments under similar conditions. This might be interesting and correct, but controls are lacking to demonstrate specificity, which would make the data more convincing.
Answer to comment 8
We addressed the possibility that the observed EB–MT co-localization reflects stochastic overlap by quantifying the fraction of the cellular area occupied by MTs at the 10-min time point. At this stage of MT recovery, MTs occupied approximately 14% of the cellular area, whereas 35% of internalized EBs co-localized with MTs. Thus, EB–MT association occurred substantially more frequently than expected from MT area coverage alone. Using the MT-covered cellular area as the probability of random overlap, the observed frequency was significantly higher than expected for a random spatial distribution (exact binomial test, p The association was also strongly biased toward a specific MT population. Of the MT-associated EBs, 71% were associated with acetylated MTs, although acetylated MTs represented only approximately 9% of the total MT population under these conditions. This strong enrichment further argues against stochastic overlap.
We deliberately performed this analysis at 10 min after shifting the cells back to 37°C, when MT re-polymerization is still incomplete and individual MT filaments are clearly distinguishable. This minimizes apparent co-localization resulting simply from the dense MT network present in untreated interphase cells.
We do not consider F-actin to provide an equivalent negative control for this question. F-actin remains extensively distributed under these conditions and, importantly, actin is directly involved in chlamydial entry; EB association with actin would therefore be biologically expected rather than a measure of nonspecific cytoskeletal overlap. We did not analyse intermediate filaments. We consider the comparison between the observed EB–MT association and the quantitatively determined probability of random overlap to provide the more direct test of stochastic association.
We have revised the Results section accordingly (lines 154-166): “Next, we analysed the subcellular localization of internalized EBs. At the 10-min time point (Fig. 1A), MT re-polymerization was still incomplete and individual MT filaments were clearly distinguished. Approximately 14% of the cellular area was occupied by MTs, whereas 35% of internalized EBs co-localized with MTs (Fig. 1E, F). Thus, EB association with MTs occurred at a substantially higher frequency than expected from MT area coverage alone. Consistently, comparison with a random spatial distribution using the fraction of MT-covered cellular area as the probability of random EB–MT overlap showed that the observed association was significantly higher than expected by chance (exact binomial test, p __ __
Comment 9
Figure 3. The cell cycle block relies upon RO-3306 which shifts the mitotic cell population from 5% to 36%. Would a thymidine block and release to synchronise the population yield a higher proportion of cells in mitosis? Did the authors consider this approach and exclude it for a defined reason?
Answer to comment 9
Achieving the highest possible proportion of mitotic cells was not the primary requirement for our experiment. Rather, we required a sufficient number of cells entering mitosis within a defined time window after release.
Thymidine arrests cells at the G1/S transition, and cells must subsequently progress through S and G2 before entering mitosis. In contrast, the CDK1 inhibitor RO-3306 arrests cells directly at the G2/M transition and therefore allows rapid and temporally defined entry into mitosis following washout. We therefore considered RO-3306 more suitable for our experimental design. Importantly, our analysis does not rely on the entire synchronized population being mitotic. Following RO-3306 release, mitotic and non-mitotic cells were identified and analysed separately at the single-cell level. Under our conditions, approximately 36% of the population was mitotic, providing sufficient numbers of mitotic cells for quantification of C. pneumoniae infection. Thus, increasing the overall percentage of mitotic cells would not alter the basis of our comparison between mitotic and non-mitotic cells.
Comment 10
Many properties change in mitotic cells in addition to MT architecture. A particular consideration is the profound reorganisation of the actin cytoskeleton and changes in the composition of the plasma membrane, which might also influence the rates of Cpn entry. It is very technically difficult to show that these effects are specifically due to the MT changes and consequently this experiment, while interesting might have many alternative interpretations.
Answer to comment 10
We agree with the reviewer that mitosis involves extensive cellular reorganization in addition to the replacement of the interphase MT network by the mitotic spindle, and that the experiment in Fig 3 cannot by itself attribute the reduced C. pneumoniae entry specifically to changes in MT architecture. Indeed, we already considered this issue in the Results section. We note that endocytosis is generally reduced during early mitosis and is reactivated from anaphase onwards. At the same time, receptor-specific internalization pathways can remain active during mitosis, including EGFR uptake, which is particularly relevant here because EGFR is utilized by C. pneumoniae for host-cell entry. In addition, Fig 3D shows the mitotic reorganization of the actin cytoskeleton by rhodamine-phalloidin staining.
Thus, we agree that changes in actin organization, membrane trafficking and other mitosis-associated cellular properties may contribute to the reduced infection efficiency observed in mitotic cells. Our intention with this experiment was not to establish that the reduction in EB entry is caused exclusively by loss of the interphase MT array. Rather, we asked whether C. pneumoniae entry is altered in a physiological cellular state in which the interphase MT architecture is absent and replaced by the mitotic spindle. We find that mitotic cells remain permissive to EB entry, but infection efficiency is strongly reduced compared with interphase cells.
The MT-specific conclusions of our study are therefore based primarily on the experiments in Figs 1 and 2, in which MT composition and stability are directly analysed or manipulated. The mitotic-cell experiment provides complementary evidence showing that a cellular state lacking the normal interphase MT architecture is associated with strongly reduced EB entry, but we agree that this experiment alone cannot distinguish the contribution of MT reorganization from other mitosis-associated changes.
To make this limitation explicit, we have changed the final sentence of this part of the results section to (lines 276-278):" Thus, EB entry is strongly reduced in mitotic cells, a cellular state characterized by loss of the interphase MT architecture but also by broader changes in cytoskeletal organization and membrane trafficking."__ __
Comment 11a (please note- we have divided this into several sub comments)
While the overexpression experiments in cells and yeast are interesting, these come with caveats about the dose of the effector and the relevance of the system to the pathological process.
Answer to comment 11a
The S. pombe experiments were designed to analyse the effects of CPn0572 on MT dynamics under controlled expression conditions, rather than to reproduce the infection process. CPn0572-mCherry is expressed from a single genome-integrated copy under an inducible TetO promoter, and MT dynamics are analysed after only 1 h of induction. This minimizes dosage heterogeneity and allows early effects of CPn0572 on the MT cytoskeleton to be analysed in living cells. This rationale is already described in the manuscript (lines 384-391).
The manuscript also explicitly acknowledges that TetO-driven expression cannot reproduce the spatially restricted delivery of an effector by the bacterial secretion system; rather, it provides a tractable system in which the consequences of CPn0572 appearance in a eukaryotic cell can be analysed (lines 577-583).
The relevance of the S. pombe system for analysing MT dynamics is addressed in detail in our response to Comment 15. Importantly, CPn0572 has independently been shown to associate with and stabilize MTs in mammalian cells in the infection (Höhler et al., 2024, doi:10.1242/jcs.263450). Thus, the S. pombe experiments are used to resolve how CPn0572 alters MT behaviour, not as a surrogate for C. pneumoniae infection.
Comment 11b
*Could Chlamydia trachomatis TARP that interacts with actin but not MT be used as a control here? *
Answer to comment 11b
We do not consider C. trachomatis TarP an appropriate matched negative control for CPn0572. Although both proteins belong to the TarP family and both modulate actin, their activities toward the actin cytoskeleton are not equivalent. TarP and CPn0572 show distinct patterns of subcellular localization and F-actin association, and CPn0572 additionally binds preassembled F-actin and protects it from cofilin-mediated destabilization (Jewett et al., 2006, doi:10.1073/pnas.0603044103; Jewett et al., 2010, doi:10.1371/journal.ppat.1000997; Zrieq et al., 2017, doi:10.3389/fcimb.2017.00511). CPn0572 additionally associates with MTs (Höhler et al., 2024, doi:10.1242/jcs.263450).
This distinction is important because the actin and MT cytoskeletons are functionally interconnected (Dogterom and Koenderink, 2019, doi:10.1038/s41580-018-0067-1). Consequently, differences in MT behaviour following expression of TarP and CPn0572 could not be attributed specifically to the presence or absence of MT-binding activity. TarP therefore would not constitute a control differing from CPn0572 only in its ability to target MTs.
Comment 11c
While there are interesting effects of CPn0572, which in part relate to the other phenotypes identified in the work, the link between the activities of Cpn0572 overexpression and the infection process are currently weak, beyond the fact that this is one of a number of effectors what have the capability of manipulating the actin and/or MT cytoskeletal networks. The current presentation is therefore speculative.
Answer to comment 11c
We believe that this concern reflects a misunderstanding of how the CPn0572 experiments are positioned within the manuscript. The study addresses three consecutive but distinct questions: (1) whether the pre-existing state of the host MT network influences C. pneumoniae entry; (2) whether C. pneumoniae itself alters the host MT network during early infection; and (3) how a chlamydial protein with MT-modulating activity can alter MT dynamics. CPn0572 is used for the third question as one experimentally tractable example of a chlamydial MT modulator. We do not propose that CPn0572 alone accounts for either the entry phenotype or the infection-induced increase in MT acetylation.
This three-part logic is already stated in the original manuscript. In the Introduction, we first define the permissive host MT state, then describe the infection-induced increase in MT acetylation, and finally introduce controlled expression of CPn0572 to analyse its effect on MT stability (lines 96-104).
The distinction between the first two parts is made particularly explicit in the Discussion: “The preferential infection of cells containing acetylated MTs needs to be distinguished from the increase in MT acetylation observed 1 hr after chlamydial infection.” The manuscript then states that the former represents a host-cell property present before infection, whereas the latter demonstrates that C. pneumoniae remodels the host MT cytoskeleton during infection (lines 531-536).
Likewise, CPn0572 is explicitly introduced as one example with which to investigate how chlamydial proteins might alter MT dynamics. The relevant results sections states that several C. pneumoniae proteins are likely to jointly manipulate the MT cytoskeleton and that CPn0572 was analysed “to start to understand how MTs might be modulated by chlamydial proteins” (lines 374-381). We then show that CPn0572 reduces MT catastrophe and depolymerization and suppresses the normal catastrophe response at the cell cortex, thereby increasing MT persistence (lines 403-410).
The manuscript furthermore explicitly argues against a single-effector model. We state that the infection-induced increase in MT acetylation is likely to result from “multiple EB-associated effectors that together remodel the host MT network” (lines 572-574).
To strengthen this concept experimentally, we have added an experiment in the revised version of the manuscript. We now analysed the combined activity of two chlamydial MT-modulating proteins namely CPn0572 and CPn0443. Thus, if we have two independent MT modulations, we would expect a phenotype intermediate between those produced by either protein alone. CPn0443 was originally identified as a C. pneumoniae protein that strongly alters the interphase MT cytoskeleton by destabilizing it (Wevers et al., 2023, doi:10.3390/ijms24087618) which is opposite to the function of CPn0572.
We therefore asked what happens when these two opposing chlamydial MT modulators are present in the same cell. CPn0572 increases MT occupancy, whereas CPn0443 strongly reduces MT occupancy and longitudinal MT organization. Importantly, simultaneous expression produces an intermediate phenotype: CPn0572 partially counteracts both the CPn0443-induced reduction in MT occupancy and the loss of longitudinal MT organization. These new data (Fig. 7) directly demonstrate that two chlamydial MT modulators can interact at the level of the same cellular MT network. They therefore provide additional experimental support for the concept that host MT remodelling may reflect the combined activities of multiple chlamydial proteins rather than the action of CPn0572 alone.
Thus, the manuscript neither establishes nor claims a one-to-one causal relationship between CPn0572 and the infection-induced MT phenotype. Rather, a pre-existing host MT state affects bacterial entry, C. pneumoniae subsequently remodels the host MT network, and CPn0572 is used as one example to determine how a chlamydial MT-modulating protein can alter MT dynamics.
Comment 12
The Discussion is extensive, and could be reduced to deal with the key findings presented in the work and potentially to address some of the limitations.
__
____Answer to comment 12__
We have shortened the discussion.
The original discussion already discusses the limitations and boundaries. For example, we state that Taxol-induced MT stabilization may not fully recapitulate the properties of naturally acetylated MTs, that the proposed contribution of MT-dependent membrane trafficking to EB entry remains to be tested, and that the infection-induced MT phenotype is likely to reflect the combined activity of multiple chlamydial proteins rather than a single effector. We also explicitly note that TetO-driven ectopic expression of CPn0572 does not reproduce the spatially restricted delivery of an effector during infection.
In addition, following the reviewer’s specific concern regarding the mitotic-cell experiment, we have clarified this in the revised results section (see comment 10).
Reviewer #1 (Significance (Required)):
*
This is an interesting and potentially important study, which will be of interest to researchers studying Cpn, related Chlamydiae and obligate intracellular bacteria, and more generally to those studying the entry of bacterial pathogens into host mammalian cells. Bacterial effectors like Cpn0572 are also of interest to the cell biology community, as studying their activities can reveal novel insights into the regulation and dynamics of the cytoskeleton, relevant to fields including immunology, developmental biology and cancer biology.*
*
The manuscript addresses key unresolved questions - for example, it tries to reconcile the potential role for the MT cytoskeleton in bacterial entry, which has been suspected but overtaken by studies of the actin cytoskeleton, where cause and effect and more straightforward. The work investigates role for the posttranslational modification of MT and how this can be reprogrammed by pathogens. Finally, it offers an opportunity to study the interplay between the actin and MT networks and how this might be bridged. This is not well understood in mammalian cells.*
*Notwithstanding the comments above, the individual experiments presented are largely well executed and support the individual conclusions drawn. The weakness of the study is that it is descriptive and correlative. It is an assembly of interesting, but potentially differentially related, experiments examining MT during Cpn infection, essentially in three separate sections i) stability of cellular MT being important for Cpn infection, ii) assessing changes to MT modifications during Cpn infection, iii) the effects of a particular effector amongst many on these processes. While broadly self-supporting in that they all address Cpn and MT, they are presented as cohesive, although the direct relationships between these different topics remains somewhat subjective.
The reviewer actively researches interactions between bacterial pathogens and the host cytoskeleton.*
Our answer to Reviewer 1 (significance)
We appreciate the reviewer’s positive assessment of the interest and potential importance of the study. We would, however, like to clarify both the conceptual connection between the experimental sections and what we consider an important aspect of the novelty of the work.
Bacterial entry into mammalian cells has overwhelmingly been studied as an actin-driven process. Although MTs have been implicated in several bacterial infection cycles, their functions have been studied much less extensively and mainly in post-entry trafficking and later stages of infection. A defined role for different MT states during bacterial entry has remained largely unexplored.
Our central finding is therefore not simply that MTs contribute to C. pneumoniae infection. We show that, within the same mammalian cell population, cells with a particular pre-existing MT state are preferentially infected. Increasing amounts of acetylated/long-lived MTs correlate with increasing entry efficiency, whereas detyrosinated MTs do not, and the Taxol/Tubacin experiments further distinguish MT stability from acetylation itself. To our knowledge, a pre-existing MT state has not previously been identified as a determinant of differential host-cell permissiveness to bacterial entry.
The subsequent experiments build directly on this finding. Having established that a long-lived interphase MT state favors entry, we ask whether C. pneumoniae itself modifies this state and show that early infection increases MT acetylation in a viability-dependent manner. We then use CPn0572 as one mechanistically tractable early effector to ask how a chlamydial MT stabilizer can generate increased MT persistence and show that it suppresses catastrophe and reduces depolymerization.
Thus, while the study does not establish a single linear molecular pathway, the experiments are not an assembly of differentially related observations. Together, they identify a previously unrecognized host-cell MT state that determines permissiveness to bacterial entry, show that Chlamydia subsequently remodels this cytoskeletal system, and provide mechanistic insight into how an early chlamydial effector can generate a persistent MT state.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Evidence, reproducibility, and clarity
*
Summary
The manuscript by Schenk et al examines how the microtubule state of U2OS cells affects the ability of Chlamydia pneumoniae to enter the host cell. The authors test whether two tubulin post-translational modifications, detyrosination and acetylation, affect bacterium entry and find that cells with higher levels of acetylation display more internalized Chlamydia particles. They also test whether the tubulin state or the post-translational modification is the important factor for Chlamydia entry and find that stabilization of microtubules with taxol treatment is sufficient for increasing the number of internalized particles. They show that entry is higher in interphase cells than mitotic cells. Finally, they show that the Chlamydia protein CPn0572, which was previously shown to alter microtubules in mammalian cells, can alter microtubule dynamics in yeast cells. Overall, this is a straight-forward set of experiments that add information about how the state of microtubules in cells impacts the entry step of Chlamydia infection.*
Response to Summary
We thank the reviewer for this accurate summary of the main findings of our study.
Major comments
Comment 13
In general, the claims and the conclusions are supported by the data. The data in Figures 1–4 address very specific questions and are straightforward. The only issue is that the microtubule immunofluorescence does not look very good. Especially the total tubulin staining. In many cells, it doesn't even look filamentous. Generally, methanol fixation preserves microtubule structures much better than PFA.
__Answer to comment 13 __
We agree that methanol fixation can provide a sharper visualization of filamentous MTs. However, the choice of fixation also depends on the biological question being addressed. Importantly, a study specifically examining fixation effects in Chlamydia trachomatis-infected cells showed that alcohol-based fixation can induce cellular shrinkage and distortion, whereas formaldehyde fixation is used to better preserve overall cellular architecture and the spatial relationships between cellular components (Kokes and Valdivia, 2015; doi: 10.1371/journal.pone.0139153). This consideration was particularly important in our experiments, because our analyses required assessment of the spatial relationship between chlamydial EBs and the host-cell MT cytoskeleton during the early stages of infection.
PFA fixation has also been used in previous studies examining early Chlamydia–host cell interactions, including studies in U2OS cells and during early C. pneumoniae infection (Nans et al., 2014; doi__: _10.1111/cmi.12310_; Mölleken and Hegemann, 2017; doi: 10.1371/journal.ppat.1006556__).
We acknowledge that the total-tubulin staining appears less sharply filamentous in some cells. Nevertheless, MT structures relevant to our analyses are distinguishable under the experimental conditions used, and identical fixation, staining and imaging conditions were applied across the respective experimental groups. Importantly, as also noted by the reviewer, the quantitative data in Figures 1–4 support the conclusions drawn from these experiments.
Comment 14
Its not clear why the inside/outside staining was only used in Fig 2. How do the authors know that the particles in the other figures are inside vs outside the host cell?
__Answer to comment 14 __
Inside/outside staining was used in Fig 2 because these experiments were designed specifically to quantify EB internalization. In contrast, Fig 1 examines changes in MT post-translational modifications, while Fig 3 examines the organization of the MT and actin cytoskeletons. For these experiments, inside/outside staining was technically not feasible because the complete staining combination would require five fluorescence channels, whereas our microscopy setup allows a maximum of four.
We therefore used a spatial approach to assign EB localization in Figs 1 and 3. We initially tested a plasma membrane marker as a means of defining the cell boundary. However, the permeabilization required for subsequent tubulin immunostaining resulted in additional intracellular staining of this marker, preventing an unambiguous identification of the plasma membrane in the final samples. We therefore used the outer boundary of the cytoplasmic α/β-tubulin signal to delineate the cellular area in interphase cells. For the mitotic cells analyzed in Fig 3, the prominent cortical F-actin signal provided a clear definition of the cell boundary.
Each optical section of the complete confocal z-stack was examined individually, and EBs were classified according to their three-dimensional position relative to the delineated cellular area. Thus, whereas Fig 2 uses inside/outside staining to directly distinguish internalized from extracellular EBs, EB localization in Figs 1 and 3 was assigned on the basis of their spatial position within the cellular volume.
Comment 15a (please note- we have divided this into several sub comments)
The use of S. pombe to test the effects of CPn0572-mCherry on microtubule dynamics seems an odd choice. It is not clear whether these findings are relevant to the story since yeast cells are very different from mammalian cells.
Answer to comment 15a
We consider S. pombe a highly appropriate system for analysing the effect of CPn0572 on MT dynamics for four reasons: (1) its simple and exceptionally well-characterized interphase MT cytoskeleton allows changes in MT bundle dynamics to be resolved particularly clearly; (2) fundamental components of the MT system are evolutionarily ancient; (3) yeast-based approaches are established for identifying functions of chlamydial proteins; and (4) Chlamydiae are themselves an ancient lineage of intracellular bacteria, making conserved eukaryotic cellular processes plausible targets for their effectors.
(1) S. pombe interphase cells contain only a small number of well-defined MT bundles whose dynamics and behaviour at the cell cortex can be followed directly and quantitatively in living cells (Drummond and Cross, 2000, doi:10.1016/S0960-9822(00)00570-4; Sawin and Tran, 2006, doi:10.1002/yea.1404). This makes changes in MT bundle dynamics considerably easier to resolve than within the dense MT network of mammalian cells.
(2) Yeast model systems have been exceptionally successful in uncovering fundamental principles of eukaryotic cell biology, as exemplified by Nobel-Prize-for- Medicine winning work on cell-cycle control, vesicle trafficking and autophagy. The tubulin-based MT cytoskeleton is likewise evolutionarily ancient: α-, β- and γ-tubulins and diverse MT motors were already present in the last eukaryotic common ancestor, before diversification of the major eukaryotic lineages (Wickstead and Gull, 2011, doi:10.1083/jcb.201102065). Many years ago, our own work provided a direct example of functional conservation: S. pombe Mal3 belongs to the EB1 family of conserved MT plus-end-tracking proteins that regulate MT dynamics, and human EB1 can substitute for Mal3 in S. pombe (Beinhauer et al., 1997, doi:10.1083/jcb.139.3.717).
__(3) __Several yeast-based approaches successfully investigated chlamydial proteins. A systematic Saccharomyces cerevisiae expression screen identified C. trachomatis proteins that affect yeast cellular functions or target eukaryotic organelles (Sisko et al., 2006, doi:10.1111/j.1365-2958.2006.05074.x), and subsequent yeast-based screening identified chlamydial proteins targeting lipid droplets (Kumar et al., 2006, doi:10.1016/j.cub.2006.06.060). In our own S. pombe screen, 13 of 116 tested C. pneumoniae proteins strongly altered the interphase MT cytoskeleton (Wevers et al., 2023, doi:10.3390/ijms24087618).
(4) Chlamydiae have a long evolutionary history of interaction with eukaryotic cells. The last common ancestor of pathogenic and symbiotic Chlamydiae was already adapted to intracellular survival approximately 700 million years ago and possessed a type III secretion system (Horn et al., 2004, doi:10.1126/science.1096330). It is therefore plausible that chlamydial effectors exploit ancient, conserved features of eukaryotic cell biology, including the MT cytoskeleton.
We therefore do not use S. pombe as a model for mammalian infection itself, but as a tractable system in which effects of CPn0572 on fundamental MT properties can be resolved clearly. Importantly, relevance to mammalian cells is independently supported by our previous demonstration that CPn0572 associates with and stabilizes MTs in mammalian cells (Höhler et al., 2024, doi:10.1242/jcs.263450).
Comment 15b
Furthermore, the Fleig group has already shown that CPn0572 binds to microtubules when ectopically expressed in mammalian cells and causes their stabilization and bundling. It would be useful to see if CPn0572 expression increases acetylation when expressed in mammalian cells
Answer to comment 15b
This experiment has already been performed. Ectopic expression of CPn0572 resulted in an approximately threefold increase in acetylated α-tubulin compared with control cells (Höhler et al., 2024, doi:10.1242/jcs.263450).
Comment 15c
And test whether it directly alters microtubule dynamics using reconstitution assays.
Answer to comment 15c
Reconstitution experiments with purified CPn0572 and tubulin could test whether CPn0572 is sufficient to alter MT dynamics in a minimal in vitro system. However, CPn0572 modulates both the actin and MT cytoskeletons, and such an assay would not establish how its MT effects arise in the cellular context, where additional host components or interactions between the two cytoskeletal systems may contribute. The aim of the present study was to determine whether CPn0572 affects MT organization and dynamics in living cells. Together with our previous demonstration that CPn0572 associates with and stabilizes MTs in mammalian cells (Höhler et al., 2024, doi:10.1242/jcs.263450), the S. pombe experiments establish that CPn0572 alters MT behaviour in a cellular context. Dissecting whether this activity is mediated by a direct interaction with tubulin/MTs or involves additional host factors will require a separate biochemical analysis and is beyond the scope of the present study.
Comment 15d
Furthermore, its role in Chlamydia infection could be tested by deleting the gene from the Chlamydia genome.
Answer to comment 15d
A CPn0572 deletion could address the contribution of this effector to C. pneumoniae infection. However, targeted gene-deletion approaches such as those available for C. trachomatis have not been established for C. pneumoniae (Shima et al., 2018, doi:10.1128/mSphere.00412-18; Wan et al., 2023, doi:10.3389/fimmu.2023.1209879). Thus, deletion of CPn0572 is currently not technically feasible.
Moreover, CPn0572 is a TarP-family effector that modulates both the actin and MT cytoskeletons (Höhler et al., 2024, doi:10.1242/jcs.263450). Consequently, even if a CPn0572 deletion mutant were available, any resulting infection phenotype would reflect the combined loss of its cellular activities and would not by itself establish the specific contribution of its MT-modulating function.
*Minor comments
*
Comment 16
The exact antibodies used for immunofluorescence and western blot should be listed. Some tubulin antibodies are not very good and the reader needs to know that the results are reliable.
__Answer to comment 16 __
We have revised the Materials and Methods section to provide the exact antibodies used for all immunofluorescence and Western blot analyses, including the respective supplier, catalogue number and antibody dilution. In addition, for experiments in which different α-tubulin antibodies were used, we now specify the antibody and its host species for each individual experiment, allowing unambiguous identification of the antibody used.
Comment 17
Fig. 4 – it would be nice to validate the increase in acetylation by immunofluorescence.
Answer to comment 17
We thank the reviewer for this valuable suggestion. We have now independently validated the infection-induced increase in MT acetylation by immunofluorescence microscopy. Representative fluorescence images together with the corresponding quantitative analysis have been added to the revised Fig. 4G, H. Consistent with the Western blot analysis, immunofluorescence showed a significant MOI-dependent increase in acetylated α-tubulin 1 h post infection.
The corresponding text has been added to the revised manuscript (lines 329–332):
“To independently validate the infection-induced increase in MT acetylation observed at 1 hpi, we additionally analysed acetylated MT levels by immunofluorescence microscopy. Consistent with the Western blot results, immunofluorescence analysis showed a significant MOI-dependent increase in MT acetylation (Fig. 4G, H).”
This independent analysis confirms the increase in MT acetylation observed by Western blotting and supports our conclusion that C. pneumoniae infection induces increased MT acetylation.
Comment 18
The term inside-out staining is confusing. I think the authors mean inside/outside staining.
Answer to comment 18
We agree that “inside/outside staining” more accurately describes the staining approach used in our experiments. We have therefore replaced “inside-out staining” with “inside/outside staining” throughout the revised manuscript.
Reviewer #2 (Significance (Required)):
*
This study provides new information about how Chlamydia alters the microtubule cytoskeleton to enter mammalian cells. Previous work had shown that Chlamydia utilizes the actin cytoskeleton so this study expands our knowledge of the entry mechanisms. The insights would be more mechanistic if the effects of CPn0572 could be shown in reconstitution assays. The work will be of interest to researchers that study the basic mechanisms of pathogen entry into mammalian cells.*
Our Response to Significance
We thank the reviewer for this assessment. The point concerning a CPn0572 reconstitution assay is addressed in our response to Comment 15c above.