Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens

Many intracellular pathogens hijack cholesterol. Inhibiting acid sphingomyelinase alters cholesterol traffic to target different intracellular bacteria, signifying a host-directed approach for treating infectious disease.

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The authors provide an impressive body of work showing inhibition of growth of (some) intracellular bacteria. They address and identify cholesterol flux as critical target for the effects of acid sphingomyelinase inhibitors on bacterial growth. The authors also provide impressive in vivo data using acid sphingomyelinase-deficient mice.
The data are of high quality and the experiments are well performed. The study identifies a novel function of the acid sphingomyelinase and provides convincing evidence for the ability to treat infections with Anaplasma phagocytophilum, Coxiella burnetii and Chlamydiae with functional inhibitors of the acid sphingomyelinase. This is of broad interest. The study is very complete and I only have a few suggestions: 1. It would be interesting to determine the residual activity of the acid sphingomyelinase after treatment with desipramine. These drugs usually do not induce complete inhibition of the enzyme and it would be important to determine the degree of acid sphingomyelinase inhibition that is required to inhibit the infections.
2. In line with the degree of acid sphingomyelinase inhibition that is sufficient to prevent bacterial growth, the authors may want to discuss whether individuals who are heterozygous for acid sphingomyelinase mutations and, thus, healthy, might be protected from infections with certain pathogens and whether this provides a survival advantage.
3. Desipramine and also many similar drugs such as amitriptyline, imipramine, fluoxetine, etc. are widely used as antidepressants. Are any data available that patients that are taking these drugs are protected from the infection with intracellular pathogens?
Reviewer #3 (Comments to the Authors (Required)): The authors present the relevant topic of intracellular bacterial pathogens and provide mechanistic evidence how functional inhibitors of acid sphingomyelinase (ASM) could serve in conjunction or as alternative to antibiotics to combat these infections. Evidence is collected in different relevant cell types for four vacuole-adapted bacteria as models -Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae -with varying degree of dependence on cholesterol trafficking and also extended to the application in mice. The effects of ASM inhibition by treatment with desipramine (amitriptyline and nortriptyline as confirmation) are shown particularly for A. phygocatophilum with respect to the generation of bacterial load, infectious progeny, vacuole maturation and expansion, and the stage of the infection cycle and are compared to those in the other model species. The authors introduce the topic with recent literature and provide sufficient background. Experiments contain mostly clear method descriptions including controls and appear to present solid results; the data are convincing, documented in detail with additional test and well visualized in informative figures. The results section frequently contains background explanations that could be transferred to the introduction but due to the utilization of four different models are helpful to understand the experiments and data at their place. The discussion is well prepared.
There are only a number of minor comments and suggestions (see below). Some additional questions to consider: Is there any data on a lower susceptibility of Niemann-Pick Disease patients to infections with the mentioned group of bacteria? How about other lysosomal storage diseases, is infectivity altered, are there epidemiological data? Or is there any information on susceptibility of patients taking medication that acts as FIASMAs such as many antidepressants -are these people protected to some degree?
Minor comments: Introduction: Lines 66-67: "Niemann-Pick disease severity correlates with decreased ASM activity (Kornhuber et al., 2010)." Why has this reference on FIASMAs instead of one on NPD (original work) been chosen here?

Results
For figures in general, it could be helpful to include a label of the cells applied (HL-60 vs. RF/6A) or a label of the bacterial species (e.g. Figures 7 A+B vs. C+D) to see this information quickly without searching through the legend. Lines 100-113: This part does not report results but is rather an introductory paragraph on A. phagocytophilum and should thus be moved to the introduction section.
Lines 114 + 119 state that "Promyelocytic HL-60 and RF/6A endothelial cells" were used in this part but the figure 1 legend only names" HL-60 cells ... or human peripheral blood neutrophils". It appears that the RF/6A cell data are shown in S1 but also some important HL-60 data. There is continuous switching between these two figures. It would be easier to follow if all relevant data (figure 1 and S1) were combined into one larger figure with a common legend. The amount of details of the two legends is not consistent (also for later figures).  Labeling of figures 2D-E-F could be improved and shortened by showing the times below the identical time points (20h etc.) and for example +/-for desipramine maintenance /removal of full and empty circle symbols with a legend for desipramine maintained or removed €, similarly for D (empty vs. full symbols) and E. It would be better to have D-E-F aligned in a column (for example swap figures C and D). The legend describes (lines 959-960) the maintenance (D) and removal (E) of desipramine but not (F) with later addition of desipramine. Lines 178-180: "Vimentin associated with all ApVs observed under both conditions (Figure 2A), indicating that this early ApV biogenesis event is not dependent on ASM activity." How do the authors prove that vimentin (despite its early recruitment and irreversible association with ApV lines described in lines 169-170) is associated with all (?) ApVs?
Line284 "Beginning on day 3, the first day on which there was any detectable increase in fluorescence signal..." -In figure 6, to which conditions are the time points compared (indicated by asterisks)? It is not obvious that there is already a clear increase at day 3 labelled "***" compared to day 0. Explain abbreviation Cb (C. burnetii) in the legend.
Line 376 explain abbreviation NSF Methods: Lines 501f: To allow replication of presented results, used commercial antibodies should be listed with their product numbers not just the company, since many companies offer different antibodies for the same target.
Lines 538f: For how long were the cells left with the FIASMA: for the entire duration of the infection or just the 1h prior to infection? The exchange of media (line 567f) suggests the latter but it's not clear from the sentence.
Line 550f: "Infection conditions were optimized for both cell type and vessel for less than one internalized bacterium per cell." Is this true only for the infections with A. phagocytophilum described above or only for the infections described below the sentence of for both? If for all, consider adding "all" and moving the sentence.
Line 576: "0.5% Triton X-100" in PBS as given for PFA? Line 584: "0.1% saponin in 1% BSA" in PBS? Line 601: "2.5% PFA" also in PBS as before? Line 602 "1% BSA" also in PBS? Please always consistently state the exact solution or provide a general sentence (all in PBS). Line 607 and later: "Hepes (Thermo Fisher Scientific)", please use HEPES buffer Lines 622f: "Bacterial load was determined using primers specific for A. phagocytophilum 16S rDNA and host cell beta-actin using SsoFast EvaGreen Supermix (BioRad, Hercules, CA) as previously described (Ojogun et al., 2012)." Please provide exact primers used in this study. The reference Ojogun et al. 2012 lists primers for RT-qPCR including Ap 16S-527F and Ap 16S-753R that could be meant but none for (species specific to the used cells) beta-actin. "Relative 16S rDNA was normalized to mouse beta-actin using the 2-DeltaDeltaCT 626 (Livak) method" -why mouse? Cell lines were not (all) from mice. Does this only refer to mouse blood samples?
Was RNA quality checked? It would be helpful to provide approximate amounts of template/dilutions used for these reactions (qPCR, cDNA synthesis, RT-qPCR). In general, MIQE guidelines should be followed where possible.
Lines 657f: "qPCR as described above. Thermal cycling conditions used were 98{degree sign} C for 2 min, followed by 40 cycles of 98{degree sign} C for 5 s and 60{degree sign} C for 30 s" Why was the thermal cycling protocol different from the one described above? Which beta-actin primers have been used for the mouse studies?

Language
Please check spelling and correct notation particularly of chemicals throughout the text, for example: Line 517f: We hereby resubmit our manuscript, "Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens" (LSA-2018-00292-TR) for your consideration for publication in Life Science Alliance. Thank you for the opportunity to do so. We are encouraged by your enthusiasm for the studies and appreciate your efforts in identifying areas for improvement. Of the 35 points raised, 33 were editorial, each of which we addressed. We performed the experiment requested by Reviewer 3 and include the results as new supplementary data ( Figure S1A). Reviewer 2 stated that it would be interesting to perform a particular experiment, but this experiment was not pursued due to numerous factors that we respectfully submit would have confounded interpretation of the results. Overall, we believe that the revised manuscript has been considerably strengthened and hope that you will now view it as acceptable for publication. Below is our pointby-point response to your critiques. Thank you for your consideration.

Reviewer #2:
The manuscript entitled "Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens" by Chelsea L. Cockburn et al. describes that inhibition of the acid sphingomyelinase by functional inhibitors of the enzyme regulates cholesterol egress from the lysosome and thereby the infection cycles of vacuole-adapted bacteria. The authors apply these concepts to cellular infection with Anaplasma phagocytophilum, Coxiella burnetii and Chlamydiae and demonstrate a marked inhibition of the infection cycles and/or an indirect bactericidal effect of functional acid sphingomyelinase inhibitors on these pathogens. They extend these studies to in vivo infections and demonstrate that A. phagocytophilum fails to productively infect in acid sphingomyelinase deficient mice or after treatment of wildtype mice with acid sphingomyelinase inhibitors.
The data are of high quality and the experiments are well performed. The study identifies a novel function of the acid sphingomyelinase and provides convincing evidence for the ability to treat infections with Anaplasma phagocytophilum, Coxiella burnetii and Chlamydiae with functional inhibitors of the acid sphingomyelinase. This is of broad interest. The study is very complete and I only have a few suggestions: 1. It would be interesting to determine the residual activity of the acid sphingomyelinase after treatment with desipramine. These drugs usually do not induce complete inhibition of the enzyme and it would be important to determine the degree of acid sphingomyelinase inhibition that is required to inhibit the infections.

RESPONSE:
We strive to be as accommodating as possible, but respectfully submit that this experiment would be confounded by the following issues. In addition to ASM that remains in the lysosome, a portion of the ASM pool is translocated to the plasma membrane, and there is also secretory ASM. FIASMAs accumulate in lysosomes to specifically inhibit ASM in that organelle, which blocks cholesterol egress to the detriment of A. phagocytophilum, C. burnetii, and C. pneumoniae. Secretory and plasma membrane-localized ASM are not inhibited by FIASMAs. Because commercially available ASM enzymatic assays (e.g. Abcam product number ab190554; ThermoFisher product number A12220) do not discriminate between lysosomal ASM activity from that of secretory ASM or plasma membrane-localized ASM, it would be impossible to correlate ASM activity with effects on intracellular bacteria. Additionally, it is well-established that many intracellular microbes (e.g. Brucella, Neisseria, ebola virus, sindbis virus, rhinovirues) utilize ASM as a receptor and/or exploit its activity to enter host cells. While it is not yet known if any of the model pathogens utilized in the current study do so, there is at least circumstantial published evidence that A. phagocytophilum does enter host cells at lipid rafts (plasma membrane sites where ASM is known to be enriched). Thus, it is reasonable to posit that the organisms studied herein might exploit or modulate plasma membrane activity, which could also confound the assay. Please note that, while due to these challenges, we did not pursue this suggested experiment, we did perform the only other suggested experiment, which was proposed by Reviewer 3.

2.
In line with the degree of acid sphingomyelinase inhibition that is sufficient to prevent bacterial growth, the authors may want to discuss whether individuals who are heterozygous for acid sphingomyelinase mutations and, thus, healthy, might be protected from infections with certain pathogens and whether this provides a survival advantage.

RESPONSE:
We have added such a statement to the Discussion. Please see lines 469-471.

3.
Desipramine and also many similar drugs such as amitriptyline, imipramine, fluoxetine, etc. are widely used as antidepressants. Are any data available that patients that are taking these drugs are protected from the infection with intracellular pathogens? RESPONSE: This is a great and insightful question. To our knowledge, the answer is no. But, we are very interested in pursuing. In fact, Dr. Cockburn (first author of this manuscript), who is returning to medical school to continue her clinical rotations in Spring 2019, has an internal grant under review by a University committee to pursue this project as part of her medical school studies. If funded, I would remain as her advisor for this project.

Reviewer #3:
The authors present the relevant topic of intracellular bacterial pathogens and provide mechanistic evidence how functional inhibitors of acid sphingomyelinase (ASM) could serve in conjunction or as alternative to antibiotics to combat these infections. Evidence is collected in different relevant cell types for four vacuoleadapted bacteria as models -Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae -with varying degree of dependence on cholesterol trafficking and also extended to the application in mice. The effects of ASM inhibition by treatment with desipramine (amitriptyline and nortriptyline as confirmation) are shown particularly for A. phagocytophilum with respect to the generation of bacterial load, infectious progeny, vacuole maturation and expansion, and the stage of the infection cycle and are compared to those in the other model species. The authors introduce the topic with recent literature and provide sufficient background. Experiments contain mostly clear method descriptions including controls and appear to present solid results; the data are convincing, documented in detail with additional test and well visualized in informative figures. The results section frequently contains background explanations that could be transferred to the introduction but due to the utilization of four different models are helpful to understand the experiments and data at their place. The discussion is well prepared.

1.
There are only a number of minor comments and suggestions (see below). Some additional questions to consider: Is there any data on a lower susceptibility of Niemann-Pick Disease patients to infections with the mentioned group of bacteria? How about other lysosomal storage diseases, is infectivity altered, are there epidemiological data? Or is there any information on susceptibility of patients taking medication that acts as FIASMAs such as many antidepressants -are these people protected to some degree?

RESPONSE:
The minor comments have been addressed point-by-point below. In regards to Niemann-Pick Disease patients or individuals taking FIASMAs being resistant to certain intracellular bacterial infections, to our knowledge such literature is lacking. But, this is an exciting line for future investigation. Per our response to Reviewer 2's third query, Dr. Cockburn (first author of this manuscript), who is returning to medical school to continue her clinical rotations in Spring 2019, has an internal grant under review by a University committee to pursue this project as part of her medical school studies. If funded, I would remain as her advisor for this project.
Minor comments: Introduction: 2. Lines 66-67: "Niemann-Pick disease severity correlates with decreased ASM activity (Kornhuber et al., 2010)." Why has this reference on FIASMAs instead of one on NPD (original work) been chosen here?

5.
For figures in general, it could be helpful to include a label of the cells applied (HL-60 vs. RF/6A) or a label of the bacterial species (e.g. Figures 7 A+B vs. C+D) to see this information quickly without searching through the legend.

RESPONSE:
We have made these recommended changes to each of the figures that include multiple cell types and/or multiple bacteria.

6.
Lines 100-113: This part does not report results but is rather an introductory paragraph on A. phagocytophilum and should thus be moved to the introduction section.

RESPONSE:
We respectfully elect to keep this material (lines 107-119) at the beginning of the results section. Since the paper describes results with four different pathogens, we submit that providing a brief introduction to each pathogen at the appropriate Results section is the best means for framing the driving rationale each section. To include all of the background information for A. phagocytophilum as well as C. burnetii and the chlamydial species would make the introduction too cumbersome and diffuse. As quoted by Reviewer 3 above, "The results section frequently contains background explanations that could be transferred to the introduction but due to the utilization of four different models are helpful to understand the experiments and data at their place.", it appears that the reviewer sympathizes with this approach.

7.
Lines 114 + 119 state that "Promyelocytic HL-60 and RF/6A endothelial cells" were used in this part but the figure 1 legend only names" HL-60 cells ... or human peripheral blood neutrophils". It appears that the RF/6A cell data are shown in S1 but also some important HL-60 data. There is continuous switching between these two figures. It would be easier to follow if all relevant data (figure 1 and S1) were combined into one larger figure with a common legend. The amount of details of the two legends is not consistent (also for later figures).

RESPONSE:
As recommended, the data originally presented in Figures 1 and S1 have been solely reorganized into Figure 1. The cognate Results section (lines 106-154) and Figure 1 legend (lines 967-991) have been updated accordingly. Figure 1: For faster grasping the information, it could be worthwhile to leave the first bar (DMSO) in Figure  1B empty (instead of full black color) to have this comparable to all other DMSO conditions in Figure 1.

RESPONSE:
We agree. The requested change has been made. Figure 2: The legend states, cells were fixed at 20, 24, 28 and 32 h in A to C, but figure A shows no 32h data -what is the reason? RESPONSE: Thank you for pointing this out. Representative IFA images for 32 h are now included in Figure  2A.

10.
Labeling of figures 2D-E-F could be improved and shortened by showing the times below the identical time points (20h etc.) and for example +/-for desipramine maintenance /removal of full and empty circle symbols with a legend for desipramine maintained or removed €, similarly for D (empty vs. full symbols) and E. It would be better to have D-E-F aligned in a column (for example swap figures C and D). The legend describes (lines 959-960) the maintenance (D) and removal (E) of desipramine but not (F) with later addition of desipramine.