The phosphatidylinositol-5′ phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus

ABSTRACT The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5′ kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process. IMPORTANCE Staphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

understanding of the study 1.The authors investigated the internalization of S. aureus by host cells by 2 different approaches: gentamicin assay and flow cytometer.However the flow cytometer-based assay of bacterial internalization is missing in the materials and methods 2. Fig. 1: Did the authors performed one statistical analysis in the fig 1 A and C? 3. The internalization of S. aureus within different cells is described in different figures.However, the "y" axis is different.In fig1 is CFU/cell, however, in the figures 3B and C, the results are expressed as "CFUx10E3 cells".Are these results not per cell?Moreover, the amount of intracellular CFU for cells such as SYNJ1-KO is different between the fig 3B and 5C. 4. Line 255: Do you mean Fig. 5D? 5. Discussion: do you think that this mechanism is common among all non-professional cells?Is this mechanism different in professional cells?Please include these answers in your discussion 6.Please check the sentence from line 469 "In parallel....".samples of? 7. Please check the references, some of them are in different format (ex #2) Reviewer #2 (Comments for the Author): Staphylococcus aureus is internalized by non-professional phagocytic cells (NPPC).This process is mediated by host integrins that bind to a fibronectin bridge, which in turn is bound by Fn-binding proteins on the staphylococcal cell wall.
In the presented study "The phosphatidylinositol-5' phosphatase synaptojanin1 limits integrin-mediated invasion of Staphylococcus aureus" by Shi, Muenzner, & Hauck the authors investigate the contribution of synaptojanin1 (SYNJ1) to this process.SYNJ1 thereby was identified by an shRNA screen that encompassed ten candidate phosphatases.Upon RNAi-based knockdown of SYNJ1 S. aureus was internalized more efficiently by HEK293.This was corroborated also in NIH 3T3 murine fibroblasts that were knocked-out in Synj1 by CRISPR/Cas9-based gene editing.Complementtation did recover the wild-type phenotypes and led to the reduction of S. aureus uptake.A non-functional mutant as well as a deletion mutant in the Sac1 domain, however, did not complement the mutant illustrating that the activity and a Sac1 domain is needed for the observed phenotype.
The paper is generally well-written, informative and covers an interesting subject.Methods are laid out well and are experiments include the proper controls.
There are only two points that should be addressed: 1) Fig. 5: The subcellular distribution of the delta-Sac1 version is missing (fluorescence microscopy).It would be interesting to see if it is altered with respect to S. aureus localization when compared to the wild-type.
2) The authors focus on possible differences in the changes of talin in presence of absence of SYNJ1 activity.However, in Fig. 6 a quantification of talin recruitment is lacking.In order to corroborate their point, quantification is indispensable.

Minor Issues:
In the Methods Section: a) Addgene sources of pLKO1, pMD2.G and pPAX2 are not listed.
b) It should be noted somewhere, that the SYNJ1 shRNA was directed axainst the 3' UTR in the last exon, which allows for the observed complementation in the KD cell lines.
c) The section on fluorescence microscopy (starting at line 474) should list the channels (Ex/Em) and further details of image acquisition.
General: d) line 469: 'which resemble the "recovered intracellular bacteria"' : Do you mean: "which constitute" or similar?e) Whenever the mouse homolog of human SYNJ1 is used, the correct gene symbol (capitalization) Synj1 should be used.f) line 213: "exaggerated endocytosis" : probably more suitably is "enhanced endocytosis" or similar.

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Editor and Reviewer comments -Response_to_Reviewers:
Reviewer #1 (Comments for the Author): Shi et al investigate the mechanism of S. aureus internalization in non-professional cells.The authors addressed specifically the role of PI-4,5-P2.They found that SYNJ1 has a negative impact on S. aureus internalization by host cells.Moreover, the integrin binding protein talin resulted enriched in SYNJ1 knock out cells.The introduction gives a sufficient background and justifies the aim to determine the relationship between SYNJ1 and the internalization of S. aureus by 293 cells and fibroblasts.The manuscript is very interesting, well written and easy to follow.However, I have some suggestions to improve the quality and understanding of the study.
1.The authors investigated the internalization of S. aureus by host cells by 2 different approaches: gentamicin assay and flow cytometer.However the flow cytometer-based assay of bacterial internalization is missing in the materials and methods We apologize for this omission and we have now included not only a description of the flowcytometer-based quantification of bacterial internalization, but also a detailed description of the fluorescence labeling procedure of the bacteria.Furthermore, we refer now to our prior publications, where we present these methods and describe the controls used to establish these procedures in more detail.

"Evaluation of bacterial internalization by flow cytometry
For flow cytometric analysis of bacterial uptake, cells were infected with CFSE-labelled bacteria and analyzed as described (53).Briefly, 4x10 5 HEK293 cells or 1x10 5 fibroblasts were seeded one day before infection in poly-L-lysine or gelatine-coated 6-well plates.PBSwashed bacteria from logarithmic growing cultures were stained with 5-( 6 Though the experimental approach depicted in Figure 1A and C was repeated multiple times (n = 4 -6), we did not perform a statistical evaluation.On the one hand, this was due to the variable outcome observed with regard to cell-associated and intracellular bacteria, which we clearly depict in Fig. 1A and in Suppl.Fig. S1A and S1B.The variable outcome might reflect differences in knock-down efficiencies between biological replicates, but also the potential additional effect of phosphatase knock-down on receptor surface expression as well as on intracellular trafficking (and therefore survival) of internalized bacteria, which we have already mentioned in the results section.
On the other hand, this first approach was intended to serve as an initial screen (and not a definitive proof) to identify potential candidate enzymes for a more detailed, consecutive analysis.Therefore, we have focussed the remainder of the study on verifying the gain-offunction phenotype observed for Synaptojanin-1-knock-down cells with independent genetic and functional analyses, rather than spending additional time and resources on determining statistical significance of an initial screen.
3. The internalization of S. aureus within different cells is described in different figures.However, the "y" axis is different.In fig1 is CFU/cell, however, in the figures 3B and C, the results are expressed as "CFUx10E3 cells".Are these results not per cell?Moreover, the amount of intracellular CFU for cells such as SYNJ1-KO is different between the fig 3B and 5C.
The reviewer is completely correct in that the different experimental approaches used to evaluate intracellular bacteria lead to different labeling of Y-axes in these Figures.E.g. in Fig. 2D and 2E, the number of intracellular bacteria is given as bacteria/cell, as in these cases the intracellular bacteria were quantified on the basis of immunofluorescence stainings of intracellular bacteria, which are counted on a cell-based basis.In other cases, the intracellular bacteria were determined by antibiotic protection assays (gentamicin protection assays), where the infected cell culture is lysed and the evaluation is based on the number of viable bacteria (cfu) recovered from the whole sample (e.g.Fig. 3B).This explains the different labeling of the y-axes.
However, we are thankful that the reviewer alerted us to the gross differences in cfu recovered from SYNJ1-KO cells in Fig. 3B and Fig. 5C.In this case, we have inadvertently used the wrong scale as the numbers on the y-axis reflected cfu x 10 3 instead of cfu x 10 4 (left panel) and cfu x 10 2 instead of cfu x 10 3 (right panel) in Fig. 5C.We have now corrected this mistake and now the numbers of recovered intracellular bacteria from SYNJ1 cells are similar in these two experiments, while the number of total extracellular bacteria differs by a factor of ~4 between Fig. 3B and Fig. 5C.We do not have a definite answer for this difference, but we would like to stress that the fibroblasts used in Fig. 3B are the Control knock-out and the SYNJ1 knock-out cells, while the cells used in Fig. 5B are the complemented cells (expressing GFP or GFP-fused SYNJ1 proteins), which are all derived from the SYNJ1 ko cells.Therefore, the cell lines used in these two experiments are related, but not identical.In addition to slight differences in the density of the bacterial inoculum between experiments, the genetic differences between the knock-out cells and the GFPcomplemented knock-out cells might contribute to the increased amount of total cellassociated bacteria found in Fig. 5C. 4. Line 255: Do you mean Fig. 5D?At this point of the results section, we indeed refered to Fig. 5C as written in the text, where results with cells re-expressing GFP-synaptojanin-1-ΔSac1 are presented.5. Discussion: do you think that this mechanism is common among all non-professional cells?Is this mechanism different in professional cells?Please include these answers in your discussion We thank the reviewer for this advice.In the discussion section on page 11, line 314ff we refer to the ubiquitous expression of synaptojanin1 and emphasize our observation of synaptojanin1-dependent effects in different cell types (murine fibroblasts, human 293 kidney epithelial cells) suggesting that this process might play a role in multiple cell types.
6. Please check the sentence from line 469 "In parallel....".samples of?This description of the gentamicin protection assay should make clear, that similarly infected cells were processed in two distinct ways: i) incubated with gentamicin to kill extracellular bacteria before lysis of the infected host cells and release of viable intracellular bacteria and ii) lysis of infected host cells without prior gentamicin treatment to release cell-associated (attached) and intracellular bacteria (= total cell-associated bacteria).These two samples are infected in parallel, using the same set of transfected cells and the same bacterial inoculum.We have now re.written this section to clarify this point.This paragraph on page 16, line 477ff now reads: "Gentamicin protection assay 2x10 5 293 cells or 5x10 4 NIH 3T3 cells were seeded into poly-L-lysine coated 24-well plates.Cells were infected at MOI 20 for 2 h at 37°C and 5% CO 2 .Two sets of triplicate wells with identically infected cells were processed in two different ways: A) to evaluate the number of viable intracellular bacteria, the medium was carefully replaced with a fresh medium containing 50 μg/ml gentamicin.After incubation for 1 hr at 37°C intracellular bacteria were released by incubation with 0.5% saponin for 15 min at 37°C.Released bacteria were diluted in PBS and plated on TSB agar plates to determine the colony-forming units (cfu).Colony counts of these samples are refered to as "recovered intracellular bacteria".B) to determine the total number of cell-associated intracellular and extracellular bacteria, the infected cells were gently washed with PBS, lysed with 0.5% saponin without prior gentamicin treatment and dilutions were plated on TSB agar plates.Colony counts of these samples are refered to as "total cell-associated bacteria".

7.
Please check the references, some of them are in different format (ex #2) The reviewer is correct in pointing out differences in reference format and we have corrected this mistake.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred )carboxyfluorescein-succinimidylester (CFSE) and added at a multiplicity of infection (MOI) of 20.After 2 hours, infected cells were detached, washed with PBS and cell-associated fluorescein fluorescence was analysed by flow cytometry.To quench signals from extracellular bacteria, trypan blue solution (0.4%; Sigma, Taufkirchen, Germany) was added to a final concentration of 0.2% directly before analysis.Samples were analysed on a LSRII (BD Biosciences) by gating on the eukaryotic cells based on forward and side scatter and cell-associated fluorescence of 10,000 cells per sample was measured in fluorescence channel 1 (FL1-H) detecting CFSE fluorescence as a measure of internalized bacteria."2. Fig. 1: Did the authors performed one statistical analysis in the fig 1 A and C?