Defining the Roles of Pyruvate Oxidation, TCA Cycle, and Mannitol Metabolism in Methicillin-Resistant Staphylococcus aureus Catheter-Associated Urinary Tract Infection

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of complicated urinary tract infection (UTI) associated with the use of indwelling urinary catheters. Previous reports have revealed host and pathogen effectors critical for MRSA uropathogenesis. Here, we sought to determine the significance of specific metabolic pathways during MRSA UTI. First, we identified four mutants from the Nebraska transposon mutant library in the MRSA JE2 background that grew normally in rich medium but displayed significantly reduced growth in pooled human urine (HU). This prompted us to transduce the uropathogenic MRSA 1369 strain with the transposon mutants in sucD and fumC (tricarboxylic acid [TCA] cycle), mtlD (mannitol metabolism), and lpdA (pyruvate oxidation). Notably, sucD, fumC, and mtlD were also significantly upregulated in the MRSA 1369 strain upon exposure to HU. Compared to the WT, the MRSA 1369 lpdA mutant was significantly defective for (i) growth in HU, and (ii) colonization of the urinary tract and dissemination to the kidneys and the spleen in the mouse model of catheter-associated UTI (CAUTI), which may be attributed to its increased membrane hydrophobicity and higher susceptibility to killing by human blood. In contrast to their counterparts in the JE2 background, the sucD, fumC, and mtlD mutants in the MRSA 1369 background grew normally in HU; however, they displayed significant fitness defects in the CAUTI mouse model. Overall, identification of novel metabolic pathways important for the urinary fitness and survival of MRSA can be used for the development of novel therapeutics. IMPORTANCE While Staphylococcus aureus has historically not been considered a uropathogen, S. aureus urinary tract infection (UTI) is clinically significant in certain patient populations, including those with chronic indwelling urinary catheters. Moreover, most S. aureus strains causing catheter-associated UTI (CAUTI) are methicillin-resistant S. aureus (MRSA). MRSA is difficult to treat due to limited treatment options and the potential to deteriorate into life-threatening bacteremia, urosepsis, and shock. In this study, we found that pathways involved in pyruvate oxidation, TCA cycle, and mannitol metabolism are important for MRSA fitness and survival in the urinary tract. Improved understanding of the metabolic needs of MRSA in the urinary tract may help us develop novel inhibitors of MRSA metabolism that can be used to treat MRSA-CAUTI more effectively.

as membrane permeability. If the main hypothesis is the impact on metabolism, can the growth defect of the LpdA mutant be complemented by acetyl-coA? Alternatively, if the main impact of the lpdA mutant is thought to be on cell membrane fluidity and subsequent susceptibility to factors such as antimicrobial peptides or complement in blood, it would be beneficial to demonstrate whether the lpdA mutant is more susceptible to membrane-permeable dyes or daptomycin.
It would be useful to include a diagram of the metabolic pathways being explored in this work, denoting which had in vitro and in vivo competitive defects.
Line 273 states that "pyruvate oxidation plays an essential role" in growth and survival in HU, but the lpdA mutant still grows in urine and achieves high CFUs. The expectation for an essential factor is that MRSA would not be able to grow at all without it. Similarly, line 321 states that "LpdA activity is required" for colonization of the lower UT. However, the LpdA mutant still colonized the bladder and catheter of all mice, so it does not appear to be required for colonization. It would be more accurate to state that LpdA "contributes" to colonization. Line 336 references the "essential" roles of LpdA and MtlD, which again is not supported by the data since the LpdA mutant still colonizes and CFUs are not provided for the MtlD mutant. Similarly, line 402 states that the TCA cycle is "required" for MRSA pathogenesis, which overstates the data. Figure 1A is confusing as presented. For instance, line 256 states that "the lpdA::Tn mutant displayed a severe growth defect at 24h" (which I believe should be corrected to 22 h), but there is only a single, slightly red line for NE1610. By eye, this appears to be similar to the single red line for a mutant that somewhere between NE701 and NE801 at 22 hours, no mutant in this range is mentioned in the text or the RNA-Seq heatmap. I suggest moving the heatmap to supplemental material and displaying growth kinetics of the selected mutants in HU vs BHI for figure 1. I also suggest displaying the actual fold change in expression values, perhaps as a table, rather than a heatmap.
For Figure 4, it would be helpful to include the CFUs for WT and each mutant, in addition to the Cis.
Line 339: Define BCFA on first use Lines 396-397 state that "sucD and fumC" did not display a competitive disadvantage during growth in human urine, but sucD had a significant defect in Figure 3A.
Reviewer #2 (Comments for the Author): The article reports on a study that investigated the importance of specific metabolic pathways during Methicillin-resistant Staphylococcus aureus (MRSA) urinary tract infection. The study identified mutations in the TCA cycle (fumC, sucD), mannitol metabolism (mtlD, and pyruvate oxidation (lpdA) that resulted in significant defects in the growth and colonization of MRSA in the urinary tract of mice, suggesting the potential use of these pathways as targets for novel therapeutics. I don't have any major issues with the methodologies and conclusions of this study. However, I do have several suggestions that will hopefully make the manuscript clearer and easier to digest.
- Fig 1A: The authors should indicate which are the 6 mutants that showed significant growth defects in HU but normal growth in BHI. Maybe the zoom-in version of 6 mutants is needed (panel 1B does not need such huge space). - Fig 1B: the colour scale is difficult to read, the authors should consider providing the logFC value for each gene presented. -Fig 2: the authors should draw plots in panel B and D in the same style as other figures in this manuscript where individual data points are shown. -In line 246, the authors should quantify the statement "significant growth defects" -does it mean showing growth ratios < 0.5 in all time points? I see quite a lot of red at 1h and 2h, even 4h in Fig 1A. -How would the authors interpret the results that out of the 4 mutants that were defective for growth in HU in JE2, only one still showed defective in the 1369 background strain? Are there any differences in the TCA cycle genes between JE2 and 1369?

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To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " We thank the reviewers for the careful review of our manuscript. We have incorporated all of their insightful comments in the revised manuscript. The major changes in the revision include: (i) data from examination of lpdA mutant growth in human urine supplemented with glucose or citrate, (ii) clarification on the selection of mutants, and (ii) the addition of a metabolic pathway schematic. The following is the point-by-point response (marked in blue) to the reviewers comments and suggestions. The line numbers are from unmarked version.
Reviewer comments: Reviewer #1 (Comments for the Author): This study by Paudel et al explores the contribution of four components of metabolic pathways to Staphylococcus aureus growth in urine and pathogenesis in a mouse model of catheter-associated urinary tract infection (CAUTI). This is an important topic, as S. aureus represents an understudied but clinically-significant urinary tract pathogen. By screening transposon mutants in the JE2 background, the authors identified mutants in fumC, mtlD, lpdA, and miaB as having growth defects in human urine but not rich laboratory medium. All four genes were next mutated in the methicillin-resistant isolate MRSA 1369 and assessed for growth and fitness in urine and during experimental CAUTI. Only the lpdA mutant had a growth defect in urine, but all four mutants had fitness defects in urine for at least one timepoint and in at least one organ during CAUTI. The authors further explored the potential contribution of mtlD and lpdA to ROS sensitivity and serum killing. The manuscript is well-written overall, but there are several places where the conclusions are not fully supported by the data. Specific comments are as follows: 1.1) The body of work would be strengthened by confirming that the indicated metabolic pathways are disrupted in each mutant and by ruling out potential off-target effects by complementation, at least for in vitro experiments. From our previous experience, complementation of knockout mutants by a plasmidborne copy of the mutated gene is possible but not always successful in Staphylococcus aureus strains. Hence, as an alternative to complementation, we ruled out potential polar effects of mutants by monitoring growth of lpdA mutant in human urine supplemented with various downstream (citrate) or alternative (glucose) carbon sources. As shown in supplemental figure S2, lpdA showed more robust growth in HU supplemented with either glucose or citrate at 24 h time point.

2)
It is unclear if the main effect of each mutation is thought to be the impact on metabolic pathways, or on downstream effect such as membrane permeability. If the main hypothesis is the impact on metabolism, can the growth defect of the LpdA mutant be complemented by acetyl-coA? Alternatively, if the main impact of the lpdA mutant is thought to be on cell membrane fluidity and subsequent susceptibility to factors such as antimicrobial peptides or complement in blood, it would be beneficial to demonstrate whether the lpdA mutant is more susceptible to membrane-permeable dyes or daptomycin. We observed that lpdA mutant growth is improved by the addition of glucose as well as citrate as explained earlier. At 30 h, compared to growth in plain human urine we observed higher lpdA CFU/ml in HU+ glucose (not significant by t test) and in HU+citrate (P=0.054, t test). These results are presented in Fig S2. In addition, we have revised narrative (lines 485-488) to mention that LpdA mutation affects metabolism as well as membrane permeability of MRSA both of which may in turn affect the urinary pathogenesis of lpdA mutants.

1.3)
It would be useful to include a diagram of the metabolic pathways being explored in this work, denoting which had in vitro and in vivo competitive defects. In the revised Fig 1, we have presented a schematic of metabolic pathways (glycolysis and TCA cycle) and have indicated specific metabolic steps catalyzed by MtlD, LpdA, FumC, and sucD.

1.4)
Line 273 states that "pyruvate oxidation plays an essential role" in growth and survival in HU, but the lpdA mutant still grows in urine and achieves high CFUs. The expectation for an essential factor is that MRSA would not be able to grow at all without it. Similarly, line 321 states that "LpdA activity is required" for colonization of the lower UT. However, the LpdA mutant still colonized the bladder and catheter of all mice, so it does not appear to be required for colonization. It would be more accurate to state that LpdA "contributes" to colonization. Line 336 references the "essential" roles of LpdA and MtlD, which again is not supported by the data since the LpdA mutant still colonizes and CFUs are not provided for the MtlD mutant. Similarly, line 402 states that the TCA cycle is "required" for MRSA pathogenesis, which overstates the data. Thank you for the suggestion. We have revised the manuscript to state that LpdA and MtlD are neither essential nor required but are important for MRSA uropathogenesis. This change is also reflected in the revised running title: "LpdA and MtlD are required for MRSA uropathogensis". Figure 1A is confusing as presented. For instance, line 256 states that "the lpdA::Tn mutant displayed a severe growth defect at 24h" (which I believe should be corrected to 22 h), but there is only a single, slightly red line for NE1610. By eye, this appears to be similar to the single red line for a mutant that somewhere between NE701 and NE801 at 22 hours, no mutant in this range is mentioned in the text or the RNA-Seq heatmap. I suggest moving the heatmap to supplemental material and displaying growth kinetics of the selected mutants in HU vs BHI for figure 1. I also suggest displaying the actual fold change in expression values, perhaps as a table, rather than a heatmap. Thank you for these suggestions. We have moved the original Fig 1A) to supplemental figure S1; the original Fig 1B) is presented as Table 2. In addition, we have significantly edited the language to avoid confusion about selection of mutants for this study. The revised narrative (Lines 257-273) explains that the four mutants were selected for this study because they were defective for growth in human urine but grew normally in nutrient rich BHI. Three of the four genes (sucD, fumC, and mtlD) were also upregulated when exposed in vitro to HU for 2h. The revision also explains that despite showing a growth ratio <0.5 at 24 h, NE751 was not selected for this study because it encodes a hypothetical protein and because it was defective for growth in nutrient rich BHI. Figure 4, it would be helpful to include the CFUs for WT and each mutant, in addition to the Cis. The revised figure for in vivo competition shows both competitive indices and raw CFU/ml data.

1.7)
Line 339: Define BCFA on first use This has been revised.

1.8)
Lines 396-397 state that "sucD and fumC" did not display a competitive disadvantage during growth in human urine, but sucD had a significant defect in Figure 3A.
This has been revised.

Reviewer #2 (Comments for the Author):
The article reports on a study that investigated the importance of specific metabolic pathways during Methicillin-resistant Staphylococcus aureus (MRSA) urinary tract infection. The study identified mutations in the TCA cycle (fumC, sucD), mannitol metabolism (mtlD, and pyruvate oxidation (lpdA) that resulted in significant defects in the growth and colonization of MRSA in the urinary tract of mice, suggesting the potential use of these pathways as targets for novel therapeutics. I don't have any major issues with the methodologies and conclusions of this study. However, I do have several suggestions that will hopefully make the manuscript clearer and easier to digest. Fig 1A: The authors should indicate which are the 6 mutants that showed significant growth defects in HU but normal growth in BHI. Maybe the zoom-in version of 6 mutants is needed (panel 1B does not need such huge space). Thank you for this suggestion. As mentioned, in a response to 1.5), we have provided log2FC values for the four genes examined in this manuscript. Fig 1B: the colour scale is difficult to read, the authors should consider providing the logFC value for each gene presented. Thank you for this suggestion. As mentioned, in a response to 1.5), we have provided log2FC values for the four genes examined in this manuscript.

2.4)
In line 246, the authors should quantify the statement "significant growth defects"does it mean showing growth ratios < 0.5 in all time points? I see quite a lot of red at 1h and 2h, even 4h in Fig 1A. We admit that "significant growth defects" was not adequately defined in the original submission. A similar concern was also raised by the reviewer 1 (please see response to 1.5). We selected sucD, fumC, and mtID mutants because they showed growth ratio<0.5 at 2 and 4 h in human urine and because their growth in BHI was not affected. These three genes were also significantly upregulated in MRSA 1369 exposed to HU for 2 h. We selected lpdA mutants because it was significantly defective for growth in human urine at 22 h. Moreover, sucD, fumC, mtID, and lpdA encode enzymes in the central carbon metabolism pathways, which is the theme for this project. The revised narrative can be found on lines 257-273.

2.5)
How would the authors interpret the results that out of the 4 mutants that were defective for growth in HU in JE2, only one still showed defective in the 1369 background strain? Are there any differences in the TCA cycle genes between JE2 and 1369? Thank you for this suggestion. We address the differences in in vitro HU growth between MRSA JE2, a skin isolate, and MRSA 1369, a urinary isolate, on lines 418-432. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): The authors have addressed all prior reviewer comments. I only note one concern regarding the conclusions drawn about the lpdA glucose and citrate experiments. Lines 297-298 state that growth of the lpdA mutant was delayed in HU+glucose, but growth appears identical to that of the lpdA mutant in HU without glucose in S2A and S2B until the 30h time point. The text here also states that growth of the lpdA mutant was partially restored in HU+citrate, but the growth curves in S2C are largely identical except for what might be a very small difference at the 24 and 30 time points (hard to tell as there are no error bars for the 30h time point, and S2D shows a P value of 0.054 for the 24 hour time point). How do the CFUs for the lpdA+glucose compare to lpdA alone and wt+glucose at the 30 h time point? This comparison would be needed to support the stated conclusion that "growth of lpdA mutant in HU can be rescued to some extent by supplementation with glucose...or citrate" Reviewer #2 (Comments for the Author): The authors have adequately addressed all of my concerns.

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We thank the reviewers for carefully reviewing our resubmission and suggesting edits. We have incorporated the reviewers' suggestions: (i) Supplementary figure #2 (examination of lpdA mutant growth in human urine supplemented with glucose or citrate) is edited to include statistical comparisons between different groups, (ii) the sentences describing S2 are edited for clarity. The following is the point-by-point response (marked in blue) to the reviewers comments and suggestions. The line numbers are from the unmarked version.
Reviewer comments: Reviewer #1 (Comments for the Author): The authors have addressed all prior reviewer comments. I only note one concern regarding the conclusions drawn about the lpdA glucose and citrate experiments. Lines 297-298 state that growth of the lpdA mutant was delayed in HU+glucose, but growth appears identical to that of the lpdA mutant in HU without glucose in S2A and S2B until the 30h time point. The text here also states that growth of the lpdA mutant was partially restored in HU+citrate, but the growth curves in S2C are largely identical except for what might be a very small difference at the 24 and 30 time points (hard to tell as there are no error bars for the 30h time point, and S2D shows a P value of 0.054 for the 24 hour time point). How do the CFUs for the lpdA+glucose compare to lpdA alone and wt+glucose at the 30 h time point? This comparison would be needed to support the stated conclusion that "growth of lpdA mutant in HU can be rescued to some extent by supplementation with glucose...or citrate" We apologize for the mistake in the figure legend for Fig S2. Panels B and D show data for 30 h time point. This has been corrected.
We have edited Figure S2 as follows-1) the y axis in edited panels B and D start from 100,000 making the differences between histograms discernible, 2) asterisks are shown for comparisons where the difference between groups is statistically significant, 3) the duplicate data points for each group are shown in Fig S2B and Fig S2D. The edited description of Fig S2 reads as follows (Lines 294-303): "Next, we compared the growth of the WT and lpdA mutant strains in HU supplemented with glucose or with citrate ( Fig S2). The lpdA mutant growth in HU+glucose (Fig S2A) or in HU+citrate (Fig S2C) was affected up to 24 h time point similar to what was observed in HU alone. However, at 30 h time point, the lpdA mutant showed ~3-fold higher CFU/ml in HU+glucose (3.2x10 7 CFU/ml, not significant, Fig S2B) and ~2-fold higher CFU/ml in HU+citrate (2.1x10 7 CFU/ml, P=0.054, unpaired t test, Fig S2D) compared to HU alone (9.4x10 6 CFU/ml). These results suggest that the lower growth of lpdA mutant in HU can be rescued modestly but statistically insignificantly by supplementation with glucose, an alternative carbon source, or citrate which is downstream of the metabolic step catalyzed by LpdA."