Targeting host deoxycytidine kinase attenuates Staphylococcus aureus virulence

Reviewer #1 (Public Review):

Aiming at the problem that Staphylococcus aureus can cause apoptosis of macrophages, the author found and verified that drug (R)-DI-87 can inhibit mammalian deoxycytidine kinase (dCK), weaken the killing effect of staphylococcus aureus on macrophages, and reduce the apoptosis of macrophages. And increase the infiltration of macrophages to the abscess, thus weakening the damage of Staphylococcus aureus to the host. This work provides new insights and ideas for understanding the effects of Staphylococcus aureus infection on host immunity and discovering corresponding therapeutic interventions.

The logic of the study is commendable, and the design is reasonable.

Some data related to the conclusion of the paper need to be supplemented, and some experimental details need to be described.

Reviewer #2 (Public Review):

Summary:

In this study, Winstel and colleagues test if the deoxycytidine kinase inhibitor, (R)-DI-87 provides therapeutic benefit during infection with Staphylococcus aureus. The premise behind the current work is a series of prior studies that found that S. aureus can disable functional immune clearance by generating NET-derived deoxyribonucleosides to induce macrophage apoptosis via purine salvage. Here, the authors use in vitro and in vivo experiments with (R)-DI-87 to demonstrate that inhibition of deoxycytidine kinase prevents S. aureus-induced deoxyribonucleoside-mediated macrophage cell death, to bolster immune cell function and promote more effective clearance during infection. The authors conclude that (R)-DI-87 represents and potentially important Host-Directed Therapy (HDT) with good potential to promote natural clearance of infection without targeting the bacterium. Overall, the study represents an important next step in the exploration of purine salvage and deoxyribonucleoside toxicity as a targetable pathway to bolster infection clearance and provides early-stage evidence of the therapeutic potential of (R)-DI-87 during S. aureus infection.

Strengths:

The study has several strengths that support its conclusions:
1. Well-controlled in vitro studies that firmly establish (R)-DI-87 is capable of blocking deoxyribonucleoside-mediated apoptosis of immune cell lines and primary cells.
2. Solid evidence to support that administration of (R)-DI-87 can have therapeutic benefits during infection (reduced number of abscesses and reduced CFU).
3. Controls included to ascertain the degree to which (R)-DI-87 might have secondary effects on immune cell distribution.
4. Controls included to ascertain whether or not (R)-DI-87 has intrinsic antibacterial properties.

Weaknesses:

However, there are several important weaknesses related to the rigor of the research and the conclusions drawn. The most relevant weaknesses noted by this reviewer are:

1. Drawing firm conclusions about the therapeutic potential of (R)-DI-87 using only S. aureus strain Newman, a methicillin-susceptible S. aureus, that while a clinical isolate is not clearly representative of the strains of S. aureus causing infection in hospitals and communities. Newman also harbors an unusual mutation in a regulator that dramatically changes virulence factor gene expression. While the data with Newman remains valuable, the absence of consideration of other strains, including MRSA, makes it more difficult to support the relatively broad conclusions about therapeutic potential made by the authors.

2. In vitro (R)-DI-87 efficacy studies with dAdo and dGuo are strong, however, the authors do not test the in vitro efficacy of (R)-DI-87 using S. aureus. They have done this type of work in prior studies (See doi: 10.1073/pnas.1805622115 - Figure 5). If included it would greatly strengthen their argument that (R)-DI-87 is directly affecting the S. aureus --> Nuclease --> AdsA macrophage-killing pathway. Without it, the evidence provided remains indirect, and several conclusions may be overstated.

3. Caspase-3 immunoblot experiments seem to suggest an alternative conclusion to what was made by the authors. They point out that Caspase-3 cleavage does not occur upon treatment with (R)-DI-87. However, the data seem to argue that there is almost no caspase-3 present in (R)-DI-87 treated cells (cleaved or uncleaved). Might this suggest that caspase-3 is not even produced when cells are not experiencing deoxyribonucleoside toxicity? Perhaps the authors could reconsider the interpretation of this data.

4. There are some concerns over experimental rigor and clarity of the experimental design in the methods. The most important points noted by this reviewer are included here. (a.) There is no description of the number of replicates or representation of the Western blots and no uncropped blots are provided. (b.) the methods describing the treatment conditions for in vivo studies are not sufficiently clear. For example, it is hard to tell when (R)-DI-87 is first administered to mice. Is it immediately before the infection, immediately after, or at the same time? This has important implications for interpreting the results in terms of therapeutic potential. (c.) There are several statements made that (R)-DI-87 does not have a negative impact on the mice however, it is not sufficiently clear that the studies conducted are sufficient to make this broader claim that (R)-DI-87 has no impact on the animal, except as it relates to the distribution of immune cells, which is directly tested. (d.) there are no quantitative measures of apoptosis or macrophage infiltration, which impacts the rigor of these imaging experiments. (d.) only female mice are used in the in vivo studies. There is no justification provided for this choice; however, the rigor of the study design and the ability to draw conclusions about therapeutic potential is impacted in the absence of consideration of both sexes.

5. Animal studies show significant disease burden (CFU) even after administration of (R)-DI-87. Given the absence of robust clearance of infection, the author's claims read as an overstatement of the data. The authors may wish to reframe their conclusions to better highlight the potential benefit of this therapy at reducing severe disease but also to point out relevant limitations, especially considering that it does not lead to clearance in this model. In general, the consideration of the limitations of the proposed therapeutic approach, as uncovered by the data, is not present. A more nuanced consideration of the data and its interpretations, including both strengths and limitations, would greatly help to frame the study.