Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia

Reviewer #1 (Public Review):

In this manuscript by Douglas et al, the investigative team seeks to identify Staphylococcus aureus genes (and associated polymorphisms) that confer altered susceptibility to human serum, with the hypothesis that such genes might contribute to the propensity of a strain to cause bacteremia, invasive disease, and/or death. Using an innovative GWAS-like approach applied to a bank of over 300 well-characterized clinical S. aureus isolates, the authors discover SNPs in seven different staphylococcal genes that confer increased survival in the setting of serum exposure. The authors then mainly focus on one gene, tcaA, and illustrate a potential mechanism whereby modification of peptidoglycan structure and WTA display leads to altered susceptibility to serum, serum-derived antimicrobial compounds, and antibiotics. One particularly significant finding is that the identified tcaA SNP is significantly associated with patient mortality, in that patients infected with the SNP bearing isolate are less likely to die from infection. It is therefore hypothesized that this SNP represents an adaptive mutation that promotes serum survival while decreasing virulence and host mortality. In a murine model of infection, the strain bearing the WT allele of tcaA is significantly more virulent than the tcaA mutant, suggesting that the role of tcaA in bacteremia is infection-phase dependent.

This manuscript has many strengths. The triangulation of genomic analysis, patient outcomes data, and in vitro and in vivo mechanistic testing adds to the significance of the findings in terms of human disease. Testing the impact of mutating tcaA in multiple staphylococcal lineages and backgrounds also increases the rigor of the study. The identification of bacterial loci that impact susceptibility to both host antimicrobial compounds and commonly used antibiotics is also a strength of this work, given the evolutionary and treatment implications for such genes.

One moderate weakness is that the impact of the identified SNP in tcaA is only tested in some of the assays, whereas the majority of the testing is performed with a whole gene knockout. Additionally, for some experiments, rigor is lacking in that statistical measures are not deployed to support the conclusions of biologically meaningful changes based on data with very modest differences between groups. In some cases this results in more speculative conclusions that will require further testing to validate. Finally, there are instances of inter-experiment variability that require further explanation. All in all, this is an exciting manuscript that will be of interest to the broader research communities focused on staphylococcal pathogenesis, bacterial evolution, and host-pathogen interactions, as well as to clinicians who care for patients with invasive staphylococcal infection.

Reviewer #2 (Public Review):

The authors embarked on a study to identify SNPs in clinical isolates of S. aureus that influence sensitivity to serum killing. Through a phenotypic screen of 300 previously sequenced S. aureus bacteremia (SAB) isolates, they identified ~40 SNPs causing altered serum survival. The remainder of the study focuses of tcaA, a gene with unknown function. They show that when tcaA is disrupted, it results in increased resistance to glycopeptides and antimicrobial components of human serum.

They perform an elegant series of experiments demonstrating how a tcaA knockout is more resistant to killing by whole serum. arachadonic acid, LL-37 and HNP-1. They provide compelling evidence that in the absence of tcaA resistance to arachidonic acid is mediated through release of wall teichoic acids from the cell wall, which acts as a decoy and sequesters the fatty acid.

Similarly, they suggest that resistance to cationic antimicrobial peptides is through alteration of the net charge of the cell wall due to loss of negatively charged WTAs based on reduced cytochrome C binding.

They continue to show that tcaA is induced in the presence of human serum, which causes increased resistance to the glycopeptide teichplanin.

They propose that tcaA disruption causes altered cell wall structure based on morphologic changes on TEM and increased sensitivity to lysostaphin and increased autolysis via triton x-100 assay.

5, Finally, they propose that tcaA influences mortality in SAB based on raw differences in 30-day morality. Interestingly they do decreased fitness during murine bacteremia model compared to wild-type.

Strengths:

1. The manuscript is well-written and easy to follow
2. The identification of SNPs leading to altered serum killing is convincing and valuable data
3. The mechanism for tcaA-mediated resistance to arachadonic acid and AMPs is compelling and novel
4. The murine infection data demonstrating that tcaA mutants exhibit reduced virulence is important data

Weaknesses:

1. Some of the conclusions are not supported by the data shown (either missing or incomplete)
2. The authors conclude that tcaA mutants show reduced peptidoglycan crosslinking. This conclusion is based on qualitative TEM images and increased sensitivity to lysostaphyin/autolysis. While these data are suggestive. it is difficult to draw such a conclusion without analysis of the cell wall by LC-MS (such as http://doi.org/10.1371/journal.ppat.1009468).
3. The authors conclude "TcaA contributes to increased disease severity in mice and humans". While it seems biologically plausible that a polymorphism known to increase glycopeptide MIC affects mortality, the human data presented is based on raw 30-day mortality numbers. It is misleading to make the association with mortality without adjusting for confounding variables known to influence mortality in SAB (e.g. age, comorbidities, presence of sepsis, endocarditis, duration of bacteremia). Also, with just 12 patients in the SNP group, this is likely underpowered to detect any difference.

Overall, I think this is a good submission and the majority of their conclusions are supported by the data. The mechanism behind the clinically relevant tcaA mutation is important, given its known role in glycopeptide resistance and therefore likely clinical outcomes. This manuscript would benefit with the inclusion of some additional experiments to help support their finding.

Reviewer #3 (Public Review):

In this manuscript by Douglas et al., the authors used a functional genomics approach to understand how Staphylococcus aureus survives in the bloodstream to cause bacteraemia. They identified seven novel genes that affect serum survival. The study focused on tcaA, a gene associated with resistance to the antibiotic teicoplanin and is activated when exposed to serum and plays a role in producing a critical virulence factor called wall teichoic acids (WTA) in the cell envelope. This protein affects the bacteria's sensitivity to cell wall attacking agents, human defense fatty acids, and antibiotics, as well as autolytic activity and lysostaphin sensitivity. The data in this study suggested that TcaA play a role in the ligation or retention of WTA within the cell wall. However, more work is needed to clarify that part. Interestingly, despite making the bacteria more vulnerable to serum killing, tcaA contributes to S. aureus virulence by altering the cell wall architecture, as demonstrated by the wild type strain outcompeting the tcaA mutant in a Mouse Co-infection model. The study raises an important point that TcaA in S. aureus may represent a system balancing two scenarios: it makes the bacteria more susceptible to serum killing, potentially limiting bacteraemia and providing long-term benefits between hosts; however, once established in the bloodstream, the bacteria survive and thrive, causing successful bacteraemia, as per the short-sighted evolution of virulence hypothesis. This duality highlights the complex interplay between within-host and between-host fitness in bacterial evolution. I strongly suggest creating a graphical abstract to illustrate the complex relationship between within-host and between-host fitness scenarios involving TcaA. Having this visual representation in the discussion will enhance comprehension and provide a concise summary of the complex system for the reader.

In this manuscript, the authors achieved their aims, and the results support their conclusions. This work will be fundamental for understanding this complex system and for developing novel therapeutics and vaccines for S. aureus.