A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

  1. Abderrahman Hachani
  2. Stefano G Giulieri
  3. Romain Guérillot
  4. Calum J Walsh
  5. Marion Herisse
  6. Ye Mon Soe
  7. Sarah L Baines
  8. David R Thomas
  9. Shane Doris Cheung
  10. Ashleigh S Hayes
  11. Ellie Cho
  12. Hayley J Newton
  13. Sacha Pidot
  14. Ruth C Massey
  15. Benjamin P Howden
  16. Timothy P Stinear

  • Curated by eLife

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    eLife assessment

    This paper describes a new method to investigate Staphylococcus aureus intracellular virulence that has produced important insights into the mechanisms of staphylococcal pathogenesis. The results are convincing and the methodology is state-of-the-art. This paper will be of interest to scientists studying microbial intracellular pathogenesis and cell biology.

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Abstract

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model , we have developed a platform called InToxSa ( in tracellular tox icity of S. a ureus ) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S . aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA , encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

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  1. Version published to 10.7554/elife.84778 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    1. There are two main 'weaknesses'. The first is the limited power that comes from only using measuring the phenotype of 387 strains. Whether this is because of the expense/ difficulty of the inToxSa is not discussed, leaving open the question of how much this assay could be scaled up in the future.

    A previous study investigating the toxicity of S. aureus culture supernatants assessed 217 clinical strains (https://doi.org/10.1371/journal.pbio.1002229).That study had sufficient power to uncover important genetic determinants of S. aureus virulence. Here, we significantly increased the throughput to 387 clinical strains combined with a sophisticated cell toxicity assay that measures the kinetics of cytotoxicity caused by intracellular S. aureus. We have investigated the S. aureus genetic associations using this rich dataset (each of the 387 strains were assessed in 3 to 15 replicates, accruing 655,005 measurements corresponding to kinetic cytotoxicity assessments of intracellular S. aureus). This rich dataset enabled the accurate identification of genomic signatures that modulate cytotoxicity; genomic signatures that we then validated by reconstructing the mutations, thus demonstrating the power of our approach. The upscaling of this method (4-fold, with adequate technical adjustments) should be possible with the adoption of a 384-well plate format instead of a 96-well plate. We will continue to investigate additional clinical isolates and explore the use of 384-well plates, but the analysis we present of data from the 96-well format is already a substantial advance for the field.

    Across this study, and as presented in the current manuscript, the maximum throughput of the InToxSa assay was of 7x 96 well plates per week, thus corresponding to 98 distinct clinical strains testable per week (encompassing 6 individual replicates, each tested across 2 different days/plates). Following the reviewer suggestion, we have added this information to the discussion (Lines: 406-409).

    1. The second is that the main output of the assay is actually reduced intracellular toxicity (PI uptake AUC), which is inferred to be strongly linked to increased intracellular persistence. The linkage between the phenotypes comes primarily from microscopic studies on a limited number of strains. It may be true of all cases, but the possibility exists that for some of the strains, reduced cytotoxicity may be associated with intracellular elimination, which would presumably be a negative outcome for systemic infection.

    Whilst the reviewer’s comment is pertinent, we note that none of the least cytotoxic S. aureus isolates identified by the InToxSa assay have resulted from bacterial clearance, intracellular bacterial growth defects or evasion from their cellular niche, as we have assessed intracellular bacterial loads at 3h and 24h (post-bacterial uptake) in experimental conditions using cell-impermeant antibiotics (which would kill extracellular bacteria and prevent over-infection of non-infected bystander HeLa cells), as shown in figures 5F and 5H and also in Figure 5 Supplementary figure 5, highlighting an inverse correlation between cytotoxicity and intracellular persistence.

    Reviewer #2 (Public Review):

    1. …Thus, my concerns are focused on further understanding the practical utility of the approach and whether or not the HeLa cell model recapitulates what happens in professional phagocytes.

    HeLa cells have proven a useful cellular model in infection and in pathogen biology to assess the ability of bacterial pathogens to invade, persist and replicate within host cells. Several studies have convincingly used HeLa cells to assess S. aureus phenotypes at the bacteria-host cell interface, as exemplified by the following recent research (DOIs: 10.1128/mBio.02250-20, 10.1371/journal.ppat.1009874, 0.138/s41598-019-51894-3, and 10.1128/mSphere.00374-18). We do also acknowledge the limitations of cell line models in the discussion (Lines 494-510).

    1. …it is not clear to me that this system has the statistical power to find novel, biologically relevant rare mutations without first being very mindful in selecting strains that are extremely genetically similar.

    As described, this is a S. aureus bacteraemia study, wherein the strains composing the collection are, by definition, closely related. We articulated this in the manuscript “We used InToxSa to identify S. aureus pathoadaptive mutations, enriched in bacterial populations that are associated with human disease (e.g., upon transit from colonising to invasive”. “We hypothesised that these mutations would support an intracellular persistence for S. aureus.”) We see no foreseeable reasons preventing this type of study of being replicated elsewhere.

    1. It is also not clear to me that the toxicity assay captures the important features of the intracellular persistence that occurs in vivo within professional phagocytic cells.

    Response: Indeed, it is possible that InToxSa using HeLa cells may not capture the features of intracellular S. aureus persistence within professional phagocytes. However, our data shows that it remains possible to uncover genomic features related to intracellular cytotoxicity and persistence, both traits relevant S. aureus-host cell biology. The cells forming physical barriers, such as the epithelial cells and endothelial cells play major roles in staphylococcal pathobiology. Whilst HeLa cells are a model cell line, their tractability makes them ideal for high throughput studies tested over longer infection times.

  3. eLife

    eLife assessment

    This paper describes a new method to investigate Staphylococcus aureus intracellular virulence that has produced important insights into the mechanisms of staphylococcal pathogenesis. The results are convincing and the methodology is state-of-the-art. This paper will be of interest to scientists studying microbial intracellular pathogenesis and cell biology.

  4. eLife

    Reviewer #1 (Public Review):

    In this study, the authors sought to develop a measure of Staphylococcus aureus intracellular virulence levels in the lab (the InToxSa assay) that more closely mimics the activity seen in vivo. They then used untargeted approaches (GWAS, homoplasy) on a set of 387 Australasian clinical isolates to identify genes with mutants associated with reduced intracellular toxicity. The authors identified several mutated genes which reduced virulence in the strains chosen for the study, demonstrating that their approach can be used to uncover virulence-related genes in S. aureus.

    The study is clearly written, with high-quality figures. The development of the InToxSa assay is carefully described and logical. InToxSa was shown to potentially be more sensitive than the tryptophan blue test in detecting reduced intracellular cytotoxicity phenotype. They also showed evidence for agrA mutants and other transposon mutants with reduced inToxSa cytotoxicity having increased bacterial cell numbers cells compared to wild-type (Fig 2, Fig5GH), which is critical to the argument that bacteremia selects for intracellular persistence as a way to escape the immune system. There was an interesting and thoughtful use of random forest to choose the most appropriate parameters of the kinetic model.

    The GWAS studies used publicly deposited genome data and clearly showed lineage effects of reduced intracellular survival of CC239 and CC22, confirming previous results. GWAS also confirmed the well-known pervasive association of agr mutants with reduced toxicity. Using a well-described homoplasy test for convergent evolution to extract more power, several other potential genes associated with enhanced intracellular toxicity were discovered or rediscovered, perhaps most significantly, the ausA gene, with biosynthesizes aureusimines (pyrazinone secondary metabolites) posited to have a role in the phagosomal escape.

    There are two main 'weaknesses'. The first is the limited power that comes from only using measuring the phenotype of 387 strains. Whether this is because of the expense/ difficulty of the inToxSa is not discussed, leaving open the question of how much this assay could be scaled up in the future. The second is that the main output of the assay is actually reduced intracellular toxicity (PI uptake AUC), which is inferred to be strongly linked to increased intracellular persistence. The linkage between the phenotypes comes primarily from microscopic studies on a limited number of strains. It may be true of all cases but the possibility exists that for some of the strains, reduced cytotoxicity may be associated with intracellular elimination, which would presumably be a negative outcome for systemic infection.

    Overall, the authors achieved their aims in terms of assay development and showing the utility of the pipeline for mutation discovery. This is a waypoint in the larger aim of understanding mutational pathways that lead to increased persistence of systemic S. aureus. Obviously, a lot more data is needed. The InToxSa intracellular screening method is interesting and could be reused/adapted by the community. This research should also spark more interest in the role of ausA and aureusimines in virulence and some of the other genes discovered through the untargeted approach.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript introduces a novel assay in a 'phenomics' approach to address an important aspect of S. aureus pathogenesis. The authors set out to identify mutations that arise during clinical S. aureus infections that cause a decrease in intracellular host-cell toxicity and increase intracellular persistence. To do this, they use a 'phenomics' approach. For phenotype, they quantify HeLa cell toxicity for each strain in a panel of 387 clinical S. aureus isolates. This is done by measuring HeLa cell death induced by intracellular S. aureus via propidium-iodide uptake. The whole genomes of each of these 387 isolates had previously been sequences. They use the genomic data and phenotype data to carry out a genome-wide association study (GWAS) looking for genetic signatures that correlate with reduced HeLa cell cytotoxicity. As expected, mutations in agr were the strongest locus-level signal, but the study did identify one agr-independent mutation in ausA, which was able to be independently validated, showing that the assay is robust enough to find causal mutations. The analysis is thoughtful, the assay appears robust, and I think the discussion of conclusions and limitations is mostly valid. Thus, my concerns are focused on further understanding the practical utility of the approach and whether or not the HeLa cell model recapitulates what happens in professional phagocytes. For example, it is not clear to me that this system has the statistical power to find novel, biologically relevant rare mutations without first being very mindful in selecting strains that are extremely genetically similar. It is also not clear to me that the toxicity assay captures the important features of the intracellular persistence that occurs in vivo within professional phagocytic cells. Thus, given these practical limitations and a somewhat artificial model system, the impact on the field is likely to be moderate in nature. However, the analysis and approach taken could be re-purposed to any robust quantitative phenotype, and this will certainly be of great interest to others that study bacterial evolution in clinical contexts.

  6. Version published to 10.1101/2022.12.11.519971v1 on bioRxiv