Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa

Author's response

Laura Camus shared

1. Could acetoin be a carbon source produced by other microbiota species which may indirectly promote aureus co-existence with P. aeruginosa?

Acetoin is indeed known to be produced by other microbial species; Bacillus subtilis is for instance a great acetoin producer (1). Regarding microorganisms colonizing CF environment, clinical isolates of Stenotrophomonas maltophilia were sometimes shown to produce the molecule, although it is not a generality (2, 3). Acetoin dosed in the CF sputa can thus arise from production by S. aureus but also by other microbial species present in the sample, as well as other microorganisms than P. aeruginosa might catabolize acetoin in these conditions (1). This question could be addressed by evaluating the abilities of acetoin production and catabolism of all microorganisms isolated from the CF sputa, which can represent a lot of work due to the number of samples.

We observed that acetoin production and catabolism were both more efficient for coexisting strains of P. aeruginosa and S. aureus. However, if acetoin can promote the coexisting interaction between P. aeruginosa and S. aureus remains unclear. Coexistence seems to be related to P. aeruginosa adaptation to CF environment, that induces a decreased production of virulence and thus anti-staphylococcal factors. This low-virulent state was shown to be selected in response to selective pressures mainly induced by the host immune system (4, 5).

In one hand, acetoin could also constitute a selective force and directly favor a non-virulent state of P. aeruginosa. In this case, acetoin presence, produced by S. aureus or other microorganisms, would play a role in the coexistence establishment. In the other hand, evolution in the CF lungs also goes along with trophic adaptation according to the available resources in this environment (6). The acetoin metabolism between our two pathogens might arise from this trophic adaptation, independently of any virulence modifications. If so, acetoin presence would probably not affect the anti-staphylococcal behavior of P. aeruginosa, and thus coexistence.

2. The changes to aeruginosa gene expression of liuA (involved in leucine metabolism) requires an interaction with S. aureus, do you have a suggestion of what role liuA may play in co-existing infections?

P. aeruginosa trophic adaptation to CF lung goes along with auxotrophy and could explain why leucine degradation seems to be an important feature of our clinical strains (4). We suppose that P. aeruginosa overexpresses genes of the leucine catabolism pathway in co-culture with S. aureus due to elevated leucine levels in these conditions, in comparison to P. aeruginosa monoculture. Although this amino acid is already part of the rich medium we use (BHI), elevated leucine levels could come from a production by S. aureus. Interestingly, leucine and acetoin biosynthesis pathways are connected and they share a direct and common precursor (1, 7). This suggests that leucine, besides acetoin, may be overproduced by S. aureus during the coexisting interaction. We hypothesize that leucine, and thus P. aeruginosa’s liuA gene, might be the factors of another trophic cooperation between the two bacteria.

Why liuA overexpression relies on S. aureus presence requires further investigations. Contrary to acetoin, leucine may not be secreted by S. aureus in the medium in absence of P. aeruginosa. This could explain why S. aureus supernatant (grown in monoculture) does not induce the liuA overexpression in P. aeruginosa. It is also possible that other signals than leucine play a role in liuA induction. In all cases, leucine dosages in mono- and co-culture would certainly answer many questions about this mechanism.

3. The production of acetoin by aureus was increased in the presence of the co-existing P. aeruginosa strain, perhaps suggesting that S. aureus senses and responds to P. aeruginosa, do you know how this effect is caused?

We indeed observed an increased acetoin production by coexisting S. aureus strains in comparison to competitive ones, but only when the strains were cultivated in P. aeruginosa supernatant.

This phenomenon could rely only on nutritional and metabolism clues. Indeed, it is likely that P. aeruginosa supernatant presents a highly different composition than unaltered rich medium, with different proportions of each resource usable by S. aureus. This specific nutrient composition may dysregulate metabolic pathways of S. aureus, including acetoin biosynthesis. Coexisting S. aureus thus appears to regulate their metabolism differentially than competitive ones according to the medium composition. Such metabolic adaptations were already described for P. aeruginosa in the CF context (6).

However, it is also possible that this effect relies on P. aeruginosa quorum-sensing (QS) molecules, that are diffused in the medium and thus in the culture supernatant. Besides their role in intra-species interactions, it is well-known that these signals can be sensed by other microorganisms and affect their activities (8). It would be possible that some QS-molecules of P. aeruginosa are able to directly modify the regulation of acetoin production in S. aureus, especially in the coexistence context. Interestingly, we observed that a lasR defective mutant of PA14 was able to catabolize acetoin more efficiently that the wild-type strain, supporting the role of QS in this activity (not published).

In all cases, the exact signals and mechanisms of this increased production of acetoin in presence of P. aeruginosa supernatant remain to be studied. By performing experimental evolution, we aim to confirm that this phenomenon arises from co-evolution of P. aeruginosa and S. aureus.

References

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