Staphylococcus aureus sRNA-mediated adaptation to metal-based host defense strategies – MetalAureus

To identify and characterize the S. aureus regulatory RNAs involved in metal homeostasis, we have developed a high-throughput sequencing approach targeting regulatory RNAs. Using a combination of experimental approaches, such as MAPS (MS2-affinity purification coupled with RNA sequencing), and in silico predictions, we have revealed the interactome of these regulatory RNAs. We then used various microbiological, biochemical, and molecular methods to characterize their respective roles in S. aureus survival and virulence.

This work was first carried out under controlled growth conditions, which allowed us to dissect the responses of S. aureus to metal-dependent stress and to facilitate the transition to more physiological and therefore more complex conditions. Subsequently, the metal-based host defense strategies and countermeasures of S. aureus were examined in the presence of effectors (calprotectin), immune cells (macrophages) and also within the host (mouse model). To this end, we have implemented the dual RNA sequencing method, which allows the simultaneous measurement of gene expression in S. aureus and host cells.

Using a small RNA sequencing approach (80-300 nucleotides), we have identified several regulatory RNAs whose expression is induced or repressed in response to different types of stress, including metal starvation or excess.

IsrR is the functional analog of the RyhB sRNA identified in E. coli. Like RyhB, IsrR synthesis is repressed by the transcription factor Fur in the presence of iron. In collaboration with Dr. Jeffrey Boyd (The State University of New Jersey - USA), we have shown that IsrR represses the expression of many iron-dependent enzymes involved in the TCA cycle, in response to iron starvation. Thus, IsrR reduces intracellular iron demand and allows its remobilization. The maintenance of iron homeostasis by IsrR is particularly important for the virulence of S. aureus.

We have also identified an unprecedented interaction between IsrR and the Mn-dependent riboswitch mnrS, enabling fine-regulation of the expression of the mntY gene, encoding a Mn efflux pump. Remarkably, deletion of mntY induces significant growth defects, decreases expression of virulence factors and drastically affects immune evasion and survival of S. aureus during infection. We have demonstrated that MntY is essential for the adaptation of S. aureus to both low and high Mn environments, due to its dual role in metalation of Mn-dependent exoenzymes and in Mn detoxification. Our findings identify MntY as a promising new therapeutic target to combat staphylococcal infections.

In addition, we have characterized the first regulatory RNA involved in a zinc-sparing response in bacteria. ZinS is negatively regulated by the transcription factor Zur in the presence of zinc. In the absence of Zn, ZinS is synthesized and reduces the production of several Zn-dependent enzymes. Similar to IsrR, the control exerted by ZinS reduces the intracellular need for zinc.

In collaboration with Dr. Thomas Kehl-Fie (University of Iowa - USA), we have demonstrated that RsaC sRNA effectively reduces the cellular demand for manganese in response to Mn starvation. However, the benefit of this sparing response depends on the environmental conditions, as RsaC suppresses the expression of the Mn-dependent superoxide dismutase, which sensitizes S. aureus to oxidative stress. Nevertheless, RsaC is necessary for S. aureus to establish infection, and its importance depends on the efficiency of manganese sequestration by the host.

This detailed characterization of the regulatory networks involved in S. aureus adaptation to metal fluctuations revealed that iron, manganese and zinc homeostasis in S. aureus is controlled by the combination of a metal-sensitive transcription factor and a regulatory RNA.

The MetalAureus project has enabled us to identify new key players in metal homeostasis and thus in the adaptive response of S. aureus to metal-dependent host defense strategies. The characterization of these regulatory networks is necessary to understand how this pathogen copes with these immune strategies during infection. Indeed, identifying the adaptive mechanisms that S. aureus uses to respond to specific stresses under controlled conditions will facilitate the transition to more complex metal-limiting environments, such as abscesses.

Our work has also revealed complex and interdependent connections between the different metallosystems. These findings open up new therapeutic opportunities. We plan to exploit the established interactions by targeting the homeostasis of a specific metal to sensitize S. aureus to the limitation or excess of another metal.

In addition, we have shown that the manganese efflux pump MntY is critical for resistance to several antimicrobial compounds, immune evasion and establishment of infection. The fact that MntY is an essential protein and is located on the surface of the bacterial membrane makes this pump a relevant and attractive target for therapeutic development against this dreadful human pathogen. The sophisticated regulation of its synthesis to avoid the toxicity associated with its overexpression and deletion suggests that it may have a reduced tolerance to mutations. We plan to use various strategies to block the function of this pump, thus mimicking the drastic effects obtained by deleting the corresponding gene. Since this membrane protein is conserved, this therapeutic strategy could be extended to other pathogenic bacteria.

Staphylococcus aureus is an opportunistic human pathogen responsible for a wide range of superficial and severe systemic infections. Even more alarming is the emergence and worldwide spread of hypervirulent multidrug-resistant isolates such as methicillin-resistant S. aureus (MRSA), which is included in the World Health Organization priority pathogens list for the research and development of new antibiotics. Hence, there is an urgent need to explore new therapeutic avenues to tackle this global health concern.
A promising strategy is to emulate and strengthen existing host defense mechanisms such as those modulating metal bioavailability. Indeed, host immune cells, especially neutrophils and macrophages, either restrict access to essential metals or poison S. aureus with toxic metals overload. Despite experiencing both metal-related feast (metal toxicity) and famine (nutritional immunity), S. aureus possesses efficient adaptative mechanisms to survive. Notably, our preliminary data point out the crucial role of small regulatory RNAs (sRNAs). Since their discovery in the early 1980’s, accumulated results have depicted sRNAs as key actors in virtually all aspect of bacterial physiology (e.g. primary metabolism, cell division or virulence). Usually described as 50 to 500 nt-long RNA fragments, sRNAs are mostly involved in the post-transcriptional regulation of a subset of messenger RNAs forming their interactome. Interestingly, our recent data support the existence of a functional analog of the well-characterized sRNA RyhB, the real orchestra conductor during iron (Fe) starvation in Escherichia coli, which quickly re-establishes Fe homeostasis. Moreover, we identified the first manganese (Mn)-responsive sRNA, RsaC. The major function of RsaC is to spare intracellular Mn2+ via the repression of non-essential Mn-containing proteins like the superoxide dismutase A (SodA). In parallel, RsaC plays a critical role in the oxidative stress response by promoting the alternative Fe-SodM-dependent pathway to detoxify reactive oxygen species (ROS), notably produced by immune cells to eradicate S. aureus. Remarkably, RsaC has a broader role in iron and zinc stress responses, but also in the synthesis of virulence factors, strengthening the crucial role of sRNAs in connecting metal-related countermeasures and pathogenicity.
Through the MetalAureus project, we intend to reveal the pivotal role of sRNAs in S. aureus survival and adaptation to both metal starvation and toxicity, typically encountered during infection. As S. aureus needs to strictly balance the cellular level of various essential metals, our working hypothesis is that additional metal-responsive sRNAs remain to be identified. For this purpose, we plan the unprecedented identification of all sRNAs involved in the homeostasis of 6 metals (i.e. Fe, Mn, Zn, Cu, Co and Ni) regarded as crucial for S. aureus physiology and virulence. Then, we will decipher their functions by revealing their whole interactome using innovative and complementary high-throughput RNA sequencing technologies (DBRI and MAPS). This will first be performed in controlled growth conditions (e.g. specific metal-depleted media or metal overload) to untangle intricate and intertwined regulatory networks and to facilitate data interpretation in more complex physiological conditions. Indeed, the ultimate goal is to scrutinize both host metal-based defense strategies and S. aureus countermeasures during host-pathogen interaction using the powerful Dual RNA-seq method.
The MetalAureus project represents an exceptional opportunity to understand the mechanisms employed by S. aureus to evade metal-based immune defenses and to cope with promising metal-related therapeutic strategies (e.g. metal chelators or metal-based antimicrobials). This is notably of paramount importance to tailor and exploit these alternatives to the classical antimicrobial compounds.