David Lalaouna

The bacterial small RNA (sRNA) OxyS is expressed in Escherichia coli during oxidative stress. The sRNA OxyS enhances cell survival by controlling genes involved in the regulation of hydrogen peroxide (H ₂ O ₂ ) and iron-sulfur (Fe-S) cluster formation. Here, we used the MS2 affinity purification coupled with RNA sequencing (MAPS) technique to identify new target mRNAs of the sRNA OxyS. Our analysis revealed a significant enrichment of mepS mRNA, which encodes a peptidoglycan endopeptidase that promotes cell growth. Our results confirm a previous report on the sRNA OxyS repressing the translation of mepS . We also found that an Δ oxyS background facilitates the emergence of mutations, conferring increased resistance to the last-resort antibiotics polymyxin B and E (colistin), but only in the presence of the target mepS gene. This suggests that the translation repression of mepS by OxyS could prevent mutations in bacterial DNA during H ₂ O ₂ -induced oxidative stress. Moreover, we show that adding the antioxidant thiourea or sequestering iron in the Δ oxyS background effectively reduces the emergence of resistance against both polymyxin B and colistin. These results suggest that reactive oxygen species (ROS), in conjunction with intracellular iron, play a key role in driving the emergence of antibiotic resistance. Overall, our work underlines a mechanism of antimicrobial emergence implicating oxidative stress, intracellular Fe, and cell wall remodeling in E. coli . IMPORTANCE: This study uncovers an underexplored link between peptidoglycan remodeling and oxidative stress responses during exposure to antibiotics. By elucidating how MepS and the sRNA OxyS interact in the presence of polymyxins and oxidative stress, our study suggests that MepS may exert an anti-mutator function. The repression of mepS translation by OxyS seems to limit the emergence of antibiotic resistance driven by DNA mutations. Together, these findings suggest cell wall remodeling and oxidative stress response pathways as promising targets to enhance antibiotic efficacy and limit the emergence of resistance.

Small regulatory RNAs (sRNAs) are key drivers of bacterial adaptation to environmental fluctuations, including iron and manganese restriction imposed by the host. This study explored the repertoire of sRNAs produced by the human pathogen Staphylococcus aureus in response to metal limitation. Two sRNAs were identified, S1077 and ZinS, that were regulated by zinc (Zn) availability. Further investigations revealed that, similar to the cnt operon from which it derives, S1077 synthesis is controlled by the transcription factors Zur and Fur. In contrast, zinS transcription is solely repressed by Zur. RNA affinity purification identified several ZinS-associated mRNAs encoding Zn-dependent enzymes, including the alcohol dehydrogenase Adh, whose synthesis is negatively regulated by ZinS. Loss of ZinS does not alter staphylococcal metal accumulation, suggesting a role in a Zn-sparing response. Remarkably, zinS also encodes a small peptide, ZinP. Genomic analysis shows that the regulatory portion of zinS emerged from the 3′ untranslated region of zinP and is conserved in S. aureus and closely related species, whereas zinP is also found in phylogenetically distant organisms. Our findings demonstrate that sRNAs facilitate bacterial adaptation to Zn limitation, and that genetic exchange and subsequent neofunctionalization have enabled S. aureus to adapt to metal-restricted environments.

The bacterial small RNA (sRNA) OxyS is expressed in Escherichia coli during oxidative stress. The sRNA OxyS enhances cell survival by controlling genes involved in the regulation of hydrogen peroxide (H2O2) and iron-sulfur (Fe-S) cluster formation. Here, we used the MS2 affinity purification coupled with RNA sequencing (MAPS) technique to identify new target mRNAs of the sRNA OxyS. Our analysis revealed a significant enrichment of mepS mRNA, which encodes a peptidoglycan endopeptidase that promotes cell growth. Our results confirm a previous report on the sRNA OxyS repressing the translation of mepS. We also found that an ΔoxyS background facilitates the emergence of mutations, conferring increased resistance to the last-resort antibiotics polymyxin B and E (colistin), but only in the presence of the target mepS gene. This suggests that the translation repression of mepS by OxyS could prevent mutations in bacterial DNA during H2O2-induced oxidative stress. Moreover, we show that adding the antioxidant thiourea or sequestering iron in the ΔoxyS background effectively reduces the emergence of resistance against both polymyxin B and colistin. These results suggest that reactive oxygen species (ROS), in conjunction with intracellular iron, play a key role in driving the emergence of antibiotic resistance. Overall, our work underlines a mechanism of antimicrobial emergence implicating oxidative stress, intracellular Fe, and cell wall remodeling in E. coli. IMPORTANCE: This study uncovers an underexplored link between peptidoglycan remodeling and oxidative stress responses during exposure to antibiotics. By elucidating how MepS and the sRNA OxyS interact in the presence of polymyxins and oxidative stress, our study suggests that MepS may exert an anti-mutator function. The repression of mepS translation by OxyS seems to limit the emergence of antibiotic resistance driven by DNA mutations. Together, these findings suggest cell wall remodeling and oxidative stress response pathways as promising targets to enhance antibiotic efficacy and limit the emergence of resistance.

Small regulatory RNAs (sRNAs) can specifically bind to their target mRNAs to inhibit their expression, either by promoting degradation or by blocking translation. However, the molecular determinants that select which repression mode an sRNA employs remain poorly understood. To investigate this, we used the model target mRNA hdeD , which is repressed by two sRNAs, CyaR and RprA, both of which use distinct modes of repression. Whereas CyaR promotes the target mRNA hdeD degradation, the sRNA RprA specifically blocks hdeD translation initiation, leaving hdeD mRNA intact. We found that mutating two nucleotides in the seed pairing region of RprA with hdeD is sufficient to switch its mode of repression from translation inhibition to degradation by recruiting RNase E. We investigated this further by using various RNase E deletion mutants of the C-terminal domain (CTD). Among these was an RNase E mutant deleted to contain a minimal C-terminal scaffold ( rne 701), a form which typically fails to promote sRNA-induced mRNA degradation. Our data indicated that even with a minimal scaffold, the mutated RprA sRNA still promoted hdeD mRNA degradation, whereas the native sRNA CyaR could not. Our data suggested that the mutated RprA sRNA relies primarily on the arginine-rich RNA-binding (ARRBD) domain of RNase E to induce target mRNA hdeD degradation. This suggests that the mutated RprA sRNA requires fewer components of the CTD of RNase E and RNA degradosome to induce target mRNA degradation.

Pseudomonas brassicacearum a la particularité de générer une diversité intraclonale aussi bien in vitro qu'en conditions naturelles dans la rhizosphère de plantes. Ce phénomène de variation phénotypique commun chez les bactéries est un processus d'adaptation aux environnements changeants. Des données de transcriptomique issues de puces à ADN, contenant aussi bien des séquences codantes que non codantes, nous ont permis d'identifier les gènes dont l'expression est altérée et surtout de relier ce phénomène à l'expression d'ARN non codants régulateurs (ARNnc) de type Rsm qui sont sous le contrôle du système à deux composants GacS/GacA. Nous avons montré que des mutations ponctuelles dans les gènes gacS ou gacA sont à l'origine de cette variation phénotypique et que l'expression de l'un des trois gènes rsmX, rsmY ou rsmZ permet de restaurer le phénotype de la souche sauvage. L'importance de ces ARNnc dans la survie de la bactérie aux fluctuations de son environnement est dénotée par la duplication de rsmX en un gène que nous avons nommé rsmX-2, dont la fonction a été validée. Nos données suggèrent une activation exclusive des gènes rsmX-1 et rsmX-2 par GacA et l'intervention de régulateurs additionnels dans le cas de rsmY et rsmZ. Au vu de la redondance fonctionnelle de ces quatre ARNnc, nous avons investigué leur niveau d'expression et leur stabilité dans différentes conditions de culture et montré des différences pour les quatre ARNnc. En réponse à une carence en nutriments, l'expression des ARNnc Rsm est fortement activée et atteint son maximum quand le ppGpp est détecté dans le milieu, suggérant un lien entre le système Gac/Rsm et la réponse « stringente ».

Bacterial regulatory RNAs (sRNAs) are commonly short non-coding RNAs that function as pleiotropic regulators by post-transcriptionally impacting mRNA stability and/or translation. They play significant roles in bacterial physiology and are typically expressed in response to specific environmental stimuli such as nutrient limitation. The bacterial pathogen Staphylococcus aureus faces decreased access to essential metal ions, including iron, in the mammalian host via a process called nutritional immunity. In response to host-mediated iron limitation, S. aureus expresses the sRNA IsrR, which coordinates an iron-sparing response by downregulating the expression of mRNAs coding for iron-requiring proteins or processes. Herein, we utilized MS2-Affinity Purification coupled with RNA Sequencing (MAPS) to reveal the in vivo IsrR interaction network. Analysis of co-purified RNAs revealed previously unpredicted putative IsrR targets coding for proteins associated with iron-requiring processes. We validated that IsrR directly interacts with nine targets in vitro . We demonstrate physiological roles for IsrR in mediating heme biosynthesis, aerobic respiration, and the detoxification of oxygen radicals. These activities are critical for pathogenesis, and this study establishes how S. aureus leverages these processes to adapt to iron scarcity, which is commonly encountered in the mammalian host. IMPORTANCE: Staphylococcus aureus causes numerous and varied infections in mammals, making it a significant public health burden and concern. The prevalence of S. aureus infections is due to its robust repertoire of virulence factors and its ability to adapt to host microenvironments. Elucidation of the metabolic processes and pathways that promote adaptation to host-promoted stressors provides information about host-pathogen interactions. It could also aid the development of new antimicrobials or unveil treatment and prevention strategies. One common stress bacteria encounter within the mammalian hosts is limited access to iron. In response to iron scarcity, S. aureus expresses the regulatory sRNA IsrR. Here, we identified mRNAs that associate with IsrR. We verified that IsrR targets mRNAs that code for proteins involved in aerobic respiration, the metabolism of reactive oxygen species, and heme synthesis. This work provides significant insight into how S. aureus responds to host-mediated iron starvation.

Small regulatory RNAs (sRNAs) are key drivers of bacterial adaptation to environmental fluctuations, including iron and manganese restriction imposed by the host. This study explored the repertoire of sRNAs produced by the human pathogen Staphylococus aureus in response to metal limitation. Two sRNAs, S1077 and ZinS (RsaX20), regulated by zinc (Zn) availability, were identified. Further investigations revealed that, similar to the cnt operon from which it derives, S1077 synthesis is controlled by the transcription factors Zur and Fur. In contrast, zinS transcription is solely repressed by Zur. Amongst the ZinS targets are several Zn-dependent enzymes, such as the alcohol dehydrogenase Adh, whose synthesis is negatively regulated by ZinS. Loss of ZinS does not alter staphylococcal metal accumulation, suggesting a role in a Zn-sparing response. Remarkably, zinS also encodes a small peptide, ZinP. Genomic analysis suggests that the regulatory portion of ZinS emerged from the 3’ untranslated region of zinP in S. aureus and closely related species after horizontal gene transfer from phylogenetically distant organisms. All our findings demonstrate that sRNAs also facilitate bacterial adaptation to Zn limitation, and that genetic exchange and subsequent neofunctionalization have enabled S. aureus to adapt to metal-restricted environments.

Responding to stress is critical to the survival of life, especially for microbes that have a limited ability to manipulate their environment. During infection, Staphylococcus aureus and other invaders must overcome both the host-imposed absence of manganese and the oxidative burst of immune cells, which increases the need for this essential metal. The current investigations revealed that a small RNA, RsaC, integrates the staphylococcal responses to manganese starvation and oxidative stress. Upon manganese limitation, RsaC activates a manganese-sparing response, which decreases the cellular demand for manganese, enabling growth in manganese-restricted environments. However, the benefit of this response is environment-dependent as RsaC suppresses the expression of the manganese-dependent superoxide dismutase SodA, sensitizing S. aureus to oxidative stress. Despite this suppression, RsaC is necessary for S. aureus to cause infection, with its importance dependent on the efficacy of the host’s manganese withholding response. These results reveal a previously unappreciated manganese-sparing response that is important for bacterial virulence, and the imperative role of sRNAs in balancing bacterial adaptation to stressors that place conflicting demands on cellular physiology. IMPORTANCE: During infection, pathogens must utilize processes that impose conflicting cellular demands. This conflict is exemplified by the need of Staphylococcus aureus to preserve essential processes and survive the oxidative burst of immune cells, both of which require manganese despite experiencing host-imposed manganese starvation. The current investigations revealed that S. aureus activates a manganese-sparing response controlled by the regulatory RNA, RsaC, in response to host-imposed manganese starvation. This small RNA sacrifices the expression of a manganese-dependent superoxide dismutase to preserve the activity of essential manganese-dependent processes. Despite this, RsaC is necessary for infection, revealing the important role of this manganese-sparing response to pathogenesis and that invaders must actively compromise ideal stress responses to cause disease.

SUMMARY: Maintaining manganese and iron homeostasis is critical for the human pathogen Staphylococcus aureus . To counteract metal-based host defense strategies (e.g., nutritional immunity, metal poisoning), S. aureus uses a combination of metal-sensing transcription factors and regulatory RNAs to maintain metal homeostasis. In this study, we uncovered an unprecedented interaction between a cis- and a trans-acting regulatory RNA controlling a conditionally essential gene, mntY , encoding a Mn efflux pump. This broadly conserved RNA-RNA interaction between a Fe-responsive sRNA and a Mn-sensing riboswitch allows the integration of Feand Mn-related stresses, notably encountered at the infection site, to fine-tune mntY expression. Remarkably, deletion of the mntY gene is strongly pleomorphic, causing growth defects, altering virulence factor expression, immune evasion, and survival during infection. We demonstrated that MntY is critical 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 Mn detoxification. These findings point to MntY as a promising new therapeutic target to combat multidrug-resistant staphylococcal infections.

Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3’ UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3’ UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3’ UTR was found to repress dapE , a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3’ UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3’ UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3’ UTR. Our findings reveal an extended regulatory network for vigR , uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate.