Biology subjects

Py, B.

Publications and source records attributed to Py, B..

6 recordsLinked to original sources

bioRxiv2023

Small RNA regulation of an essential process induces bacterial resistance to aminoglycosides during oxidative stress

Fe-S clusters are essential cofactors involved in many reactions across all domains of life. In Escherichia coli and other enterobacteria, Fe-S cluster synthesis involves two machineries: Isc and Suf. While Isc functions as a housekeeping system, Suf is activated under stress conditions such as iron starvation or oxidative stress. Interestingly, cells functioning under Suf show reduced entry of aminoglycosides, leading to resistance to these antibiotics. The transcriptional regulator IscR, itself an Fe-S cluster containing protein, controls the transition between Isc and Suf machineries. Noteworthy, IscR has a critical impact on the virulence of various bacterial pathogens by regulating both Fe-S biogenesis and other pathways directly linked to host adaptation. Here, we discovered that two small regulatory RNAs (sRNAs), FnrS and OxyS, control iscR expression by base-pairing to the 5 UTR of the iscR mRNA. Remarkably, these sRNAs act in opposite ways and in opposite conditions: FnrS, expressed in anaerobiosis, represses the expression of iscR while OxyS, expressed during oxidative stress, activates it. Using an E. coli strain experiencing protracted oxidative stress, we further demonstrate that iscR expression is rapidly and significantly enhanced in the presence of OxyS. Strikingly, we further show that OxyS induces resistance to aminoglycosides during oxidative stress through this unexpected regulation of Fe-S clusters biogenesis, revealing a new role for this sRNA. Significance StatementThis study sheds light on the regulatory mechanisms controlling the synthesis of essential Fe-S clusters in bacteria, revealing unexpected roles for two small RNAs (FnrS and OxyS) in modulating the expression of the transcriptional regulator IscR. The findings suggest that this regulatory network could lead bacterial resistance to aminoglycoside antibiotics during oxidative stress, a condition associated with chronic infections. Ultimately, this work highlights the importance of understanding the intricate regulatory networks controlling bacterial metabolism and adaptation to stress, which could have significant implications for public health.

microbiology↗

bioRxiv2023

An optogenetic approach to control and monitor inflammasome activation.

Inflammasomes are multiprotein platforms which control caspase-1 activation, leading to the processing of proinflammatory cytokines into mature and active cytokines IL-1{beta} and IL-18, and to pyroptosis through the cleavage of gasdermin-D (GSDMD). Inflammasomes assemble upon activation of specific cytosolic pattern recognition receptors (PRRs) by damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs). They converge to the nucleation of apoptosis-associated speck-like containing a caspase activation and recruitment domain (ASC) to form hetero-oligomers with caspase-1. Studying inflammasome encoding activities remains challenging because PAMPs and DAMPs are sensed by a large diversity of cytosolic and membranous PRRs. To bypass the different signals required to activate the inflammasome, we designed an optogenetic approach to temporally and quantitatively manipulate ASC assembly (i.e. in a PAMP- or DAMP-independent manner). We reveal that controlling light-sensitive oligomerization of ASC is sufficient to recapitulate the classical features of inflammasomes within minutes, and enabled us to decipher the complexity of volume regulation and pore opening during pyroptosis. Overall, this approach offers interesting perspective to decipher PRR signaling pathways in the field of innate immunity.

immunology↗

bioRxiv2023

Characterisation of the SUF FeS cluster machinery in the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is the first eukaryotic organism described as a complete amitochondriate, yet it shares common features with heterotrophic anaerobic/microaerophilic protists, some of which bear divergent mitochondrion-related organelles or MROs. It has been postulated that the retention of these organelles stems from their involvement in the assembly of essential cytosolic and nuclear FeS proteins, whose maturation requires the evolutionarily conserved mitochondrial ISC and cytosolic CIA machineries. The amitochondriate M. exilis lacks genes encoding the ISC machinery yet contains a bacteria-derived SUF system (MeSuf), composed of the cysteine desulphurase SufS fused to SufD and SufU, as well as the FeS scaffolding components MeSufB and MeSufC. Here, we show that expression of the M. exilis SUF genes, either individually or in tandem, can restore the maturation of the FeS protein IscR in the Escherichia coli double mutants of {Delta}sufS {Delta}iscS and {Delta}sufB {Delta}iscUA. In vivo and in vitro studies indicate that purified MeSufB, MeSufC and MeSufDSU proteins interact suggesting that they act as a complex in the protist. MeSufBC can undergo conformational changes in the presence of ATP and assemble FeS clusters under anaerobic conditions in presence and absence of ATP in vitro. Altogether, these results indicate that the dynamically interacting MeSufDSUBC proteins may function as an FeS cluster assembly complex in M. exilis thereby being capable of replacing the organelle-enclosed ISC system of canonical eukaryotes.

biochemistry↗

bioRxiv2022

The AAA+ ATPase RavA-ViaA complex sensitizes Escherichia coli to aminoglycosides under anaerobic low energy conservation conditions

Aminoglycosides have been used against Gram-negative bacteria for decades. Yet, uncertainties remain about various aspects of their uptake mechanism. Moreover their killing efficiency is well known to vary as a function of growth conditions and types of metabolism used by the targeted bacterium. Here we show that RavA, an AAA+ ATPase from the MoxR subfamily, associated with its VWA-containing partner, ViaA sensitize E. coli to lethal concentrations of AG, including gentamycin (Gm) and tobramycin, but not of antibiotics of other classes. We show this sensitizing effect to be due to enhanced Gm uptake in a proton motive force dependent manner. We evaluated the influence of RavA ViaA throughout a series of growth conditions, including aerobiosis and anaerobiosis. This led us to observe that the sensitizing effect of RavA ViaA varies with the respiratory chain used, i.e. RavA ViaA influence was prominent in the absence of exogenous electron acceptor or with fumarate, i.e. in poor energy conservation conditions, and dispensable in the presence of nitrate or oxygen, i.e. in high level of energy conservation. We propose RavA ViaA to be able to sense energetic state of the cell and to be used under low energy conditions for facilitating uptake of chemicals across the membrane, including Gm. Author SummaryAntibiotic resistance is a major public health, social and economic problem. Aminoglycosides are known for their high efficiency against Gram-negative bacteria but their use is restricted to life threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG sensitization mechanisms in bacteria will allow the use of a decreased effective dose of AGs. Here we identified new molecular actors, RavA and ViaA, which sensitize E. coli to AG under anaerobiosis. RavA belongs to the AAA+ ATPase family while ViaA bears a VWA motif. Moreover we show here that the influence of RavA ViaA on AG sensitivity varies with growth conditions and respiratory metabolism used by E. coli. This is a significant step forward as anaerobiosis is well known to reduce antibacterial activity of AG. This study emphasizes the crucial importance of the relationships between culture conditions, metabolism and antibiotic resistance.

microbiology↗

bioRxiv2021

Resolving phylogenetic and biochemical barriers to functional expression of heterologous iron-sulphur cluster enzymes

Many of the most promising applications of synthetic biology, including engineering of microbes for renewable chemical production, relies upon the ability of genetically-tractable hosts to express heterologous enzymes from foreign species. While countless methods for facilitating heterologous enzyme expression have been developed, comparable tools for facilitating heterologous enzyme activity are generally lacking. Such tools are needed to fully exploit the biosynthetic potential of the natural world. Here, using the model bacterium Escherichia coli, we investigate why iron-sulphur (Fe-S) enzymes are often inactive when heterologously expressed. By applying a simple growth complementation assay with collections of Fe-S enzyme orthologs from a wide range of prokaryotic diversity, we uncover a striking correlation between phylogenetic distance and probability of functional expression. Moreover, co-expression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be functionally expressed. On the other hand, we find that heterologous Fe-S enzymes that require specific electron carrier proteins within their natural host are rarely functionally expressed unless their specific reducing partners are identified and co-expressed. We demonstrate in vitro that such selectivity in part derives from a need for low-potential electron donors. Our results clarify how phylogenetic distance and electron transfer biochemistry each separately impact functional heterologous expression and provide insight into how these barriers can be overcome for successful microbial engineering involving Fe-S enzymes.

synthetic biology↗

bioRxiv2020

NLRP3 controls ATM activation in response to DNA damage.

The DNA damage response (DDR) is essential to preserve genomic integrity and acts as a barrier to cancer. The ATM pathway orchestrates the cellular response to DNA double strand breaks (DSBs), and its attenuation is frequent during tumorigenesis. Here, we show that NLRP3, a Pattern Recognition Receptor known for its role in the inflammasome complex formation, interacts with the ATM kinase to control the early phase of DDR, independently of its inflammasome activity. NLRP3 down-regulation in human bronchial epithelial cells impairs ATM pathway activation as shown by an altered ATM substrate phosphorylation profile, and due to impaired p53 activation, confers resistance to acute genomic stress. Moreover, we found that NLRP3 is down-regulated in Non-Small Cell Lung Cancer (NSCLC) tissues and NLRP3 expression is correlated with patient overall survival. NLRP3 re-expression in NSCLC cells restores appropriate ATM signaling. Our findings identify a non-immune function for NLRP3 in genome integrity surveillance and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways.

cancer biology↗