Biology subjects

Ravins, M.

Publications and source records attributed to Ravins, M..

8 recordsLinked to original sources

bioRxiv2026

A small RNA guides a post-transcriptional regulatory protein to its target

RNA-binding proteins are central to post-transcriptional regulation and are generally thought to recognize their target RNAs through direct binding. Here, we uncover a previously unrecognized mechanism in which a small RNA (sRNA) guides a protein regulator to specific target mRNAs, enabling programmable post-transcriptional control. By mapping in pathogenic Escherichia coli the RNA interactome of CsrA, a global post-transcriptional regulator, we identify ~800 ternary complexes in which CsrA simultaneously binds an sRNA and an mRNA. Focusing on one class of these complexes, we show that PasE, a newly discovered sRNA, directs CsrA to virulence-associated mRNAs that are otherwise not targeted by CsrA, resulting in their repression. Altering the PasE seed sequence redirects CsrA to selected mRNAs, establishing a modular RNA-guided platform for gene regulation. Together, our findings reveal a new and likely widespread mechanism of bacterial post-transcriptional regulation and provide a framework for a new type of RNA-based synthetic regulation.

microbiology↗

bioRxiv2026

Bacterial nanotubes extend from a tubeosome organelle to establish an intercellular bridge

Bacterial nanotubes are widespread intercellular membranous bridges mediating molecular exchange among neighboring cells, yet their formation and dynamics remain largely unexplored. By combining live-cell imaging with in situ cryo-electron tomography, we describe nanotube biogenesis in the Gram-positive Bacillus subtilis. Nanotubes form elaborate, highly dynamic networks, with extending nanotubes exhibiting multidirectional movement toward nearby cells, repeatedly scanning their surface. Ultimately, nanotubes penetrate through the recipient cell wall to establish an intercellular bridge. Strikingly, nanotubes emerge within milliseconds from lipid-enriched sites representing a previously unrecognized membranous organelle, termed tubeosome, located in an unusual pseudo-periplasmic space beneath the cell wall. We present evidence that tubeosomes comprise proto-nanotube reservoirs that unfold to project nanotubes. Our study uncovers the tubeosome as a novel bacterial organelle and provides the first real-time visualization of intercellular bridge formation. As connecting nanotubes are found across bacteria, archaea, and eukaryotes, this mechanism may reflect a broadly conserved strategy for cellular communication.

microbiology↗

bioRxiv2025

A family of endonucleases that block nanotube-mediated plasmid dissemination

Small non-conjugative plasmids constitute a substantial portion of the bacterial mobile genome, driving the dissemination of beneficial genes, with their transfer primarily attributed to transformation, transduction, or co-mobilization with conjugative elements1-3. Here we explore an understudied plasmid spread route among bacteria, mediated by intercellular membranous nanotube conduits4. We reveal that, unlike traditional donor-to-recipient delivery, Nanotube-dependent Plasmid exchange (NPex) operates bidirectionally, enabling both plasmid donation and, to a lesser extent, plasmid acquisition. By identifying a Bacillus subtilis natural isolate deficient in NPex, we discovered a prophage-encoded factor, YokF, that blocks plasmid transmission, chiefly acting within the donor cell to inhibit plasmid donation. YokF is a nuclease that localizes to the membrane, where it interacts with a nanotube component to selectively impede plasmid transfer through degradation. Importantly, YokF homologs from various bacterial species were found to exhibit anti-NPex activity. Given their prevalence in Gram-positive bacteria, we propose that YokF homologs represent a conserved family of NPex gatekeepers that restrict plasmid flow within bacterial communities.

microbiology↗

bioRxiv2025

Ectopic expression of the sensor CovS cytosolic domain confers phosphatase activity and full virulence to the M1T1 CovSY39H attenuated variant of group A Streptococcus

Group A Streptococcus (GAS) causes a wide variety of diseases ranging from mild, noninvasive, such as pharyngitis and impetigo, to life-threatening infections, such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The two-component CovR/S system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence. An attenuated pharyngeal colonizing variant (S126) possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H) was recovered in France from a member of a family in which another individual developed NF and STSS caused by the M1T1 WT strain (S119). We employed transcriptome analyses (RNA-seq), quantitative determinations of CovR phosphorylation, measurements of virulence factor activity, and a murine model of human NF to demonstrate that CovS of strain S126 almost lost its entire phosphatase activity but retained its kinase and phosphotransfer activities. Moreover, we reversed its attenuated phenotype by ectopically expressing the cytosolic domain of wild-type CovS. Culturing the corresponding strain S126covS-3 in a chemically defined medium (CDM) supplemented with asparagine (Asn), conditions that produce an excess of cytosolic ADP over ATP, stimulated the ectopically expressed phosphatase activity. Consequently, this led to dephosphorylation of CovR[~]P and increased the expression of virulence factors. Most importantly, S126covS-3 reverted to the wild-type phenotype of S119 in the mouse model of human GAS NF. Our study provides a new mechanistic tool that enables the manipulation of CovS phosphatase activity both in vitro and in vivo. IMPORTANCEThe shift from high to low virulence and back in GAS is critical for understanding its pathogenesis and developing new treatments to control GAS infections. The two-component CovR/S system, comprising the sensor kinase/phosphatase CovS and the transcription regulator CovR, regulates the degree of GAS virulence. In the M1T1 serotype, covR/S mutations are typically associated with a loss of CovR/S function, leading to hypervirulent phenotypes. However, an attenuated pharyngeal-colonizing variant possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H strain S126) was isolated in France. Here, we demonstrate that S126 is attenuated because the mutation in CovS inhibits its phosphatase activity but preserves its kinase and phosphotransfer activities. By expressing the cytosolic domain of WT CovS in the S126 background, we endowed the resulting strain, S126covS-3, with phosphatase activity, which was further stimulated by ADP when formed in chemically defined medium (CDM) supplemented with asparagine (Asn). Most importantly, S126covS-3 switched to full virulence, similar to that of S119 in a mouse model of human GAS NF. These findings underscore the importance of comprehensive analyses of disease-related covR/S mutants in understanding the virulence and persistence of GAS.

microbiology↗

bioRxiv2024

A conjugative plasmid exploits flagella rotation as a cue to facilitate its transfer

Conjugation-mediated DNA delivery is the primary mode for antibiotic resistance spread; yet, molecular mechanisms regulating the process remain largely unexplored. While conjugative plasmids typically rely on solid surfaces to facilitate donor-to-recipient proximity, the pLS20 conjugative plasmid, prevalent among Gram-positive Bacillus spp., uniquely requires fluid environments to motivate its transfer. Here we unveiled that pLS20, carried by B. subtilis, induces adhesin-promoted multicellular clustering, which can accommodate various species, offering a stable platform for DNA delivery in liquid milieu. We further discovered that induction of pLS20 promoters, governing crucial conjugative genes, hinges on the presence of donor cell flagella, the major bacterial motility organelle. Moreover, pLS20 regulatory circuit is strategically integrated into a mechanosensing signal transduction pathway responsive to flagella rotation, harnessing propelled flagella to activate conjugation genes exclusively during the host motile phase. This flagella-conjugation coupling strategy, provides the plasmid with the benefit of disseminating into remote destinations, infiltrating new niches.

microbiology↗

bioRxiv2024

Asparagine couples group A Streptococcal metabolism to virulence

Streptococcus (GAS) is a highly adapted and human-restricted pathogen causing a wide variety of infections, some life-threatening1. This ability is linked to the expression of many virulence factors, whose transcription is regulated by the two-component system, CovR/S2-5. Here, we show that genome transcription of GAS cultured in a chemically defined medium (CDM) is globally affected when supplemented with asparagine (Asn), including increased expression of many virulence genes. For the first time, we report that GAS solely depends on asparagine synthetase (AsnA) for Asn synthesis, on the ABC transporter (GlnPQ) to import Asn, and on the asparaginase (AsnB) to maintain a precisely balanced intracellular Asn concentration. Furthermore, we show that mutants defective in either asnA, glnP, or asnB express significantly lower levels of virulence factors in CDM and are severely attenuated in the sublethal murine model of human GAS soft-tissue infection. We further show that the synthesis and import of Asn in GAS are ATP-dependent and negatively regulated by intracellular Asn. Thus, Asn availability controls the intracellular ATP level. When ATP becomes limiting, CovR phosphorylation decreases. This augments GAS growth rate, virulence production, metabolism, and vice versa when the ATP level increases. Furthermore, excess Asn accumulates inside GAS in AsnB mutant, destroying the balance between Asn and ATP. We discuss the high similarity between these mechanistic principles of the Asn-mediated control of GAS virulence and metabolism to the Asn-mediated control of tumor growth6, indicating evolutionary significance.

microbiology↗

bioRxiv2023

Type VII secretion system and its effect on Group B Streptococcus virulence

GBS may cause a devasting disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to understand better understand T7SS contribution to virulence in these different clinical scenarios. GBS isolates were obtained from colonized mother prior to delivery and from newborns with EOD. DNA was analyzed for T7SS genes. A mutant EOD strain (ST17) was created by knocking out the essC gene encoding a T7SS protein. Galleria mellonella larvae were used to compare virulence of colonizing, EOD, and mutant EOD isolates. 33 GBS genomes were tested, 17 EOD isolates and 16 colonizing isolates. The T7SS locus encoded 8 genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030). ST17 isolates encode two copies of the essC gene and esxA gene encoding putative effectors but were present only in 23.5% of isolates. In ST1 isolates three copies of esxA gene were identified, but in ST6 and ST19 isolates all T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella model compared to colonizing isolates. The 118659{Delta}essC strain was attenuated in its killing ability, and the larvae were more effective in eradicating 118659{Delta}essC infection. essC gene deletion was associated with reduced bacterial growth. We demonstrated that T7SS plays an essential role during infection and contributes to GBS pathogenicity. Author SummaryType VII secretion system (T7SS) is related to virulence in various bacteria but is not well characterized in Group B Streptococci (GBS). GBS may cause sepsis, meningitis, and death in newborns. The bacteria rarely cause disease in pregnant mothers. Newborns acquire GBS from the colonized mother during delivery. We studied the role of T7SS in GBS isolates obtained from newborns with GBS sepsis in the first week of life and in colonized pregnant mothers. By studying T7SS genes we discovered that the genetic structure of the T7SS differs between isolates causing severe disease and colonizing isolates. To study the virulence of different GBS isolates we injected them into larvae and monitored larvae survival. Isolates causing severe disease in the newborn caused a more severe disease in larvae compared to colonizing isolates. We then deleted T7SS genes in GBS isolates causing severe disease. The killing activity of GBS isolates without T7SS genes was attenuated. The larva responded to these bacteria similarly to the response found when injecting the larva with GBS isolates from colonized mothers. These results support our hypothesis that T7SS is important for causing severe infection in the newborn and that this system contributes to GBS pathogenicity.

microbiology↗

bioRxiv2022

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable sterilizing immunity at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 [≤] 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting. Significance statementWe unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.

microbiology↗