Biology subjects

Majdalani, N.

Publications and source records attributed to Majdalani, N..

8 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into the Rcs phosphorelay

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

biophysics↗

bioRxiv2025

Gut microbial interaction networks control autoimmunity to neuroretina

The gut microbiome influences the development of immune-mediated inflammatory diseases, including autoimmune uveitis, a sight-threatening ocular inflammation driven by retina-specific T cells1. Using a model of spontaneous autoimmune uveitis (sEAU) we showed that gut commensals provide immune stimuli that trigger disease2. Here we report that uveitis-promoting microbes are present in human gut flora and that colonization of germ-free (GF) mice with commensals from healthy human donors was sufficient to provoke disease. Severity of sEAU correlated with expansion of Akkermansia and contraction of short-chain fatty acid (SCFA)-producing Firmicutes, followed by decreased SCFA levels and a dominant gut Th1 effector response. Mechanistic gain-of-function experiments, enriching GF sEAU mice with Akkermansia, reproduced these microbiome, metabolite and immune phenotype shifts, and exacerbated disease, suggesting that Akkermansia promotes autoimmunity by outcompeting SCFA-producers and enhancing Th1-type responses. An inverse correlation between Akkermansia (Verrucomicrobia) and Firmicutes was also present in patients with uveitis, multiple sclerosis and Crohns disease. These findings reveal a stereotypic gut microbial interaction network that regulates systemic immune balance, and may represent an ecologically conserved mechanism through which the gut microbiome modulates autoimmune and inflammatory diseases.

immunology↗

bioRxiv2024

A highly conserved sRNA downregulates multiple genes, including a sigma54 transcriptional activator, in the virulence mode of Bordetella pertussis

Bacterial sRNAs together with the RNA chaperone Hfq post-transcriptionally regulate gene expression by affecting ribosome binding or mRNA stability. In the human pathogen Bordetella pertussis, the causative agent of whooping cough, hundreds of sRNAs have been identified, but their roles in B. pertussis biology are mostly unknown. Here we characterize a Hfq-dependent sRNA (S17), whose level is dramatically higher in the virulence (Bvg+) mode. We show that transcription from a {sigma}A-dependent promoter yields a long form of 190 nucleotides (nts) that is processed by RNase E to generate a shorter, more stable form (S17S) of 67 nts. Using RNA-seq and RT-qPCR, we identify 92 genes whose expression significantly increases in the absence of S17. Of these genes, 70 contain sequences at/near their ribosome binding sites (RBSs) that are complementary to single-stranded (ss) regions (Sites 1 or 2) of S17S. The identified genes include those encoding multiple transporters and 3 transcriptional regulators. Using a lacZ translational reporter system, we demonstrate that S17S directly represses one of these genes, BP2158, a {sigma}54- dependent transcriptional regulator, suggesting the repression of a {sigma}54 regulon in the Bvg+ mode. We find that the S17S region containing Sites 1 and 2 is 100% conserved throughout various Betaproteobacteria species, and the S17S target sites are often conserved in the homologs of the B. pertussis target genes. We speculate that S17S regulation represents a highly conserved process that fine-tunes gene expression in the Bvg+ mode of B. pertussis and perhaps under other conditions in related bacteria. IMPORTANCERegulation of gene expression involves controlling transcription, translation, and transcript degradation. sRNAs with short sequences complementary to an mRNA sequence are involved in post-transcriptional regulation by aiding or interfering with either ribosome binding or nuclease attack. In the human pathogen Bordetella pertussis, the causative agent of whooping cough, hundreds of sRNA have been identified, but their functions remain largely unknown. We have characterized a sRNA that is abundant in the virulence mode of B. pertussis and serves to down-regulate multiple genes including transcriptional regulators and various transporters. We demonstrate that this sRNA directly represses a transcriptional factor, suggesting that it influences the regulation of specific B. pertussis regulons. The high conservation of this sRNA and its targets within Betaproteobacteria suggests a conserved pathway for gene regulation.

microbiology↗

bioRxiv2024

RcsF-independent mechanisms of signaling within the Rcs Phosphorelay

The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducting signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF and characterized the underlying regulatory mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs signaling can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator in E. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for signaling; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that RcsC acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms. Author summaryThe Rcs phosphorelay signaling cascade regulates the expression of genes related to capsule synthesis, biofilm formation, virulence, and cell division in Enterobacteria and is critical for cell membrane integrity and response to beta-lactam antibiotics and antimicrobial peptides. RcsF is the sole known sensor, but other proteins have been reported to activate this pathway in the absence of RcsF. We have discovered a novel RcsF-independent Rcs activator and found that each of three RcsF-independent proteins activate the system differently. Most significantly, we find that the histidine kinase RcsC can be involved in signal sensing independently of RcsF. Our study sheds light into the complex mechanisms of Rcs activation and adds to our knowledge of non-orthodox signaling systems across organisms.

microbiology↗

bioRxiv2023

A negative feedback loop is critical for recovery of RpoS after stress in Escherichia coli.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels. Author summaryIn their native environments, bacteria are exposed to constant changes in nutrient availability, as well as other biotic and abiotic stressors. To adjust to these changes, bacteria must rewire gene expression to adapt to or avoid stress-induced damage. A key player in the global response to general stresses is the alternative sigma factor RpoS, a promoter-specificity determining subunit of RNA polymerase. RpoS levels increase with stress, due to increased translation and stabilization of the otherwise unstable RpoS protein. Here, we examine how the cell restores homeostasis after the stress has passed. We show that a negative feedback loop in which RpoS regulates the transcription of an adaptor for proteolysis poises the cell to rapidly resume RpoS degradation upon the exit from stress.

microbiology↗

bioRxiv2021

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

microbiology↗

bioRxiv2021

A fluorescence-based genetic screen reveals diverse mechanisms silencing small RNA signaling in E. coli

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation as well as virulence. sRNAs can alter target mRNA translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify new regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in E. coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX and McaS, previously found to act through Hfq competition, RNase T, a 3'-5' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two new specific regulators were identified. AspX, a novel 3'end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria. SignificanceThe ability to promptly switch genes on and off allows bacteria to adapt rapidly to changing environments for better survival. Many sRNAs, including RyhB, a sRNA made in response to iron starvation, are important switches in bacteria. We discovered new factors that can keep the sRNA switch off by using a facile genetic screen platform. These new factors include a new RNA sponge and an adaptor protein for a ribonuclease, providing new perspectives on controlling sRNA signaling in bacteria.

microbiology↗

bioRxiv2020

Negative regulation of the Rcs phosphorelay via IgaA contact with the RcsD phosphotransfer protein

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in changing bacterial behavior in response to antimicrobial peptides, beta-lactam antibiotics, and other challenges to the cell surface. The Rcs system is unusual in that IgaA, an inner membrane protein, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it has previously been shown that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay was unknown. Here we use in vivo interaction assays and genetic dissection of the critical proteins to demonstrate that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions in periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a weaker interaction between cytoplasmic loop 1 of IgaA and a truncated PAS domain in RcsD. A point mutation in the PAS domain increases interactions between the two proteins and is sufficient to abolish induction of this phosphorelay. RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, appears to be indirectly regulated by IgaA via the contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is not necessary for the response to inducing signals. The multiple contacts between IgaA and RcsD form a poised sensing system, preventing over-activation of this apparently toxic phosphorelay but allowing it to be rapidly and quantitatively responsive to signals. Author SummaryThe Rcs phosphorelay plays a central role in allowing enterobacteria to sense and respond to antibiotics, host-produced antimicrobials, and interactions with surfaces. A unique negative regulator, IgaA, keeps signaling from this pathway under control when it is not needed, but how it controls the phosphorelay has been unclear. We define a set of critical interactions between IgaA and the phosphotransfer protein RcsD. A periplasmic contact between IgaA and RcsD provides a necessary inhibition of Rcs signaling, modulated further by regulated interactions in the cytoplasmic domains of each protein. This multipartite interaction provides a sensitive regulatory switch.

microbiology↗