Biology subjects

Raivio, T. L.

Publications and source records attributed to Raivio, T. L..

8 recordsLinked to original sources

bioRxiv2025

YfgH is a transmembrane glycine-zipper containing lipoprotein that stabilizes excess cardiolipin and outer membrane proteins during envelope stress

The outer membrane (OM) of Escherichia coli is an essential, asymmetric bilayer composed of lipopolysaccharide and phospholipids that protects cells from environmental stress. While several systems maintain OM integrity during stress, the roles of many outer membrane lipoproteins are less well characterized. Here, we identify YfgH, a glycine-zipper domain containing lipoprotein, as a novel OM-stabilizing factor that functions distinctly from the previously studied SlyB. Deletion of yfgH increases sensitivity to detergents and metal chelators, enhances OM permeability, and leads to global loss of OM proteins under stress. YfgH is essential for survival during CRISPR interference-mediated depletion of OM biogenesis genes, and its overexpression rescues these phenotypes. AlphaFold modeling predicts YfgH forms oligomeric ring-like structures similar to SlyB, but genetic suppressor screens indicate distinct mechanisms: clsA (cardiolipin synthase) mutations suppress yfgH deletion, while slyB suppressors map to the Mla lipid transport pathway. Biochemical analyses reveal YfgH interacts with multiple OM proteins, especially under stress. Together these findings suggest YfgH functions by stabilizing cardiolipin-rich nanodomains, representing a novel response to OM stress. This work expands our understanding of glycine-zipper lipoproteins as stress-specific membrane stabilizers and highlights the critical role of YfgH in preserving OM integrity during envelope perturbation. ImportanceThe bacterial outer membrane is essential for survival in harsh environments, yet how Gram-negative cells maintain its stability under stress remains less understood. We identify YfgH, a previously uncharacterized outer membrane lipoprotein, as a key factor that preserves membrane integrity during envelope stress. Our findings show that YfgH stabilizes cardiolipin-rich nanodomains and protects outer membrane proteins when biogenesis pathways are compromised. This work reveals a new mechanism of outer membrane maintenance and expands the functional repertoire of glycine zipper- containing lipoproteins, highlighting a broader family of stress-specific membrane stabilizers that may be conserved across Gram-negative bacteria.

molecular biology↗

bioRxiv2023

The sensor of the bacterial histidine kinase CpxA is a novel dimer of extracytoplasmic Per-ARNT-Sim (PAS) domains

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA* alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologues revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation. SignificanceBacterial two-component systems play an essential role in sensing environmental cues, mitigating stress, and regulating virulence. We approach the study of a key Gram-negative sensor kinase CpxA with both classical methods in structural biology and genetic analysis and emerging protein-folding prediction software. This approach provides a wholistic perspective on the structure and function of histidine kinases as proteins with modular and cellular compartment-spanning domain architectures. We report a novel organization of PAS domains in CpxA, highlighting the versatility and diversity of this sensory fold. Ultimately, these studies will facilitate the continued development of novel antimicrobials against sensor kinases, including CpxA, which is a previously studied target for antimicrobials.

molecular biology↗

bioRxiv2022

NlpE is an OmpA-associated outer membrane sensor of the Cpx envelope stress response

Gram-negative bacteria utilize several envelope stress responses (ESRs) to sense and respond to diverse signals within a multi-layered cell envelope. The CpxRA ESR responds to multiple stresses that perturb envelope protein homeostasis. Signaling in the Cpx response is regulated by auxiliary factors such as the outer membrane (OM) lipoprotein NlpE, an activator of the response. NlpE communicates adhesion to surfaces to the Cpx response; however, the mechanism by which NlpE accomplishes this remains unknown. In this study, we report a novel interaction between NlpE and the abundant OM protein OmpA. Both NlpE and OmpA are required to activate the Cpx response in surface-adhered cells. Furthermore, NlpE senses OmpA overexpression and the NlpE C-terminal domain transduces this signal to the Cpx response, revealing a novel signaling function for this domain. Overall, these findings reveal NlpE to be a versatile envelope sensor that takes advantage of its structure, localization, and cooperation with other envelope proteins to initiate adaptation to diverse signals. SignificanceThe envelope is not only a barrier that protects bacteria from the environment but also a crucial site for the transduction of signals critical for colonization and pathogenesis. The discovery of novel complexes between NlpE and OmpA contributes to an emerging understanding of the key contribution of complexes of {beta}-barrel proteins and lipoproteins to envelope stress signaling. Overall, our findings provide mechanistic insight into how the Cpx response senses signals relevant to surface adhesion and biofilm growth to facilitate bacterial adaptation.

microbiology↗

bioRxiv2022

Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium cpxRA mutants and uncovers additive effects of Cpx regulated genes on fitness

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used to model enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. During colonization, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. Here, we utilized simulated colonic fluid (SCF) to mimic the gastrointestinal environment which we show strongly induces the Cpx ESR and highlights a fitness defect specific to the {Delta}cpxRA mutant. While investigating genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, we found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler, and the genes yebE, ygiB, bssR, and htpX relied on CpxRA for proper expression. We then determined that CpxRA and select gene mutants were essential for proper growth in SCF when in the presence of extraneous stressors and in competition. Although none of the Cpx-regulated gene mutants exhibited marked virulence phenotypes in vivo, the {Delta}cpxRA mutant had reduced colonization and attenuated virulence, as previously determined, which replicated the in vitro growth phenotypes specific to SCF. Overall, these results indicate that the {Delta}cpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

microbiology↗

bioRxiv2021

Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium mutants and highlights a critical role for the Cpx envelope stress response in mediating stressors encountered in the gastrointestinal tract

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. While in the host, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. To investigate genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, RNA-Seq, SILAC, and microarray data from previous research was mined and the genes yebE, ygiB, bssR, and htpX were confirmed to be strongly upregulated by the presence of CpxRA using lux reporter constructs. To determine the function of these genes in vivo, knockout mutants were tested in C57Bl/6J and C3H/HeJ mice. Although none of the mutants exhibited marked virulence phenotypes, the {Delta}cpxRA mutant had reduced colonization and attenuated virulence, as previously determined. We also found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler. In addition, we determined that the {Delta}cpxRA mutant had a growth defect in medium simulating the colon, as did several of the mutants bearing deletions in Cpx-upregulated genes. Overall, these results indicate that the {Delta}cpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

microbiology↗

bioRxiv2021

Isolation and characterization of a novel temperate Escherichia coli bacteriophage, Kapi1, which modifies the O-antigen and contributes to the competitiveness of its host during colonization of the murine gastrointestinal tract.

In this study, we describe the isolation and characterization of novel bacteriophage vB_EcoP_Kapi1 (Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the seroconverting phages of Shigella flexneri, and clusters taxonomically with P22-like phages. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways. Kapi1 displays unstable lysogeny, and we find that lysogeny is favored during growth in simulated intestinal fluid. Furthermore, Kapi1 lysogens have a competitive advantage over their non-lysogenic counterparts both in vitro and in vivo, suggesting a role for Kapi1 during colonization. We thus report the use of MP1 and Kapi1 as a model system to explore the molecular mechanisms of mammalian colonization by E. coli to ask what the role(s) of prophages in this context might be. ImportanceAlthough research exploring the microbiome has exploded in recent years, our understanding of the viral component of the microbiome is lagging far behind our understanding of the bacterial component. The vast majority of intestinal bacteria carry prophages integrated into their chromosomes, but most of these bacteriophages remain uncharacterized and unexplored. Here, we isolate and characterize a novel temperate bacteriophage infecting a commensal strain of Escherichia coli. We aim to explore the interactions between bacteriophages and their hosts in the context of the gastrointestinal tract, asking what role(s) temperate bacteriophage may play in growth and survival of bacteria in the gut. Understanding the fundamental biology of gut commensal bacteria can inform the development of novel antimicrobial or probiotic strategies for intestinal infections.

microbiology↗

bioRxiv2021

Isolation and characterization of a novel bacteriophage, Kapi1, capable of O-antigen modification in commensal Escherichia coli.

In this study, we describe the isolation and characterization of novel bacteriophage Kapi1 (vB_EcoP_Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the phages of Shigella flexneri, and clusters taxonomically with P22-like phages. Investigation of the lifestyle of Kapi1 shows that this phage displays unstable lysogeny and influences the growth of its host. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways. We hope to use MP1 and Kapi1 as a model system to explore molecular mechanisms of mammalian colonization by E. coli and ask what the role(s) of prophages in this context might be. ImportanceAlthough research exploring the microbiome has exploded in recent years, our understanding of the viral component of the microbiome is lagging far behind our understanding of the bacterial component. The vast majority of intestinal bacteria carry prophages integrated into their chromosomes, but most of these bacteriophages remain uncharacterized and unexplored. Here, we isolate and characterize a novel temperate bacteriophage infecting a commensal strain of Escherichia coli. We aim to explore the interactions between bacteriophages and their hosts in the context of the gastrointestinal tract, asking what role(s) temperate bacteriophage may play in growth and survival of bacteria in the gastrointestinal tract. Understanding the fundamental biology of commensal bacteria in the gastrointestinal tract can inform the development of novel antimicrobial or probiotic strategies for intestinal infections.

microbiology↗

bioRxiv2019

Impaired Efflux of the Siderophore Enterobactin Induces Envelope Stress in Escherichia coli

The Cpx response is one of several envelope stress responses that monitor and maintain the integrity of the gram-negative bacterial envelope. While several conditions that are known or predicted to generate misfolded inner membrane proteins activate the Cpx response, the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. Enterobactin accumulation in the tolC mutant reduces activity of the NADH oxidation arm of the aerobic respiratory chain. However, NADH dehydrogenase I, NADH dehydrogenase II, and cytochrome bo3 do not contribute to Cpx pathway activation in the E. coli tolC mutant. We show that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. These results suggest that the Cpx response promotes adaptation to envelope stress in enteric bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging compounds and conditions.

microbiology↗