Biology subjects

Uhlin, B. E.

Publications and source records attributed to Uhlin, B. E..

8 recordsLinked to original sources

bioRxiv2026

Regulatory plasticity of the rdar biofilm morphotype in clinical uropathogenic Escherichia coli and its modulation by ciprofloxacin

Red, dry and rough (rdar) biofilm formation in Escherichia coli is characterized by the coordinated production of extracellular cellulose and amyloid curli fimbriae. Although rdar biofilm formation has been extensively characterized in laboratory strains, its clinical relevance and implications for antimicrobial treatment remain poorly understood. Here, we systematically investigate rdar biofilm formation of 150 consecutively isolated E. coli strains recovered from patients with urinary tract infections and correlate it to antimicrobial resistance. Genetic analysis of rdar regulation revealed distinct nucleotide signatures within the csgD promoter region that discriminate semi-constitutive rdar expression from temperature-dependent phenotypes, highlighting regulatory plasticity among clinical isolates. Whole-genome sequencing-based phylogenetic analysis further demonstrated that rdar-positive isolates are distributed across diverse E. coli phylogroups and sequence types, indicating that rdar biofilm formation is not restricted to specific clonal lineages. Strikingly, phenotypic assays revealed that the fluoroquinolone antibiotic ciprofloxacin suppresses rdar biofilm formation and associated extracellular matrix architecture in ciprofloxacin-resistant isolates at subinhibitory concentrations, suggesting that ciprofloxacin modulates biofilm-associated pathways beyond its canonical bactericidal targets. Together, our findings establish the rdar morphotype as a clinically relevant biofilm phenotype in uropathogenic E. coli and reveal an antibiofilm activity of ciprofloxacin that is uncoupled from antibiotic resistance. These results underscore the importance of considering antibiotic-mediated modulation of biofilm behavior when interpreting treatment responses and designing strategies to combat persistent urinary tract infections.

microbiology↗

bioRxiv2025

Mechanisms involved in the lipid membrane tubulation activity of the pH-responsive MakA protein from Vibrio cholerae

Bacterial pore-forming toxins (PFTs) are key virulence factors that disrupt host cell membranes. The Vibrio cholerae cytotoxin MakA, a member of the ClyA structural family of -PFTs, exhibits a unique, pH-dependent ability to induce lipid membrane tubulation. This study elucidates the molecular mechanisms underlying this process. We identify the N-terminal histidine residue (His30) of MakA as a critical pH sensor that is indispensable for membrane tubulation but not required for membrane binding and cell lysis. Substitution of His30 with lysine, MakAH30K , abrogated tubulation without affecting membrane insertion, instead triggering aberrant membrane fusion events. Circular dichroism spectroscopy analysis revealed that the MakAH30K substitution attenuates the robust -helical transition observed in wild-type MakA upon lipid membrane binding, providing a molecular explanation for its functional defect. Furthermore, we established that membrane cholesterol contributes to MakA-induced tubulation of lipid membranes, as its absence or mutation of a putative cholesterol-binding motif MakAI236D&I237D abolishes both membrane association and tubule formation. A key implication of our work is the mechanistic uncoupling of membrane tubulation from cytolysis, revealing that MakA can perturb membranes via distinct pathways. These findings define a sophisticated activation mechanism where His30 protonation in the acidic host environment and specific cholesterol binding synergistically drive the conformational change necessary for helical oligomerization and membrane tubulation. We discuss our findings from the viewpoint of possible convergent evolution, where bacterial PFTs like MakA partly may mimic ancient membrane remodelers involved in membrane repair pathways, such as ESCRT-III, but for bacterial fitness in host interactions and pathogenesis. Moreover, the present study provides a foundation for possible development of therapeutic strategies aimed at inhibiting these critical interactions.

biochemistry↗

bioRxiv2025

Bacteroidales T6SS minor Hcp subunits form heteromers recognising effectors

The type VI secretion system (T6SS) is a macromolecular protein complex found in Gram-negative bacteria that mediates intercellular antagonism. T6SSs are gut colonization factors that influence gut biodiversity. Hemolysin-coregulated proteins (Hcp) are major structural proteins in these systems. Hcp forms hexameric rings that stack to create an inner tube structure essential for translocating effector proteins into target cells. In Bacteroidales, T6SS loci encode multiple Hcp proteins with unknown function. The gut commensal Bacteroides fragilis encodes five Hcp subunits (sHcp and Hcp1-4) that have low sequence similarity. In this study, we investigated the roles of these proteins. Interaction studies showed that sHcp forms homohexamers, which is consistent with a major role of forming the bulk of the inner tube. In contrast, the less abundant minor Hcp1-4 were shown to form an interaction network involving heteromeric complexes. Biochemical analyses demonstrated that Hcp1 and Hcp2 assemble into heterohexamers and that this complex recognizes the secreted effector Bte1. Finally, we showed that Hcp modules, which are encoded in highly syntenic regions in T6SS loci of Bacteroidales, cluster with effectors. These results imply that the minor Hcps genetically cosegregate with cognate effectors, contributing to effector cassette variability. Thus, minor Hcp subunits function as recognition particles for effectors to mediate secretion, which appears to be a conserved trait in Bacteroidales T6SSs. Exploiting these features could facilitate the characterization of unknown effectors by copurifying them with their cognate Hcps. This approach may reveal new insights into bacterial interactions and the mechanisms that establish gut biodiversity.

biochemistry↗

bioRxiv2023

Emergence of Acinetobacter baumannii International Clone 10 predominantly found in the Middle East

Acinetobacter baumannii is a globally distributed human pathogen. Infections caused by carbapenem-resistant isolates of A. baumannii (CRAB) are of great concern, as treatment options are very limited. Despite having among the highest rates reported worldwide, there exists limited genomic data from CRAB strains isolated in the Middle East. Here we report epidemiological, phenotypic, and genome sequencing data (short reads and long reads) on a set of 60 A. baumannii isolates belonging to Sequence Type ST158 (Pasteur MLST scheme). They represent a novel international clone (IC), designated IC10, with limited geographic spread beyond the Middle East. Specific antibiotic-resistance genes associated with this clone were identified and data on the plasmid content associated with this lineage are presented.

microbiology↗

bioRxiv2021

Protein-lipid interaction at low pH induces oligomerisation of the MakA cytotoxin from Vibrio cholerae

Many pathogenic bacteria produce protein toxins that target and perturb host cell membranes. The secreted -pore-forming toxins (-PFTs) cause membrane damage via pore formation. This study demonstrates a remarkable, hitherto unknown mechanism by an -PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other -PFTs. In contrast, MakA protein alone induces tube-like structures in the acidic lysosomal host cell compartment. In vitro studies unravel the dynamics of tubular growth, which occur in a pH-, lipid- and concentration-dependent manner. A 3.7-[A] cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. In its active -PFT conformation, MakA embeds its transmembrane helices into a thin annular lipid bilayer and spirals around a central cavity. Our study provides molecular insights into a novel tubulation mechanism of an -PFT protein, revealing a new mode of action by a secreted bacterial toxin.

cell biology↗

bioRxiv2021

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae, Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD and MakE. The MakA, MakB and MakE proteins were readily detected in culture supernatants of wild type V. cholerae whereas secretion was very much reduced from a flagellum deficient mutant. Crystal structures of MakA, MakB and MakE revealed structural relationship to a superfamily of bacterial pore-forming proteins. Cloning and expression of MakA/B/E in Escherichia coli resulted in toxicity of the bacteria towards Caenorhabditis elegans used as a predatory organism model. None of these Mak proteins alone or in pairwise combinations were cytolytic but an equimolar mixture of MakA, MakB and MakE acted as a tripartite cytolytic toxin in vitro causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining in formation of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity towards C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a novel genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen V. anguillarum. We suggest that the hitherto unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms. Significance StatementVibrio cholerae, responsible for outbreaks and pandemics of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe that the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto unrecognized novel genomic island. The proteins MakA/B/E can form a tripartite cytolytic toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the Mak protein cytolytic activity was obtained by X-ray crystallography. Flagellum-facilitated secretion, remarkably ensuring spatially co-ordinated delivery of Mak proteins, revealed a new role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of new diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

biochemistry↗

bioRxiv2020

The Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy

Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy subversion and immune modulation employed by V. cholerae during bacterial infection.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Polar mutagenesis of bacterial transcriptional units using Cas12a

Bacterial genes are often organized in functionally related transcriptional units or operons. One such example is the fimAICDFGH operon, which codes for type I fimbriae in Escherichia coli. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in the fim operon. Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 30%, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Our results showed that some of the obtained mutants, including one with a single base substitution at the fim locus, had decreased mRNA levels of fimA, suggesting that the regulation of the fim operon was disrupted. We corroborated the polar effect of these mutants by phenotypic assays and quantitative PCR, showing up to a 43 fold decrease in expression of genes downstream fimA. We believe this strategy could be useful in engineering the transcriptional shut-down of multiple genes in one single step. For bio-production in E. coli, this opens the possibility of inhibiting competing metabolic routes.

microbiology↗