Biology subjects

Sondermann, H.

Publications and source records attributed to Sondermann, H..

9 recordsLinked to original sources

bioRxiv2025

Robust antibiotic sensitization of pathogenic Pseudomonas aeruginosa via negative hysteresis in the cell envelope

Antibiotic combination in time and space is a key strategy to combat antimicrobial resistance. The success of such treatment designs requires their robust efficacy across treatment conditions and a pathogens genomic diversity. This study found that an initial treatment with a {beta}-lactam antibiotic causes robust cellular sensitization towards an aminoglycoside antibiotic across the high-risk human pathogen Pseudomonas aeruginosa, including resistant strains. This phenomenon of cellular sensitization, termed negative hysteresis, is modulated by the Cpx envelope stress response system and linked to membrane stress during growth. The increase in efficacy is achieved through a {beta}-lactam induced elevated cellular uptake of the subsequently administered aminoglycoside. Negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions, thus enhancing efficacy of antibiotic combinations. Overall, our study identifies the phenomenon of negative hysteresis as a robustly inducible phenotype and thus a unique focus for optimizing antimicrobial therapy.

evolutionary biology↗

bioRxiv2025

Ancestral proteins trace the emergence of substrate specificity and oligomerization within bacterial DEDDy dinucleases

Nucleases play a crucial role in bacterial physiology, influencing processes such as DNA repair, genome maintenance, and host-pathogen interactions. We recently identified a class of nucleases, diDNases, which are encoded on mobile genetic elements and homologous to the house-keeping nanoRNase C (NrnC). Despite their shared structural fold, diDNases and NrnC orthologs exhibit differences. DiDNases form dimers and preferably cleave DNA dinucleotides, whereas NrnC homologs assemble into octamers that do not discriminate between RNA or DNA dinucleotides. Here, we investigate the evolutionary divergence of these enzymes using ancestral sequence reconstruction. Our results show that both diDNases and NrnC orthologs originated from a dimeric ancestor with intermediate substrate preferences. Structural analyses of ancestral and extant dinucleases provide a molecular rational for how gradual changes in conformation gave rise to substrate preferences, oligomeric state, and catalytic efficiency of these related, yet distinct enzyme clades. These findings provide insights into how small structural modifications enable large-scale changes in molecular assembly and functional specialization harnessing a conserved protein fold. In addition, the preference of the early ancestors for DNA dinucleotides and preservation of this activity in all extant enzymes strongly argues for a biological function of DNA dinucleotides.

evolutionary biology↗

bioRxiv2025

A class of secreted retropepsin-like enzymes is required for osmotic stress tolerance, antibiotic resistance and biofilm formation in Pseudomonas aeruginosa

Proteases regulate important biological functions. Here we present the structural and functional characterization of three previously uncharacterized aspartic proteases in Pseudomonas aeruginosa. We show that these proteases have structural hallmarks of retropepsin peptidases and play redundant roles for cell survival under hypoosmotic stress conditions. Consequently, we named them retropepsin-like osmotic stress tolerance peptidases (Rlo). Our research shows that while Rlo proteases are homologous to RimB, an aspartic peptidase involved in rhizosphere colonization and plant infection, they contain N-terminal signal peptides and perform distinct biological functions. Mutants lacking all three secreted Rlo peptidases show defects in antibiotic resistance, biofilm formation, and cell morphology. These defects are rescued by mutations in the inactive transglutaminase transmembrane protein RloB and the cytoplasmic ATP-grasp protein RloC, two previously uncharacterized genes in the same operon as one of the Rlo proteases. These studies identify Rlo proteases and rlo operon products as critical factors in clinically relevant processes, making them appealing targets for therapeutic strategies against Pseudomonas infections. IMPORTANCEBacterial infections have become harder to treat due to the ability of pathogens to adapt to different environments and the rise of antimicrobial resistance. This has led to longer illnesses, increased medical costs, and higher mortality rates. The opportunistic pathogen Pseudomonas aeruginosa is particularly problematic because of its inherent resistance to many antibiotics and its capacity to form biofilms, structures that allow bacteria to withstand hostile conditions. Our study uncovers a new class of retropepsin-like proteases in P. aeruginosa that are required for biofilm formation and bacterial survival upon stress conditions, including antibiotic exposure. By identifying critical factors that determine bacterial fitness and adaptability, our research lays the foundation for developing new therapeutic strategies against bacterial infections.

microbiology↗

bioRxiv2024

Malaria parasite HOPS/CORVET complexes are critical for endocytosis and invasion organelles function

The tethering complexes HOPS/CORVET are central for vesicular fusion through the eukaryotic endolysosomal system, but the functions of these complexes in the intracellular development of malaria parasites are unknown. Here we show that early inactivation of core HOPS/CORVET complex subunits in Plasmodium falciparum leads to developmental arrest and accumulation of cytosolic vesicles, indicating a role of HOPS/CORVET in parasite endocytosis and fusion of endosomes to the digestive vacuole membrane. Late inactivation of the core HOPS/CORVET subunits led to the mislocalization of luminal rhoptry and microneme proteins, and to a severe defect in merozoite invasion. Ultra-expansion microscopy revealed a reduced rhoptry volume and the accumulation of numerous vesicles, further supporting a role of HOPS/CORVET in protein trafficking to the apical organelles. Malaria parasites have therefore repurposed HOPS/CORVET to perform dual functions consistent with a canonical endocytosis pathway for delivery of host cell material to the digestive vacuole in trophozoite stages and a parasite specific role in trafficking of protein cargo to the apical organelles required for invasion in schizont stages.

cell biology↗

bioRxiv2023

Reconstitution of a Biofilm Adhesin System from a Sulfate-Reducing Bacterium in Pseudomonas fluorescens

Biofilms of the sulfate reducing bacterium (SRB) Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses; however, the mechanisms of biofilm formation by DvH are not yet well-understood. Evidence suggests that a large adhesin, DvhA, may be contributing to biofilm formation in DvH. The dvhA gene and its neighbors encode proteins that resemble the Lap system, which regulates biofilm formation by Pseudomonas fluorescens, including a LapG-like protease DvhG and effector protein DvhD, which has key differences from the previously described LapD. By expressing the Lap-like adhesion components of DvH in P. fluorescens, our data support the model that the N-terminal fragment of the large adhesin DvhA serves as an adhesin "retention module" and is the target of the DvhG/DvhD regulatory module, thereby controlling cell-surface location of the adhesin. By heterologously expressing the DvhG/DvhD-like proteins in a P. fluorescens background lacking native regulation ({Delta}lapG{Delta}lapD) we also show that cell surface regulation of the adhesin is dependent upon the intracellular levels of c-di-GMP. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems. SignificanceCorrosion leads to [~]2.5 trillion US dollars in economic losses, 20% of which are estimated to be microbially induced. Biofilms of sulfate reducing bacteria (SRB), especially the genus Desulfovibrio, are important members of the corrosion consortium and accelerate deterioration of metals. Understanding how biofilms are formed by SRB can provide important clues to mitigate this challenge. In this study, we used genetic and biochemical tools to investigate the mechanism of biofilm formation by Desulfovibrio vulgaris Hildenborough. Our study reveals critical genes responsible for regulating the secretion of a large adhesin known to be required for biofilm formation.

microbiology↗

bioRxiv2019

Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

Degradation of RNA polymers is a multistep process catalyzed by specific subsets of RNases. In all cases, degradation is completed by exoribonucleases that recycle RNA fragments into nucleotide monophosphate. In {gamma}-proteobacteria, a group of up to eight 3-5 exoribonucleases have been implicated in RNA degradation. Oligoribonuclease (Orn) is unique among them as its activity is required for clearing short RNA fragments, a function important for cellular fitness. However, the mechanistic basis for this substrate selectivity remained unclear. Here we show that Orns activity as a general exoribonuclease has been vastly overestimated by demonstrating that the enzyme exhibits a much narrower substrate preference for diribonucleotides. Co-crystal structures of Orn with substrates reveal an active site optimized for diribonucleotides that does not accommodate longer substrates. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, our functional studies demonstrate that Orn is the one and only diribonucleotidase that completes the final stage of the RNA degradation pathway. Together, these results indicate that Orn is a dedicated diribonucleotidase that clears the diribonucleotide pool that otherwise affects cellular physiology and viability.

microbiology↗

bioRxiv2018

Coopting the Lap system of Pseudomonas fluorescens to reversibly customize bacterial cell surfaces

Initialattachment to a surface is a key and highly regulated step in biofilm formation. In this study, we present a platform for reversibly functionalizing bacterial cell surfaces, with an emphasis on designing biofilms. We engineered the Lap system of Pseudomonas fluorescens Pf0-1, which is normally used to regulate initial cell surface attachment, to display various protein cargo at the bacterial cell surface and control extracellular release of the cargo in response to changing levels of the second messenger cdi-GMP. To accomplish this goal, we fused the protein cargo between the N-terminal retention module and C-terminal secretion signal of LapA, and controlled surface localization of the cargo with natural signals known to stimulate or deplete c-di-GMP levels in P. fluorescens Pf0-1. We show this system can tolerate large cargo in excess of 500 amino acids, direct P. fluorescens Pf0-1 to surfaces it does not typically colonize, and program this microbe to sequester the toxic medal cadmium.

bioengineering↗

bioRxiv2017

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction Between a Diguanylate Cyclase and Its Receptor

The second messenger, cyclic dimeric GMP (c-di-GMP) regulates biofilm formation for many bacteria. The binding of c-d{inverted exclamation}-GMP by the inner-membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbCs calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present GcbC activity is significantly enhanced ~8-fold, indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the l-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of P. fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.\n\nImportanceIn many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP, however it is unclear how undesired crosstalk is mitigated in the context of this soluble signal, and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-d{inverted exclamation}-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the l-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a \"cloud\" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling and biofilm formation.

microbiology↗

bioRxiv2017

Cell surface associated LapA of Pseudomonas fluorescens is anchored inside its Type-1 Secretion TolC-like Pore

The type-1 secretion system (T1SS) of gram-negative bacteria enables a one-step translocation strategy known to move functionally diverse proteins from the cytoplasm into the extracellular environment without a periplasmic intermediate. LapA of Pseudomonas fluorescens Pf0-1 is a giant type-1 secreted (T1S) adhesin that facilitates biofilm formation only when displayed at the cell surface. A LapA-targeting periplasmic protease, LapG, connects intracellular cyclic diguanylate (c-di-GMP) levels with cell surface-associated LapA by cleaving and absolving LapA from the cell surface under conditions unsuitable for biofilm formation. Here, we demonstrate that LapA contains a novel N-terminal element, called the retention module (RM), which prohibits classical one-step T1S of LapA. We provide evidence that the RM of LapA tethers LapA at the cell surface through its outer membrane TolC-like pore, LapE, where LapA is accessible to the periplasmic protease LapG. We also demonstrate that this unusual retention strategy is likely conserved among LapA-like proteins and represents a new subclass of T1SS ABC transporters exclusively involved in transporting LapA-like adhesins.\n\nSignificance StatementBacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is often key for initiating contact with a preferred substrate to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence these adhesins are secreted by a new subclass of T1SS ABC transporters.

microbiology↗