Biology subjects

Kielkopf, C. S.

Publications and source records attributed to Kielkopf, C. S..

5 recordsLinked to original sources

bioRxiv2025

Structure of a novel contractile injection system in subsp.

Extracellular contractile injection systems (eCISs) are phage-derived nanomachines used by bacteria to deliver effectors into target cells. Well-studied examples include the Photorhabdus asymbiotica virulence cassettes and the antifeeding prophage from Serratia entomophila, which have been engineered for heterologous cargo delivery. Recent genomic analyses identified previously uncharacterized eCIS gene clusters in the opportunistic human pathogen Salmonella enterica subspecies salamae, but their structure, function, and biotechnological potential remain unexplored. Here, we report a high-resolution cryo-electron microscopy structure of the S. enterica eCIS. Our atomic models reveal a unique sheath architecture, an expansive cage-like shell around a central spike, and an associated transmembrane hydrolase. We identify a putative effector encoded within the operon exhibiting periplasmic toxicity and provide evidence that the S. enterica eCIS deviates from canonical eCISs by interacting with the inner membrane. Guided by these structural features, we uncover a previously unannotated cluster of contractile injection systems (CISs). Together, our findings expand the known diversity of CISs structures and functions and lay the groundwork for engineering customizable protein delivery platforms.

microbiology↗

bioRxiv2024

Towards a complete phage tail fiber structure atlas.

Bacteriophages use receptor-binding proteins (RBPs) to adhere to bacterial hosts. Understanding the structure of these RBPs can provide insights into their target interactions. Tail fibers, a prominent type of RBP, are typically elongated, flexible, and trimeric proteins, making it challenging to obtain high-resolution experimental data of their full-length structures. Recent advancements in deep learning-based protein structure prediction, such as AlphaFold2-multimer (AF2M) and ESMfold, allow for the generation of high-confidence predicted models of complete tail fibers. In this paper, we introduce RBPseg, a method that combines monomeric ESMfold predictions with a novel sigmoid distance pair (sDp) protein segmentation technique. This method segments the tail fiber sequences into smaller fractions, preserving domain boundaries. These segments are then predicted in parallel using AF2M and assembled into a full fiber model. We demonstrate that RBPseg significantly improves AF2M v2.3.2 in terms of model confidence, running time, and memory usage. To validate our approach, we used single-particle cryo-electron microscopy to analyze five tail fibers from three phages of the BASEL collection. Additionally, we conducted a structural classification of 67 fibers and their domains, which identified 16 well-defined tail fiber classes and 89 domains. Our findings suggest the existence of modular fibers as well as fibers with different sequences and shared structure, indicating possible sequence convergence, divergence, and domain swapping. We further demonstrate that these structural classes account for at least 24% of the known tail fiber universe.

microbiology↗

bioRxiv2024

T6SS-associated Rhs toxin containers: Structural and functional insights into bacterial weaponry and self-protection

Bacteria use the type VI secretion system (T6SS) to secrete a variety of toxins into pro- and eukaryotic cells via a machinery consisting of a contractile sheath and a rigid tube. Rearrangement hotspot (Rhs) proteins represent one of the most common T6SS-secreted effectors. The C-terminal toxin domain of Rhs proteins displays great functional diversity, while the large Rhs core is characterised by YD repeats. T6SS- associated Rhs proteins are attached to the VgrG spike protein, often via an N- terminal PAAR-repeat domain. Using X-ray crystallography, we here elucidate the Rhs core structures of PAAR- and VgrG-linked Rhs proteins from Salmonella bongori and Advenella mimigardefordensis, respectively. The Rhs core forms a large container made up of {beta}-sheets that has a negatively charged interior and encloses a large volume. The presence of the toxin domain of S. bongori PAAR-linked Rhs does not lead to ordered density in the Rhs container, suggesting the toxin domain is at least partially unfolded. Together with bioinformatics analysis showing that Rhs toxins predominantly act intracellularly, this suggests that the Rhs core domain functions two-fold, as safety feature for the producer cell and as delivery mechanism for the toxin domain. Our results strengthen our knowledge of Rhs structure and function.

biochemistry↗

bioRxiv2023

N-terminal toxin signal peptides efficiently load therapeutics into a natural nano-injection system

Targeted delivery of therapeutics to specific cells is a major bottleneck towards personalized medicine. The extracellular injection system (eCIS) of Serratia entomophila, the antifeeding prophage (Afp), promises potential for drug delivery purposes. However, the precise mechanism of action, toxin location, and Afp loading remain unclear. Here, we reveal a minimal N-terminal signal peptide (NtSP) of the toxin Afp18, that plays a key role in toxin packing. By engineering fusion proteins, we demonstrate that Afp18s NtSP can shuttle effectors for Afp loading. We packed non-eCIS effectors, including CRISPR-Cas protein Cas{Phi}-2 from Biggiephage, and a human antimicrobial peptide, LL37, into Afp. Additionally, NtSPs from eCIS effectors of other species facilitate loading of Cas{Phi}-2 into Afp. We observed cargo being packed inside the Afp tail tube through cryo-EM single particle analysis. The presented results enhance our understanding of eCIS toxin packing and contribute to their development as targeted delivery systems. TeaserA novel use of the Afp nano injection systems N-terminal signal peptide in targeted therapeutics delivery

molecular biology↗

bioRxiv2018

Identification of a novel tetrameric structure for human apolipoprotein-D

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded {beta}-barrel with a single adjacent -helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimers disease, where apolipoprotein-D overexpression reduces the amyloid-{beta} burden in Alzheimers disease mouse models.\n\nOligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids.\n\nHere, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a {bsim}95 to {bsim}100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

biochemistry↗