Biology subjects

Krupka, M.

Publications and source records attributed to Krupka, M..

3 recordsLinked to original sources

bioRxiv2026

Biochemical and Immunological Properties of Engineered Low-Immunogenic Staphylokinases for Next-Generation Thrombolytic Therapy

Staphylokinase (SAK) is a highly fibrin-specific plasminogen activator with significant potential as a safe and affordable thrombolytic. Yet, its clinical translation can be limited by potential immunogenicity. To accelerate the development of improved thrombolytics, a critical step is identifying the most suitable molecular template. Therefore, we performed a comparative analysis of biochemical and immunological properties of three engineered low-immunogenic variants (SAK SY155, SAK THR174, and SAK STAR FRIDA) and two wild-types (SAK STAR and SAK 42D), using a newly established panel of assays. All variants retained potent thrombolytic activity, with SAK SY155 displaying the highest catalytic efficiency and fibrin-clot permeability. However, this advantage did not fully translate into improved clot reduction under flow conditions. Comprehensive immunological profiling, including T lymphocyte proliferation, dendritic cell maturation, mouse immunization models, and human serum reactivity tests, confirmed decreased immunogenicity for two low-immunogenic variants. Overall, low-immunogenic SAK SY155 emerged as the most promising template for rational engineering of next-generation thrombolytics.

biochemistry↗

bioRxiv2023

Concurrent infection of human brain with multiple species of Lyme disease spirochetes

Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infected hosts, including humans. The diverse strategies used by spirochetes to avoid the host immune system and persist in the host include active immune suppression, induction of immune tolerance, phase and antigenic variation, intracellular seclusion, and, importantly, incursion into immune privileged sites such as the brain. Invasion of immune privileged sites, like the brain allows the spirochetes not only escape from the host immune system but also can reduce the efficacy of antibiotic therapy. Here we present a case of the detection of DNA of spirochetes from Borrelia burgdorferi sensu lato complex from multiple loci of LD patients post-mortem brain. The presence of co-infection with Borrelia burgdorferi sensu stricto and Borrelia garinii in LD patients brain was confirmed by PCR. The presence of atypical spirochete morphology was confirmed by immunohistochemistry of the brain samples and also in tissues of experimental mice, infected with Borrelia by simultaneous injection of spirochetes subcutaneously and intraperitoneally. Even though both spirochete species were simultaneously present in brain, the brain regions where the two species were detected were different and non-overlapping.

microbiology↗

bioRxiv2018

Escherichia coli ZipA organizes FtsZ polymers into dynamic ring-like protofilament structures

ZipA is an essential cell division protein in Escherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfaces in vitro, visible as GTP-dependent chiral vortices several hundred nm in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with some in vitro studies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also used E. coli mutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filaments in vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.\n\nIMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at mid-cell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane during E. coli cell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surface in vitro. Importantly, these swirls are only observed when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundling in vitro. In addition, we present several lines of in vivo evidence indicating that ZipA does not act to directly bundle FtsZ polymers.

microbiology↗