Biology subjects

Kisthardt, S. C.

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

5 recordsLinked to original sources

bioRxiv2026

Bile acid dependent attenuation of toxin mediated disease is independent of colonization resistance against C. difficile

Clostridioides difficile infection (CDI) is a severe antibiotic associated disease and a major cause of morbidity and mortality worldwide. CDI is thought to arise from the loss of protective gut microbes that mediate functions such as secondary bile acid metabolism and nutrient competition, yet the relative contributions of these mechanisms remain unclear. To determine how these processes influence C. difficile growth, virulence, and disease, we performed in vitro and in vivo experiments using two Clostridia strains previously associated with colonization resistance against C. difficile. Neither organism prevented colonization or growth through nutrient competition alone. In contrast, secondary bile acid metabolism significantly reduced toxin-mediated disease in vivo in a strain dependent manner. These findings demonstrate that secondary bile acid modulation is an important component of CDI prevention independent of nutrient competition and suggest that attenuating virulence, in addition to limiting colonization, may represent a key strategy for next-generation CDI therapeutics.

microbiology↗

bioRxiv2025

Assessing contamination in DNA extraction kits commonly used for microbiome research

Sequencing-based measurements are now routinely used to investigate the microbial world, however, contamination by DNA outside the intended sample remains a problem. Contaminants obscure the true microbial signal and can lead to misleading scientific interpretations. Much work has been done to address the effects of these contaminants including best practices as outlined in Eisenhofer et al. and Fierer et al. Yet, even with best practices in place, the current literature consensus is that contaminants remain impactful, at least in low biomass environments (5, 7, 11, 13, 16). One well-known source of contaminants are those found within DNA extraction kits, as was shown clearly in the pioneering work of Salter et al. 2012 and Karstens et al. 2019. However, given the rapid evolution of DNA sequencing methods, it would be worthwhile to revisit the issue of contaminants in contemporary DNA extraction kits (the "kitome"). Here we provide an updated characterization of the kitomes of DNA extraction kits commonly used for microbiome research. ImportanceMicrobial contamination in commonly used DNA extraction kits has not been recently assessed. Here we evaluate the contamination in DNA extraction kits commonly used in microbiome studies over the past several years, and provide actionable guidance on appropriate DNA extraction kits for low biomass microbiome measurements.

molecular biology↗

bioRxiv2023

The microbial derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota

C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration (MIC) against C. difficile R20291, and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Here we show that epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile. LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. ImportanceIn the search for a novel therapeutic that targets C. difficile, bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that iLCA and iaLCA specifically are potent inhibitors of C. difficile, affecting key virulence factors including growth, toxin expression and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

microbiology↗

bioRxiv2021

Flavonols and dihydroflavonols inhibit the main protease activity of SARS-CoV-2 and the replication of human coronavirus 229E

Since December 2019, the deadly novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 pandemic. To date, vaccines are available in the developed countries to prevent the infection of this virus, however, medicines are necessary to help control COVID-19. Human coronavirus 229E (HCoV-229E) causes the common cold. The main protease (Mpro) is an essential enzyme required for the multiplication of these two viruses in the host cells, and thus is an appropriate candidate to screen potential medicinal compounds. Flavonols and dihydroflavonols are two groups of plant flavonoids. In this study, we report docking simulation with two Mpro enzymes and five flavonols and three dihydroflavonols, in vitro inhibition of the SARS-CoV-2 Mpro, and in vitro inhibition of the HCoV 229E replication. The docking simulation results predicted that (+)-dihydrokaempferol, (+)-dihydroquercetin, (+)-dihydromyricetin, kaempferol, quercetin, myricentin, isoquercetin, and rutin could bind to at least two subsites (S1, S1, S2, and S4) in the binding pocket and inhibit the activity of SARS-CoV-2 Mpro. Their affinity scores ranged from -8.8 to -7.4. Likewise, these compounds were predicted to bind and inhibit the HCoV-229E Mpro activity with affinity scores ranging from -7.1 to -7.8. In vitro inhibition assays showed that seven available compounds effectively inhibited the SARS-CoV-2 Mpro activity and their IC50 values ranged from 0.125 to 12.9 {micro}M. Five compounds inhibited the replication of HCoV-229E in Huh-7 cells. These findings indicate that these antioxidative flavonols and dihydroflavonols are promising candidates for curbing the two viruses.

biochemistry↗

bioRxiv2020

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms

SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola virus and demonstrated activity against SARS-CoV-2 in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in vitro in HeLa cells and of mouse adapted Ebola virus in mouse in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with Kd values of 339 nM and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 hence justifying in vivo evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.

pharmacology and toxicology↗