Stream, A. | bioRxiv Science

bioRxiv2025

Caspase Cleavage of Kaposi Sarcoma-Associated Herpesvirus Proteins: A role for K5 in Preventing Caspase-Mediated Cell Death during Lytic Replication

We previously reported that Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) acts as a pseudo-substrate for caspases-1 and 3, thereby interfering with their inflammatory and apoptotic activity, respectively. To determine if other KSHV proteins undergo caspase cleavage, we screened the KSHV proteome for potential caspase cleavage sites. Using SitePrediction (SP), 30 KSHV proteins with potential caspase-cleavage sites were identified. Among those with highest SP score was an early lytic protein, K5. Treatment of BJAB K5-FLAG cells with Fas, an apoptotic stimulus, led to caspase-processing of full length K5-FLAG and generation of a C-terminal peptide fragment. Using mass spectrometry, we determined that K5-FLAG undergoes caspase cleavage at D222. K5 was also cleaved by caspases in KSHV infected cells induced to lytic replication. Importantly, the expression of K5-FLAG significantly inhibited Fas-induced caspase-mediated cell death. To determine if K5 plays a protective role in KSHV infected cells, iSLKK cells infected with wild type or K5 knockout BAC16 virus were induce to lytic replication to activate caspases. Although lytic induction showed little effect on the viability of WT infected cells, the viability of K5-knockout cells decreased by 25%. Thus, K5 may protect KSHV-infected cells from caspase-mediated cell death during lytic replication. Interestingly, cleavage of K5 by caspases did not affect its ability to downregulate MHC-1 surface expression. Overall, these data suggest that K5 not only downregulates immunologic surface marker expression to avoid immune recognition but may also play an additional role in mitigating caspase-mediated cell death during KSHV lytic replication.

biochemistry↗

bioRxiv2024

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacteriums poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based sgRNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just two days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ-inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. Significance statementWhile GAS remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS, and together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

microbiology↗