Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

Widera, M.; Wilhelm, A.; Toptan, T.; Raffel, J. M.; Kowarz, E.; Roesmann, F.; Siemund, A. L.; Luciano, V.; Kuelp, M.; Reis, J.; Bracharz, S.; Pallas, C.; Ciesek, S.; Marschalek, R.

doi:10.1101/2021.04.27.441606

bioRxiv · 10.1101/2021.04.27.441606

Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

Widera, M.·Wilhelm, A.·Toptan, T.·Raffel, J. M.·Kowarz, E.·Roesmann, F.·Siemund, A. L.·Luciano, V.·Kuelp, M.·Reis, J.·Bracharz, S.·Pallas, C.·Ciesek, S.·Marschalek, R.

View original source ↗Export metadata ↓

Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Widera, M., Wilhelm, A., Toptan, T., Raffel, J. M., Kowarz, E., Roesmann, F., Siemund, A. L., Luciano, V., Kuelp, M., Reis, J., Bracharz, S., Pallas, C., Ciesek, S., Marschalek, R.. 2021-04-27. Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors. https://doi.org/10.1101/2021.04.27.441606

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A conserved cysteine-histidine-glutamate metal site identifies DUF501 (Rv1025), an essential uncharacterised protein family of Mycobacterium tuberculosis, as a candidate metalloenzyme and drug target

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

microbiology↗

bioRxiv2026

Mycoplasmal endosymbionts of Trichomonas vaginalis are associated with reduced risk for Chlamydia trachomatis endometrial infection in asymptomatic, coinfected, women.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most common curable non-viral sexually transmitted infection, and Chlamydia trachomatis is a bacterial pathogen that can ascend to the upper genital tract and cause pelvic inflammatory disease, infertility, and ectopic pregnancy. T. vaginalis harbors bacterial endosymbionts, including Candidatus Malacoplasma girerdii, an obligate symbiont, and Metamycoplasma hominis, which can live freely or symbiotically. In a 16S rRNA sequencing study of the cervicovaginal microbiome of women at high risk for chlamydial infection, Ca. M. girerdii abundance was one of 13 features predicting lack of chlamydial spread to the endometrium, despite no direct association between T. vaginalis infection and reduced chlamydial ascension. Investigating the relationship between these microorganisms further, we found that T. vaginalis vaginal abundance correlated positively with chlamydial burden in women whose infection was confined to the cervix, while a nonsignificant inverse relationship was seen in women with endometrial spread. Among participants with high chlamydial burden, Ca. M. girerdii was detected exclusively in women without endometrial infection. Both endosymbionts trended toward more frequent detection, and higher abundance, in coinfected women without endometrial spread, while M. hominis abundance correlated strongly with T. vaginalis burden in this group. These findings suggest that mycoplasmal endosymbionts of T. vaginalis, rather than T. vaginalis itself, are microbial factors limiting chlamydial ascension, and point to a three-way interaction between parasite, endosymbiont, and bacterial pathogen that shapes upper genital tract C. trachomatis infection risk.

microbiology↗

bioRxiv2026

Understanding the physiological alterations of Vibrio cholerae upon exposure to L-ascorbic acid

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

microbiology↗