A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance

Chengan, K.; Hind, C.; Nagappa, L.; Wand, M. E.; Hanson, T.; Escolano, R. M.; Tsaousis, A.; Bengoechea, J. A.; Sutton, J. M.; Smales, C. M.; Moore, S. J.

doi:10.1101/2023.04.13.536698

bioRxiv · 10.1101/2023.04.13.536698

A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance

Chengan, K.·Hind, C.·Nagappa, L.·Wand, M. E.·Hanson, T.·Escolano, R. M.·Tsaousis, A.·Bengoechea, J. A.·Sutton, J. M.·Smales, C. M.·Moore, S. J.

View original source ↗Export metadata ↓

Abstract

Antimicrobial resistance (AMR) is a pandemic spread across multiple priority infectious disease threats. While the cell envelope plays a key role in AMR, this also makes it challenging to study how antibiotics function inside the cell. Herein, we present a Klebsiella pneumoniae cell-free gene expression (CFE) platform for the rapid profiling of intracellular antibiotic sensitivity and resistance. This cell-free approach provides the unique macromolecular and metabolite components from this microbe, which include multiple antibiotic targets from transcription, translation, and metabolic processes. First, we compare the K. pneumoniae CFE system to whole cell antimicrobial assays. We find that several antibiotic classes show higher sensitivity in the CFE system, suggesting limitations in antibiotic transport in the whole cell assay. Next, we evolved K. pneumoniae strains with resistance to specific antibiotics and use whole genome sequencing analysis for genotyping. As an exemplary case, we show that a single RNA polymerase beta subunit variant H526L (also frequently found in multidrug resistant Mycobacterium tuberculosis) confers a 58-fold increase in CFE resistance to rifampicin. Overall, we describe a safe (i.e., non-living, non-pathogenic) platform suitable for studying an infectious disease model in a Containment Level 1 laboratory. Our CFE strategy is generalisable to laboratory and clinical K. pneumoniae strains and provides a new experimental tool to profile intracellular AMR variants. In conclusion, our CFE tool provides a significant advance towards understanding AMR and complements wider infectious disease studies.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Chengan, K., Hind, C., Nagappa, L., Wand, M. E., Hanson, T., Escolano, R. M., Tsaousis, A., Bengoechea, J. A., Sutton, J. M., Smales, C. M., Moore, S. J.. 2023-04-13. A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance. https://doi.org/10.1101/2023.04.13.536698

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↗