Active bacterial modification of the host environment through RNA polymerase II inhibition
Inès Ambite, … , Ulrich Dobrindt, Catharina Svanborg
Inès Ambite, … , Ulrich Dobrindt, Catharina Svanborg
Published December 15, 2020
Citation Information: J Clin Invest. 2021;131(4):e140333. https://doi.org/10.1172/JCI140333.
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Research Article Inflammation Microbiology

Active bacterial modification of the host environment through RNA polymerase II inhibition

Abstract

Unlike pathogens, which attack the host, commensal bacteria create a state of friendly coexistence. Here, we identified a mechanism of bacterial adaptation to the host niche, where they reside. Asymptomatic carrier strains were shown to inhibit RNA polymerase II (Pol II) in host cells by targeting Ser2 phosphorylation, a step required for productive mRNA elongation. Assisted by a rare, spontaneous loss-of-function mutant from a human carrier, the bacterial NlpD protein was identified as a Pol II inhibitor. After internalization by host cells, NlpD was shown to target constituents of the Pol II phosphorylation complex (RPB1 and PAF1C), attenuating host gene expression. Therapeutic efficacy of a recombinant NlpD protein was demonstrated in a urinary tract infection model, by reduced tissue pathology, accelerated bacterial clearance, and attenuated Pol II–dependent gene expression. The findings suggest an intriguing, evolutionarily conserved mechanism for bacterial modulation of host gene expression, with a remarkable therapeutic potential.

Authors

Inès Ambite, Nina A. Filenko, Elisabed Zaldastanishvili, Daniel S.C. Butler, Thi Hien Tran, Arunima Chaudhuri, Parisa Esmaeili, Shahram Ahmadi, Sanchari Paul, Björn Wullt, Johannes Putze, Swaine L. Chen, Ulrich Dobrindt, Catharina Svanborg

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Figure 6

Membrane interaction and transfer of rNlpD into host cells.

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Membrane interaction and transfer of rNlpD into host cells. (A) Sequence...
(A) Sequence alignment of NlpD and the human MS4D protein. (B) Membrane interaction of rNlpD (Alexa Fluor 488, green) with giant unilamellar vesicles, labeled with rhodamine (red). Membrane colocalization (yellow) was detected in 6 of 13 vesicles. PC, phosphatidyl choline. (C–F) NlpD was detected in lysates and was internalized by human kidney cells exposed to rNlpD-His protein (0–250 μg/mL). (C) Western blot stained with anti-His antibodies. (D) Confocal imaging of cells stained with anti-His antibodies. Nuclei were counterstained with DRAQ5. Data are presented as mean ± SEM (n = 3 experiments). (E) NlpD was detected in the cytoplasm, membrane, and nuclei of treated cells. Western blot of cellular fractions stained with anti-NlpD antibodies. (F) NlpD internalization and nuclear translocation in cells exposed to Alexa Fluor 633–labeled rNlpD (magenta, 250 μg/mL) for 1 or 3 hours. Live-cell imaging: the nuclear plane of each image is shown. NlpD levels were quantified from 3 Z-stacks surrounding the nuclear plane. Nuclei were counterstained with Hoechst 33342. Data are presented as mean ± SEM (n = 18–24 cells). (G and H) Detection of NlpD in human kidney cells after infection with E. coli 83972 or the nlpD-reconstituted strain E. coli SN25-pRH320. (G) Western blot of whole cell lysates stained with anti-NlpD antibodies. Data are representative of 2 independent experiments. (H) NlpD uptake after infection. Staining was quantified by confocal imaging using anti-NlpD antibodies. E. coli SN25 served as a negative control. Scale bars: 10 μm. Data are presented as mean ± range (n = 2 experiments). *P < 0.05, **P < 0.01, ***P < 0.001 compared with 25 μg/mL (D) or compared with blank (F) by Kruskal-Wallis test with Dunn’s multiple-comparison test.