Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

Abstract

Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease-associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that enhances their persistence.

Keywords: RNA polymerase II; asymptomatic bacteriuria; gene expression; phages; transcriptional modulation.

Figure 1

Suppression of gene expression in inoculated patients and infected human kidney cells. (A) Schematic of therapeutic inoculation with the ABU strain E. coli 83972, which established ABU in the inoculated patients. RNA was extracted from peripheral blood mononuclear cells (PBMCs) before and 24 h after intravesical inoculation. Significantly regulated genes were identified by transcriptomic analysis. Genes in the Pol II network were suppressed, by the ABU strain; (B) Human kidney epithelial cells (A498) were infected with the ABU strain E. coli 83972 and significantly regulated genes were identified by transcriptomic analysis, compared to uninfected cells. Pol II transcription cycle with gene categories regulated by ABU indicated in red. ABU infection affected different steps in the Pol II cycle, including chromatin opening, escape from pausing and pre-mRNA splicing. The ABU strain failed to activate/inhibit the pathology-associated TLR4 and IFN-β pathways. Adapted from Lutay et al. [14].

Figure 2

The ABU strain inhibits Pol II Ser 2 phosphorylation in vitro. (A) Immunoperoxidase staining of human kidney epithelial cells (A498), using HRP-conjugated antibodies. Pol II phosphorylation (brown) was reduced by the ABU strain compared to uninfected cells. A scale representing each staining intensity is shown (4 = brown, 0 = colorless). Inhibition of Pol II phosphorylation was expressed in percent of the uninfected controls (0% = control, no inhibition; 100% = complete inhibition). The cells were exposed to 2 × 10⁹ CFU/mL of E. coli CFT073 (APN) or 83972 (ABU), (n = 100 cells per sample, p-value by χ² test). Scale bar = 50 μm; (B) Phospho-Pol II in human kidney epithelial cells, infected with the ABU or APN strains. Western blots of whole cell extracts. The phospho-specific staining was normalized against total Pol II or GAPDH; (C) Competitive infection. The strain to which the cells were first exposed, was shown to determine Pol II activation. Pol II Ser 2 staining of human kidney epithelial cells after 2 h + 2 h infection with the ABU strain followed by the APN strain or the APN strain followed by the ABU strain (n ≥ 167 cells, p-values by χ² test).

Figure 3

Induction of Prophage 3 by E. coli 83972 and construction of the E. coli 83972 Prophage 3 deletion mutant. (A) Electron microscopy of lambda-like Prophage 3 released by E. coli 83972 during in vitro growth in pooled human urine; (B) Quantification of lytic phages by plaque formation (plaque forming units, PFU) on E. coli C600. E. coli 83972 released lytic phages during growth in urine and Luria Bertani (LB) broth. Moderate release of lytic phages by CFT073 was induced by mytomicin C; (C) The Prophage 3 sequence was deleted from the E. coli 83972 chromosome. The ABU—p3 mutant strain is unable to produce and release lytic Prophage 3 particles; (D) The Prophage 3 sequence was replaced by a chloramphenicol acetyltransferase (cat) cassette, using homologous Lambda red recombination technology and chloramphenicol resistance for selection. The bacterial sit gene, involved in iron uptake, was conserved in the mutant strain. In the construct, the chloramphenicol resistance gene is surrounded by two flippase recognition target (FRT) sites.

Figure 4

Pol II phosphorylation is not altered by the Prophage 3 deletion. (A) The Pol II Ser 2 staining intensity (brown) did not differ between cells infected with the ABU strain or the -Prophage 3 deletion mutant (10⁹ and 2 × 10⁹ CFU/mL, n = 100 cells, p-values by χ² test). Histograms show the distribution of human kidney epithelial cells according to their Pol II Ser 2 staining intensity (+++ = highly stained, - = no staining); (B) Supernatants of A498 cells infected with the ABU strain or the ABU-Prophage 3 deletion mutant. Similar effects on Pol II phosphorylation (10⁹ CFU/mL, n = 100 cells, p-values by χ² test). Pol II Ser 2 staining was quantified by immunoperoxidase staining using Pol II Ser2specific antibodies; (C) Pol II Ser 2 phosphorylation was not inhibited by lytic phage particles released by the ABU strain during growth in human urine, in vitro.