Pilicide ec240 disrupts virulence circuits in uropathogenic Escherichia coli - PubMed (original) (raw)

Pilicide ec240 disrupts virulence circuits in uropathogenic Escherichia coli

Sarah E Greene et al. mBio. 2014.

Abstract

Chaperone-usher pathway (CUP) pili are extracellular organelles produced by Gram-negative bacteria that mediate bacterial pathogenesis. Small-molecule inhibitors of CUP pili, termed pilicides, were rationally designed and shown to inhibit type 1 or P piliation. Here, we show that pilicide ec240 decreased the levels of type 1, P, and S piliation. Transcriptomic and proteomic analyses using the cystitis isolate UTI89 revealed that ec240 dysregulated CUP pili and decreased motility. Paradoxically, the transcript levels of P and S pilus genes were increased during growth in ec240, even though the level of P and S piliation decreased. In contrast, the most downregulated transcripts after growth in ec240 were from the type 1 pilus genes. Type 1 pilus expression is controlled by inversion of the fimS promoter element, which can oscillate between phase on and phase off orientations. ec240 induced the fimS phase off orientation, and this effect was necessary for the majority of ec240's inhibition of type 1 piliation. ec240 increased levels of the transcriptional regulators SfaB and PapB, which were shown to induce the fimS promoter phase off orientation. Furthermore, the effect of ec240 on motility was abolished in the absence of the SfaB, PapB, SfaX, and PapX regulators. In contrast to the effects of ec240, deletion of the type 1 pilus operon led to increased S and P piliation and motility. Thus, ec240 dysregulated several uropathogenic Escherichia coli (UPEC) virulence factors through different mechanisms and independent of its effects on type 1 pilus biogenesis and may have potential as an antivirulence compound.

Importance: CUP pili and flagella play active roles in the pathogenesis of a variety of Gram-negative bacterial infections, including urinary tract infections mediated by UPEC. These are extremely common infections that are often recurrent and increasingly caused by antibiotic-resistant organisms. Preventing piliation and motility through altered regulation and assembly of these important virulence factors could aid in the development of novel therapeutics. This study increases our understanding of the regulation of these virulence factors, providing new avenues by which to target their expression.

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Figures

FIG 1

Pilicide ec240 affects type 1 pili. (A) Structure of ec240. (B) Growth curve of UTI89 in LB, LB-DMSO, or LB-250 μM ec240. There is no significant difference in the final bacterial density under these conditions, as measured by Kruskal-Wallis test. (C) Type 1 piliation in UTI89 grown with DMSO or ec240 as measured by HA. Titers measured with no mannose and which are abrogated in the presence of mannose indicate agglutination mediated by type 1 pili. Titers in the presence of mannose indicate mannose-resistant or non-type-1-pilus-mediated agglutination. Error bars represent standard errors of the means. Averages shown are from at least three biological replicates.

FIG 2

Global transcriptional and proteomic responses to ec240. (A) Genes whose expression is dysregulated by growth in ec240 rather than DMSO, as identified by RNA-Seq analysis. Genes were classified by using KEGG (

http://www.genome.jp/kegg/

) and EcoCyc (

http://www.ecocyc.org

). The genes in each functional category are shown as percentages of the total genes dysregulated. (B) MA plots of UTI89 gene abundance following treatment with ec240. Each dot represents an annotated UTI89 gene with the log2 of its relative abundance in ec240 versus that in DMSO (M) plotted against the average log2 of its abundance under both conditions (A). M and A values are based on data from three biological replicates under each growth condition. The dotted lines indicate 3-fold changes. (C) Fold changes in CUP gene transcripts altered in UTI89 grown with ec240 rather than the DMSO vehicle control, as determined by RNA-Seq. Genes are shown in their order within the operon. (D) Proteins dysregulated after growth in ec240 rather than DMSO, as determined by iTRAQ analysis. Proteins were classified by using KEGG (

http://www.genome.jp/kegg/

) and EcoCyc (

http://www.ecocyc.org

). The proteins in each functional category are shown as percentages of the total proteins dysregulated and include proteins with increased and decreased abundances. (E) Fold changes in CUP proteins altered in UTI89 grown with ec240 rather than the DMSO vehicle control, as determined by iTRAQ analysis. (F) Dysregulation by ec240 of siderophore-related and iron transport genes by both RNA-Seq and iTRAQ analysis. Shown are fold changes in transcript or protein levels in UTI89 grown in ec240 rather than DMSO. Genes are shown in their order within the operon.

FIG 3

Pilicide ec240 alters CUP pilus expression. (A) qPCR of CUP pilin subunit transcripts in UTI89 grown with ec240. One subunit was used as a readout for the overall transcript level of the operon. Shown is a log transform of the fold change in the transcript level in UTI89 grown with ec240 compared to UTI89 grown with DMSO. Error bars represent standard errors of the means. Averages shown are from at least three biological replicates. Dotted lines indicates 2-fold changes in the transcript level for the qPCR data. (B) Immunoblot assay of CUP levels in UTI89 grown with DMSO or ec240. Antibodies against type 1 pili identified FimA and SfaA bands, while antibodies against PapD and SfaE identified PapD and SfaE, respectively.

FIG 4

CUP pilus response to fim mutation. (A) qPCR of each CUP pilin subunit transcript in UTI89Δ_fim_ as a readout of the overall transcript level of the CUP operon. Shown is a log transform of the fold change in the transcript level in UTI89Δ_fim_ compared to UTI89. (B) Pili removed from the surface of UTI89 or UTI89Δ_fim_ and depolymerized into pilin subunits were separated by SDS-PAGE and stained. Bands subjected to N-terminal sequencing are numbered, and the CUP pilin subunits they contain are FimA (band 1), PapA (band 2), and SfaA (band 3). (C) Type 1 piliation in UTI89 and CUP operon mutants as measured by HA titer and S piliation as measured by HA titer with desialylated guinea pig erythrocytes. (D) qPCR of each CUP pilin subunit transcript in UTI89 or UTI89Δ_fim_ grown with ec240 as a readout of the overall transcript level of the CUP operon. Shown as a log transform of the fold change in transcript levels in each strain grown with ec240 compared to that strain grown with DMSO. Error bars represent standard errors of the means. Averages shown in are from at least three biological replicates. The dotted lines in panels A and D indicate 2-fold changes in the transcript level for the qPCR data.

FIG 5

Pilicide ec240 alters CUP piliation. (A) Type 1 and S piliation as determined by HA titers. UTI89Δ_fim_ produces S pili; thus, the HA titer of UTI89Δ_fim_ grown with ec240 demonstrates ec240-reduced S piliation. (B) HA with human erythrocytes performed with C600/pFJ29 (encoding the pap operon from J96) grown alone or with DMSO or ec240. (C) ImageJ quantification of immunoblot assays quantifying PapA and PapD, shown as fold changes over C600/pFJ29. Error bars represent standard errors of the means. Averages shown are from at least three biological replicates. Statistically significant differences were determined by t test. *, P < 0.05.

FIG 6

Mechanism of action of pilicide ec240. (A) HA titer of UTI89 and the UTI89Δ_fimC_ chaperone mutant, alone and transformed with the pTRC99a vector control or p_fimC_ (pTRC99a-fimC). (B) Phase assay of UTI89 and the UTI89Δ_fimC_ chaperone mutant and each strain transformed with the pTRC99a vector control or p_fimC_ (pTRC99a-fimC) (see Materials and Methods). (C) Phase off or on UTI89 inoculum was back diluted in LB, LB-DMSO, and LB-ec240. Shown is a phase assay after growth of the back-diluted culture under type 1 pilus-inducing conditions. (D) Structure of ec342. (E) Piliation of UTI89 or LIR (fimS phase locked on strain) grown under type 1 pilus-inducing conditions with DMSO, ec240, or ec342, as measured by HA. (F) qPCR of recombinase fimB, fimE, and fimX transcript levels in UTI89 or UTI89Δ_fim_ grown with ec240, shown as the log transform of fold change in transcript levels in each strain grown with ec240 compared to each strain grown with DMSO. (G) Phase assay of UTI89 mutants after growth under type 1 pilus-inducing conditions, either alone or with DMSO or ec240. Error bars represent standard errors of the means. Averages shown in panels A, E, and F are from at least three biological replicates. Dotted lines indicate 2-fold changes in transcript levels for the qPCR data.

FIG 7

Effects of pilicide ec240 on motility. (A) Swimming motility in soft agar of UTI89Δ_fim_ as determined by the fold change in the diameter of the growth zone compared to that of UTI89 after the growth of each strain under type 1 pilus-inducing conditions. (B) qPCR of fliC transcript levels in UTI89Δ_fim_, shown as a log transform of the fold change relative to UTI89 after growth under type 1 pilus-inducing conditions. (C) Immunoblot assay of flagellar levels in UTI89 grown with DMSO and ec240 with anti-H7 antibody. (D) Swimming motility in soft agar of UTI89 or UTI89 mutants grown with ec240 as determined by the fold change in the diameter of the growth zone compared to that of UTI89 or UTI89 mutants grown with the DMSO vehicle control. *, P < 0.05. Statistically significant differences were determined by one-sample t test. Error bars represent standard errors of the means. The dotted lines in panel B indicate 2-fold changes in transcript levels for the qPCR data.

FIG 8

Model of pilicide ec240 action on CUP pili and flagella.

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Pilicide ec240 disrupts virulence circuits in uropathogenic Escherichia coli - PubMed (original) (raw)