Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits

Abstract

Uropathogenic Escherichia coli (UPEC) utilizes a complex community-based developmental pathway for growth within superficial epithelial cells of the bladder during cystitis. Extracellular DNA (eDNA) is a common matrix component of organized bacterial communities. Integration host factor (IHF) is a heterodimeric protein that binds to double-stranded DNA and produces a hairpin bend. IHF-dependent DNA architectural changes act both intrabacterially and extrabacterially to regulate gene expression and community stability, respectively. We demonstrate that both IHF subunits are required for efficient colonization of the bladder, but are dispensable for early colonization of the kidney. The community architecture of the intracellular bacterial communities (IBCs) is quantitatively different in the absence of either IhfA or IhfB in the murine model for human urinary tract infection (UTI). Restoration of Type 1 pili by ectopic production does not restore colonization in the absence of IhfA, but partially compensates in the absence of IhfB. Furthermore, we describe a binding site for IHF that is upstream of the operon that encodes for the P-pilus. Taken together, these data suggest that both IHF and its constituent subunits (independent of the heterodimer), are able to participate in multiple aspects of the UPEC pathogenic lifestyle, and may have utility as a target for treatment of bacterial cystitis.

Figure 1. Promoter orientation.

The promoter region (fimS) from UTI89, ROL745 (UTI89 ihfA) and ROL603 (UTI89 ihfB) were amplified by PCR and digested as previously described . The size of the fragments following digestion indicates the orientation of the promoter (“off”  = 539 & 187 bp, while “on”  = 433 & 293 bp). A representative gel is shown to demonstrate the different orientations observed in the UTI89 wild type and mutant strains. The mutated genes are indicated above each lane.

Figure 2. IhfAB subunits are required for UPEC pathogenesis.

Each symbol represents single infected murine bladder or combined kidney pair. Female mice were infected with wild type UTI89 (black filled squares), ROL745 (UTI89 ihfA11; gray filled circles), ROL745/pHNα (UTI89 ihfA11 complemented with ihfA; black open circles), ROL603 (UTI89 ΔihfB; gray filled triangles), or ROL603/pHNβ (UTI89 ihfB::Cam complemented with ihfB; black open triangles) for 6 or 48 hours post inoculation. Bacterial burden of bladders and kidneys was enumerated as colony forming units ( CFUs). Statistical significance determined using non-parametric Mann Whitney (*<0.04, ** = 0.018, *** = 0.0017, ****<0.0006).

Figure 3. Architecture of Intracellular Bacteria.

Female mice were infected with UTI89/pANT4, ROL745/pANT4 (UTI89 ihfA11), ROL603/pANT4 (UTI89 ΔihfB), SJ1000/pANT4 (UTI89 surA), or UTI89 Δkps/pANT4 for 6 hours. Bladders were prepared for visualization by fluorescent microscopy . The intracellular characteristics of each of the strains indicated were visualized using strains that constitutively produce green fluorescent protein. Images were taken as an optical section (upper panel UPEC) or as total fluorescence of entire community (all other panels). The strains are indicated above each panel. Scale bar  = 10 µm and scale is unchanged between images.

Figure 4. Community architecture is restored upon complementation in trans.

Female mice were infected with UTI89, ROL745/ pHNá (UTI89 ihfA11), or ROL603/ pHNâ (UTI89 ΔihfB) for 6 hours. Bladders were prepared for visualization by immune-fluorescent microscopy . Images were taken as an optical section. The strains are indicated above each panel. Scale bar  = 10 µm and scale is unchanged between images.

Figure 5. Quantitative Assessment of Intracellular Bacteria.

The individual statistics of the bacteria growing within superficial mouse bladder epithelial cells as depicted in Figure 3 were determined. (A) The cell length of each bacterium was evaluated within individual images (NIH Image J) or 3-dimensional rendering . The cell length distribution for each strain is represented. (B) The maximum dimension in the x and y axes, the ratio of the axes as well as the overall area of the intracellular bacteria were determined using Prokarymetrics.

Figure 6. Type 1 piliation does not restore bladder colonization in the absence of IhfA.

Each symbol represents single infected murine bladder or combined kidney pair. Female mice were infected with wild type UTI89/pMMB66 (black filled squares), ROL745/pMMB66 (UTI89 ihfA11; gray filled circles), or ROL603/pMMB66 (UTI89 ΔihfB; gray filled triangles) for 6 hours post inoculation. Bacterial burden of bladders and kidneys was enumerated as colony forming units ( CFUs). Statistical significance determined using non-parametric Mann Whitney (**<0.002, *** = 0.0006).

Figure 7. IhfAB is extrabacterial within the IBC milieu.

Bladders were removed at 16 hours following transurethral introduction of UTI89/pANT4 (green). At 16 hours post introduction of bacteria, the bladders were harvested, bisected, fixed, host cells permeabilized and eIhfAB was observed with antibody (red) and Hoescht counterstained (blue). (A, D) No staining is observed when naïve rabbit serum is used as primary antibody. (B,E) eIhfAB is observed between the bacteria within the epithelial cell. (C, F) eIhfAB remains associated with UPEC following egress onto the surface of the bladder. Scale bar  = 10 µm.

Figure 8. Antiserum against IhfAB reduces UPEC binding to cultured bladder epithelial cells.

HTB-4 human bladder transitional carcinoma cell monolayers were infected with UTI89 treated with naïve antiserum (N; black) or specific antiserum directed against IhfAB (αIHF; gray). Each symbol represents the average of three experiments performed on the same day. Each experiment was replicated on three separate occasions. The number of bacteria bound is reported as a percentage of the bacteria bound in the presence of naïve serum. Statistical significance was determined by two-tailed Mann-Whitney test (**, p = 0.003).

Figure 9. IHF binds the upstream region of papB.

A) Lane 1–2: 0 & 50 nM IHF were incubated with labeled probe and subjected to DNase I digestion. The sequence protected by IHF is marked with the dashed line on the gel and the actual sequence is shown on the right side of the figure. The solid line on the sequence and gel represent the IHF recognition site, bold bases indicate the hypersensitive sites (< on the gel) and lower case letters are not protected. The numbers indicate the position from the transcription start (+1) of papB. B) Organization of the divergent papB and papI promoter region with the IHF protected region indicated by the dashed line.