Multispecies bacterial invasion of human host cells

Abstract

Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here, using clinically relevant bacteria, human epithelial bladder cells and human urine, we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.

Keywords: E. faecalis; Infection; K. Pneumoniae; Multispecies; UPEC; UTI.

Figure 1.

Single-species invasion of human epithelial bladder cells using Gram-negative and Gram-positive uropathogens. Immortalized epithelial bladder cells PD07i (cytoplasmic membranes labelled with CellBright650, magenta) grown on a 35-mm glass bottom petri-dish or in a flow chamber were infected by either A, UPEC (strain UTI89 expressing cytoplasmic mOrange2); B, K. pneumoniae (strain TOP52 expressing cytoplasmic mCerulean3); or c, E. faecalis (strain SD234 expressing cytoplasmic GFP). White arrows indicate internalised bacteria. Representative cross-sectional views, confocal 3D renderings and Z-stacks confirming that bacteria are intracellular are presented in Supplementary Fig. S1 and Supplementary Movies SM1–SM3. D, Proportion of bladder cells infected (colour) and uninfected (grey) in various conditions and bacterial species. Graphs are based on values from 3 infections for each condition. n.s. = not statistically significant. P < 0.05 = statistically significant. K. p = K. pneumoniae. E. f = E. faecalis. Dish = 35 mm glass bottom petri-dish. Flow = 15 µl min⁻¹ in flow channel (IBIDI µ-Slide I^0.2). E, PD07i cells maintained without bacteria. Images are from petri-dish experiments. Scale bars = 20 µm. Note that the bottom layers containing only epithelial membrane of the Z-stacks were omitted for better visualization of internal bacteria. These infection experiments were done using a standard gentamycin protection assay, see methods for further details.

Figure 2.

Bladder cell invasion during UPEC and E. faecalis co-infections. A, PD07i (membranes labelled with CellBright 405 or 650) cells co-infected with UPEC (orange) and E. faecalis (green). Scale bar 20 = µm. Representative cross-sectional views and a confocal Z-stack are presented in Fig. S2 and Supplementary Movie SM4. B, ratio of invaded PD07i cells. E. f = E. faecalis C, Typical intracellular UPEC (orange) and E. faecalis (green) clusters. UPEC appeared more tightly packed than E. faecalis. Scale bar 1 µm. D and E, Low resolution Z-stacks showing UPEC surrounding E. faecalis in co-invaded PD07i cells. Scale bar D = 5 µm. The graph in E shows fluorescence intensity traces for the white dash line in the image above. UPEC (orange) and E. faecalis (green). Dotted scale line = 12 µm. F, G and H, Representative single images and confocal 3D reconstructions of E. faecalis (green) surrounded by UPEC (orange). A confocal Z-stack of F is presented in Supplementary Movie SM5. Scale bar F = 10 µm. Scale bars G, H = 2 µm. I, Percentage of dual invaded PD07i cells with UPEC cells enclosing E. faecalis cells (as shown in G and H). Note various orientations of reconstructed Z-stacks. These infection experiments were done using a standard gentamycin protection assay, see methods for further details.

Figure 3.

Prevalence of bacterial invasion in dual- and triple-species infections. Representative images of PD07i cells invaded by A, UPEC and K. pneumoniae (2D confocal image); % of invasion (n = 519), Dish: UPEC 6.1 ± 5.4% (mean ± S.D.), K.p. 3.7 ± 4.2%, both 2 ± 2.2%. Flow: UPEC 4.6 ± 2.6%, K.p. 1.9 ± 1.8%, both 1.35 ± 1.9%. B, E. faecalis and K. pneumoniae (deconvolved 3D-reconstruction of a confocal Z-stack, n = 463). % of invasion: E.f 2.7 ± 3%, K.p. 6.8 ± 4.9%, both 3.7 ± 5.8%. C, UPEC, E. faecalis and K. pneumoniae (deconvolved 3D-reconstruction of a confocal Z-stack, n = 349). % of invasion: UPEC 13.3 ± 6.7%, E.f 1.3 ± 3%, K.p. 3.4 ± 3.6%, Any 2 species 8.2 ± 6.4%, all 3 strains 3.7 ± 4.4%. PD07i membranes labelled with CellBright 650. Scale bars = 4 µm. E. f = E. faecalis. K. p = K. pneumoniae. Note, different scales on y-axis. Representative cross-sectional views are presented in Figs S3 and S4. These infection experiments were done using a standard gentamycin protection assay, see methods for further details.

Figure 4.

Gram-negative, but not Gram-positive, species undergo infection-related filamentation in both single and multi-species infections. Representative images of bacteria after being exfoliated from PD07i cells during single species infections. A, UPEC, B, K. pneumoniae, C, E. faecalis. Note all strains retain their fluorescence throughout invasion and exfoliation. D, Average lengths of bacterial filaments (UPEC and K. pneumoniae) and cells (E. faecalis) after 20 h of urine exposure during infections. The average length of UPEC filaments was 56.0 ± 59.9 µm (n = 158) in dishes, and 43.3 ± 40.05 µm (n = 122) in flow. K. pneumoniae filaments were 47.3 ± 45.3 µm (n = 98) in dishes and 54 ± 57.8 µm (n = 156) in flow and. UPEC and K. pneumoniae rods grown in LB media were on average 3.46 ± 0.69 µm (n = 187) and 3.62 ± 0.76 µm (n = 192), respectively. Average lengths of E. faecalis after an infection were 1.42 ± 0.4 µm (n = 215) from petri-dish infection and 1.59 ± 0.41 µm (n = 102) from flow chambers. Lengths for E. faecalis cells grown in BHI media only was 1.74 ± 0.31 µm (n = 238). Dish = 35-mm glass bottom petri-dish. Flow = 15 µl min⁻¹ in flow chamber (IBIDI I^0.2). E, Filamentation of UPEC and K. pneumoniae but not E. faecalis during a representative three species infections. Scale bars A, B and E = 10 µm, C = 4 µm.