Uropathogenic Escherichia coli proliferate as a coccoid morphotype inside human host cells

Abstract

Escherichia coli is arguably one of the most studied bacterial model systems in modern biology. While E. coli are normally rod-shaped gram-negative bacteria, they are known to undergo conditional morphology changes under environmental and nutrient stress. In this study, using an infection-based in-vitro infection model system combined with advanced dynamical imaging, we present the first molecular details of uropathogenic E. coli (UPEC) dividing to form and proliferate as coccoid-shaped cells inside human host cells. For these intracellular UPEC cells, the frequency of cell division outpaced the rate of cell growth, resulting in a morphological transition from traditional rod-shape to coccobacilli. We also visualized the subcellular protein dynamics in these cells and noted that the division proteins follow the similar localization and constriction patterns that have been demonstrated for vegetative growth. However, unlike for fast-growing rod-shaped cells, FtsZ constriction in intracellular UPEC occurs prior to visual nucleoid segregation. Our results suggest that the modulation of division rate contributes to morphological adaptability of intracellular UPEC at the single-cell level.

Fig 1. Intracellular UPEC actively divide into coccobacilli during UTI model infection.

a, PD07i human epithelial bladder cells (membranes shown in blue [CellBrite Steady Membrane 405] and nuclei shown in gold [NucSpot Live Cell 650]) were challenged with fluorescently labeled UTI89 expressing cytoplasmic mCherry (pseudo coloured magenta). Imaged by confocal microscopy. Scale bar = 4 μm. b, Representative images of cells before (T = 0, start of imaging, 6-h post infection) and after division (T = 80); note that one daughter cell has left the field of view. Times (T) are shown in minutes. Images acquired using HiLo illumination. Scale bar = 1 μm. c, d, Average cell length (c) and area (d) of extracellular and intracellular bacteria (before any division event) (n_Intra = 326, n_Extra = 117). e, Length vs. width of extracellular (‘Extra’) and intracellular (‘Intra’) bacteria (before any division event). Error bars indicate S.D. f–h, Lengths (f), widths (g), and aspect ratio (h) of intracellular bacteria before and after division, (n_before = 126, n_after = 200). Red dotted line in f represents average coccobacilli width after division. Blue dotted line in h represents an aspect ratio of 1, i.e., a perfect circle. Box plots: box edge equals S.D., midline represents average, and whiskers indicate 1%–99% interval. Statistical significance was determined by student T test, where **** stars indicate p values less than 0.0001. The data underlying this figure can be found in S1 Data.

Fig 2. Divisome dynamics in intracellular UPEC cells during infection.

a, Representative confocal image of intracellular UTI89 expressing a division marker (FtsZ-mCitrine; pseudo coloured green) with mCherry (pseudo coloured magenta) as cytoplasmic marker, bladder cell membranes shown in blue (CellBrite Steady Membrane 405) and nuclei shown in gold (NucSpot Live Cell 650). b, Still images of a time-lapse sequence showing FtsZ-mCitrine (pseudo coloured green) driven coccobacilli divisions imaged using HiLo illumination (S3 Movie). c, Average division times of intracellular bacteria over multiple generations. Midline represents average, n_first = 88, n_second= 42, n_third= 10. *** p = 0.0027. **** p < 0.0001. d, mCitrine-FtsN (pseudo coloured green) midcell constriction during coccobacilli divisions (S4 Movie). e, Subcellular FtsZ-mCherry (pseudo coloured magenta) and mCitrine-FtsN (pseudo coloured green) dynamics during coccobacilli divisions. Fluorescence intensity plots highlight a larger apparent radius of FtsN (Mid, T = 70) and that it remains longer at septum than FtsZ (Late, T = 100). Times (T) are shown in minutes since start of imaging (6 h p.i.). Scale bars a = 2 μm; b, d and e = 1 μm. The data underlying this figure can be found in S1 Data.

Fig 3. EYFP-MinD dynamics in UPEC coccobacilli during infection.

a, Still images from an EYFP-MinD time-lapse series showing dynamics in intracellular UTI89 coccobacilli (S6 Movie). b, c, Time-averaged fluorescence intensity of EYFP-MinD. Dotted line in plots (c) represent cell boundaries. d, Time-averaged fluorescence intensity of EYFP-MinD (pseudo coloured green) and FtsZ-mCherry (pseudo coloured magenta), indicating that FtsZ localize at MinD minima also in coccobacilli. e, Representative kymograph of typical EYFP-MinD oscillations of a cell longer than 1.3 μm. Yellow dotted line represents where intensity plot was generated. f, Average EYFP-MinD oscillation rate in intracellular bacteria was 0.055 ± 0.036 μm s⁻¹ (n = 71) and in extracellular 0.095 ± 0.035 μm s⁻¹ (n = 60) cells. g, Typical kymographs of EYFP-MinD oscillations of cells shorter than 1.3 μm show complex nonstandard pole-to-pole oscillation dynamics. Kymographs in g were generated from the two lower cells in S6 Movie. Scale bars a and b = 1 μm; d = 2 μm. The data underlying this figure can be found in S1 Data.

Fig 4. Chromosomal partitioning is maintained during intracellular UPEC coccobacilli divisions.

a, b, DNA (HupA-RFP) partitioning in coccobacilli (msfGFP (green) as cytoplasmic marker). DNA segregates equally into daughter coccobacilli (n = 98). c, Average chromosome lengths in extracellular (n = 216) and intracellular bacteria (n = 98). d, Chromosomes partitions into equal size after division in both extracellular (gray) and intracellular (black) bacteria. Blue dashed line indicates linear dependence. Red dots indicate average, with x–y bars representing S.D. e, f, FtsZ-mNeonGreen (green) rings assemble and, partly, constrict over undivided chromosomes (HupA-RFP; psudo coloured magenta) in both WT and ΔslmA coccobacilli. Intensity plot in f, shows FtsZ constriction over 60 min (inset show HupA-RFP fluorescence profiles taken perpendicular to the Z-ring, with no intensity decrease at midcell over the same time interval). Cerulean dotted line indicate from where the FtsZ-mNeonGreen intensity plot is generated. g, Representative image sequence of UTI89ΔslmA expressing FtsZ-mNeonGreen (green) and HupA-RFP (pseudo coloured magenta) showing FtsZ-ring constriction over unsegregated chromosomes. h, Average levels of FtsZ-mNeonGreen constriction over unsegregated chromosomes from 3 independent infections (n_cell= 30). i, FtsZ-mNeonGreen (green) rings do not constrict over unsegregated chromosomes (HupA-RFP, pseudo-coloured magenta) in extracellular rods. Statistical significance was determined by student T test, where **** stars indicate p values less than 0.0001., n.s. = not significant. Box plots: box edge equals S.D., midline represents average, and whiskers indicate 1%–99% interval. Times (T) are shown in minutes since start of imaging (6 h p.i.). Scale bars a, e, f and g = 1 μm; i = 2 μm. The data underlying this figure can be found in S1 Data.