Remodeling of the Z-Ring Nanostructure during the Streptococcus pneumoniae Cell Cycle Revealed by Photoactivated Localization Microscopy

Abstract

Ovococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring's morphology during the division cycle at the nanoscale level. We show that changes in the ring's axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci.

Importance: The Gram-positive human pathogen S. pneumoniae is responsible for 1.6 million deaths per year worldwide and is increasingly resistant to various antibiotics. FtsZ is a cytoskeletal protein polymerizing at midcell into a ring-like structure called the Z-ring. FtsZ is a promising new antimicrobial target, as its inhibition leads to cell death. A precise view of the Z-ring architecture in vivo is essential to understand the mode of action of inhibitory drugs (see T. den Blaauwen, J. M. Andreu, and O. Monasterio, Bioorg Chem 55:27-38, 2014, doi:10.1016/j.bioorg.2014.03.007, for a review on FtsZ inhibitors). This is notably true in ovococcoid bacteria like S. pneumoniae, in which FtsZ is the only known cytoskeletal protein. We have used superresolution microscopy to obtain molecular details of the pneumococcus Z-ring that have so far been inaccessible with conventional microscopy. This study provides a nanoscale description of the Z-ring architecture and remodeling during the division of ovococci.

FIG 1

Development of spDendra2 for PALM studies of S. pneumoniae. Live cells expressing the ectopic Dendra2-FtsZ (D2-FtsZ) or spDendra2-FtsZ (spD2-FtsZ) fusions were visualized at an OD₆₀₀ of 0.3 using conventional microscopy. Dendra2 and spDendra2 green epifluorescence, phase-contrast, and merged images are shown. Scale bars = 1 µm. (B) Expression levels of native FtsZ (FtsZ) and of ectopic Dendra2-FtsZ (D2-FtsZ) and spDendra2-FtsZ (spD2-FtsZ) fusions in strains R6 (left lane), spCM18 (middle lane), and spCM72 (right lane). Samples of cultures were harvested at an OD₆₀₀ of 0.3 for the preparation of protein extracts. Equivalent amounts of total proteins for each sample were separated by SDS-PAGE, blotted to membranes, and analyzed by immunoblotting using anti-FtsZ serum. Positions of molecular mass markers are indicated by the numbers (in kilodaltons) to the left of the blots.

FIG 2

FtsZ displays specific septal patterns in fixed S. pneumoniae cells. Pneumococcal cells expressing an endogenous FtsZ-spDendra2 fusion (spMJ40) (A) or free spDendra2 in their cytoplasm (spMJ33) (B) were fixed and imaged at an OD₆₀₀ of 0.3. Bright fields (a) and reconstructed PALM images (b) are shown. Red arrowheads point at cells in one of the three stages described in Fig. 3 or at a cell displaying a double ring (DR) of FtsZ as illustrated in Fig. 6. Gray-boxed inset images are 5-fold magnifications of one predivisional PALM pattern displayed in panel b. Dashed gray lines represent the periphery of the cell. Scale bars = 2 µm.

FIG 3

PALM images of representative patterns of the Z-ring throughout the pneumococcal cell cycle. Typical pneumococcal cells expressing the endogenous FtsZ-spDendra2 fusion (spMJ40) (A and C) or the ectopic spDendra2-FtsZ fusion (spCM72) (B) were classified into three stages defined by the diameter of the Z-ring, the cell length, and the distributions of FtsZ at the division site of the mother cell and the division sites of the two daughter cells. Cells were either fixed (A and B) or directly imaged (C) at an OD₆₀₀ of 0.3. Bright-field (a), diffraction-limited (b), and reconstructed PALM (c) images are shown. Scale bars = 250 nm.

FIG 4

The pneumococcal Z-ring is made of heterogeneous clusters. (A) Reconstructed PALM image of the Z-ring in fixed cells expressing the endogenous FtsZ-spDendra2 fusion at stages i, ii, and iii or in a tilted orientation (iv). Scale bars = 250 nm. (B) Cluster analysis of the PALM images shown in panel A using the DBSCAN algorithm (38). Different colors are used to visualize the different clusters. Nonclustered molecules are shown as black dots. (C) Cluster analysis of simulated PALM imaging of continuous and homogeneous Z-rings. The cluster diameter threshold was set at 22 nm.

FIG 5

The Z-ring’s axial thickness increases during constriction. (A) Mean values for the Z-ring diameter (A), the number of imaged septal FtsZ molecules (B), and the Z-ring’s axial thickness (C) are reported for fixed cells in the early (Z-ring diameter > 800 nm [dark gray]) or late (Z-ring diameter <700 nm [black]) divisional stage. During constriction, the Z-ring’s axial thickness increases by ~32 nm while the number of imaged FtsZ-spDendra2 molecules at the division site shows no significant variation. Error bars represent standard deviation values.

FIG 6

Pneumococcal FtsZ displays double-ring patterns. Pneumococcal cells expressing the endogenous FtsZ-spDendra2 fusion (spMJ40) (A and C) or the ectopic spDendra2-FtsZ fusion (spCM72) (B) were either fixed (A and B) or directly imaged (C) at an OD₆₀₀ of 0.3. Bright-field (a), diffraction-limited (b), and reconstructed PALM (c) images are shown. Scale bars = 250 nm.

FIG 7

Model of the nanostructure of the Z-ring in the cell cycle of ovococci. At the onset of a division cycle, FtsZ molecules (represented as light-pink squares) polymerize and form heterogeneous clusters, which are distributed within an ~95-nm-thick ring-like structure (the so-called Z-ring) at midcell. Three alternative paths are then proposed. In the first path (1), the predivisional Z-ring directly proceeds to constriction. In the second path (2), the single predivisional ring duplicates into two functional rings that progressively separate as the cell wall is synthesized at the division site (interpeak distances ranging from 50 to 300 nm). Upon constriction, the distance separating the double Z-rings decreases, and the unresolved rings are eventually imaged as an ~127-nm-thick single ring at midcell. In both paths, the FtsZ molecules start disassembling from the parental division site at stage iii and two new predivisional Z-rings appear at the future division sites of the daughter cells. Each newborn daughter cell will eventually display a single stage i Z-ring. In the third path (3), double rings are not functional for division and they are potentially resolved by an unknown proofreading or stress response mechanism. Blue boxes frame regions that have been magnified to illustrate the heterogeneous and clustered arrangement of the Z-ring, the splitting of the predivisional Z-ring before constriction, and the thickening of the apparent single Z-ring at stage ii. Dimensions measured in this work are indicated. Purple-red areas represent regions with high FtsZ density (FtsZ clusters). These regions have been magnified to illustrate the presence of FtsZ filaments (pink rectangles), potentially connected by FtsZ partners (green triangles). Gray areas represent regions of cell wall synthesis.