Nascent teichoic acids insertion into the cell wall directs the localization and activity of the major pneumococcal autolysin LytA

Abstract

The bacterial cell wall is in part composed of the peptidoglycan (PG) layer that maintains the cell shape and sustains the basic cellular processes of growth and division. The cell wall of Gram-positive bacteria also carries teichoic acids (TAs). In this work, we investigated how TAs contribute to the structuration of the PG network through the modulation of PG hydrolytic enzymes in the context of the Gram-positive Streptococcus pneumoniae bacterium. Pneumococcal TAs are decorated by phosphorylcholine residues which serve as anchors for the Choline-Binding Proteins, some of them acting as PG hydrolases, like the major autolysin LytA. Their binding is non covalent and reversible, a property that allows easy manipulation of the system. In this work, we show that the release of LytA occurs independently from its amidase activity. Furthermore, LytA fused to GFP was expressed in pneumococcal cells and showed different localization patterns according to the growth phase. Importantly, we demonstrate that TAs modulate the enzymatic activity of LytA since a low level of TAs present at the cell surface triggers LytA sensitivity in growing pneumococcal cells. We previously developed a method to label nascent TAs in live cells revealing that the insertion of TAs into the cell wall occurs at the mid-cell. In conclusion, we demonstrate that nascent TAs inserted in the cell wall at the division site are the specific receptors of LytA, tuning in this way the positioning of LytA at the appropriate place at the cell surface.

Keywords: Choline-Binding Proteins; Major autolysin LytA localization; Peptidoglycan hydrolases; Streptococcus pneumoniae cell wall; Teichoic acids localization.

Graphical abstract

Fig. 1

Effect of choline on pneumococci sensitivity to LytA-induced lysis. A. Growth of wild-type pneumococci in CY, a choline-rich medium, in the absence or in the presence of exogenous LytA at 10 µg/ml. B. Growth in CDM medium, containing a choline concentration of 10 µg/ml, in the absence or in the presence of exogenous LytA at 10 µg/ml. C. Growth in CDM medium, containing a choline concentration of 5 µg/ml, in the absence or in the presence of exogenous LytA at 10 µg/ml. The addition of exogenous LytA is indicated by an arrow in all three panels.

Fig. 2

Scanning (A, B and C) and transmission (D and E) electron micrographs of normal (A and D) and tarIJ depleted (B, C and E) S. pneumoniae cells. Cells grown in the presence of 2 µM ComS^* (A and D) displayed the typical ovococcal shape of wild-type pneumococci. Depletion of tarIJ expression by removal of ComS^* from the growth medium (B, C and E) gave rise to severe morphological abnormalities and multiple false or aborted division zones (indicated by arrows). All cell samples were collected during exponential growth. Scale bars in A, B, C: 1 µm; in D and E: 0.5 µm.

Fig. 3

Reduced expression of tarIJ triggers LytA sensitivity in growing S. pneumoniae. A. Impact of decreasing TarIJ expression on the growth of SPH146. TarIJ expression was regulated by growing the SPH146 cells in medium containing concentration of ComS^* ranging from 0.125 to 0.041 µM. B. LytA-sensitivity of SPH146 cells growing exponentially in medium containing 0.125, 0.100 and 0.080 µM ComS^*. Purified LytA (5 µg ml⁻¹) was added to the cultures when they reached OD₄₉₂ 0.2–0.3, as indicated by the arrow.

Fig. 4

Localization of LytA varies according to the culture growth phase. A. Fluorescence images of LytA-sfGFPop expressed in ΔlytA cells withdrawn at different growth phase. Phase contrast (grey), GFP fluorescent signal (green) and merge (right) images are shown. Scale bars: 2 µm. B. Distribution of bacteria displaying homogeneous, heterogeneous and mid-cell localization of LytA-sfGFPop during exponential phase (OD_600 nm 0,40), stationary phase (OD_600 nm 0,97), early (OD_600 nm 0,76) and late (OD_595 nm 0,23) lytic phase. N = 98–170 bacteria per OD point were analyzed. C. Demograph representation of the fluorescence signal profile of LytA-sfGFPop in cells sorted by increasing cell length (MicrobeTracker, MATLAB). Images analysis was performed on cells withdrawn during the exponential phase (upper demograph) and the late lytic phase (bottom demograph). N = 30–120 bacteria per demograph were analyzed.

Fig. 5

LytA is released in the medium and binds at the surface of non-lyzed cells at mid-cell. A. Distribution of bacteria displaying homogeneous, heterogeneous and mid-cell localization of LytA-sfGFPop during lytic phase before and after treatment with 2% choline. B. Pneumococcal wild-type cells expressing LytA-sfGFPop were mixed with cells deleted from lytA and expressing FtsZ fused to a red fluorescent protein (mKate2) at the ratio 2:1. The growth of the mixed culture was pursued at 37 °C until and aliquots were withdrawn when the culture entered the lytic phase. Phase contrast (grey), GFP fluorescent signal (green), mKate2 (red) and merge (right) images are shown. Scale bars: 2 µm. C. Growth curves of the ΔlytA strain transformed with the Zn-inducible plasmid encoding the inactive form of LytA, LytAE₈₇Q, in the absence or the presence of 0.15 mM Zn. D. Growth curves of the ΔlytA strain transformed with the Zn-inducible plasmid encoding the amidase domain of LytA, in the absence or the presence of 0.15 mM Zn. E. Growth curves of the ΔlytA strain transformed with the Zn-inducible plasmid encoding the CBD domain of LytA, in the absence or the presence of 0.15 mM Zn. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.)

Fig. 6

Localization of LytA and cell wall cleavage sites. A. The CBD is sufficient to trigger localization of LytA. Wild-type cells in exponential growth phase were incubated with sfGFPop-LytA or sfGFPop-LytA^CBD recombinant proteins (10 µg/mL). Merge image of fluorescence and phase contrast views are showed. Scale bars: 2 µm. Demograph representation of the signal profile of sfGFPop-LytA and sfGFPop-LytA^CBD in cells sorted by increasing cell length (MicrobeTracker, MATLAB). B. Electron micrographs of S. pneumoniae sacculi incubated 1 min at 37 °C with buffer or with 5 µg/mL of recombinant purified LytA. Cleavage of the cell wall by LytA at both sides of the mid-cell sites are indicated by arrows and at the cell pole by arrow heads.

Fig. 7

LytA is associated to newly synthesized cell wall in growing cells. sfGFPop-LytA (10 µg/ml) and fluorescent amino-acid HADA (500 µM) were added to pneumococcal cells (OD_600 nm 0.3) expressing FtsZ-mKate2 and incubated for 4 min at 37 °C before washing and fluorescence microscopy imaging. Phase contrast (grey) and fluorescent signals of mKate2 (red), GFP (green) and HADA images are shown. Scale bars: 1 µm. Cell division process is divided into i to v stages. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.)

Fig. 8

Association of LytA to the nascent cell wall. Time-lapse images of WT cells labelled by sfGFPop-LytA (green) in a microfluidic system. sfGFPop-LytA was pulse-injected at 50 µg/ml on cells before extensive wash with CY medium (see Methods). The movie shows an overlay of GFP (green) and phase-contrast (grey) images. Scale bar: 1 µm. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.)

Fig. 9

LytA binds to newly synthesized TAs positioned at the septal cross-wall site. Pneumococcal cells grown in the presence of normal choline were washed at OD_600 nm 0.3 and incubated for 4 min with choline-N3, DIBO and sfGFPop-LytA. After two washes, cell growth was pursued in CY at 37 °C. Microscopy observations were performed immediately after the 4-min pulse period (0 min) and after a chase duration of 20 min and 40 min. Phase contrast (grey), fluorescent signals of TAs labelled with DIBO-Alexa594 (red), GFP (green) and merge images are shown. The parental cross-wall septal site is indicated by yellow arrows and the daughter new septal site by cyan arrows. Scale bars: 2 µm. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.)