Polysaccharide II Surface Anchoring, the Achilles' Heel of Clostridioides difficile

Abstract

Cell wall glycopolymers (CWPGs) in Gram-positive bacteria have been reported to be involved in several bacterial processes. These polymers, pillars for proteins and S-layer, are essential for the bacterial surface setup, could be essential for growth, and, in pathogens, participate most often in virulence. CWGPs are covalently anchored to peptidoglycan by proteins that belong to the LytR-CpsA-PSr (LCP) family. This anchoring, important for growth, was reported as essential for some bacteria such as Bacillus subtilis, but the reason why CWGP anchoring is essential remains unknown. We studied LcpA and LcpB of Clostridioides difficile and showed that they have a redundant activity. To delete both lcp genes, we set up the first conditional-lethal mutant method in C. difficile and showed that polysaccharide II (PSII) anchoring at the bacterial surface is essential for C. difficile survival. In the conditional-lethal mutant, C. difficile morphology was impaired, suggesting that peptidoglycan synthesis was affected. Because Lcp proteins are transferring CWPGs from the C₅₅-undecaprenyl phosphate (also needed in the peptidoglycan synthesis process), we assumed that there was competition between PSII and peptidoglycan synthesis pathways. We confirmed that UDP-MurNAc-pentapeptide precursor was accumulated, showing that peptidoglycan synthesis was blocked. Our results provide an explanation for the essentiality of PSII anchoring in C. difficile and suggest that the essentiality of the anchoring of CWPGs in other bacteria can also be explained by the blocking of peptidoglycan synthesis. To conclude, our results suggest that Lcps are potential new targets to combat C. difficile infection. IMPORTANCE Cell wall glycopolymers (CWGPs) in Gram-positive bacteria have been reported to be involved in several bacterial processes. CWGP anchoring to peptidoglycan is important for growth and virulence. We set up the first conditional-lethal mutant method in Clostridioides difficile to study LcpA and LcpB involved in the anchoring of CWPGs to peptidoglycan. This study offers new tools to reveal the role of essential genes in C. difficile. LcpA and LcpB activity was shown to be essential, suggesting that they are potential new targets to combat C. difficile infection. In this study, we also showed that there is competition between the polysaccharide II synthesis pathway and peptidoglycan synthesis that probably exists in other Gram-positive bacteria. A better understanding of these mechanisms allows us to define the Lcp proteins as a therapeutic target for potential design of novel antibiotics against pathogenic Gram-positive bacteria.

Keywords: Clostridium difficile; essentiality; pathogens; peptidoglycan; polysaccharide anchoring; polysaccharides; surface structures.

FIG 1

The ΔlcpB mutant (JMV4) presents curved, inflated, longer, and larger cells than the parental strain JMV1. (A) JMV3 (ΔlcpA) and JMV4 (ΔlcpB) single mutants were analyzed by optical microscopy and complemented with pMTL84222 (vector), plcpA (plasmid carrying lcpA expressed with its own promoter), or plcpB (plasmid carrying lcpB expressed with its own promoter); scale bar, 20 μm. (B) The percentage of abnormal (curved, thick, or inflated) cells among total cells was calculated by measuring more than 100 cells for each strain. (C) Cell length of the parental strain JMV1, JMV3, and JMV4 complemented with the vector (pMTL84222), plcpA, or plcpB. (D) Cell width of the parental strain JMV1, JMV3, and JMV4 complemented with the vector (pMTL84222), plcpA, or plcpB. For B to D, the number above each bar represents the number of cells counted. Data were analyzed by Student’s t test; ****, P < 0.0001.

FIG 2

(A and B) Both lcpA and lcpB are constitutively expressed. GusA activity measured for lcpA (A) and lcpB (B) promoters is shown. GusA activity (blue curve) was measured in Miller units, and growth (red curve) was followed by measuring the optical density at 600 nm (OD_{600 nm}) for 8 h.

FIG 3

Both ΔlcpA and ΔlcpB exhibit an altered PSII layer at the surface. An immunofluorescence assay of JMV1, JMV3, and JMV4 strains was performed using a superresolution microscope. Bacteria were stained for DNA (DAPI, blue) and PSII (anti-PSII, green). The merged pictures show both localizations simultaneously; scale bars, 20 μm. Insets show magnifications of the respective images; scale bars, 2 μm.

FIG 4

The lcp conditional-lethal mutant (JMV6) is not able to grow without ATc induction of the P_tet-lcpB copy. (A) JMV1 and JMV2 grown in liquid culture were diluted and plated on BHI agar petri dishes. JMV6 grown in liquid culture in the presence of 10 (JMV6 [ATC 10]) or 50 ng mL⁻¹ ATc (JMV6 [ATc 50]) was diluted and plated on BHI agar petri dishes. The control strain, JMV6 harboring the plasmid plcpA grown in liquid culture in the presence of 50 ng mL⁻¹ ATc (JMV6 + plcpA [ATc 50]), was diluted and plated on BHI agar petri dishes. Petri dishes contained ATc from 0 to 250 ng mL⁻¹ in the BHI agar medium. (B) JMV1, JMV2, JMV6, and JMV6 + plcpA were grown in the presence of 50 ng mL⁻¹ ATc, and growth was measured for 20 h (1,200 min) without ATc (JMV1 and JMV6 + plcpA), in the presence of 10 ng mL⁻¹ ATc (JMV6 [ATc 10]), or in the presence of 50 ng mL⁻¹ ATc (JMV2 [ATc 50], JMV6 [ATc 50], and JMV6 + plcpA [ATc 50]). The graph represents the mean of three independent experiments.

FIG 5

In the presence of 10 ng mL⁻¹ ATc, the lcp conditional-lethal mutant (JMV6) loses its rod shape. (A) JMV1, JMV6 + plcpA, JMV6 + plcpB, JMV2 in the presence of 50 ng mL⁻¹ ATc (JMV2 [ATc 50]), and JMV6 in the presence of 10 (JMV6 [ATc 10]) or 50 ng mL⁻¹ ATc (JMV6 [ATc 50]) were observed by optical microscopy; scale bar, 20 μm. (B and C) Cell length (B) and cell width (C) of bacteria from each strain observed in A were measured. The number above each column represents the number of cells counted. Data were analyzed by Student’s t test; ****, P < 0.0001.

FIG 6

In the presence of 10 ng mL⁻¹ ATc, the lcp conditional-lethal mutant (JMV6) loses its rod shape, but PSII is still detected at the surface. Immunofluorescence assays of JMV1, JMV2 in the presence of 50 ng mL⁻¹ (JMV2 [ATc 50]), JMV6 in the presence of 10 ng mL⁻¹ (JMV6 [ATc 10]), JMV6 in the presence of 50 ng mL⁻¹ (JMV6 [ATc 50]), and JMV6 + plcpA in the presence of 50 ng mL⁻¹ (JMV6 + plcpA [ATc 50]) strains were performed using a superresolution microscope. Bacteria were stained for DNA (DAPI, blue) and PSII (anti-PSII, green). The merged images show both localizations simultaneously; scale bars, 20 μm. The insets show magnifications of parts of the images; scale bars, 2 μm.

FIG 7

PSII is released into the supernatant of the JMV6 strain in the presence of 10 ng mL⁻¹ ATc. Dot blot analysis using specific antibodies targeting PSII was performed on the bacterial surface content (pellet) and supernatant content (culture supernatant) from JMV1, JMV2 JMV3, JMV4, JMV6 grown in the presence of 10 ng mL⁻¹ ATc (JMV6 [ATc 10]), JMV6 grown in the presence of 50 ng mL⁻¹ ATc (JMV6 [ATc 50]), and JMV6 plcpA grown in the presence of 50 ng mL⁻¹ ATc (JMV6 + plcpA [ATc 50]). Each content was diluted up to 1:64. PG-PSII was used as a positive control, and PG was used as a negative control.

FIG 8

(A to F) PSII anchoring impairment is associated with Cwp proteins released in the culture supernatant. Characterization of surface (A, C, and E) and supernatant (B, D, and F) protein profiles from JMV1, JMV2, JMV6, and JMV6 + plcpA grown in the absence of ATc (−) or in the presence of 10 ng mL⁻¹ ATc (10) or in the presence of 50 ng mL⁻¹ ATc (50). Coomassie blue staining (A and B), anti-Cwp66 Western blots (C and D), and anti-SlpA Western blots (E and F) were performed. The protein ladder is graduated in kiloDaltons; MW, molecular weight.

FIG 9

PSII anchoring mutants present an autolysis phenotype. (A) Autolysis of JMV1, JMV3, and JMV4 harboring the empty plasmid pMTL84222 (+ vector), the plcpA plasmid (+ plcpA), or the plcpB plasmid (+ plcpB) were measured. (B) Autolysis of JMV1, JMV2 grown in the presence of 50 ng mL⁻¹ ATc (JMV2 [ATc 50]), JMV6 + plcpA, JMV6 grown in the presence of 10 ng mL⁻¹ ATc (JMV6 [ATc 10]), JMV6 grown in the presence of 50 ng mL⁻¹ ATc (JMV6 [ATc 50]), and JMV6 + plcpA grown in the presence of 50 ng mL⁻¹ ATc (JMV6 + plcpA [ATc 50]) was measured. The optical density was measured for 3 h (180 min), and the result is presented as a cell survival percentage. The graph represents the mean of three independent experiments.

FIG 10

PG cytoplasmic precursors accumulate when PSII anchoring is impaired. (A to C) Purification and quantification of UDP-MurNAc-pentapeptide from JMV1 (A), JMV6 grown in the presence of 10 ng mL⁻¹ ATc (JMV6 [ATc 10]) (B), and JMV6 + plcpA grown in the presence of 50 ng mL⁻¹ ATc (JMV6 + plcpA [ATc 50]) (C) were performed. (D) Table showing each peak area and area/optical density ratio. In addition, the observed and calculated monoisotopic masses obtained after mass spectrometry analysis are presented in the two last columns; mAU, milli-arbitrary units; Da, Dalton.