The molecular switch that activates the cell wall anchoring step of pilus assembly in gram-positive bacteria - PubMed (original) (raw)

The molecular switch that activates the cell wall anchoring step of pilus assembly in gram-positive bacteria

Anjali Mandlik et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Cell surface pili in gram-positive bacteria orchestrate the colonization of host tissues, evasion of immunity, and the development of biofilms. Recent work revealed that pilus assembly is a biphasic process wherein pilus polymerization is catalyzed by a pilus-specific sortase followed by cell wall anchoring of the pilus that is promoted by the housekeeping sortase. Here, we present molecular genetic and biochemical studies of a heterotrimeric pilus in Corynebacterium diphtheriae, uncovering the molecular switch that terminates pilus polymerization in favor of cell wall anchoring. The prototype pilus contains a major pilin (SpaA) forming the shaft, a tip pilin (SpaC), and another minor pilin (SpaB). Cells lacking SpaB form pilus fibers, but they are largely secreted in the medium, a phenotype also observed when cells lack the housekeeping sortase. Furthermore, the average pilus length is greatly increased in the absence of SpaB. Remarkably, a SpaB mutant that lacks the cell wall sorting signal but contains a critical lysine residue is incorporated in the pilus. However, the resulting pili fail to anchor to the cell wall. We propose that a specific minor pilin acts as the terminal subunit in pilus assembly. Cell wall anchoring ensues when the pilus polymer assembled on the pilus-specific sortase is transferred to the minor pilin presented by the housekeeping sortase via lysine-mediated transpeptidation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

SpaB minor pilin promotes the efficient cell wall anchoring of SpaA pili. Equivalent samples collected from culture medium (M) and cell wall fragments (W) were analyzed on 4 to 12% gradient gels and detected by immunoblotting with α-SpaA (A) or α-SpaB (B). An antibody against penta-histidine (α-His) that detects a protein found in both culture medium and cell wall was used as loading controls. The high-molecular weight pilus products (SpaHMW) and the position of molecular weight markers are indicated.

Fig. 2.

Fig. 2.

SpaB sequences required for cell wall anchoring of SpaA pili and SpaB incorporation into pilus. (A) Truncated derivatives of SpaB used, along with signal peptide (hatched), the LAFTG motif (white box), hydrophobic domain (black box), and positively charged tail (+). Samples from various corynebacterial strains (B and C) were treated and analyzed as in Fig. 1. Slot blot of total SpaB pilins in each strain is shown in (C).

Fig. 3.

Fig. 3.

SpaB K139 is essential for covalent SpaB-SpaA linkage and cell wall anchoring of pilus. Relative position of the lysine 139 in SpaB sequence is shown in A. Samples from corynebacterial strains (B and C) were treated and analyzed as in Fig. 1.

Fig. 4.

Fig. 4.

Minor pilin governs both pilus length and cell wall anchoring. Electron microscopic images (A–H) of pili on whole cells and in culture medium from various corynebacterial mutants are shown (see Materials and Methods). (Scale bar: 0.2 μm.)

Fig. 5.

Fig. 5.

The revised model of pilus assembly in C. diphtheriae. Details are described in text. (A and B) Depicted is the biphasic mode of pilus assembly: pilus polymerization (A) followed by cell wall anchoring of pilus polymer (B). (C) presents our view of how SpaB might be interspersed in the pilus. SpaB is linked to the major pilus shaft SpaA via transpeptidation that joins the lysine of the SpaA pilin motif to threonine of the SpaB LAFTG motif. Sortases and pilins are color-coded. Straight arrows show the flow of assembly steps, whereas lysine-to-cysteine arrows indicate the nucleophilic attacks catalyzing transpeptidation.

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References

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