Dancing to another tune-adhesive moonlighting proteins in bacteria

Abstract

Biological moonlighting refers to proteins which express more than one function. Moonlighting proteins occur in pathogenic and commensal as well as in Gram-positive and Gram-negative bacteria. The canonical functions of moonlighting proteins are in essential cellular processes, i.e., glycolysis, protein synthesis, chaperone activity, and nucleic acid stability, and their moonlighting functions include binding to host epithelial and phagocytic cells, subepithelia, cytoskeleton as well as to mucins and circulating proteins of the immune and hemostatic systems. Sequences of the moonlighting proteins do not contain known motifs for surface export or anchoring, and it has remained open whether bacterial moonlighting proteins are actively secreted to the cell wall or whether they are released from traumatized cells and then rebind onto the bacteria. In lactobacilli, ionic interactions with lipoteichoic acids and with cell division sites are important for surface localization of the proteins. Moonlighting proteins represent an abundant class of bacterial adhesins that are part of bacterial interactions with the environment and in responses to environmental changes. Multifunctionality in bacterial surface proteins appears common: the canonical adhesion proteins fimbriae express also nonadhesive functions, whereas the mobility organelles flagella as well as surface proteases express adhesive functions.

Figure 1

Adherence of L. crispatus ST1 and binding of His₆-GS of ST1 are affected by pH. (A) Adherence of L. crispatus ST1 at pH 4 and pH 8 to the immobilized basement membrane preparation Matrigel and to BSA. Prior to the assays, the bacteria from an overnight culture were washed with the pH 4 or the pH 8 buffer, and the adhesion assays were performed at the corresponding pH. Means of adherent bacteria in 20 randomly chosen microscopic fields of 1.6 × 10⁴ μm² and standard deviations are shown. (B) Binding of purified His₆-GS to immobilized laminin was tested by surface plasmon resonance at pH 5.5, 6.0 and 6.5 (Adapted from [28] Copyright^© American Society for Microbiology).

Figure 2

Release of moonlighting proteins from L. crispatus ST1 surface and reassociation of recombinant proteins onto the cell wall. (A) Western blotting of GS, GPI, enolase, and GAPDH from the surface of L. crispatus ST1 cells and from the cell-free buffer. The cells were incubated at pH 4 or pH 8 for 1 h, and the moonlighting proteins in neutralized samples were visualized by immunoblotting. (B) Release of moonlighting proteins in the presence of increasing concentrations of the cationic peptide LL-37. L. crispatus ST1 cells were treated with LL-37 at the indicated concentrations, and the moonlighting proteins in the buffer were visualized by Western blotting. (C) Binding of His₆-GPI proteins of L. crispatus ST1 and L. rhamnosus GG to bacterial cells at pH 4. The binding was visualized by indirect immunofluorescence, phase contrast images of the microscopic field are also shown. The arrows indicate protein binding to bacterial cell surface. Size bar, 3 µm. (Adapted from [28] Copyright^© American Society for Microbiology).

Figure 3

Schematic illustration of the association of moonlighting proteins with the cell surface of Lactobacillus. (A) Moonlighting proteins (shown in red, blue, yellow, purple and turquoise) of Lactobacillus associate to the cell surface via electrostatic or ionic interactions (B) and they are released into surroundings in stress situations, such as neutral or alkaline pH or presence of cathelidicins or bile acids. The surface location of the S-layer protein (shown in green) with the pI of 10, is not affected by environmental changes. (C) The extracellularly released moonlighting proteins associate back onto the cell surface in favorable environmental conditions, i.e., at acidic pH. The proteins associate with the cell surface of the same lactobacillar species where the proteins were originally released, but also with the cell surface of other lactobacillar species. The binding is not evenly distributed around the cells but is concentrated to cell division areas as well as to the cell poles. The arrows indicate the moonlighting proteins and arrowheads the S-layer protein.