Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes

Abstract

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.

Streptococcus pneumoniae Type 1 Pilus – A Multifunctional Tool for Optimized Host Interaction

“…(iii) Regulation of P1 length: based on a model of “pilus chain terminator role” of anchor pilin SpaB in C. diphtheriae SpaA pilus ( Mandlik et al, 2008 ), it should be investigated whether the step of RrgC anchor incorporation into the SrtC-activated RrgB n RrgA fiber has a similar role in defining P1 final length. Importantly proper regulation of P1 RrgB mediated length determines the relative position of major adhesin RrgA and might be crucial for P1 functionality as recently demonstrated in other Gram-positive pilus systems ( Chang et al, 2019 ).…”

Magnetic tweezers meets AFM: ultra-stable protein dynamics across the force spectrum

“…Here, with magnetic tweezers, we have fully addressed the dynamics of FimA. Around 100-150 FimA subunits compose the structure of a mature type 2 pilus 60,61 . The isopeptide bonds in FimA prevent the mechanical unfolding of most of the protein, and only the IDL motif extends at very high forces.…”

A Cell-based Screen in Actinomyces oris to Identify Sortase Inhibitors

“…A. oris uses three distinct sortases to elaborate its cell envelope. Ao SrtA is a class E housekeeping sortase that attaches GspA and other proteins containing the LAQTG sorting signal to the cell wall 49 . Ao SrtA catalyzes a transpeptidation reaction that attaches GspA to lipid II, a cell wall precursor.…”

“…These sortases catalyze transpeptidation by linking protein subunits of the pilus together via lysine-isopepide bonds. Subsequent cell wall anchoring of pilus polymers to peptidoglycan requires Ao SrtA 49 . SrtC1 produces type 1 fimbriae that are comprised of the fimbrial shaft FimP protein and the tip fimbrillin FimQ protein.…”

“…5A-D). Since Ao SrtA has been implicated in anchoring of the pilus to the peptidoglycan, one might still expect to see diminished pili display in the absence of the housekeeping sortase; however, it is not uncommon for class C enzymes to compensate for loss of the housekeeping sortase and independently display pili as SrtC2 is capable of catalyzing pilus polymerization and cell wall anchoring of pilus polymers 49,52 . Thus, the data supports molecule 6 as being more selective for Ao SrtA in intact cells.…”