Protein and Sugar Export and Assembly in Gram-positive Bacteria by Fabio Bagnoli & Rino Rappuoli

Author:Fabio Bagnoli & Rino Rappuoli
Language: eng
Format: epub
Publisher: Springer International Publishing, Cham

4 Essentiality of Sortase SrtA in Cell Wall Anchoring of Surface Proteins in Actinomyces oris

Actinomyces oris is an oral biofilm-forming actinobacterium that possesses two class C sortases SrtC1 and SrtC2 and a class A sortase, i.e., SrtA. While the former are involved in pilus assembly (Mishra et al. 2007; Wu et al. 2011), the latter is predicted to catalyze cell wall anchoring of pilus polymers and fourteen LPXTG-containing surface proteins (Reardon-Robinson et al. 2014). In contrast to srtA in other Gram-positive bacteria studied to date, A. oris srtA is an essential gene. To investigate its role in cell wall anchoring of surface proteins, Wu and colleagues made several failed attempts to generate a srtA deletion mutant. Essentiality of A. oris srtA was demonstrated by a conditional gene deletion method, whereby chromosomal srtA can be removed when SrtA is ectopically provided under the control of a tetR-inducible promoter (Wu et al. 2014). When grown in the absence of inducers for SrtA expression, the srtA mutant ceased to grow. Strikingly, srtA depleted cells exhibited aberrant cell morphology with multiple septa and expansion of the cell envelope. Interestingly, envelope expansion was only observed at one end of the cells.

Using transposon mutagenesis, Wu and coworkers found five sets of mutants that suppress the lethal phenotypes of srtA deletion (Wu et al. 2014). The first set of suppressors was mapped to genes coding for a LytR-CpsA-Psr (LCP)-like protein and a LPXTG-containing protein named GspA. It has been proposed that LCP attaches glycan strands to a membrane-bound form of GspA, which is then anchored to the cell wall by sortase SrtA. In the absence of SrtA, the glycosylated GspA polymers accumulated in the membrane, which was proposed to be toxic. This could be possibly due to membrane jamming of the SecA-dependent transport system that causes cell stress, growth arrest, and ultimately cell death (Wu et al. 2014). This conjecture is supported by the fact that A. oris cells expressing a GspA mutant devoid of the CWSS are able to survive without srtA. It still remains unclear how other suppressor mutants are involved in this lethality; many of the targeted genes encode transporters and ATPases. Perhaps, they may be required for transport of glycan precursors that are substrates for LCP-catalyzed glycosylation of GspA. Without glycan strands, a stalled membrane-bound form of GspA may not so be detrimental.

The unexpected finding that A. oris srtA is essential in A. oris provided a convenient cell-based assay to identify inhibitors that can penetrate the Gram-positive cell envelope and inactivate sortase activities, hence bacterial virulence. This class of inhibitors—anti-virulence inhibitors—may not impose a selective pressure on other Gram-positive pathogens like inhibitors targeting essential genes or pathway.

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