Abstract
The dlt operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes dltXABCD but the involvement of the first ORF (dltX) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in Bacillus thuringiensis. We, therefore constructed an in frame deletion mutant of dltX, without affecting the expression of the other genes of the operon. The growth characteristics of the dltX mutant and those of the wild type strain were similar under standard in vitro conditions. However, disruption of dltX drastically impaired the resistance of B. thuringiensis to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the dltX mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of B. thuringiensis in insects. To our knowledge, this work is the first report examining the involvement of dltX in the D-alanylation of TAs.
Highlights
The cell wall of gram-positive bacteria contains highly charged anionic polymers called teichoic acids (TAs) that consist of alditol phosphate repeats
We report here that the dltX of B. thuringiensis is essential for the incorporation of D-alanyl esters into TAs and is required for resistance to cationic antimicrobial peptides (CAMPs) and for full virulence of the bacterium following its injection into the model insects Galleria mellonella and Drosophila melanogaster
In strain Bacillus thuringiensis strain 407 (Bt 407), transcripts of dltX have been detected by mapping RNA-seq datasets, obtained in vitro 2 and 5 h after the end of exponential phase and in vivo in infected G. mellonella cadavers, 36 h post mortem, FIGURE 1 | In silico analysis of DltX by Phobius, a combined transmembrane protein topology and signal peptide predictor
Summary
The cell wall of gram-positive bacteria contains highly charged anionic polymers called teichoic acids (TAs) that consist of alditol phosphate repeats. These polymers are either anchored to the cytoplasmic membrane via a lipid anchor [lipoteichoic acid (LTA)] (Fischer, 1988; Fischer et al, 1990) or covalently linked to N-acetylmuramic acid residues of the peptidoglycan layer. The function of TAs is not fully understood; they have long been considered to be essential for Gram-positive bacteria This is no longer the case since mutants of S. aureus or B. subtilis devoided of WTA have been obtained (D’Elia et al, 2006a,b). The incorporation of D-alanine esters into TAs to partially neutralize the negative charge of their cell walls represents one of the most common bacterial resistance mechanisms that depend on such charge modifications (Fischer, 1994; Collins et al, 2002; Poyart et al, 2003; Kovács et al, 2006; Abi Khattar et al, 2009; McBride and Sonenshein, 2011)
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