DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects

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.

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

“…Although dltX was previously identified as being part of the operon and has been linked to DLT function in Bacillus thuringiensis (17,27), these results identify an additional small ORF in the S. aureus dlt operon. It is unknown whether these ORFs encode peptides.…”

“…The D-alanine modification of lipoteichoic acid is ubiquitous among Grampositive bacteria, which include the human pathogens Streptococcus pneumoniae (5), Enterococcus faecalis (6,7), and Staphylococcus aureus (8 -11). This modification modulates cell surface electrostatics as measured by cell-surface affinity for cytochrome c (12)(13)(14)(15)(16) and whole-cell electrophoretic mobility (17). Fluorescence polarization with S. aureus cells also showed an impact of D-alanylation on cell membrane fluidity (14).…”

Allyl rhodanine azo dye derivatives: Potential antimicrobials target d‐alanyl carrier protein ligase and nucleoside diphosphate kinase

“…The morphological changes involved malformation and incomplete separation of the cells; HL 3 ‐treated B. cereus showed significant short irregular rods with multiple cross walls (Table and Figure ); and HL 3 ‐treated S. aureus showed some filament‐like cells rather than normal cocci cells (Figure ). These malformations and multiple cross walls can be associated with teichoic acids lacking d ‐alanine, in cell walls, when DltA is inhibited …”

Resistance Mechanisms to Antimicrobial Peptides in Gram-Positive Bacteria

“…Staphylococcus Enterococcus GAS GBS Pneumococci D-alanylation of the membrane X X X X X Peschel et al, 1999;Poyart et al, 2001Poyart et al, , 2003Abachin et al, 2002;Frick et al, 2003;Cao and Helmann, 2004;Kristian et al, 2005;May et al, 2005;Fabretti et al, 2006;Fisher et al, 2006;Kovacs et al, 2006;Palumbo et al, 2006;Walter et al, 2007;Beiter et al, 2008;Abi Khattar et al, 2009;Cox et al, 2009;Jann et al, 2009;McBride and Sonenshein, 2011a;Saar-Dover et al, 2012;Simanski et al, 2013;Carvalho et al, 2015;Wydau-Dematteis et al, 2015;Kamar et al, 2017;Hirt et al, 2018 Lysinylation of the membrane X X X Oku et al, 2004;Staubitz et al, 2004;Kraus and Peschel, 2006;Thedieck et al, 2006;Ernst et al, 2009;Samant et al, 2009;Bao et al, 2012;Shireen et al, 2013;Kumariya et al, 2015;Nasser et al, 2019 O-acetylation of the peptidoglycan X X Crisostomo et al, 2006;Pfeffer et al, 2006;Hebert et al, 2007;Aubry et al, 2011;Laaberki et al, 2011;…”

“…Among those changes, the insertion of D-alanine in the lipoteichoic acids, a process named D-alanylation, is used to reduce the negative membrane charge, thus inhibiting interaction with AMPs. This resistance mechanism is regulated by the dlt operon, and it has been described in several Bacillus species, such as B. cereus (Abi Khattar et al, 2009), B. anthracis (Fisher et al, 2006), B. thuringiensis (Kamar et al, 2017) and B. subtilis (Cao and Helmann, 2004;May et al, 2005), L. monocytogenes (Abachin et al, 2002;Carvalho et al, 2015), C. difficile (McBride and Sonenshein, 2011a), C. butyricum (Wydau-Dematteis et al, 2015), Lactobacillus plantarum (Palumbo et al, 2006), and Lactobacillus reuteri (Walter et al, 2007). Another mechanism of envelope modification is called lysinylation of the membrane; it consists of addition of L-lysine to the phosphatidylglycerol.…”