d -Alanyl Ester Depletion of Teichoic Acids in Lactobacillus plantarum Results in a Major Modification of Lipoteichoic Acid Composition and Cell Wall Perforations at the Septum Mediated by the Acm2 Autolysin

Abstract

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.Teichoic acids (TAs) are essential polymers found in grampositive bacteria and represent up to 50% of the cell wall dry weight (17). Lactobacillus plantarum contains two types of TAs: lipoteichoic acids (LTA) and wall teichoic acids (WTA). The L. plantarum LTA are polyglycerophosphate polymers anchored in the membrane through a glycolipid. They are highly substituted with D-alanyl esters (D-Ala:phosphate [P] ratio of 0.89) and to a minor extent with glucose (Glc:P ratio of 0.11) (2). L. plantarum WTA are polyribitolphosphates covalently bound to the peptidoglycan via a linkage unit (22). They also carry D-Ala and Glc residues but in strain-dependent variable ratios (14).Despite their essentiality, our understanding of the physiological role(s) of TAs is still incomplete (17). D-Alanyl substitutions strongly contribute to the function of TAs, and the study of mutants deficient in D-alanylation is of interest to expand our insight into the physiological role of these substitutents. The positively charged amino groups of D-alanyl esters partially counteract the negative charges of the backbone phosphate groups. Consequently, D-alanyl esters can modulate cell envelope properties and the function of several extracellular proteins (for a review, see reference 24). The biosynthesis of D-alanyl-LTA was extensively studied with Lactobacillus. rhamnosus (formerly casei) (10,18,19,24,25) and with Bacillus subtilis (27). D-Alanylation requires four proteins (DltA/Dcl, DltB, DltC/Dcp, and DltD) encoded in the dlt operon. In B. subtilis, which contains both LTA and WTA, inactivation of dlt genes prevents D-alanylation of both types of TAs (27). The dlt mutants exhibit an extended variety of phenotypes (for a review, see reference 24). For L. plantarum, it was recently demonstrated that the dlt mutant analyzed in this work displayed an enhanced anti-inflammatory capacity in a murine model of colitis (16). A large number of phenotypes can be associated with the higher-density of negative charges in the cell wall resulting from the lack of D-alanyl esters (for a review, see reference 24). As an example, TAs are thought to be involved in the control of autolysins through electrostatic interactions (12,29). Autolysins play an important role in autolysis, cell separation, and peptidoglycan...

Molecular Control of Bacterial Death and Lysis

“…For example, dlt mutations in B. subtilis resulted in enhanced autolysis and increased susceptibility to methicillin (270,271). Furthermore, the cell walls of the mutants were more negatively charged, as indicated by the increased binding of the cationic protein cytochrome c. Effects of D-Ala ester content on autolysis were also observed in a variety of other bacterial species including S. aureus (64,140,181,197), Lactococcus lactis (234), and Lactobacillus plantarum (188). Other consequences of D-Ala depletion include increased susceptibility to a variety of different antimicrobial compounds (61,132,136,137,196,197), decreased survival in neutrophils (49,137), decreased virulence (61,138), decreased epithelial cell invasion (137), and decreased biofilm formation (61).…”

GtfA and GtfB Are Both Required for Protein O -Glycosylation in Lactobacillus plantarum

“…The TagE proteins of L. plantarum WCFS1 are annotated according to their originally predicted function in teichoic acid glycosylation [poly(glycerolphosphate)-␣-glucosyltransferases]. The glucose substitution levels in lipoteichoic acids (LTA) of L. plantarum WCFS1 are very low (59,60), while glucose is a backbone constituent into the repeating unit of wall teichoic acids (WTA) that does not have additional glucose substitutions in this strain (60,61). Neither LTA nor WTA of L. plantarum WCFS1 contain N-acetylhexosamine (59,61), the glycan transferred by TagE5 and TagE6.…”

Increased d-alanylation of lipoteichoic acid and a thickened septum are main determinants in the nisin resistance mechanism of Lactococcus lactis

“…Nisin has a net positive charge and the increase of positive charge in the cell wall probably hampers nisin from reaching lipid II in the cytoplasmic membrane by electrostatic repulsion. In a Lactobacillus plantarum strain, LTA showed higher levels of glucose when the dlt operon was deleted (Palumbo et al, 2006). This suggests that by deleting the dlt operon, other sugar moieties increase in number in the LTA.…”