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.

Keywords: B. thuringiensis; D-alanylation; antimicrobial peptides; dltX; innate immunity; insects; virulence.

FIGURE 1

In silico analysis of DltX by Phobius, a combined transmembrane protein topology and signal peptide predictor. Transmembrane, cytoplasmic, and non-cytoplasmic domains are represented in the chart. The plot shows the posterior probabilities of cytoplasmic/non cytoplasmic/TM, helix/signal peptide. Predicted transmembrane (amino acids 12 to 33: LTQWVAKTVYYLAILFALLWLY) regions are shown in gray, cytoplasmic and non-cytoplasmic regions are shown in green and blue respectively (amino acids 1 to 11: MERLKEIWSRP and amino acids 34 to 48: GFHDTNTSTFIYNEF). The prediction gives the most probable location and orientation of transmembrane helices in the sequence.

FIGURE 2

Schematic diagram showing the construction of an in frame deletion of the dltX gene by Splicing by Overlap Extension (SOE). The top part of the diagram shows the DNA fragment containing the upstream and downstream DNA regions flanking dltX. Synthetic oligonucleotides are represented by lines with arrows indicating the 5′–3′ orientation. Oligo dltX-b 3′ and oligo dltX-c 5′ match their respective template DNA sequences in their 3′ portions and are complementary to each other in their 5′ portions. The double stranded DNA products generated in separate PCR reactions were denatured, allowed to anneal at their overlap and were 3′ extended by DNA polymerase. The fusion product was then further amplified by PCR in the presence of oligo dltX-a and oligo dltX-d.

FIGURE 3

Effects of dltX deletion on Bt 407 cells. Scanning electron micrographs show exponential growth phase cells of WT (A), ΔdltX (B), and ΔdltX complemented cells (C) in Y buffer (pH 5.6). Magnifications are x20k and x30k. The dotted lines on pictures in the top panel represent a scale of 1.5 μm (x20K magnification) and those in the bottom panel represent a scale of 1 μm (x30K magnification).

FIGURE 4

Half inhibitory concentration (IC₅₀) of WT 407 (solid line) and ΔdltX complemented strains (CO) (broken line) (A), and the ΔdltX mutant (B). Bacterial growth was scored after 6 h of inoculation. The results shown are the means of at least three independent experiments performed in duplicate.

FIGURE 5

In vivo virulence of WT, ΔdltX, and complemented strains in Galleria mellonella. Bacterial cells were collected at OD = 1 and 1 × 10⁴ cfu were injected into the hemocoel of last instar G. mellonella larvae weighing about 250 mg. Infected larvae were kept at 30°C in individual Petri dishes and mortality was recorded 48 h following infection. The means and standard errors of the mean (bars) of four biological replicates are shown. Asterisks indicate a significant difference (p < 0.0001) determined by the χ² test.

FIGURE 6

Survival analysis of Drosophila melanogaster adult flies infected with WT, ΔdltX, and complemented bacterial strains. Relish (Rel) flies were used as immunocompromised mutants of the Imd pathway and wild type (WT) Oregon (Or) flies were used as an immunocompetent control in these experiments. These flies were infected with WT, ΔdltX, or complemented strains and survival was recorded at various time points post-infection. The means and standard errors of the mean (bars) of three independent experiments are shown.

FIGURE 7

Amounts of D-alanine released from whole cells by alkaline hydrolysis for WT, mutant, and complemented strains. The mean values and standard deviations (black bars) of results from three independent experiments are shown. Asterisks indicate a significant difference (p < 0.0001) determined by the χ² test.

FIGURE 8

Electrophoretic mobility EM (10^-8 m²s^-1V^-1) of WT, mutant, and complemented strains. Bacteria were suspended in 1.5 × 10^-3 M sodium chloride and the values shown correspond to the results obtained at pH7. The mean values and standard deviations (bars) were calculated from two independent experiments with technical duplicates for each. Asterisks indicate a significant difference (p = 0.0007) determined by the χ² test.

FIGURE 9

Models of D-alanine substitution of LTA. (A) Fischer model: DltA ligates D-alanine (small blue circle) onto the carrier protein DltC. DltB then transfers D-alanine from DltC to undecaprenyl-phosphate (C₅₅-P) to produce D-Ala-P-C_55. This lipid linked intermediate is, then, flipped accross the membrane, whereas DltD, active at the outer side of the membrane, transfers D-Ala to LTA (adapted from reference 5). (B) Neuhaus and Baddiley model. DltA ligates D-alanine (small blue circle) onto the carrier protein DltC. DltD is thought to facilitate D-alanine ligation to DltC and DltB is believed to be involved in the translocation of Alanylated-DltC across the membrane where it may then transfer D-alanine directly onto LTA (adapted from reference 5). (C) Revised model including DltX: our results show that DltX is essential for D-alanylation of TAs. Therefore, DltX was included onto the existing models to obtain a more complete picture of the mechanism of D-alanine incorporation into the cell wall polymers of Gram-positive bacteria. DltX, due to its alpha-helical structure prediction and the presence of both extracellular and cytosolic domains, can theoretically interact with the four other proteins of the operon (see hypotheses in the text). However, the exact functions of DltX as well as DltB and DltD in this process remain to be elucidated.