Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

Abstract

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

Keywords: Lactobacillus acidophilus; S-layer; cell surface; lipoteichoic acid; probiotic; proteomics; transcriptomics.

FIGURE 1

XY plots of log2 transformed, normalized transcripts per kilobase million (TPM) metric of open reading frames in Lactobacillus acidophilus. Black data points meet the significance thresholds p < 1.36e^-5, α = 0.05) and fold change (>2) in each pairwise comparison. Colored data points indicate differentially expressed genes in operons.

FIGURE 2

(A) RNA-seq transcriptional profiles of L. acidophilus derivatives visualized by two-way hierarchical clustering of trimmed mean of M-values (TMM) normalized reads per gene. Black circles denote differentially expressed genes and colored circles denote differentially expressed operons. (B) RNA-seq transcriptional profiles of predicted peptidoglycan turnover proteins in L. acidophilus. Values are TMM gene expression levels. Non-solid bars indicate LTA-deficient strains.

FIGURE 3

(A) Microscopy images of wild-type L. acidophilus and LTA derivatives at 400x magnification and (B) comparison of stationary phase cultures for average chain lengths. 40 cells from three independent biological replicates were measured and their means compared using a two-tailed, equal variance Student’s t-test. Asterisk denotes p < 0.05. (C) Growth profiles of L. acidophilus NCFM derivatives in normal glucose semi-defined medium (left) and high manganese glucose semi-defined medium (right). Data are reported as the average of three biological replicates and open markers indicate LTA-deficient strains.

FIGURE 4

Extracellular protein profiles of L. acidophilus LTA derivatives. (A) SDS-PAGE images of S-layer (left) and S-layer associated protein fractions (right) and (B) two-way hierarchical clustering of spectral counts per extracellular protein generated by liquid chromatography tandem mass spectrometry. The molecular weight marker denotes kDa size.

FIGURE 5

(A) Distribution of normalized spectral counts for proteins with (left) and without (right) secretion signals. Asterisk denotes p < 0.05. (B) Comparison of normalized spectral counts of select extracellular proteins across L. acidophilus derivatives. Non-solid bars indicate LTA-deficient strains.