AcmB Is an S-Layer-Associated β-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM

Abstract

Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a β-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species.

Importance: Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.

FIG 1

(A) The modular domain organization of the 11 predicted autolysins of L. acidophilus NCFM. The numbers indicate the corresponding amino acid length. (B) The corresponding specific activity of each catalytic domain on the peptidoglycan structure. (C) RNA-seq transcriptional analysis of the gene encoding each autolysin, with colors corresponding to mRNA transcripts from genes with the following catalytic activities: blue, endopeptidase; red, β-N-acetylglucosaminidase; green, β-N-acetylmuramidase; and purple, N-acetylmuramoyl-l-alanine amidase.

FIG 2

acmB orthologs were found in various S-layer-forming strains of Lactobacillus, including L. amylovorus GRL1112, L. helveticus H10, L. kefiranofaciens ZW3, L. crispatus ST1, L. melliventris, L. amylolyticus DSM 11664, and L. gigeriorum DSM 23908. The genetic region surrounding acmB was highly syntenic in all species examined. Arrows represent genes, while the colors represent specific genes, as follows: red, acmB; purple, amidase; green, Na⁺/H⁺ ion transporter; blue, oxidoreductase; orange, GMP synthetase; teal, conserved hypothetical protein (HP); pink, gpmA; dark gray, pyrrolidine carboxypeptidase; and yellow, conserved HP. Genes in white are divergent genes unique to each species where indicated. Ψ indicates a truncated pseudogene.

FIG 3

The gene encoding AcmB was deleted from the chromosome of L. acidophilus NCFM. (A) RNA-seq analysis demonstrates that acmB is polycistronically expressed with lba0177, which encodes an S-layer-associated N-acetylmuramoyl-l-alanine amidase. (B) Gel electrophoresis of PCR products using the primers indicated in panel A for the parent strain (WT) compared to the ΔacmB mutant. The deletion was confirmed by sequencing. L, DNA ladder. (C) SDS-PAGE of the noncovalently bound extracellular S-layer proteins (SLP) and S-layer-associated proteins (SLAPs) isolated from both the WT and ΔacmB strains.

FIG 4

(A) The cellular morphologies of the wild-type (WT), mutant (ΔacmB), and acmB complemented (CΔacmB) strains were assessed using phase-contrast light microscopy over a 24-h growth period. (B) Chain length measurements were taken for the WT, ΔacmB, and CΔacmB strains. The chain length for the ΔacmB mutant (n = 611 cells) was significantly higher than those for the WT (n = 661 cells) and CΔacmB (n = 316 cells) strains across all time points measured (P < 0.001). Error bars represent confidence intervals.

FIG 5

(A) Autoaggregation of WT, mutant (ΔacmB), and complemented (CΔacmB) cells. Asterisks indicate statistical significance (P < 0.001). (B) Triton X-100-induced autolysis assays for WT, ΔacmB, and complemented strains. The differences between the ΔacmB mutant and the WT and complemented strains are statistically significant (P < 0.001). Each assay was performed in triplicate; all error bars represent confidence intervals.

FIG 6

The ability of the ΔacmB mutant to bind to mucin and extracellular matrices (ECM) was assessed. Compared the wild-type (WT) reference (dotted line), the ΔacmB strain showed a significant reduction in binding to mucin, collagen, fibronectin, and laminin. Asterisks indicate statistical significance (P < 0.001). Adherence assays were performed in triplicate; all error bars represent confidence intervals.