Zif, the Zoocin A Immunity Factor, Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Abstract

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a D-alanyl-L-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three L-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.

Identification of CiaR Regulated Genes That Promote Group B Streptococcal Virulence and Interaction with Brain Endothelial Cells

“…Zoocin A has two functional domains, an N-terminal catalytic domain and a C-terminal substrate-binding or target recognition domain [ 43 , 44 ]. Zoocin A has been determined to act as a ᴅ-alanyl- l -alanine endopeptidase which hydrolyses the cross bridge of peptidoglycan of certain Streptococcus species [ 45 ]. Utilization of peptidoglycan hydrolases for both peptidoglycan rearrangement and pathogenicity in host cells has been described in several bacteria.…”

Bacteriocin Protein BacL1of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

“…The cross bridge in peptidoglycan of S. equi is an L-Ala-L-Ala peptide and is susceptible to the peptidoglycan hydrolase activity of zoocin A (57). Zif, an immunity factor of zoocin A, belongs to the FemABX-like protein family (58). It additively increases L-Ala residues in the cross bridges of peptidoglycans and converts L-Ala-L-Ala into L-Ala-L-Ala-L-Ala, resulting in resistance to zoocin A activity.…”

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

“…In the S. equi strains carrying the zooA gene, the gene is always accompanied by a neighboring immunity gene termed zif. The zif gene product protects the zoocin A producer strain by inserting an extra alanine residue in the peptidoglycan cross bridges of its cell wall (11). There is no zif immunity gene flanking the S. agalactiae zooA gene, suggesting that the two zoocin A homologues carry out different biological functions in their respective species.…”