The streptococcolytic enzyme zoocin A is a penicillin-binding protein

Heath, Lucie S.; Heath, Harry E.; LeBlanc, Paul A.; Smithberg, S. Rochelle; Dufour, Muriel; Simmonds, Robin S.; Sloan, Gary L.

doi:10.1111/j.1574-6968.2004.tb09648.x

Cited by 18 publications

(13 citation statements)

References 20 publications

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“…Zoocin A is produced by Streptococcus equi subsp. zooepidemicus 4881 and has been shown to be a penicillin binding protein acting as a D-alanyl endopeptidase (16). Thus, we speculate that a general mechanism related to cell wall stabilization or restructuring might contribute to the observed multiple-stress tolerance mediated by CiaR.…”

Section: Discussionmentioning

confidence: 96%

The CiaR Response Regulator in Group BStreptococcusPromotes Intracellular Survival and Resistance to Innate Immune Defenses

et al. 2009

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Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS ⌬ciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and ⌬ciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.

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Section: Discussionmentioning

confidence: 96%

The CiaR Response Regulator in Group BStreptococcusPromotes Intracellular Survival and Resistance to Innate Immune Defenses

et al. 2009

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“…Since Zif increases the number of alanines within the cross bridge from primarily two to primarily three, the chirality of this extra alanine may affect sensitivity to zoocin A. Zoocin A has been shown previously to be a penicillin-binding protein that only very slowly hydrolyzes a D-alanyl-D-alanine analog, nitrocefin (14). If Zif inserts a D-alanine onto the N terminus of the cross bridge, zoocin A may be unable to hydrolyze a D-alanyl-D-alanyl peptide bond.…”

Section: Resultsmentioning

confidence: 99%

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

Gargis

Heath

et al. 2009

Appl Environ Microbiol

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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.

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“…Strain 4881.1KOK, which was constructed to facilitate genetic manipulations not re-ported here, was made by replacing the erythromycin resistance marker from strain 4881.1KOE (8) with the kanamycin resistance gene from ⍀-Km2 (13). Plasmid p3-10 DNA (8) was transformed into strain 4881.1KOE, and kanamycinresistant transformants were selected.…”

Section: Methodsmentioning

confidence: 99%

“…Third, zoocin A covalently binds penicillin and slowly hydrolyzes a chromogenic cephalosporin, both of which are D-alanyl-D-alanine analogs. These three lines of evidence suggest that zoocin A hydrolyzes the bond between the terminal D-alanine of the stem peptide and the first Lalanine of the cross bridge (8).…”

mentioning

confidence: 99%

Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A

Gargis

Heath

et al. 2009

Appl Environ Microbiol

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Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a D-alanyl-L-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive-and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.Streptococcal peptidoglycans (16) have a repeating backbone of the amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Connected to the lactyl group of MurNAc is a tetrapeptide (stem peptide) composed of L-alanine, D-isoglutamine (or D-isoglutamate), L-lysine, and D-alanine. The stem peptides are connected by peptide cross bridges that typically contain two or three L-alanine residues ( Fig. 1). During peptidoglycan synthesis, the stem peptides contain an additional C-terminal D-alanine ( Fig. 1) that is removed during cross-bridge formation by a transpeptidase (11). In some cases, the cross bridges are not formed and the terminal D-alanine may or may not be removed by a carboxypeptidase (11).Zoocin A is a streptococcolytic enzyme produced by Streptococcus equi subsp. zooepidemicus 4881. The genes for zoocin A and for the zoocin A immunity factor (zif) are adjacent on the chromosome and are divergently transcribed (1). Zoocin A hydrolyzes the cell walls of some closely related streptococci, including S. pyogenes and S. mutans, the main causative agents of group A streptococcal sore throat and dental caries, respectively (20). Zoocin A is presumed to target peptidoglycan cross bridges based on three lines of evidence. First, the catalytic domain of zoocin A has a high degree of similarity to the catalytic domains of several bacteriolytic endopeptidases (lysostaphin [21], ALE-1 [22], LytM [23], and millericin B [3]). Second, the introduction of the gene for lysostaphin resistance into a zoocin A-sensitive streptococcus resulted in the insertion of serines into its peptidoglycan cross bridges and a decrease in sensitivity to zoocin A (6). Third, zoocin A covalently binds penicillin and slowly hydrolyzes a chromogenic cephalosporin, both of which are D-alanyl-D-alanine analogs. These three lines of evidence suggest that zoocin A hydrolyzes the bond between the terminal D-alanine of the stem peptide and the first Lalanine of the cross bridge (8).If zoocin A is an endopeptidase, determination of its site of action on streptococcal peptidoglycans only by the mass of the products is not possible beca...

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The streptococcolytic enzyme zoocin A is a penicillin-binding protein

Cited by 18 publications

References 20 publications

The CiaR Response Regulator in Group BStreptococcusPromotes Intracellular Survival and Resistance to Innate Immune Defenses

The CiaR Response Regulator in Group BStreptococcusPromotes Intracellular Survival and Resistance to Innate Immune Defenses

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

Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A

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