New insights in the coordinated amidase and glucosaminidase activity of the major autolysin (Atl) in Staphylococcus aureus

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (μSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

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“…mutants having rougher cell wall compared to WT, which has been shown in previous work (Nega et al, 2020).…”

Section: In Vitro Characterization Of Staphylococcus Aureus Mutantssupporting

confidence: 82%

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

Masters

Muthukrishnan

et al. 2021

Front. Microbiol.

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“…The cell wall of S. epidermidis contains protein, nucleic acid, and peptidoglycan components ( Büttner et al, 2015 ), and a major protein component of the cell wall is the peptidoglycan hydrolase, autolysin (AtlE). The AtlE protein plays an essential role in bacterial cell wall cleavage and is therefore critical during cell division ( Nega et al, 2020 ). The R2ab and amidase domains in AtlE are responsible for enzyme targeting in the septum region ( Yamada et al, 1996 ).…”

Section: Introductionmentioning

confidence: 99%

Understanding How Staphylococcal Autolysin Domains Interact With Polystyrene Surfaces

et al. 2021

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Biofilms, when formed on medical devices, can cause malfunctions and reduce the efficiency of these devices, thus complicating treatments and serving as a source of infection. The autolysin protein of Staphylococcus epidermidis contributes to its biofilm forming ability, especially on polystyrene surfaces. R2ab and amidase are autolysin protein domains thought to have high affinity to polystyrene surfaces, and they are involved in initial bacterial attachment in S. epidermidis biofilm formation. However, the structural details of R2ab and amidase binding to surfaces are poorly understood. In this study, we have investigated how R2ab and amidase influence biofilm formation on polystyrene surfaces. We have also studied how these proteins interact with polystyrene nanoparticles (PSNPs) using biophysical techniques. Pretreating polystyrene plates with R2ab and amidase domains inhibits biofilm growth relative to a control protein, indicating that these domains bind tightly to polystyrene surfaces and can block bacterial attachment. Correspondingly, we find that both domains interact strongly with anionic, carboxylate-functionalized as well as neutral, non-functionalized PSNPs, suggesting a similar binding interaction for nanoparticles and macroscopic surfaces. Both anionic and neutral PSNPs induce changes to the secondary structure of both R2ab and amidase as monitored by circular dichroism (CD) spectroscopy. These changes are very similar, though not identical, for both types of PSNPs, suggesting that carboxylate functionalization is only a small perturbation for R2ab and amidase binding. This structural change is also seen in limited proteolysis experiments, which exhibit substantial differences for both proteins when in the presence of carboxylate PSNPs. Overall, our results demonstrate that the R2ab and amidase domains strongly favor adsorption to polystyrene surfaces, and that surface adsorption destabilizes the secondary structure of these domains. Bacterial attachment to polystyrene surfaces during the initial phases of biofilm formation, therefore, may be mediated by aromatic residues, since these residues are known to drive adsorption to PSNPs. Together, these experiments can be used to develop new strategies for biofilm eradication, ensuring the proper long-lived functioning of medical devices.

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“…We next analyzed whether TfNamZ1 and TfNamZ2 can also cleave the PGN-derived disaccharide MurNAc-GlcNAc, which constitutes the minimal natural substrate of the exo-β- N -acetylmuramidase BsNamZ [Müller, 2021 #18933]. MurNAc-GlcNAc was obtained by enzymatic digestion of PGN from S. aureus with recombinant Atl N -acetylmuramyl-L-Ala amidase SaAtl AM and recombinant Atl N -acetylglucosaminidase SaAtl Glc [Oshida, 1995 #15478;Nega, 2020 #19874]. LC-MS analyses revealed that Atl releases not only the disaccharide MurNAc-GlcNAc, which is the main carbohydrate product, as recently described [Nega, 2020 #19874], but also the trisaccharide GlcNAc-MurNAc-GlcNAc in about 15 times lower abundance (data not shown).…”

Section: Resultsmentioning

confidence: 99%

NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificity

Borisova

Balbuchta

Lovering

et al. 2021

Preprint

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The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-β-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves non-reducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-β-1,4-N-acetylglucosamine (GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic β-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of pNP-MurNAc and MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic β-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-β-N-acetylmuramidases.IMPORTANCETwo exo-β-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the non-reducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

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New insights in the coordinated amidase and glucosaminidase activity of the major autolysin (Atl) in Staphylococcus aureus

Cited by 36 publications

References 47 publications

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis

Understanding How Staphylococcal Autolysin Domains Interact With Polystyrene Surfaces

NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificity

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