Structure Activity Relationship of the Stem Peptide in Sortase A Mediated Ligation from Staphylococcus aureus**

Abstract

The surfaces of most Gram-positive bacterial cells, including that of Staphylococcus aureus (S. aureus), are heavily decorated with proteins that coordinate cellular interactions with the extracellular space. In S. aureus, sortase A is the principal enzyme responsible for covalently anchoring proteins, which display the sorting signal LPXTG, onto the peptidoglycan (PG) matrix. Considerable efforts have been made to understand the role of this signal peptide in the sortase-mediated reaction. In contrast, much less is known about how the primary structure of the other substrate involved in the reaction (PG stem peptide) could impact sortase activity. To assess the sortase activity, a library of synthetic analogs of the stem peptide that mimic naturally existing variations found in the S. aureus PG primary sequence were evaluated. Using a combination of two unique assays, we showed that there is broad tolerability of substrate variations that are effectively processed by sortase A. While some of these stem peptide derivatives are naturally found in mature PG, they are not known to be present in the PG precursor, lipid II. These results suggest that sortase A could process both lipid II and mature PG as acyl-acceptor strands that might reside near the membrane, which has not been previously described.

Measurement of Accumulation of Antibiotics toStaphylococcus aureusin Phagosomes of Live Macrophages

“…Our lab 42-52 and others 53-56 have extensively demonstrated the ability to metabolically remodel bacterial PG with synthetic analogs of PG precursors. Further, we demonstrated that such analogs can be incorporated in vivo into the cell wall of S. aureus infected C. elegans 50 and various diverse bacterial species residing in the gut microbiome of a mouse model.…”

“…[37][38][39] Given that the PG is a critical component to the fitness of the pathogen, many antibiotics target PG biosynthesis enzymes. [40][41] Our lab [42][43][44][45][46][47][48][49][50][51][52] and others [53][54][55][56] have extensively demonstrated the ability to metabolically remodel bacterial PG with synthetic analogs of PG precursors. Further, we demonstrated that such analogs can be incorporated in vivo into the cell wall of S. aureus infected C. elegans 50 and various diverse bacterial species residing in the gut microbiome of a mouse model.…”

“…27 Given that the PG is a critical component to the fitness of the pathogen, many antibiotics target PG biosynthesis enzymes. 28 Our lab 22,29,30 and others 31 have extensively demonstrated the ability to metabolically remodel bacterial PG with synthetic analogs of PG precursors. For most of these modifications, the viability and overall cellular structure of the PG remain unaffected.…”

Non-invasive Analysis of Peptidoglycan from Living Animals

“…More specifically, SrtA recognizes the LPXTG (where X is any amino acid) motif to link the third position amino acid on the stem peptide between T and G on the anchored protein. We 35,46,47 and others 48 have previously used synthetic analogs of LPXTG conjugated to a fluorophore on the N -terminus to metabolically tag isolated bacterial sacculi from pure culture. Here, we incubated fecal sacculi with Fl-LPMTG (fluorescein linked LPMTG, Figure 3 ) in the presence of SrtA and saw a 46-fold increase in fluorescence associated with the sacculi ( Figure 3A ).…”

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…SrtA was also utilized to probe intracellular contact using a PEG‐lipid conjugate to a triglycine peptide followed by fluorescence labelling [126] . It has been shown to have broad substrate tolerance allowing it to modify peptidoglycan as well as its precursor lipid II [127] . SrtA has found applications in protein modifications and immobilization on surfaces and synthesis of protein polymer conjugates [128] …”