SaccuFlow: A High-Throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

Apostolos, Alexis J.; Ferraro, Noel J.; Dalesandro, Brianna E.; Pires, Marcos M.

doi:10.1021/acsinfecdis.1c00255

Cited by 20 publications

(28 citation statements)

References 66 publications

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“…4A ). 43 Sacculi from M. smegmatis treated with TriSeLAN showed high levels of fluorescence, which was consistent with the results obtained with whole cells ( Fig. 4B ).…”

Section: Resultssupporting

confidence: 91%

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

Apostolos

Koyasseril-Yehiya

Santamaria

et al. 2021

Preprint

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The bacterial cell wall supports cell shape and prevents lysis due to internal turgor pressure. A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is comprised of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are often the target of antibiotics as they are essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study bacterial cell growth and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to build synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of a m-DAP analogue, selenolanthionine. We show that selenolanthionine is installed within the PG of live bacteria by the native cell wall crosslinking machinery in several mycobacteria species. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP containing organisms.

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“…4A ). 43 Sacculi from M. smegmatis treated with TriSeLAN showed high levels of fluorescence, which was consistent with the results obtained with whole cells ( Fig. 4B ).…”

Section: Resultssupporting

confidence: 91%

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

Apostolos

Koyasseril-Yehiya

Santamaria

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

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“…Additionally, a number of experiments were performed to demonstrate the incorporation of the synthetic stem peptide analogue within the PG of cells. We initially turned to SaccuFlow, a protocol we described recently to quantitatively measure the fluorescence of isolated PG (sacculi, Figure A) . Sacculi from M.…”

Section: Resultsmentioning

confidence: 99%

Metabolic Processing of Selenium-Based Bioisosteres of meso-Diaminopimelic Acid in Live Bacteria

et al. 2022

Self Cite

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A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is composed of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are targets of potent antibiotics as proper assembly of the PG is essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study the bacterial cell structure, growth, and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to access synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of m-DAP (selenolanthionine) and show that it is installed within the PG of live bacteria by the native cell wall crosslinking machinery in mycobacterial species. This PG probe has an orthogonal release mechanism that could be important for downstream proteomics studies. Finally, we describe a bead-based assay that is compatible with high-throughput screening of cell wall enzymes. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP-containing organisms.

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“…Our lab has recently demonstrated a novel assay, SaccuFlow, to evaluate binding to bacterial PG using isolated, intact sacculi from a variety of bacterial strains ( Figure 4A ). 75 To further demonstrate the binding specificity of P1fl to D-ala on PG, sacculus from E. faecalis and E. faecium isolated and individually incubated with P1fl and cell binding was assessed via flow cytometry ( Figure 4B ). As expected, P1fl bound to the sacculi of both E. faecalis and E. faecium , with E. faecium exhibiting higher binding levels as indicated by a higher fluorescence signal.…”

Section: Resultsmentioning

confidence: 99%

Immuno-targeting of Gram-positive Pathogens via a Cell Wall Binding Tick Antifreeze Protein

Dalesandro

Pires

2022

Preprint

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The human immune system employs several mechanisms to defend against pathogenic bacteria. However, pathogenic bacterial cells have evolved means to counter these responses, rendering our immune system less effective. Immunological agents that supplement or modulate the host immune response have proven to have powerful therapeutic potential, although this modality is less explored against bacterial pathogens. We describe the application of a bacterial binding protein to re-engage the immune system towards pathogenic bacteria. More specifically, a hapten was conjugated to a protein expressed by Ixodes scapularis ticks, called Ixodes scapularis antifreeze glycoprotein (IAFGP), that has high affinity for the D-alanine residue on the peptidoglycan of the bacterial cell wall. We showed that a fragment of this protein retained high surface binding affinity. Moreover, when conjugated to a hapten this conjugate led to the display of haptens on the cell surface of vancomycin-resistant Enterococcus faecalis. Hapten display then induced the recruitment of antibodies and promoted immune-cell mediated uptake of bacterial pathogens. These results demonstrate the feasibility in using cell wall binding agents as the basis of a class of bacterial immunotherapies against bacterial pathogens.

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SaccuFlow: A High-Throughput Analysis Platform to Investigate Bacterial Cell Wall Interactions

Cited by 20 publications

References 66 publications

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

Metabolic Processing of Selenium-Based Bioisosteres of meso-Diaminopimelic Acid in Live Bacteria

Immuno-targeting of Gram-positive Pathogens via a Cell Wall Binding Tick Antifreeze Protein

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