Compositions for in situ labeling of bacterial cell walls with fluorophores and methods of use thereof

Abstract

Disclosed herein are compositions for assessing peptidoglycan biosynthesis in bacteria, for identifying bacteria, and for screening for bacterial cell wall-acting and/or cell wall-disrupting agents via modified D-amino acids and methods of use thereof. Also disclosed are live bacteria having one or more modified D-amino acids as described herein incorporated into peptidoglycan of a bacterial cell wall.

The invention claimed is: 
     
         1 . A modified amino acid comprising a D-amino acid covalently attached to a fluorescent label. 
     
     
         2 . The modified amino acid of  claim 1 , wherein the fluorescent label is 7-hydroxycoumarin 3-carboxylic acid (HCC-OH), 7-nitrobenzofurazan (NBD), 4-chloro-7-nitrobenzofurazan (NBD-Cl), ADA, Cy 3B ADA, AF 647 ADA, AF 350 ADA, BADA, FADA, HADA, NADA, TADA, YADA, FDL, HDL, NDL, TDL, AHA, EDA, ETDA, fluorescein (F) or carboxytetramethylrhodamine (T). 
     
     
         3 . The modified amino acid of  claim 1  or  2 , wherein the D-amino acid is selected from the group consisting of 3-amino-D-Ala, D-Ala, D-Asp, D-Cys, D-Glu and D-Lys. 
     
     
         4 . A muramylpentapeptide precursor unit comprising an N-acetyl muramic acid (NAM) moiety having a stem peptide of three to five amino acids, wherein one or more of the amino acids in the stem peptide comprises a modified amino acid of any one of the preceding claims and optionally an additional modified amino acid, wherein the additional modified amino acid comprises a clickable D-amino acid. 
     
     
         5 . A peptidoglycan unit comprising the muramylpentapeptide precursor unit of  claim 4  covalently linked to an N-acetyl glucosamine (NAG) moiety. 
     
     
         6 . A live bacterial organism comprising a bacterium having a modified cell wall comprising modified peptidoglycan containing at least one modified amino acid according to any one of  claims 1  to  3 , and optionally at least one additional modified amino acid, wherein the at least one additional amino acid comprises a clickable D-amino acid. 
     
     
         7 . A method of assessing bacterial cell wall synthesis in real time, the method comprising:
 providing live bacteria with a first amount of at least one modified amino acid of any one of  claims 1  to  3 , and optionally a second amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for bacterial cell wall synthesis, wherein the bacteria covalently incorporate the at least one modified amino acid and optionally the at least one additional modified amino acid into a stem peptide of peptidoglycan of the bacterial cell wall.   
     
     
         8 . The method of  claim 7 , wherein the first amount and second amount comprise a first concentration and a second concentration, respectively, wherein the first and second concentrations range from about and including 0.10 μM to about and including 1 mM. 
     
     
         9 . The method of  claim 7  or  8 , further comprising detecting the at least one modified amino acid, and optionally the at least one additional modified amino acid incorporated into the stem peptide. 
     
     
         10 . The method of any one of  claims 7  to  9 , wherein the bacteria are Gram-positive bacteria or Gram-negative bacteria. 
     
     
         11 . A method of screening for a putative cell wall-acting agent, the method comprising:
 co-contacting bacteria with an effective amount of an agent and an amount of at least one modified amino acid of any one of  claims 1  to  3 , and optionally an amount at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient to permit ongoing peptidoglycan biosynthesis in a bacterial cell wall, wherein the agent comprises a cell wall-acting agent if the agent interferes with ongoing peptidoglycan biosynthesis in the bacterial cell wall.   
     
     
         12 . The method of  claims 11 , further comprising: detecting one or more D-amino acids incorporated in the bacterial cell wall. 
     
     
         13 . The method of  claim 12 , further comprising: comparing the amount and/or identity of incorporated D-amino acids in the bacterial cell wall resulting from contacting the bacteria with the agent with the corresponding amount and/or identity of incorporated D-amino acids in a bacterial cell wall resulting from contacting the bacteria with a known cell wall-acting agent. 
     
     
         14 . A method of screening for a putative cell wall-disrupting agent, the method comprising: contacting modified bacteria with an amount of an agent, wherein the agent comprises a cell wall-disrupting agent if the agent weakens integrity of peptidoglycan in an existing bacterial cell wall, and wherein the modified bacteria have a modified cell wall containing modified peptidoglycan having at least one stem peptide containing at least one modified amino acid of any one of  claims 1  to  3 , and optionally at least one additional modified amino acid comprising a clickable D-amino acid. 
     
     
         15 . The method of  claims 14 , further comprising: detecting one or more D-amino acids disrupted in the bacterial cell wall. 
     
     
         16 . The method of  claim 15 , further comprising: comparing the amount and/or identity of disrupted D-amino acids in the bacterial cell wall resulting from contacting the bacteria with the agent with the corresponding amount and/or identity of disrupted D-amino acids in a bacterial cell wall resulting from contacting the bacteria with a known cell wall-disrupting agent. 
     
     
         17 . The method of any one of  claims 11  to  16 , wherein the bacteria are Gram-positive bacteria or Gram-negative bacteria. 
     
     
         18 . A method of identifying bacteria, the method comprising:
 contacting live bacteria with an amount of at least one modified amino acid of any one of  claims 1  to  3 , and optionally an amount of at least one additional modified amino acid comprising a clickable D-amino acid, under conditions sufficient for ongoing bacterial cell wall synthesis, wherein the bacteria covalently incorporate into peptidoglycan of a bacterial cell wall the at least one modified amino acid, and optionally the at least one additional modified amino acid, wherein each of the least one modified amino acid and optionally the at least one additional modified amino acid comprises spectrally distinct fluorescent label; and   visualizing the spectrally distinct fluorescent labels to determine an incorporation pattern of the at least one modified amino acid, and optionally the at least one additional modified amino acid, wherein the incorporation pattern identifies the bacteria.   
     
     
         19 . A kit for incorporating labeled D-amino acids into live bacteria, the kit comprising:
 at least one modified amino acid of any one of  claims 1  to  3 ; and   a positive bacterial control and optionally a negative bacterial control, wherein the positive bacterial control has at least one modified amino acid of any one of  claims 1  to  3  incorporated into a stem peptide of peptidoglycan of the bacterial cell wall,   wherein the optional negative bacterial control, if included, does not have the modified amino acid of any one of  claims 1  to  3  incorporated into a stem peptide of peptidoglycan of the bacterial cell wall.   
     
     
         20 . The kit of  claim 17 , further comprising at least one clickable D-amino acid.