Methods, compositions and kits for assaying mitochondrial function

Abstract

The invention provides methods, compositions, devices, and kits relating to the use of cholesterol-dependent cytolysins (e.g., PFOs) for measuring intracellular mitochondrial activity.

1 . A method comprising:
 (a) contacting a preparation comprising a cell with a cholesterol-dependent cytolysin; and   (b) determining the level of a molecule in the preparation, wherein the level of the molecule is indicative of an intracellular function of the cell.   
     
     
         2 . The method of  claim 1 , wherein the level of the molecule is indicative of mitochondrial function. 
     
     
         3 . The method of  claim 1 , wherein the cholesterol-dependent cytolysin comprises an amino acid sequence selected from Table 1. 
     
     
         4 . The method of  claim 1 , wherein the cholesterol-dependent cytolysin is Perfringolysin O (PFO). 
     
     
         5 . The method of  claim 4 , wherein the PFO comprises an amino acid sequence as set forth in any one of SEQ ID NOS: 1-12. 
     
     
         6 . The method of  claim 4 , wherein the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a cysteine to alanine substitution at amino acid position 459. 
     
     
         7 . The method of  claim 4 , wherein the PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with a threonine to cysteine substitution at amino acid position 319 and a valine to cysteine substitution at amino acid position 334. 
     
     
         8 . The method of  claim 6 , wherein the amino acid sequence further comprises an aspartate to serine substitution at amino acid position 434. 
     
     
         9 - 42 . (canceled) 
     
     
         43 . A method comprising:
 (a) contacting a preparation, comprising a cell, with a cholesterol-dependent cytolysin; and   (b) measuring an intracellular function of the cell.   
     
     
         44 . The method of  claim 43 , wherein the intracellular function is a metabolic rate of the cell, a respiratory rate of the cell, a proportion of aerobic to anaerobic respiration, a rate of consumption of a molecule, or a production rate of a molecule. 
     
     
         45 - 54 . (canceled) 
     
     
         55 . A kit comprising:
 a container housing a cholesterol-dependent cytolysin; and   a container housing a reagent for evaluating an intracellular function of a cell or a cellular-respiration effector.   
     
     
         56 . The kit of  claim 55 , wherein the intracellular function is a mitochondrial function. 
     
     
         57 - 86 . (canceled) 
     
     
         87 . An isolated perfringolysin O (PFO) comprising an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with an aspartate to serine substitution at amino acid position 434 or comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 8-12. 
     
     
         88 - 89 . (canceled) 
     
     
         90 . The isolated PFO of  claim 87 , wherein the isolated PFO comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2 with an aspartate to serine substitution at amino acid position 434, and further comprises a cysteine to alanine substitution at amino acid position 459. 
     
     
         91 . (canceled) 
     
     
         92 . A composition comprising the isolated PFO of  claim 87  and a carrier. 
     
     
         93 . A kit comprising a container housing the isolated PFO of  claim 87 . 
     
     
         94 . A method of transfecting a cell with an exogenous nucleic acid, the method comprising:
 (a) contacting the cell with a cholesterol-dependent cytolysin; and   (b) contacting the cell with the exogenous nucleic acid.   
     
     
         95 - 101 . (canceled) 
     
     
         102 . A method comprising:
 contacting a preparation comprising a cell obtained from a subject with a cholesterol-dependent cytolysin; and   determining the level of a molecule in the preparation, wherein the level of the molecule is indicative of the presence or absence of a respiratory chain deficiency in the cell.   
     
     
         103 . The method of  claim 102 , wherein presence of the respiratory chain deficiency in the cell indicates that the subject has a mitochondrial disorder, optionally wherein the mitochondrial disorder is selected from the group consisting of: Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, and Stroke (MELAS); Diabetes mellitus and deafness (DAD); Maternally Inherited Diabetes and Deafness (MIDD), Leber's Hereditary Optic Neuropathy (LHON); chronic progressive external ophthalmoplegia (CPEO); Leigh Disease; Kearns-Sayre Syndrome (KSS); Friedreich's Ataxia (FRDA); Co-Enzyme QIO (Co-QIO) Deficiency; Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); Myoneurogenic gastrointestinal encephalopathy (MNGIE); Complex I Deficiency; Complex II Deficiency; Complex III Deficiency; Complex IV Deficiency; Complex V Deficiency; another mitochondrial disease; and other myopathies that effect mitochondrial function. 
     
     
         104 . (canceled)