title: Neuropathogenic Escherichia coli K1 does not exhibit proteolytic activities to exert its pathogenicity.
abstract: BACKGROUND
  Proteases are well-known virulence factors that promote survival, pathogenesis and immune evasion of many pathogens. Several lines of evidence suggest that the blood-brain barrier permeability is a prerequisite in microbial invasion of the central nervous system. Because proteases are frequently associated with vascular permeability by targeting junctional proteins, here it is hypothesized that neuropathogenic Escherichia coli K1 exhibit proteolytic activities to exert its pathogenicity.
  
  
  METHODS
  Zymographic assays were performed using collagen and gelatin as substrates. The lysates of whole E. coli K1 strain E44, or E. coli K-12 strain HB101 were tested for proteolytic activities. The conditioned media were prepared by incubating bacteria in RPMI-1640 in the presence or absence of serum. The cell-free supernatants were collected and tested for proteases in zymography as mentioned above. Additionally, proteolytic degradation of host immune factors was determined by co-incubating conditioned media with albumin/immunoglobulins using protease assays.
  
  
  RESULTS
  When collagen or gelatin were used as substrates in zymographic assays, neither whole bacteria nor conditioned media exhibited proteolytic activities. The conditioned media of neuropathogenic E. coli K1 strain E44, or E. coli K-12 strain HB101 did not affect degradation of albumin and immunoglobulins using protease assays.
  
  
  CONCLUSIONS
  Neither zymographic assays nor protease assays detected proteolytic activities in either the whole bacteria or conditioned media of E. coli K1 strain E44 and E. coli K-12 strain HB101. These findings suggest that host cell monolayer disruptions and immune evasion strategies are likely independent of proteolytic activities of neuropathogenic E. coli K1.
authors: Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan.
authors: Junaid
authors: J
authors: Iqbal
authors: Mehak
authors: M
authors: Rajani
authors: Ruqaiyyah
authors: R
authors: Siddiqui
authors: Naveed Ahmed
authors: NA
authors: Khan
mesh_terms: D000818:Animals; D002417:Cattle; D017077:Culture Media, Conditioned / Q000737:chemistry; D057075:Enzyme Assays; D004926:Escherichia coli / Q000472:pathogenicity*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D020814:Meningitis, Escherichia coli / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology*; D010447:Peptide Hydrolases / Q000378:metabolism; D059748:Proteolysis; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine; D010447:Peptide Hydrolases
references: J Pediatr. 1974 Jul;85(1):128-30
references: 4604810
references: PLoS One. 2011 Mar 17;6(3):e17921
references: 21437287
references: Curr Opin Infect Dis. 2012 Jun;25(3):273-8
references: 22395761
references: Arch Dis Child. 1991 May;66(5):603-7
references: 2039250
references: Pediatr Infect Dis J. 1990 Nov;9(11):819-25
references: 2263432
references: J Innate Immun. 2009;1(2):109-17
references: 20375569
references: J Pediatr. 1993 Jan;122(1):15-21
references: 8419603
references: Microbiol Immunol. 1987;31(1):13-25
references: 3295490
references: Reprod Nutr Dev. 2005 Jul-Aug;45(4):485-96
references: 16045896
references: Eur J Neurol. 2006 Dec;13 Suppl 4:1-9
references: 17112344
references: Infect Immun. 1995 Nov;63(11):4470-5
references: 7591087
references: Annu Rev Biochem. 2007;76:243-65
references: 17335404
references: Res Microbiol. 2009 Nov;160(9):704-10
references: 19778606
references: Annu Rev Genet. 1996;30:465-506
references: 8982462
references: FEMS Microbiol Lett. 2008 Sep;286(1):9-15
references: 18616591
references: Rev Infect Dis. 1983 Nov-Dec;5 Suppl 5:S998-1004
references: 6419322
references: J Innate Immun. 2009;1(2):98-108
references: 20375568