Characterization of the Zinc-Containing Metalloprotease Encoded by zpx and Development of a Species-Specific Detection Method for Enterobacter sakazakii

Abstract

Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37°C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.

Cronobacter spp. – opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits

“…Kothary et al (2007) reported the presence of a zinc metalloprotease that deforms CHO cells, eventually causing their rounding and leading to cell damage. The genes for a haemolysin (hly) have been identified, but no activity has been found (Kucerova et al, 2010;Cruz et al, 2011;Grim et al, 2013).…”

“…Kothary et al (2007) identified the gene locus of zinc-containing metalloprotease (zpx) -a protein that caused rounding of CHO cells in tissue culture. In addition, the presence of putative sodA genes might provide resistance for Cronobacter against intracellular macrophage oxidase and acidic conditions, and may contribute to its intracellular persistence (Townsend et al, 2007b).…”

Insights into virulence factors determining the pathogenicity ofCronobacter sakazakii

“…93 Other potential factors Among the minor but important virulent factors, the proteolytic enzymes of Cronobacter strains have been found to cause deformation of the tissue cells in mice. 74 Kothary et al 94 isolated and characterized a cell-bound zinc-containing metalloprotease encoded by a nucleotide sequence (zpx), unique among all the 135 Cronobacter strains tested. The protease was active in against azocasein, caused rounding of Chinese's hamster ovarian cells.…”

Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen

“…6 Furthermore, C. sakazakii possess zpx, a gene which encodes a zinc containing metalloprotease which is responsible for the lysis of collagen and may allow the pathogen to cross the blood brain barrier. 89 It is important to note however that not all C. sakazakii strains have been shown to cause meningitis. An extensive multilocus sequence typing (MLST) method identified C. sakazakii sequence type 4 as the predominant sequence type found in cerebro-spinal fluid from cases of neonatal meningitis.…”