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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2009, Vol. 3 Issue (3) : 304-310     DOI: 10.1007/s11703-009-0064-4
The identification of MacSe in Streptococcus equi ssp. equi
Jiande YANG(), Yanfei LIU, Jun XU, Jifei MA
Department of Animal Science, Tianjin Agricultural University, Tianjin 300384, China
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Streptococcus equi subsp. equi (S. equi ssp. equi) causes equine strangles, a highly contagious and widespread purulent lymphadenitis of the head and neck. We have identified MacSe, a novel protein of S. equi, by screening a phage library of 3-8 kb random DNA fragments of S. equi CF32. MacSe shares 62% and 67.5% amino acid homology with Mac5005 and Mac8345 of S. pyogenes respectively. Expression during infection was shown by strong reactivity of the protein with convalescent sera and mucosal wash IgA of ponies infected by commingling exposure. Release into the culture medium was detected during the log phase of growth. Dose dependent anti-phagocytic activity for equine neutrophils involved interaction of MacSe with C3 and neutrophils.

Keywords Streptococcus equi ssp. equi      MacSe      anti-phagocytic activity     
Corresponding Authors: YANG Jiande,   
Issue Date: 05 September 2009
URL:     OR
Fig.1  The deduced amino acid sequences of MacSe
Note: Boxed sequence means a signal sequence.
Fig.2  SDS-PAGE of a lysate of expressing recombinant MacSe
Note: Lanes a-e represent lysate, containing MacSe, MacSe in eluate following nickel exchange chromatography, molecular ladder and immunoblot showing reaction of MacSe with convalescent horse serum, respectively.
Fig.3  Serum antibody and mucosal IgA response to MacSe
Note: The data obtained from 6 horses before, during and following commingling exposure to an ssp.infected horse on day 0.
Fig.4  Binding of biotinylated MacSe to equine PMNs and to C3
Note: Binding of biotinylated MacSe to PMNs was measured after incubation of a suspension of equine PMNs with MacSe. The control showed the value for sonicated PMNs in the absence of MacSe. Log phasessp.(CF32, Irish1 and E23) were incubated with fresh horse na?ve serum, washed and incubated with biotinylated MacSe for 1 h. One mol·L hydroxylamine was added to the cells after washing to elute bound C3. ELISA was performed on the eluted supernatant to measure associated MacSe using HRP-avidin. The control showed an amount of MacSe recovered when fresh serum (C3) was omitted.
Fig.5  Fluorescent staining and flow cytometry analysis of binding of biotinylated MacSe to equine neutrophils
Note: (a) and (c) represent neutrophils incubated with FITC-conjugated streptavidin. (b) and (d) represent neutrophils incubated with biotinylated MacSe for 1 h, washed, and incubated with FITC-conjugated streptavidin for 30 min. Biotinylated MacSe binding to neutrophils results in an increase in fluorescence ((b) and (d)) compared to control (a) and (c). Data representatives of three separate experiments are shown.
Fig.6  Relation of secretion of MacSe into culture supernatant (OD490 nm) with the growth of ssp.CF32 (OD600 nm)
Note: MacSe in the supernatant was measured by ELISA with rabbit antiserum to recombinant MacSe.
Fig.7  Dose-dependent inhibition of PMN phagocytic activity by MacSe (μg)
Note: PMNs were incubated for 60 min separately with preopsonized ssp. CF32, e23 and 35683 in the presence of 0.5, 5, 20 μg·mL of MacSe. Bactericidal activities of PMNs for each treatment (after 60 min) were expressed as percentages of CFU of ssp. at 60 minutes at 0 μg·mL of MacSe. The opsonin was SeM specific rabbit antiserum.
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