Please wait a minute...
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
RESEARCH ARTICLE |
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
Download: PDF(208 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract  

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,Email:Jiande.yang@gmail.com   
Issue Date: 05 September 2009
URL:  
http://academic.hep.com.cn/fag/EN/10.1007/s11703-009-0064-4     OR     http://academic.hep.com.cn/fag/EN/Y2009/V3/I3/304
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.
1 Agniswamy J, Lei B, Musser J M, Sun P D (2004). Insight of host immune evasion mediated by two variants of group a Streptococcus Mac protein. J Biol Chem , 279(50): 52789-52796
doi: 10.1074/jbc.M410698200
2 Artiushin S, Timoney J F, Nally J, Verma A (2004). Host-inducible immunogenic phingomyelinase-like protein, Lk73.5, of Leptospira interrogans. Infect Immun , 72(2): 742-749
doi: 10.1128/IAI.72.2.742-749.2004
3 Boschwitz J S, Timoney J F (1994a). Inhibition of C3 deposition on Streptococcus equi subsp. equi by M protein: a mechanism for survival in equine blood. Infect Immun , 62(8): 3515-3520
4 Boschwitz J S, Timoney J F (1994b). Characterization of the antiphagocytic properties of fibrinogen for Streptococcus equi subsp. equi. Microb Pathog , 17(2): 121-129
doi: 10.1006/mpat.1994.1058
5 Corbi A L, Kishimoto T K, Miller L J, Springer T A (1988). The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) alpha subunit. Cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B. J Biol Chem , 263(25): 12403-12411
6 Cunningham M W (2000). Pathogenesis of group A streptococcal infection. Clinical Microbi Rev , 13(3): 470-511
doi: 10.1128/CMR.13.3.470-511.2000
7 Harrington D J, Sutcliffe I C, Chanter N (2002). The molecular basis of Streptococcus equi infection and disease. Microb Infect , 4(4): 501-510
doi: 10.1016/S1286-4579(02)01565-4
8 Lei B, DeLeo F R, Hoe N P, Graham M R, Mackie S M, Cole R L, Liu M, Hill H R, Low D E, Federle M J, Scott J R, Musser J M (2001). Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis. Nature Medicine , 7(12): 1298-1305
9 Lei B, DeLeo F R, Reid S D, Voyich J, Magoun M L, Liu M, Braughton K R, Ricklefs S, Hoe N P, Cole R L, Leong J M, Musser J M (2002). Opsonophagocytosis-inhibiting mac protein of group a streptococcus: identification and characteristics of two genetic complexes. Infect Immun , 70(12): 6880-6890
doi: 10.1128/IAI.70.12.6880-6890.2002
10 Nilsson M, Weineisen M, Andersson T, Truedsson L, Sjobring U (2005). Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum. Eur J Immunol , 35(5): 1472-1481
doi: 10.1002/eji.200424850
11 Sheoran A S, Artiushin S, Timoney J F (2002). Nasal mucosal immunogenicity for the horse of a SeM peptide of Streptococcus equi genetically coupled to cholera toxin. Vaccine , 20(11-12): 1653-1659
doi: 10.1016/S0264-410X(01)00488-1
12 Sheoran A S, Sponseller B T, Holmes M A, Timoney J F (1997). Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses. Vet Immunol Immunopathol , 59(3-4): 239-251
doi: 10.1016/S0165-2427(97)00074-3
13 Timoney J F (2004). The pathogenic equine streptococci. Vet Res , 35(4): 397-409
doi: 10.1051/vetres:2004025
14 Timoney J F, Trachman J (1985). The immunologically reactive protein of Streptococcus equi. Infect Immun , 48(1): 29-38
15 Timoney J F, Walker J, Zhou M, Ding J (1995). Cloning and sequence analysis of a protective M-like protein gene from Streptococcus equi subsp. zooepidemicus. Infect Immun , 63(4): 1440-1445
16 Verma A, Artiushin S, Matsunaga J, Haake D A, Timoney J F (2005). LruA and LruB, novel lipoproteins of pathogenic Leptospira interrogans associated with equine recurrent uveitis. Infect Immun , 73(11): 7259-7266
doi: 10.1128/IAI.73.11.7259-7266.2005
No related articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed