Analysis of immunoreactivity to a Streptococcus equi subsp. zooepidemicus M-like protein To confirm an outbreak of poststreptococcal glomerulonephritis, and sequences of M-like proteins from isolates obtained from different host species

Abstract

The etiologic agent of a large 1998 outbreak of poststreptococcal acute glomerulonephritis (PSGN) in Nova Serrana, Brazil, was found likely to be a specific strain of Streptococcus equi subsp. zooepidemicus from contaminated cheese (S. Balter et al., Lancet 355:1776-1780, 2000). In the present study, we used a serologic screen for a known surface-exposed virulence factor to confirm the epidemiologic findings. Using primers flanking a previously characterized M-like protein gene (J. F. Timoney et al., Infect. Immun. 63:1440-1445, 1995), we amplified and sequenced the M-like protein (designated Szp5058) gene and found it to be identical among four independent acute-phase PSGN patient isolates. Convalescent-phase sera from 33 of 44 patients in the PSGN outbreak were found to contain antibodies highly reactive to a purified Szp5058 fusion protein, compared with 1 of 17 control sera (P < 0. 0001), suggesting that Szp5058 was expressed during infection and further implicating this strain as the cause of the PSGN outbreak. The predicted signal sequence and cell wall association motif of Szp5058 were highly conserved with the corresponding sequence from S. equi subsp. zooepidemicus SzpW60, while the predicted surface-exposed portions differed markedly between these two proteins. The 5' end of the szp5058 gene, including its variable region, was identical to the szp gene from another strain associated with a previous PSGN outbreak in England (M. Barham et al., Lancet i:945-948, 1983), and the corresponding szp sequence found from the Lancefield group C type strain isolated from a guinea pig. In addition, the hypervariable (HV) portion of szp5058 was identical to a previously published HV sequence from a horse isolate (J. A. Walker and J. F. Timoney, Am. J. Vet. Res. 59:1129-1133, 1998). Three other strains of S. equi subsp. zooepidemicus, including another strain previously associated with a PSGN outbreak, were each found to contain a distinct szp gene. Two of these szp genes had HV regions identical to szp regions from isolates recovered from different host species.

FIG. 1

Sequence comparison of the Szp5058 N terminus with corresponding regions of Szp proteins from three S. equi subsp. zooepidemicus strains. The inverted arrow indicates the predicted signal sequence cleavage site. Shaded segments represent shared identity among at least three of the four proteins. The entire deduced Szp5058 sequence shown was identical to the deduced Szp sequence from strains SS1215 (England PSGN outbreak strain) and SS189 (guinea pig isolate) (Table 1). The bold segment of Szp5058 (Brazil PSGN outbreak strain) indicates a region identical to that deduced from a partial szp gene sequence obtained from a horse pleuritic fluid isolate (GenBank accession no. AF021907). The bold segment of Szp1345 (bovine mastitis isolate) indicates a region identical to that deduced from a partial szp gene from a horse nasal aspirate (GenBank accession no. AF021907).

FIG. 2

Immunoblot analysis of patient and control sera with purified His6-Szp5058 fusion protein. Lanes: 1 to 4, controls; 5 to 8, nephritis patient sera; 9, nonreactive group C rabbit polyclonal antibody to S. equisimilis control; 10, CTB control strip with no added antibody. The blot shows an upper band of 54 kDa and a lower band of 52 kDa.

FIG. 3

Logarithmic IDVs reflecting immunoblot reactivity of nephritis patient and control sera against purified His6-Szp5058 fusion protein. The solid lines represent the mean and the standard deviation for the control and patient groups.