The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (KD, 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcαRI (CD89) and to a soluble form of the FcαRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (KD, 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.

A new genetic locus associated with Mycoplasma pneumoniae cytadherence was previously identified by transposon mutagenesis with Tn4001. This locus maps approximately 160 kbp from the genes encoding cytadherence-associated proteins HMW1 and HMW3, and yet insertions therein result in loss of these proteins and a hemadsorption-negative (HA ؊ ) phenotype, prompting the designation cytadherence-regulatory locus (crl). In the current study, passage of transformants in the absence of antibiotic selection resulted in loss of the transposon, a wild-type protein profile, and a HA ؉ phenotype, underscoring the correlation between crl and M. pneumoniae cytadherence. Nucleotide sequence analysis of crl revealed open reading frames (ORFs) orfp65, orfp216, orfp41, and orfp24, arranged in tandem and flanked by a promoter-like and a terminator-like sequence, suggesting a single transcriptional unit, the P65 operon. The 5 end of orfp65 mRNA was mapped by primer extension, and a likely promoter was identified just upstream. The product of each ORF was identified by using antisera prepared against fusion proteins. The previously characterized surface protein P65 is encoded by orfp65, while the 190,000 M r cytadherence-associated protein HMW2 is a product of orfp216. Proteins with sizes of 47,000 and 41,000 M r and unknown function were identified for orfp41 and orfp24, respectively. Structural analyses of HMW2 predict a periodicity highly characteristic of a coiled-coil conformation and five leucine zipper motifs, indicating that HMW2 probably forms dimers in vivo, which is consistent with a structural role in cytadherence. Each transposon insertion mapped to orfp216 but affected the levels of all products of the P65 operon. HMW2 is thought to form a disulfide-linked dimer, formerly designated HMW5, and examination of an hmw2 deletion mutant confirms that HMW5 is a product of the hmw2 gene.Mycoplasma pneumoniae attachment to host respiratory epithelium is a multifactorial process, the complexity of which is only beginning to be appreciated (20). This cell wall-less prokaryote is a leading cause of pneumonia in older children and young adults. Colonization requires binding to host cell receptors (cytadherence) by mycoplasma surface proteins localized primarily to a differentiated terminal structure termed the attachment organelle (reviewed in reference 34). The membrane proteins P1 (169 kDa [19]) and P30 (30 kDa [2]) are thought to be directly involved in receptor recognition (34), although the evidence for P30 is limited. Their functionality is dependent upon accessory proteins, including HMW1-HMW3 and A, B, and C (20, 34), which collectively maintain the proper distribution and/or disposition of the adhesins in the mycoplasma membrane (1, 20). In addition, proteins P65 (32) and P200 (33) share characteristic structural features with HMW1 and HMW3, suggesting a common function. Most are elements of a cytoskeleton-like network in M. pneumoniae, consistent with a possible scaffolding role (20). In particular, HMW3 is a major componen...

Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta-and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated ␤-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain.Mycoplasma pneumoniae is an extracellular pathogen of the human respiratory tract (26, 46) causing histopathological changes of lung epithelial cells, usually in older children and young adults (18). A critical step in bacterial colonization of the host cells is the specific adhesion to host cell receptors, mediated by bacterial adhesins. In M. pneumoniae, the P1 adhesin (3,17,25,26) and the adhesin-related 30-kDa protein (4, 10) have been identified. Both proteins are located mainly in a tip structure that functions as the attachment organelle of the bacterium. It could be demonstrated, by nearestneighbor analysis with a hydrophilic chemical cross-linker, that the product of open reading frame 6 (ORF6) of the P1 operon (28), a 40-kDa protein and 90-kDa protein (9, 34, 50) are located in close proximity to the P1 adhesin on the cell surface (35).Scanning and transmission electron microscope analyses of M. pneumoniae cells grown on grids and pretreated with Triton X-100 revealed a rodlike tip structure and a network of filamentous strands (22,41). Krause and coworkers identified a set of high-molecular-weight proteins (HMW1 to HMW5) which play an important role in cytadherence (30,31,(51)(52)(53) and appear to be involved in formation of a cytoskeletonlike structure (38). These proteins, ...

The gene encoding streptococcal mitogenic exotoxin Z (SMEZ) was disrupted in Streptococcus pyogenes. Despite the presence of other superantigen genes, mitogenic responses in human and murine HLA-DQ transgenic cells were abrogated when cells were stimulated with supernatant from the smez− mutant compared with the parent strain. Remarkably, disruption of smez led to a complete inability to elicit cytokine production (TNF-α, lymphotoxin-α, IFN-γ, IL-1 and -8) from human cells, when cocultured with streptococcal supernatants. The potent effects of SMEZ were apparent even though transcription and expression of SMEZ were barely detectable. Human Vβ8+ T cell proliferation in response to S. pyogenes was SMEZ-dependent. Cells from HLA-DQ8 transgenic mice were 3 logs more sensitive to SMEZ-13 than cells from HLA-DR1 transgenic or wild-type mice. In the mouse, SMEZ targeted the human Vβ8+ TCR homologue, murine Vβ11, at the expense of other TCR T cell subsets. Expression of SMEZ did not affect bacterial clearance or survival from peritoneal streptococcal infection in HLA-DQ8 mice, though effects of SMEZ on pharyngeal infection are unknown. Infection did lead to a rise in Vβ11+ T cells in the spleen which was partly reversed by disruption of the smez gene. Most strikingly, a clear rise in murine Vβ4+ cells was seen in mice infected with the smez− mutant S. pyogenes strain, indicating a potential role for SMEZ as a repressor of cognate anti-streptococcal responses.

Superantigens produced by Streptococcus pyogenes have been implicated with streptococcal toxic shock syndrome (STSS). We analyzed 19 acute-phase serum samples for mitogenic activity from patients with severe streptococcal disease. The serum samples from two patients in the acute phase of STSS showed strong proliferative activity. Streptococcal mitogenic exotoxin (SME) Z-1 and streptococcal pyrogenic exotoxin (SPE)-J were identified in one patient with peritonitis who recovered after 2 weeks in intensive care. SMEZ-16 was found in a second patient who died on the day of admission. Sequential serum samples taken on day 3 after admission from patient 1 showed clearance of mitogenic activity but absence of neutralizing anti-SMEZ antibodies. Serum samples taken on day 9 from this patient showed evidence of seroconversion with high levels of anti-SMEZ antibodies that neutralized SMEZ-1 and 12 other SMEZ-variants. These results imply that a high level of SMEZ production by group A streptococcus is a causative event in the onset and subsequent severity of STSS.