Zehava Eichenbaum

Shr Is a Broad-Spectrum Surface Receptor That Contributes to Adherence and Virulence in Group A Streptococcus

ABSTRACT Group A streptococcus (GAS) is a common hemolytic pathogen that produces a range of suppurative infections and autoimmune sequelae in humans. Shr is an exported protein in GAS, which binds in vitro to hemoglobin, myoglobin, and the hemoglobin-haptoglobin complex. We previously reported that Shr is found in association with whole GAS cells and in culture supernatant. Here, we demonstrate that cell-associated Shr could not be released from the bacteria by the muralytic enzyme mutanolysin and was instead localized to the membrane. Shr was available, however, on the exterior of GAS, exposed to the extracellular environment. In vitro binding and competition assays demonstrated that in addition to hemoprotein binding, purified Shr specifically interacts with immobilized fibronectin and laminin. The absence of typical fibronectin-binding motifs indicates that a new protein pattern is involved in the binding of Shr to the extracellular matrix. Recombinant Lactococcus lactis cells expressing Shr on the bacterial surface gained the ability to bind to immobilized fibronectin, suggesting that Shr can function as an adhesin. The inactivation of shr resulted in a 40% reduction in the attachment to human epithelial cells in comparison to the parent strain. GAS infection elicited a high titer of Shr antibodies in sera from convalescent mice, demonstrating that Shr is expressed in vivo. The shr mutant was attenuated for virulence in an intramuscular zebrafish model system. In summary, this study identifies Shr as being a new microbial surface component recognizing adhesive matrix molecules in GAS that mediates attachment to epithelial cells and contributes to the infection process.

Shr of group A streptococcus is a new type of composite NEAT protein involved in sequestering haem from methaemoglobin

A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in haem acquisition and trafficking across the cell envelope of Gram‐positive bacteria. Group A streptococcus (GAS), a β‐haemolytic human pathogen, expresses a NEAT protein, Shr, which binds several haemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane‐anchored protein, with a unique N‐terminal domain (NTD) and two NEAT domains separated by a central leucine‐rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains haem in solution and furthermore reduces the haem iron; this is the first report of haem reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding haem, although they are missing a critical tyrosine residue found in the ligand‐binding pocket of other haem‐binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methaemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methaemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester haem directly from methaemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methaemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methaemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in haem uptake found in pyogenic streptococci and Clostridium novyi.

A foreign protein incorporated on the Tip of T3 pili in Lactococcus lactis elicits systemic and mucosal immunity.

ABSTRACT The use of Lactococcus lactis to deliver a chosen antigen to the mucosal surface has been shown to elicit an immune response in mice and is a possible method of vaccination in humans. The recent discovery on Gram-positive bacteria of pili that are covalently attached to the bacterial surface and the elucidation of the residues linking the major and minor subunits of such pili suggests that the presentation of an antigen on the tip of pili external to the surface of L. lactis might constitute a successful vaccine strategy. As a proof of principle, we have fused a foreign protein (the Escherichia coli maltose-binding protein) to the C-terminal region of the native tip protein (Cpa) of the T3 pilus derived from Streptococcus pyogenes and expressed this fusion protein (MBP*) in L. lactis. We find that MBP* is incorporated into pili in this foreign host, as shown by Western blot analyses of cell wall proteins and by immunogold electron microscopy. Furthermore, since the MBP* on these pili retains its native biological activity, it appears to retain its native structure. Mucosal immunization of mice with this L. lactis strain expressing pilus-linked MBP* results in production of both a systemic and a mucosal response (IgG and IgA antibodies) against the MBP antigen. We suggest that this type of mucosal vaccine delivery system, which we term UPTOP (for unhindered presentation on tips of pili), may provide an inexpensive and stable alternative to current mechanisms of immunization for many serious human pathogens.

Use of Tn917 to generate insertion mutations in the group A streptococcus

The Enterococcus faecalis transposon Tn917 is functional in a broad range of bacteria, including both Gram-positive and Gram-negative species. We cloned Tn917-LTV3, a derivative carrying a promoterless lacZ (beta-galactosidase gene), into the thermosensitive shuttle replicon pG+host4 and assayed for chromosomal insertions in group A streptococcus (GAS). Tn917 transposed into the GAS chromosome at a frequency of (2.8 +/- 3.2) x 10(-5) per colony forming unit (cfu). Transposition products were predominantly simple insertions and no target site preference was detectable. Some transcriptional fusions were identified in which the promoterless lacZ of the transposon appeared to be expressed from an external promoter.

MtsR is a dual regulator that controls virulence genes and metabolic functions in addition to metal homeostasis in GAS

MtsR is a metal‐dependent regulator in the group A streptococcus (GAS) that directly represses the transcription of genes involved in haem and metal uptake. While MtsR has been implicated in GAS virulence, the DNA recognition and full regulatory scope exerted by the protein are unknown. In this study we identified the shr promoter (Pshr) and mapped MtsR binding to a 69 bp segment in Pshr that overlaps the core promoter elements. A global transcriptional analysis demonstrated that MtsR modulates the expression of 64 genes in GAS, 44 of which were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including metal homeostasis, nucleic acid and amino acid metabolism, and protein fate. Importantly, the MtsR regulon includes mga, emm49 and ska, which are central for GAS pathogenesis. MtsR binding to the promoter region of both negatively and positively regulated genes demonstrates that it functions as a dual regulator. MtsR footprints are large (47–130 bp) and vary between target promoters. A 16 bp motif that consists of an interrupted palindrome is implicated in the DNA recognition by the metalloregulator. In conclusion, we report here that MtsR is a global regulator in GAS that shapes the expression of vital virulence factors and genes involved in metabolic functions and metal transport, and we discuss the implications for the GAS disease process.

MtsR is a dual regulator that controls virulence genes and metabolic functions in addition to metal homeostasis in the group A streptococcus: Regulation of gene expression by MtsR

MtsR is a metal‐dependent regulator in the group A streptococcus (GAS) that directly represses the transcription of genes involved in haem and metal uptake. While MtsR has been implicated in GAS virulence, the DNA recognition and full regulatory scope exerted by the protein are unknown. In this study we identified the shr promoter (Pshr) and mapped MtsR binding to a 69 bp segment in Pshr that overlaps the core promoter elements. A global transcriptional analysis demonstrated that MtsR modulates the expression of 64 genes in GAS, 44 of which were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including metal homeostasis, nucleic acid and amino acid metabolism, and protein fate. Importantly, the MtsR regulon includes mga, emm49 and ska, which are central for GAS pathogenesis. MtsR binding to the promoter region of both negatively and positively regulated genes demonstrates that it functions as a dual regulator. MtsR footprints are large (47–130 bp) and vary between target promoters. A 16 bp motif that consists of an interrupted palindrome is implicated in the DNA recognition by the metalloregulator. In conclusion, we report here that MtsR is a global regulator in GAS that shapes the expression of vital virulence factors and genes involved in metabolic functions and metal transport, and we discuss the implications for the GAS disease process.