Inhibition of Cell Surface Export of Group A Streptococcal Anchorless Surface Dehydrogenase Affects Bacterial Adherence and Antiphagocytic Properties

Abstract

Surface dehydrogenase (SDH) is an anchorless, multifunctional protein displayed on the surfaces of group A Streptococcus (GAS) organisms. SDH is encoded by a single gene, sdh (gap or plr) that is essential for bacterial survival. Hence, the resulting nonfeasibility of creating a knockout mutant is a major limiting factor in studying its role in GAS pathogenesis. An insertion mutagenesis strategy was devised in which a nucleotide sequence encoding a hydrophobic tail of 12 amino acids ( 337 IVLVGLVMLLLS 348 ) was added at the 3 end of the sdh gene, successfully creating a viable mutant strain (M1-SDH HBtail ). In this mutant strain, the SDH HBtail protein was not secreted in the medium but was retained in the cytoplasm and to some extent trapped within the cell wall. Hence, SDH HBtail was not displayed on the GAS surface. The mutant strain, M1-SDH HBtail , grew at the same rate as the wild-type strain. The SDH HBtail protein displayed the same GAPDH activity as the wild-type SDH protein. Although the whole-cell extracts of the wild-type and mutant strains showed similar GAPDH activities, cell wall extracts of the mutant strain showed 5.5-fold less GAPDH activity than the wild-type strain. The mutant strain, M1-SDH HBtail , bound significantly less human plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity. These results indicate that the prevention of the cell surface export of SDH affects the virulence properties of GAS. The anchorless SDH protein, thus, is an important virulence factor.

Interaction with human plasminogen system turns on proteolytic activity in Streptococcus agalactiae and enhances its virulence in a mouse model

“…GAPDH has also been described as a plasminogen receptor in Streptococcus suis [25] and as a surface-displayed plasminogen-binding protein in Streptococcus pneumoniae [26]. Recently, Boel et al constructed a GAS mutant unable to secrete GAPDH, the enzyme being retained in the cytoplasm and/or in the membrane [27]. This mutant strain displayed 5.5-fold less GAPDH activity, bound significantly less plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity [27].…”

“…Recently, Boel et al constructed a GAS mutant unable to secrete GAPDH, the enzyme being retained in the cytoplasm and/or in the membrane [27]. This mutant strain displayed 5.5-fold less GAPDH activity, bound significantly less plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity [27]. Further studies will ascertain whether the S. agalactiae GAPDH is involved in the surface acquired plasmin-like activity in vivo.…”

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

“…The N-terminal His-tagged expression vector pET14B (Novagen) was used to make recombinant His 6 -tagged proteins. Vector pFW5 containing the spectinomycin gene (aad9) was used to transform M1-SF370 as described before (27,13,28). Vector pDC123 (29) and pCN40tet (26) were used for the complementation experiments within M1-SF370 and S. aureus RN4220, respectively.…”

“…Rabbit polyclonal antibody against rCdhA was custom-raised by Lampire Biological Laboratories (Pipersville, PA) and was purified by sequential Protein G and CdhA-DADPA affinity column chromatography as described previously (13). Fab-specific anti-CdhA antibody fragment after pepsin digestion was obtained as described previously (28).…”

The Expanding Landscape of Moonlighting Proteins in Yeasts

“…Different structural characteristics appear to be involved in the process, as demonstrated by the work on the glyceraldehyde-3-phosphate dehydrogenase of S. pyogenes and on the enolase of Bacillus subtilis. Addition of a C-terminal hydrophobic tail to the glyceraldehyde-3-phosphate dehydrogenase retained it in the cytosol without affecting its enzymatic activity and the strain carrying it lost many of its adhering activities (118).…”