Surface Proteins on Gram-Positive BacteriaSurface Proteins on Gram-Positive Bacteria

Surface Proteins:

One of the surface molecules we study is the surface M protein from group A streptococci. Our lab in collaboration with the June Scott laboratory at Emory University was the first to sequence an M protein molecule which was the first intact surface protein from a gram-positive bacterium. M protein is the major virulence factor of this organism by virtue of its ability to impede attack by human phagocytes. Physicochemical and sequence analysis from this laboratory revealed that M protein is an alpha-helical coiled-coil rope-like structure extending nearly 60 nm from the cell surface. DNA sequence analysis of the 3’ end of the M protein gene (the region involved in attachment to the cell) revealed that it is highly homologous to comparable regions of nearly all known surface proteins from gram-positive bacteria. This indicates that the mechanism of anchoring surface proteins in these bacteria also may be conserved.
Because gram-positive bacteria use their surface molecules to colonize or invade tissues, knowledge of the anchoring process will enable us to devise strategies to prevent their attachment to the cell and thus block infection, since naked bacteria cannot cause infection. During our studies to understand this attachment mechanism, we identified a membrane-associated enzyme, termed LPXTGase (see below), responsible for cleaving a highly conserved motif (LPXTG) within the anchor region of these surface proteins. Our results suggest that inhibition of this enzyme will prevent the proper attachment of most surface proteins resulting in nearly naked bacteria.


Vaccines and Surface Display of Proteins:

Capitalizing on the conservation of the anchoring process for surface proteins, we discovered that active polypeptides or proteins genetically fused to the common anchor region of the M protein could be used to deliver the active molecule to the surface of gram-positive bacteria (i.e., for vaccine purposes). A number of proteins from a wide range of sources (bacterial, viral, human, parasites) have been engineered to be expressed and displayed on the surface of a human commensal bacterium (Streptococcus gordonii). When placed into the nasopharynx of mice, these recombinant bacteria remained there for up to 12 weeks. During that time the colonized mice produced antigen-specific serum IgG, salivary IgA and T-cell responses to the surface expressed proteins. We anticipate that this live vaccine approach may be used for a variety of antigens to protect against invasion by disease organisms. 

We also focus on the mechanisms by which gram-positive bacteria, particularly streptococci, cause disease and use this information in the development of methods to induce a protective mucosal immune response.  Development of a cross-protective vaccine for group A streptococcal infection has been one of the major objectives of this laboratory for many years. The identification of a conserved region within the M protein of all of the >125 different serotypes of group A streptococci enabled us to design experiments to determine if a vaccine comprising this region would protect against infection by multiple serotypes of streptococci.  Synthetic peptides based on the carboxyl half of the M6 protein linked to the B subunit of cholera toxin (CTB) were shown by this laboratory to be effective in significantly reducing colonization upon intranasal challenge with a heterologous serotype in a mouse model.  This finding suggests that local immunization with the conserved region can protect at the mucosa and may be the first step in designing an anti-streptococcal vaccine.


Surface protein anchoring in Gram-positive bacteria:

We discovered that the precursors of most surface proteins on gram-positive bacteria have a C-terminal hydrophobic domain and charged tail, preceded by a conserved LPXTG motif that signals the anchoring process. This motif is the substrate for an enzyme, termed sortase, which has transpeptidation activity resulting in the cleavage of the LPXTG sequence and ultimate attachment of the protein to the peptidoglycan. While screening a group A streptococcal extract for other cleavage activities towards the LPXTG motif we identified an enzyme (which we term "LPXTGase") that differs significantly from sortase but also cleaves this motif at a different bond. The enzyme is heavily glycosylated, which is required for its activity. Amino acid composition and sequence analysis revealed that LPXTGase differs from other enzymes, in that the molecule, which is about 14 kDa in size, has no aromatic amino acids, is rich in alanine and D-alanine, and is 30% composed of uncommon amino acids, suggesting a non-ribosomal synthesis. A similar enzyme found in the membrane extract of Staphylococcus aureus, and S. pneumoniae, indicates that this unusual molecule may be common among gram-positive bacteria. Whereas peptide antibiotics have been reported from bacillus species that also contain unusual amino acids and are synthesized non-ribosomally on amino acid-activating polyenzyme templates, this would be the first reported enzyme that may be similarly synthesized. However, non-ribosomal peptide synthetases have not been found in the streptococcal genome. Studies are in progress to both further define the LPXTGase enzyme and its role in the attachment process and to identify inhibitors, because such inhibitors may be considered a new class of antibiotic.

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