The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein

Abstract

Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff’s staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl residues can stimulate biofilm formation. Thus, sugar modifications may represent promising new targets for novel therapeutic or prophylactic measures against life-threatening S. aureus infections.

Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins

“…Many virulence factors associated with initial colonization, and/or invasion of host cells have been demonstrated to undergo posttranslational modification through glycosylation 1 . The importance of protein glycosylation in virulence has been demonstrated in many bacteria including Gram-positive pathogens 1 , 38 – 41 . In S .…”

Metabolic inhibitors of bacterial glycan biosynthesis

“…Pathogenic bacteria that cannot synthesize glycoproteins have attenuated virulence. For example, Pseudomonas aeruginosa and Haemophilus inuenzae glycosylation mutants have reduced adhesion to host cells, 13,14 Staphylococcus aureus and H. inuenzae glycosylation mutants are defective at forming biolms, 15 and C. jejuni, H. pylori, and P. aeruginosa mutants with interruptions in agellin glycosylation are immotile and cannot colonize the host. 9,16,17 Small molecules that inhibit glycosylation enzymes and recapitulate the effects of genetics experiments have the potential to expand our antibiotic arsenal with agents that are minimally disruptive to benecial microora.…”

Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands

“…Two glycosyltransferase genes were observed adjacent to the sesJ gene. Glycosyltransferase genes in close proximity to Pls genes and genes for other MSCRAMMs have been observed before (26). Two glycosyltransferase genes, sdgA and sdgB, are encoded by a region adjacent to the sdrC, sdrD, and sdrE gene cluster in S. aureus.…”

“…GtfC and GtfD add N-acetylhexosamine to the serine residues in SD repeats of the Pls protein. Sugar modification of Pls by GtfC and GtfD leads to enhanced biofilm formation (26). We hypothesize that the SesJ protein on the surface of S. epidermidis is glycosylated by GtfA and GtfB.…”

“…One such virulence factor, plasmin-sensitive protein (Pls), a CWA protein from S. aureus, is present in the SCCmec type I J region (25). Pls plays a role in biofilm formation through the G5 repeats as well as the glycosylated serine aspartate dipeptide repeat region (26). In a mouse septic arthritis model, the presence of the pls gene also led to more frequent joint infection and severe arthritis (27).…”