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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.
Highlights
Usually being a common inhabitant of the human skin and mucous membranes, Staphylococcus aureus is a human pathogen that can cause diseases ranging from mild skin infections to serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, meningitis, and sepsis [1, 2]
Staphylococcus aureus is a serious pathogen that causes life-threatening infections due to its ability to attach to surfaces, form biofilms, and persist inside the host
We found that the glycosyltransferases GtfC and GtfD carry out the glycosylation reactions
Summary
Usually being a common inhabitant of the human skin and mucous membranes, Staphylococcus aureus is a human pathogen that can cause diseases ranging from mild skin infections to serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, meningitis, and sepsis [1, 2]. Due to the increasing use of various medical devices and implants in modern medicine, the number of nosocomial S. aureus infections is constantly rising [3, 4]. In the past three decades, the emergence of antibioticresistant staphylococci, such as methicillin-resistant S. aureus (MRSA) represents an increasing problem in the treatment of S. aureus infections. Alternative therapeutic or prophylactic measures against S. aureus infections are urgently required. It is widely accepted that bacteria can glycosylate proteins. Bacteria have two basic systems to glycosylate proteins: N-linked and O-linked glycosylation [8,9,10]. The N-linked glycosylation pathways have been well characterized in Gram-negative bacteria [5,6,7, 9]
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