The νSaα Specific Lipoprotein Like Cluster (lpl) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells

Abstract

All Staphylococcus aureus genomes contain a genomic island, which is termed νSaα and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the νSaα islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I νSaα island. Since the contribution of the lpl gene cluster encoded in the νSaα island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the νSaα encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes highlights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor.

Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence

“…However, more important was the finding that the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin. This was confirmed when the lpl gene cluster was transformed into S. carnosus, which became invasive in the presence of the lpl genes (50). In a murine kidney abscess model, the bacterial burden in the kidneys was decreased with the lpl deletion mutant.…”

“…The genetic organization of Sa␣ is highly conserved and is composed of two gene clusters: one cluster carries a number of highly homologous exotoxin-encoding genes (set), and the other one encodes lipoproteins, referred to as lipoprotein-like (lpl), with a typical lipobox-containing signal sequence (49). When the entire lpl gene cluster was deleted in S. aureus USA300, the mutant showed a decreased TLR2-dependent stimulation of proinflammatory cytokines in human monocytes, macrophages, and keratinocytes (50). However, more important was the finding that the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin.…”

Skin-Specific Unsaturated Fatty Acids Boost the Staphylococcus aureus Innate Immune Response

“…S4C); there was no apparent difference in Lpl1 amount in total protein extraction. Lpl1 is encoded by the first gene of the tandem lipoprotein cluster and can be regarded as a prototype lipoprotein (39). Altogether, the enhanced innate immune stimulation due to feeding with unsaturated FAs is indeed caused by the incorporation of the UFAs in lipoproteins; it is not due to altered growth, increased release of proteins into the supernatant due to increased membrane leakiness, or enhanced expression of lipoproteins.…”

“…Then the pellet was resuspended with Tris buffer containing a protease inhibitor (Merck, Darmstadt, Germany) and lysostaphin (30 g/ml) and incubated at 37°C for 2 h to disrupt the cell wall. Membrane proteins were pelleted by ultracentrifugation (235,000 ϫ g for 45 min at 4°C) and extracted according to a previous study (39). SitC18 was purified by affinity chromatography with Strep-Tactin Superflow beads (Qiagen, Germany) overnight in cold condition (6°C) under mild rotation (20 rpm).…”

“…The total proteins were subsequently extracted by the methanol-chloroform method, dissolved in SDS running buffer, and separated by SDS-PAGE. The amount of Lpl1 protein was detected by Western blotting using anti-Lpl1 antibody as described previously (39).…”