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

Nguyen MT, Kraft B, Yu W, Demircioglu DD, Demicrioglu DD, Hertlein T, Burian M, Schmaler M, Boller K, Bekeredjian-Ding I, Ohlsen K, Schittek B, Götz F - PLoS Pathog. (2015)

Bottom Line: The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Genetics, University of Tübingen, Tübingen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Genetic organization of the lpl cluster operon.(A) Shows a genomic section of the lpl cluster with five predicted promoters (P1-5) and three transcription termination sites (indicated by hair-pin symbols). The genes 0410–0422 comprise most likely an operon, as they are not interrupted by a transcription terminator sequence. The lpl operon is composed of ten genes (0410–0419) encoding lipoproteins (grayish arrows) and of three non-lipoprotein encoding genes 0420–0422 (black arrows). By Northernblot analysis two transcripts of 2 and 3.2 knt could be verified; most likely there is a 10.5 knt read-through transcript for the entire lpl operon (indicated by dotted arrow). (B) Shows a cluster dendrogram based on the sequence similarity of lipoproteins (Lpl). (C) Alignment of all Lpl proteins revealed a highly conserved (92%) core sequence while the flanking sequences are less conserved. LSP, lipo signal peptide.
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ppat.1004984.g002: Genetic organization of the lpl cluster operon.(A) Shows a genomic section of the lpl cluster with five predicted promoters (P1-5) and three transcription termination sites (indicated by hair-pin symbols). The genes 0410–0422 comprise most likely an operon, as they are not interrupted by a transcription terminator sequence. The lpl operon is composed of ten genes (0410–0419) encoding lipoproteins (grayish arrows) and of three non-lipoprotein encoding genes 0420–0422 (black arrows). By Northernblot analysis two transcripts of 2 and 3.2 knt could be verified; most likely there is a 10.5 knt read-through transcript for the entire lpl operon (indicated by dotted arrow). (B) Shows a cluster dendrogram based on the sequence similarity of lipoproteins (Lpl). (C) Alignment of all Lpl proteins revealed a highly conserved (92%) core sequence while the flanking sequences are less conserved. LSP, lipo signal peptide.

Mentions: The lipoprotein-like cluster within the νSaα island of USA300 comprises ten lpl genes (lpl 0410–0419). All of them contain a classical lipo-box motif in the signal sequence (Fig 2A). Based on protein sequence similarity the corresponding Lpl proteins can be grouped into three clusters: Lpls encoded by the genes 0410, 0411, 0413, 0414, 0417 share > 80% similarity; the two Lpls encoded by 0416 and 0418 also share > 80% similarity but the similarity between 0410 and 0416 is only approximately 60% (Fig 2B). The similarity of the Lpls suggests that they might have redundant functions. The lpl cluster contains several potential promoters located upstream of the genes 0410, 0413, 0417 and 0419 (Fig 2A). By Northern blot analysis (S1 Fig) we could verify the transcripts starting from P2, P4 and P5 promoters. However, we assume that a run-through transcript of approximately 10.5 knt is generated because there is no conspicuous transcription terminator sequence in the lpl cluster (Fig 2A). There are two predicted rho-independent transcription terminators, Ω1 and Ω2, downstream of 0422 and 0424, respectively. Downstream of the lpl cluster, but upstream of the Ω2, there are three genes (0420–0422) with unknown function. These highly conserved genes are present in all presently sequenced νSaα islands. We, therefore, predict that they might be involved in Lpl biosynthesis/modification. The third transcription terminator, Ω3, is inserted in opposite direction, downstream of 0409 (Fig 2A).


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

Nguyen MT, Kraft B, Yu W, Demircioglu DD, Demicrioglu DD, Hertlein T, Burian M, Schmaler M, Boller K, Bekeredjian-Ding I, Ohlsen K, Schittek B, Götz F - PLoS Pathog. (2015)

Genetic organization of the lpl cluster operon.(A) Shows a genomic section of the lpl cluster with five predicted promoters (P1-5) and three transcription termination sites (indicated by hair-pin symbols). The genes 0410–0422 comprise most likely an operon, as they are not interrupted by a transcription terminator sequence. The lpl operon is composed of ten genes (0410–0419) encoding lipoproteins (grayish arrows) and of three non-lipoprotein encoding genes 0420–0422 (black arrows). By Northernblot analysis two transcripts of 2 and 3.2 knt could be verified; most likely there is a 10.5 knt read-through transcript for the entire lpl operon (indicated by dotted arrow). (B) Shows a cluster dendrogram based on the sequence similarity of lipoproteins (Lpl). (C) Alignment of all Lpl proteins revealed a highly conserved (92%) core sequence while the flanking sequences are less conserved. LSP, lipo signal peptide.
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Related In: Results  -  Collection

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ppat.1004984.g002: Genetic organization of the lpl cluster operon.(A) Shows a genomic section of the lpl cluster with five predicted promoters (P1-5) and three transcription termination sites (indicated by hair-pin symbols). The genes 0410–0422 comprise most likely an operon, as they are not interrupted by a transcription terminator sequence. The lpl operon is composed of ten genes (0410–0419) encoding lipoproteins (grayish arrows) and of three non-lipoprotein encoding genes 0420–0422 (black arrows). By Northernblot analysis two transcripts of 2 and 3.2 knt could be verified; most likely there is a 10.5 knt read-through transcript for the entire lpl operon (indicated by dotted arrow). (B) Shows a cluster dendrogram based on the sequence similarity of lipoproteins (Lpl). (C) Alignment of all Lpl proteins revealed a highly conserved (92%) core sequence while the flanking sequences are less conserved. LSP, lipo signal peptide.
Mentions: The lipoprotein-like cluster within the νSaα island of USA300 comprises ten lpl genes (lpl 0410–0419). All of them contain a classical lipo-box motif in the signal sequence (Fig 2A). Based on protein sequence similarity the corresponding Lpl proteins can be grouped into three clusters: Lpls encoded by the genes 0410, 0411, 0413, 0414, 0417 share > 80% similarity; the two Lpls encoded by 0416 and 0418 also share > 80% similarity but the similarity between 0410 and 0416 is only approximately 60% (Fig 2B). The similarity of the Lpls suggests that they might have redundant functions. The lpl cluster contains several potential promoters located upstream of the genes 0410, 0413, 0417 and 0419 (Fig 2A). By Northern blot analysis (S1 Fig) we could verify the transcripts starting from P2, P4 and P5 promoters. However, we assume that a run-through transcript of approximately 10.5 knt is generated because there is no conspicuous transcription terminator sequence in the lpl cluster (Fig 2A). There are two predicted rho-independent transcription terminators, Ω1 and Ω2, downstream of 0422 and 0424, respectively. Downstream of the lpl cluster, but upstream of the Ω2, there are three genes (0420–0422) with unknown function. These highly conserved genes are present in all presently sequenced νSaα islands. We, therefore, predict that they might be involved in Lpl biosynthesis/modification. The third transcription terminator, Ω3, is inserted in opposite direction, downstream of 0409 (Fig 2A).

Bottom Line: The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Genetics, University of Tübingen, Tübingen, Germany.

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.

No MeSH data available.


Related in: MedlinePlus