André N. Tiaden

The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA

Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum‐derived ‘Legionella‐containing vacuole’ (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild‐type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt‐resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER‐derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two‐component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS‐ and LetA‐controlled interactions of L. pneumophila with phagocytes.

The Legionella Autoinducer Synthase LqsA Produces an α-Hydroxyketone Signaling Molecule

The opportunistic pathogen Legionella pneumophila replicates in human lung macrophages and in free-living amoebae. To accommodate the transfer between host cells, L. pneumophila switches from a replicative to a transmissive phase. L. pneumophila harbors a gene cluster homologous to the Vibrio cholerae cqsAS quorum sensing system, encoding a putative autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a response regulator (lqsR). LqsR is an element of the L. pneumophila virulence regulatory network, which promotes pathogen-host cell interactions and inhibits entry into the replicative growth phase. Here, we show that lqsA functionally complements a V. cholerae cqsA autoinducer synthase deletion mutant and, upon expression in L. pneumophila or Escherichia coli, produces the diffusible signaling molecule LAI-1 (Legionella autoinducer-1). LAI-1 is distinct from CAI-1 (Cholerae autoinducer-1) and was identified as 3-hydroxypentadecan-4-one using liquid chromatography coupled to high resolution tandem mass spectrometry. The activity of both LqsA and CqsA was abolished upon mutation of a conserved lysine, and covalent binding of the cofactor pyridoxal 5′-phosphate to this lysine was confirmed by mass spectrometry. Thus, LqsA and CqsA belong to a family of pyridoxal 5′-phosphate-dependent autoinducer synthases, which produce the α-hydroxyketone signaling molecules LAI-1 and CAI-1.

Bacterial gene regulation by α-hydroxyketone signaling

Bacteria produce diffusible, small signaling molecules termed autoinducers to promote cell-cell communication. Recently, a novel class of signaling molecules, the alpha-hydroxyketones (AHKs), was discovered in the facultative human pathogens Legionella pneumophila and Vibrio cholerae. In this review, we summarize and compare findings on AHK signaling in these bacteria. The L. pneumophila lqs (Legionella quorum sensing) and V. cholerae cqs (cholera quorum sensing) gene clusters synthesize and detect Legionella autoinducer 1 (3-hydroxypentadecan-4-one) or cholera autoinducer-1 (3-hydroxytridecan-4-one), respectively. In addition to the autoinducer synthase and cognate sensor kinase encoded in the cqs locus, the lqs cluster also harbors a prototypic response regulator. AHK signaling regulates pathogen-host cell interactions, bacterial virulence, formation of biofilms or extracellular filaments, and expression of a genomic island. The lqs/cqs gene cluster is present in several environmental bacteria, suggesting that AHKs are widely used for cell-cell signaling.

The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila

The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and LqsS participate in the same regulatory circuit.

Telomere length, telomerase activity and osteogenic differentiation are maintained in adipose-derived stromal cells from senile osteoporotic SAMP6 mice

Adipose tissue provides for a rich and easily accessible source of multipotent stromal cells and thus offers the potential for autologous cell‐based therapy for a number of degenerative diseases. Senile osteoporosis is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow stromal cell (BMSC) differentiation. In the present study, we have characterized adipose‐derived stromal cells (ASCs) isolated from aged osteoporotic mice and evaluated their suitability as a source of osteogenic precursor cells. Significant reductions in both tibia bone quality and telomere length in liver tissue were observed in the senescence‐accelerated mouse prone 6 strain (SAMP6), as compared to the control age‐matched senescence‐accelerated mouse resistant 1 strain (SAMR1), thus confirming osteoporosis and accelerated ageing traits in this model. ASCs isolated from inguinal fat expressed mesenchymal surface markers and were capable of differentiating along the osteoblast, adipocyte and chondrocyte lineages. Telomere length was not compromised in ASCs from SAMP6 mice but was actually found to be significantly increased as compared to control SAMR1 mice. Furthermore, ASCs from both strains were comparable in terms of telomerase activity, p21 mRNA expression, SA–β‐gal activity and proliferative capacity. The overall osteogenic and adipogenic potential of ASCs was comparable between SAMP6 and SAMR1 strains, as determined by quantitative molecular, biochemical and histological analyses. In conclusion, adipose tissue may represent a promising autologous cell source for the development of novel bone regenerative therapeutic strategies in the treatment of age‐related osteoporosis. Copyright

Detrimental Role for Human High Temperature Requirement Serine Protease A1 (HTRA1) in the Pathogenesis of Intervertebral Disc (IVD) Degeneration

Background: HTRA1 has been associated with intervertebral disc (IVD) degeneration although its role is unknown. Results: HTRA1 up-regulated matrix metalloproteinase (MMP) production by IVD cells via the generation of fibronectin fragments. Conclusion: HTRA1 plays a detrimental role in the pathogenesis of IVD degeneration. Significance: HTRA1 may represent a novel therapeutic target for the treatment of spinal disc degeneration. Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.

Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

IntroductionIntervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators.MethodsHuman IVD cells isolated from patient biopsies were stimulated with fHAs (6 to 12 disaccharides) and their effect on cytokine and matrix degrading enzyme production was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The involvement of specific cell surface receptors and signal transduction pathways in mediating the effects of fHAs was tested using small interfering RNA (siRNA) approaches and kinase inhibition assays.ResultsTreatment of IVD cells with fHAs significantly increased mRNA expression levels of interleukin (IL)-1β, IL-6, IL-8, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1 and -13. The stimulatory effects of fHAs on IL-6 protein production were significantly impaired when added to IVD cells in combination with either Toll-like receptor (TLR)-2 siRNA or a TLR2 neutralizing antibody. Furthermore, the ability of fHAs to enhance IL-6 and MMP-3 protein production was found to be dependent on the mitogen-activated protein (MAP) kinase signaling pathway.ConclusionsThese findings suggest that fHAs may have the potential to mediate IVD degeneration and discogenic back pain through activation of the TLR2 signaling pathway in resident IVD cells.

Human Serine Protease HTRA1 Positively Regulates Osteogenesis of Human Bone Marrow‐derived Mesenchymal Stem Cells and Mineralization of Differentiating Bone‐forming Cells Through the Modulation of Extracellular Matrix Protein

Mammalian high‐temperature requirement serine protease A1 (HTRA1) is a secreted member of the trypsin family of serine proteases which can degrade a variety of bone matrix proteins and as such has been implicated in musculoskeletal development. In this study, we have investigated the role of HTRA1 in mesenchymal stem cell (MSC) osteogenesis and suggest a potential mechanism through which it controls matrix mineralization by differentiating bone‐forming cells. Osteogenic induction resulted in a significant elevation in the expression and secretion of HTRA1 in MSCs isolated from human bone marrow‐derived MSCs (hBMSCs), mouse adipose‐derived stromal cells (mASCs), and mouse embryonic stem cells. Recombinant HTRA1 enhanced the osteogenesis of hBMSCs as evidenced by significant changes in several osteogenic markers including integrin‐binding sialoprotein (IBSP), bone morphogenetic protein 5 (BMP5), and sclerostin, and promoted matrix mineralization in differentiating bone‐forming osteoblasts. These stimulatory effects were not observed with proteolytically inactive HTRA1 and were abolished by small interfering RNA against HTRA1. Moreover, loss of HTRA1 function resulted in enhanced adipogenesis of hBMSCs. HTRA1 Immunofluorescence studies showed colocalization of HTRA1 with IBSP protein in osteogenic mASC spheroid cultures and was confirmed as being a newly identified HTRA1 substrate in cell cultures and in proteolytic enzyme assays. A role for HTRA1 in bone regeneration in vivo was also alluded to in bone fracture repair studies where HTRA1 was found localized predominantly to areas of new bone formation in association with IBSP. These data therefore implicate HTRA1 as having a central role in osteogenesis through modification of proteins within the extracellular matrix. STEM Cells2012;30:2271–2282

ARTD1 deletion causes increased hepatic lipid accumulation in mice fed a high-fat diet and impairs adipocyte function and differentiation

ADP‐ribosyltransferase Diphtheria toxinlike 1 [ARTD1; formerly called poly‐ADP‐ribose polymerase 1 (PARP1)] is a chromatin‐associated enzyme involved in regulating metabolic homeostasis. The liver is at the core of glucose and lipid metabolism and is significantly affected by obesity and the metabolic syndrome. Here, we show that when fed a high‐fat diet (HFD), mice lacking ARTD1 developed exacerbated hepatic steatosis. ARTD1–/– mice had a 19% higher liver weight than wild‐type (WT) animals and exhibited a significantly increased serum concentration of cholesterol (38%) and impaired glucose tolerance. In addition, adipocyte function and size were significantly reduced in ARTD1–/– mice fed an HFD (7794 μm2 for WT and 5579 μm2 for ARTD1–/– mice). The significantly reduced adipogenic differentiation of adipose‐derived stromal cells (ASCs) isolated from ARTD1–/–mice (28 vs. 11% Oil red O‐positive cells in WT and ARTD1–/– ASCs, respectively) suggested that impaired adipogenesis as the underlying cause for this adipose tissue malfunction. This function of ARTD1 was specific for adipogenesis, since osteogenic differentiation was not affected by the ARTD1 deletion. In summary, we show that ARTD1–/– mice fed an HFD display impaired adipogenesis and show exacerbated hepatic steatosis, which can have important implications for nonalcoholic fatty liver disease.—Erener, S., Mirsaidi, A., Hesse, M., Tiaden, A. N., Ellingsgaard, H., Kostadinova, R., Donath, M. Y., Richards, P. J., Hottiger, M. O. ARTD1 deletion causes increased hepatic lipid accumulation in mice fed a high‐fat diet and impairs adipocyte function and differentiation. FASEB J. 26, 2631‐2638 (2012). www.fasebj.org

The Emerging Roles of HTRA1 in Musculoskeletal Disease

High-temperature requirement serine protease A1 (HTRA1) is one of four known proteases belonging to the broadly conserved family of HTRA proteins. Although it was originally considered as representing an important modulator of tumorigenesis, an increasing number of reports have suggested that its influence on human disease may extend beyond cancer. HTRA1 has the capacity to degrade numerous extracellular matrix proteins, and as such, its potential involvement in diseases of the musculoskeletal system has been gaining increased attention. Musculoskeletal disease constitutes a wide variety of degenerative conditions that can manifest themselves in different ways such as joint and back pain, as well as deficiencies in skeletal bone quality, and ultimately result in significant suffering and reduced quality of life. Convincing data now exist to support a detrimental role for HTRA1 in the pathogenesis of joint and intervertebral disk degeneration. However, the function of HTRA1 in other closely related musculoskeletal diseases affecting bone and muscle remains unclear and largely unexplored. To help set the stage for future research, we discuss here some of the recent advances in our understanding of the role played by HTRA1 in musculoskeletal pathology.