Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Activation of peroxisome proliferator-activated receptor delta stimulates the proliferation of human breast and prostate cancer cell lines.

The nuclear receptor peroxisome proliferator-activated receptor delta [PPARdelta/beta (NR1C2)] has been implicated in colorectal carcinogenesis by various molecular genetic observations. These observations have recently been supported by studies of activation of PPARdelta by pharmacological agents. Here we present the first report of the stimulation of breast and prostate cancer cell growth using PPARdelta selective agonists. Activation of PPARdelta with compound F stimulated proliferation in breast (T47D, MCF7) and prostate (LNCaP, PNT1A) cell lines, which are responsive to sex hormones. Conversely, we have found that several steroid-independent cell lines, including colon lines, were unresponsive to compound F. These findings were confirmed with an additional high-affinity PPARdelta agonist, GW501516. Conditional expression of PPARdelta in MCF7 Tet-On cells resulted in a doxycycline-enhanced response to GW501516, thus providing direct genetic evidence for the role of PPARdelta in the proliferative response to this drug. Activation of PPARdelta in T47D cells resulted in increased expression of the proliferation marker Cdk2 and also vascular endothelial growth factor alpha (VEGFalpha) and its receptor, FLT-1, thus, suggesting that PPARdelta may initiate an autocrine loop for cellular proliferation and possibly angiogenesis. Consistent with this hypothesis, we demonstrated a pro-proliferative effect of GW501516 on human umbilical vein endothelial cell cultures and found that GW501516 also regulated the expression of VEGFalpha and FLT-1 in these cells. Our observations provide the first evidence that activation of PPARdelta can result in increased growth in breast and prostate cancer cell lines and primary endothelial cells and supports the possibility that PPARdelta antagonists may be of therapeutic value in the treatment of breast and prostate cancer.

Sequence variability is correlated with weak immunogenicity in Streptococcus pyogenes M protein.

The M protein of Streptococcus pyogenes, a major bacterial virulence factor, has an amino-terminal hypervariable region (HVR) that is a target for type-specific protective antibodies. Intriguingly, the HVR elicits a weak antibody response, indicating that it escapes host immunity by two mechanisms, sequence variability and weak immunogenicity. However, the properties influencing the immunogenicity of regions in an M protein remain poorly understood. Here, we studied the antibody response to different regions of the classical M1 and M5 proteins, in which not only the HVR but also the adjacent fibrinogen-binding B repeat region exhibits extensive sequence divergence. Analysis of antisera from S. pyogenes-infected patients, infected mice, and immunized mice showed that both the HVR and the B repeat region elicited weak antibody responses, while the conserved carboxy-terminal part was immunodominant. Thus, we identified a correlation between sequence variability and weak immunogenicity for M protein regions. A potential explanation for the weak immunogenicity was provided by the demonstration that protease digestion selectively eliminated the HVR-B part from whole M protein-expressing bacteria. These data support a coherent model, in which the entire variable HVR-B part evades antibody attack, not only by sequence variability but also by weak immunogenicity resulting from protease attack.

Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

Recent studies indicate that defective activity of complement factor H (FH) is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR) of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD) and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

Conditional Expression of Human PPARδ and a Dominant Negative Variant of hPPARδ In Vivo.

The nuclear receptor, NR1C2 or peroxisome proliferator-activated receptor (PPAR)-δ, is ubiquitously expressed and important for placental development, fatty acid metabolism, wound healing, inflammation, and tumour development. PPARδ has been hypothesized to function as both a ligand activated transcription factor and a repressor of transcription in the absence of agonist. In this paper, treatment of mice conditionally expressing human PPARδ with GW501516 resulted in a marked loss in body weight that was not evident in nontransgenic animals or animals expressing a dominant negative derivative of PPARδ. Expression of either functional or dominant negative hPPARδ blocked bezafibrate-induced PPARα-dependent hepatomegaly and blocked the effect of bezafibrate on the transcription of PPARα target genes. These data demonstrate, for the first time, that PPARδ could inhibit the activation of PPARα in vivo and provide novel models for the investigation of the role of PPARδ in pathophysiology.

The Hypervariable region of Streptococcus pyogenes M protein escapes antibody attack by antigenic variation and weak immunogenicity.

Sequence variation of antigenic proteins allows pathogens to evade antibody attack. The variable protein commonly includes a hypervariable region (HVR), which represents a key target for antibodies and is therefore predicted to be immunodominant. To understand the mechanism(s) of antibody evasion, we analyzed the clinically important HVR-containing M proteins of the human pathogen Streptococcus pyogenes. Antibodies elicited by M proteins were directed almost exclusively against the C-terminal part and not against the N-terminal HVR. Similar results were obtained for mice and humans with invasive S. pyogenes infection. Nevertheless, only anti-HVR antibodies protected efficiently against infection, as shown by passive immunizations. The HVR fused to an unrelated protein elicited no antibodies, implying that it is inherently weakly immunogenic. These data indicate that the M protein HVR evades antibody attack not only through antigenic variation but also by weak immunogenicity, a paradoxical observation that may apply to other HVR-containing proteins.

Activation of PPARbeta/delta causes a psoriasis-like skin disease in vivo.

BACKGROUND: Psoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) beta/delta, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARbeta/delta is sufficient to trigger psoriasis-like changes in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Using immunohistochemistry, we define the distribution of PPARbeta/delta in the skin lesions of psoriasis. By expression profiling, we confirm that PPARbeta/delta is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARbeta/delta in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARbeta/delta, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARbeta/delta regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARbeta/delta and show that inhibition of STAT3 phosphorylation blocks disease development. CONCLUSIONS: Activation of PPARbeta/delta in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

Ligand modulated antagonism of PPARgamma by genomic and non-genomic actions of PPARdelta.

BACKGROUND: Members of the Peroxisome Proliferator Activated Receptor, PPAR, subfamily of nuclear receptors display complex opposing and overlapping functions and a wide range of pharmacological and molecular genetic tools have been used to dissect their specific functions. Non-agonist bound PPARdelta has been shown to repress PPAR Response Element, PPRE, signalling and several lines of evidence point to the importance of PPARdelta repressive actions in both cardiovascular and cancer biology. METHODOLOGY/PRINCIPAL FINDINGS: In this report we have employed transient transfections and luciferase reporter gene technology to study the repressing effects of PPARdelta and two derivatives thereof. We demonstrate for the first time that the classical dominant negative deletion of the Activation Function 2, AF2, domain of PPARdelta show enhanced repression of PPRE signalling in the presence of a PPARdelta agonist. We propose that the mechanism for the phenomenon is increased RXR heterodimerisation and DNA binding upon ligand binding concomitant with transcriptional co-repressor binding. We also demonstrated ligand-dependent dominant negative action of a DNA non-binding derivative of PPARdelta on PPARgamma1 signalling. This activity was abolished upon over-expression of RXRalpha suggesting a role for PPAR/cofactor competition in the absence of DNA binding. CONCLUSIONS/SIGNIFICANCE: These findings are important in understanding the wide spectrum of molecular interactions in which PPARdelta and PPARgamma have opposing biological roles and suggest novel paradigms for the design of different functional classes of nuclear receptor antagonist drugs.

Reduced toll-like receptor 4 expression in children with asymptomatic bacteriuria.

Toll-like receptor (TLR) 4 is essential for the defense against infection with gram-negative pathogens, but reduced TLR4 expression has not been linked to altered disease susceptibility in humans. In mice, Tlr4 controls the mucosal response to Escherichia coli urinary tract infections. Inactivation of mouse Tlr4 causes an asymptomatic carrier state resembling asymptomatic bacteriuria (ABU). The present study compared neutrophil TLR4 expression levels between children with ABU (n=17) and age-matched control subjects (n=24), and significantly lower levels were detected in the patients with ABU. We also found elevated levels of the TLR4 adaptor protein TRIF and reduced levels of the TLR4-inhibitor SIGIRR in the patients with ABU, but MyD88 and TRAM levels were not significantly altered. Altered TLR4 and adaptor protein expression might impair TLR4 signaling and explain the weak mucosal response to urinary tract infection in patients who develop ABU rather than symptomatic disease.

A genetic basis of susceptibility to acute pyelonephritis.

BACKGROUND: For unknown reasons, urinary tract infections (UTIs) are clustered in certain individuals. Here we propose a novel, genetically determined cause of susceptibility to acute pyelonephritis, which is the most severe form of UTI. The IL-8 receptor, CXCR1, was identified as a candidate gene when mIL-8Rh mutant mice developed acute pyelonephritis (APN) with severe tissue damage. METHODS AND FINDINGS: We have obtained CXCR1 sequences from two, highly selected APN prone patient groups, and detected three unique mutations and two known polymorphisms with a genotype frequency of 23% and 25% compared to 7% in controls (p<0.001 and p<0.0001, respectively). When reflux was excluded, 54% of the patients had CXCR1 sequence variants. The UTI prone children expressed less CXCR1 protein than the pediatric controls (p<0.0001) and two sequence variants were shown to impair transcription. CONCLUSIONS: The results identify a genetic innate immune deficiency, with a strong link to APN and renal scarring.

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium.

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.