Search

Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life-threatening infections particularly in immune-compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune-evasive proteins and develop new therapies. Here, we identified Pra1, the pH-regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a-like peptide lacked the C-terminal arginine residue needed for C3a-receptor binding and activation. Moreover, Pra1 also blocked C3a-like antifungal activity as shown in survival assays, and the C3b-like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor-expressing HEK cells, activation of Fura2-AM loaded cells, intracellular Ca 2+ signaling, IL-8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3-cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune-escape protein., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Proline-rich antimicrobial peptides (PrAMPs) represent promising alternative therapeutic options for the treatment of multidrug-resistant bacterial infections. PrAMPs are predominantly active against Gram-negative bacteria by inhibiting protein expression via at least two different modes of action, i.e., blocking the ribosomal exit tunnel of 70S ribosomes (oncocin-type binding) or inhibiting the assembly of the 50S ribosomal subunit (apidaecin-type binding). The in vivo efficacy and favorable biodistribution of oncocins confirmed the therapeutic potential of short PrAMPs for the first time, whereas the in vivo evaluation of apidaecins is still limited despite the promising efficacy of apidaecin-analog Api88 in an intraperitoneal murine infection model. Here, the in vivo efficacy of apidaecin-analog Api137 was studied, which rescued all NMRI mice from a lethal intraperitoneal infection with E. coli ATCC 25922 when administered three times intraperitoneal at doses of 0.6 mg/kg starting 1 h after infection. When Api88 and Api137 were administered intravenous or intraperitoneal at doses of 5 and 20 mg/kg, their plasma levels were similarly low (<3 μg/mL) and four-fold lower than for oncocin-analog Onc72. This contradicted earlier expectation based on the very low serum stability of Api88 with a half-life time of only ~5 min compared to ~6 and ~3 h for Api137 and Onc72, respectively. Pharmacokinetic data relying on a sensitive mass spectrometry method utilizing multiple reaction monitoring and isotope-labeled peptides revealed that Api88 and Api137 were present in blood, urine, and kidney, and liver homogenates at similar levels accompanied by the same major metabolites comprising residues 1-16 and 1-17. The pretended discrepancy was solved, when all peptides were incubated in peritoneal lavage. Api137 was rapidly degraded at the C-terminus, while Api88 was rather stable despite releasing the same degradation products. Onc72 was very stable explaining its higher plasma levels compared to Api88 and Api137 after intraperitoneal administration illuminating its good in vivo efficacy. The data indicate that the degradation of therapeutic peptides should be studied in serum and further body fluids. Moreover, the high efficacy in murine infection models and the fast clearance of Api88 and Api137 within ~60 min after intravenous and ~90 min after intraperitoneal injections indicate that their in vivo efficacy relates to the maximal peptide concentration achieved in blood.

Objectives: To evaluate the efficacy of antimicrobial peptide Onc112 in a lethal Escherichia coli infection model and the pharmacokinetics of Onc72 and Onc112 administered intravenously or intraperitoneally in mice., Methods: Onc72, Onc112 and their major metabolites in blood, kidneys, liver, brain and urine were quantified by MS using multiple reaction monitoring (MRM) and isotope-labelled peptides., Results: Onc112 rescued all animals when administered intraperitoneally at a dose of 2.5 mg/kg and was thus slightly more efficient than Onc72. The MRM method provided limits of quantification in plasma, urine and kidney, liver and brain homogenates of 7-80 μg/L, well below the MICs of 2-4 mg/L. Onc72 and Onc112 reached all organs within 10 min when administered intraperitoneally (5 mg/kg). Their initial concentrations in plasma were 11.9 and 22.6 mg/L, respectively, with elimination t1/2 values of ∼14 and 21 min. The peptide concentrations in blood remained above their MICs for 20 min for Onc72 and 80 min for Onc112. The highest peptide concentrations were detected in kidney homogenates, which also contained the highest content of metabolites, indicating, together with the results from analysis of urine samples, that both peptides are cleared through the kidneys., Conclusions: Onc72 and Onc112 reach organs, including the brain, within 10 min after intravenous and intraperitoneal administration. Onc112 remained in blood at concentrations above its MIC for 80 min. The pharmacokinetic profiles explain the high in vivo efficacies in models of systemic infection and indicate the potential use of these agents for the treatment of urinary tract infections., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein-coupled receptors: namely, the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, upregulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte-derived macrophages (HMDMs). A bioluminescence resonance energy transfer (BRET) assay was used to assess the recruitment of β-arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated β-arrestin 2 recruitment levels in comparison with C5a-des Arg, whereas no significant difference was observed at C5a2. A constitutive complex that formed between β-arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for β-arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDMs, inhibition of ERK1/2 was observed only at concentrations at or above the EC50 for heteromer formation. This suggested that increased recruitment of the β-arrestin-C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis.

Inflammatory bowel diseases are a critical public health issue, and as treatment options remain limited, there is a need to unravel the underlying pathomechanisms in order to identify new therapeutic targets. Complement activation was found in patients suffering from inflammatory bowel disease, and the complement anaphylatoxin C5a and its receptor C5aR have been implicated in disease pathogenesis in animal models of bowel inflammation. To further characterize complement-related pathomechanisms in inflammatory bowel disease, we have investigated the role of the anaphylatoxin C3a receptor in acute dextran sulfate sodium-induced colitis in mice. For this, colitis was induced in C3a receptor-deficient BALB/c and C57BL/6 mice, and disease severity was evaluated by clinical and histological examination, and by measuring the mRNA expression or protein levels of inflammatory mediators in the tissue. C3a receptor deficiency was partially protective in BALB/c mice, which had significantly reduced weight loss, clinical and histological scores, colon shortening, and CXCL-1/KC mRNA, myeloperoxidase and interleukin-6 tissue levels compared to the corresponding wild type mice. In C57BL/6 mice the differences between wild type and C3a receptor-deficient animals were much smaller and reached no significance. Our data demonstrate that the contribution of C3a receptor to disease pathogenesis and severity of dextran sulfate sodium-induced colitis in mice depends on the genetic background. Further studies will be required to clarify whether targeting of C3a receptor, possibly in combination with C5a receptor, might be considered as a therapeutic strategy for inflammatory bowel disease.

The activation of the complement cascade, a cornerstone of the innate immune response, produces a number of small (74-77 amino acid) fragments, originally termed anaphylatoxins, that are potent chemoattractants and secretagogues that act on a wide variety of cell types. These fragments, C5a, C4a, and C3a, participate at all levels of the immune response and are also involved in other processes such as neural development and organ regeneration. Their primary function, however, is in inflammation, so they are important targets for the development of antiinflammatory therapies. Only three receptors for complement peptides have been found, but there are no satisfactory antagonists as yet, despite intensive investigation. In humans, there is a single receptor for C3a (C3a receptor), no known receptor for C4a, and two receptors for C5a (C5a₁ receptor and C5a₂ receptor). The most recently characterized receptor, the C5a₂ receptor (previously known as C5L2 or GPR77), has been regarded as a passive binding protein, but signaling activities are now ascribed to it, so we propose that it be formally identified as a receptor and be given a name to reflect this. Here, we describe the complex biology of the complement peptides, introduce a new suggested nomenclature, and review our current knowledge of receptor pharmacology.

Background: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells., Results: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes., Conclusions: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.

Background: Inflammatory bowel disease (IBD) is a critical public health issue; more and more people are affected, but treatment options remain limited. Complement activation and the anaphylatoxin C5a have been shown to play a role in IBD. In this study, mouse models of acute and chronic dextran sulfate-induced colitis were used to further elucidate the impact of C5a and its receptor (C5aR) on disease development., Methods: In C57BL/6J wildtype and C5aR(-/-) mice the extent of complement activation, changes in weight, and water/food consumption were determined. Disease severity was evaluated via a clinical score, histology, cytokine- and myeloperoxidase-determination as well as real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of anaphylatoxin receptors and inflammatory mediators., Results: C5aR(-/-) mice showed milder disease symptoms, less histological damage, and a lower expression of inflammatory mediators in acute colitis, a setting where complement was activated. In chronic colitis the knockout mice exhibited aggravated weight loss, a higher degree of histological damage and granulocyte infiltration. Intriguingly, increases in C3a-receptor and C5L2 mRNA were dependent on C5aR. Compared to wildtype mice, C5aR(-/-) animals displayed smaller lymph nodes in acute colitis, but extensive swelling and diminished IL-4 and IFN-γ responses in the chronic disease, demonstrating that C5aR modifies T-helper cell polarization., Conclusions: C5aR exerts detrimental functions in acute colitis, strongly supporting the idea that a C5aR-antagonist might be useful for IBD treatment. However, since the absence of C5aR was no longer protective and in some regards disadvantageous in chronic IBD, future studies should address the efficacy and the possible side effects of a sustained antagonist treatment., (Copyright © 2009 Crohn's & Colitis Foundation of America, Inc.)

One of the most prominent features of malignant melanoma is the fast generation of metastasizing cells, resulting in the poor prognosis of patients with this tumor type. For this process, cells must gain the ability to migrate. The oncogenic receptor Xmrk (Xiphophorus melanoma receptor kinase) from the Xiphophorus melanoma system is a mutationally activated version of the epidermal growth factor receptor that induces the malignant transformation of pigment cells. Here, we show that the activation of Xmrk leads to a clear increase of pigment cell motility in a fyn-dependent manner. Stimulation of Xmrk induces its interaction with the focal adhesion kinase (FAK) and the interaction of active, receptor-bound fyn with FAK. This results in changes in FAK activity and induces the modulation of stress fibers and focal adhesions. Overexpression of dominant-negative FAK shows that the activity of innate FAK and a receptor-induced focal adhesion turnover are a prerequisite for pigment cell migration. Our findings show that in our system, Xmrk is sufficient for the induction of pigment cell motility and underlines a role of the src family protein tyrosine kinase fyn in melanoma development and progression.

In an attempt to obtain bacteria growing on 1,2-dimethylbenzene as sole carbon and energy source two different strains were isolated. One was identified as an Arthrobacter strain, the other as a Corynebacterium strain. Corynebacterium strain C125 was further investigated. The organism was not capable to grow on 1,3- and 1,4-dimethylbenzene. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 3,4-dimethylcatechol-2,3-dioxygenase activity was found in cell extracts. When 3,4-dimethylcatechol was added to cell extract of 1,2-dimethylbenzene-grown cells, first a compound with the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate was formed and subsequently acetate was produced. It is proposed that dioxygenases are involved in the initial steps of 1,2-dimethylbenzene degradation, and ring opening proceeds via meta-cleavage.