A protein with protective properties against the cellular defense reactions in insects

The molecular mechanism of how insects recognize intruding microorganisms and parasites and distinguish them from own body structures is not well known. We explored evolutionary adaptations in an insect parasitoid host interaction to identify components that interfere with the recognition of foreign objects and cellular encapsulation. Because some parasitoids provide protection for the developing wasp in the absence of an overt suppression of the insect host defense, we analyzed the surface of eggs and symbiotic viruses for protective properties. Here we report on the molecular cloning of a 32-kDa protein (Crp32) that is one of the major protective components. It is produced in the calyx cells of the female wasp ovaries and attached to the surface of the egg and other particles including polydnaviruses. The recombinant protein confers protection to coated objects in a cellular encapsulation assay suggesting that a layer of Crp32 may prevent cellular encapsulation reactions by a local inactivation of the host defense system.

A continent of plant defense peptide diversity: Cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym : Braconidae)

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparositoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidose. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C rubecula PDV-protein (Crp32). Arch. Insect Biochem. Physiol. 53:92-100, 2003. (C) 2003 Wiley-Liss, Inc.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

Discovery, structure and biological activities of the cyclotides

The cyclotides are a family of small disulfide rich proteins that have a cyclic peptide backbone and a cystine knot formed by three conserved disulfide bonds. The combination of these two structural motifs contributes to the exceptional chemical, thermal and enzymatic stability of the cyclotides, which retain bioactivity after boiling. They were initially discovered based on native medicine or screening studies associated with some of their various activities, which include uterotonic action, anti-HIV activity, neurotensin antagonism, and cytotoxicity. They are present in plants from the Rubiaceae, Violaceae and Cucurbitaccae families and their natural function in plants appears to be in host defense: they have potent activity against certain insect pests and they also have antimicrobial activity. There are currently around 50 published sequences of cyclotides and their rate of discovery has been increasing over recent years. Ultimately the family may comprise thousands of members. This article describes the background to the discovery of the cyclotides, their structural characterization, chemical synthesis, genetic origin, biological activities and potential applications in the pharmaceutical and agricultural industries. Their unique topological features make them interesting from a protein folding perspective. Because of their highly stable peptide framework they might make useful templates in drug design programs, and their insecticidal activity opens the possibility of applications in crop protection.

Functional analysis of the a-defensin disulfide array in mouse cryptdin-4

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Capsule and fimbria interaction in Klebsiella pneumoniae

The capsular polysaccharide and type I fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate D-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type I fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

Nitric oxide synthase inhibition in sepsis? Lessons learned from large-animal studies

Nitric Oxide (NO) plays a controversial role in the pathophysiology of sepsis and septic shock. Its vasodilatory effects are well known, but it also has pro- and antiinflammatory properties, assumes crucial importance in antimicrobial host defense, may act as an oxidant as well as an antioxidant, and is said to be a vital poison for the immune and inflammatory network. Large amounts of NO and peroxynitrite are responsible for hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis, and ultimately multiorgan failure. Therefore, NO synthase (NOS) inhibitors were developed to reverse the deleterious effects of NO. Studies using these compounds have not met with uniform success however, and a trial using the nonselective NOS inhibitor N-G-methyl-L-arginine hydrochloride was terminated prematurely because of increased mortality in the treatment arm despite improved shock resolution. Thus, the issue of NOS inhibition in sepsis remains a matter of debate. Several publications have emphasized the differences concerning clinical applicability of data obtained from unresuscitated, hypodynamic rodent models using a pretreatment approach versus resuscitated, hyperdynamic models in high-order species using posttreatment approaches. Therefore, the present review focuses on clinically relevant large-animal studies of endotoxin or living bacteria-induced, hyperdynamic models of sepsis that integrate standard day-today care resuscitative measures.

Enhanced clearance of Candida albicans from the oral cavities of mice following oral administration of Lactobacillus acidophilus

Orally administered live Lactobacillus acidophilus was assessed for its capacity to enhance clearance from the oral cavity of DBA/2 mice shown previously to be 'infection prone'. L. acidophilus fed to DBA/2 mice significantly shortened the duration of colonization of the oral cavity compared to controls. Enhanced clearance of Candida albicans correlated with both early mRNA gene expression for interleukin (IL)-4 and interferon (IFN)-gamma and expression of their secreted products in cultures of cervical lymph nodes stimulated with Candida antigen. In addition rapid clearance correlated with higher levels of IFN-gamma and nitric oxide in saliva. Delayed clearance, less pronounced levels of the cytokine response, saliva IFN-gamma and nitric oxide, and later mRNA expression for IL-4 and IFN-gamma relative to feeding with the L. acidophilus isolate were noted in mice fed a different Lactobacillus isolate (L. fermentum). These observations indicate significant variations in individual isolates to activate the common mucosal system.

S100A8 chemotactic protein is abundantly increased, but only a minor contributor to LPS-induced, steroid resistant neutrophilic lung inflammation in vivo

Neutrophilic lung inflammation is an essential component of host defense against diverse eukaryotic and prokaryotic pathogens, but in chronic inflammatory lung diseases, such as chronic obstructive lung disease (COPD), severe asthma, cystic fibrosis, and bronchiolitis, it may damage the host. Glucocorticosteroids are widely used in these conditions and in their infectious exacerbations; however, the clinical efficacy of steroids is disputed. In this study, we used a proteomic approach to identify molecules contributing to neutrophilic inflammation induced by transnasal administration of lipopolysaccharide (LPS) that were also resistant to the potent glucocorticosteroid dexamethasone (Dex). We confirmed that Dex was biologically active at both the transcript (suppression of GM-CSF and TNFalpha transcripts) and protein levels (induction of lipocortin) and used 2D-PAGE/MALDI-TOF to generate global expression profiles, identifying six LPS-induced proteins that were Dex resistant. Of these, S100A8, a candidate neutrophil chemotactic factor, was profiled in detail. Steroid refractory S100A8 expression was highly abundant, transcriptionally regulated, secreted into lung lavage fluid and immunohistochemically localized to tissue infiltrating neutrophils. However, in marked contrast to other vascular beds, neutralizing antibodies to S100A8 had only a weak anti-neutrophil recruitment effect and antibodies against the related S100A9 were ineffective. These data highlight the need for extensive in vivo profiling of proteomically identified candidate molecules and demonstrates that S100A8, despite its abundance, resistance to steroids and known chemotactic activity, is unlikely to be an important determinant of LPS-induced neutrophilic lung inflammation in vivo.

Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene-targeting

Cell-mediated immunity is important for anti-Candida host defence in mucosal tissues. In this study we used cytokine-specific gene knockout mice to investigate the requirement for T helper type 1 (Th1) and Th2 cytokines in recovery from oral candidiasis. Knockout mice used in this study included interleukin-4 (IL-4), IL-10, IL-12p40, interferon-gamma (IFN-gamma), and tumour necrosis factor (TNF). The mice were challenged either orally or systemically with Candida albicans yeasts, and levels of colonization were determined. IL-12p40 knockout mice developed chronic oropharyngeal candidiasis, but were not more susceptible to systemic challenge. On the other hand, TNF knockout mice displayed increased susceptibility to both oral and systemic challenge, but only in the acute stages of infection. TNF apparently has a protective effect in the acute stages of both oral and systemic candidiasis, whereas IL-12p40 is essential for recovery from oral but not systemic candidiasis. The role of IL-12p40, and its relation to T-cell-mediated responses remain to be determined.

Structural and functional characterization of the conserved salt bridge in mammalian Paneth cell alpha-defensins - Solution structures of mouse cryptdin-4 AND (E15D)-cryptdin-4

Defensins are mediators of mammalian innate immunity, and knowledge of their structure-function relationships is essential for understanding their mechanisms of action. We report here the NMR solution structures of the mouse Paneth cell α-defensin cryptdin-4 (Crp4) and a mutant (E15D)-Crp4 peptide, in which a conserved Glu15 residue was replaced by Asp. Structural analysis of the two peptides confirms the involvement of this Glu in a conserved salt bridge that is removed in the mutant because of the shortened side chain. Despite disruption of this structural feature, the peptide variant retains a well defined native fold because of a rearrangement of side chains, which result in compensating favorable interactions. Furthermore, salt bridge-deficient Crp4 mutants were tested for bactericidal effects and resistance to proteolytic degradation, and all of the variants had similar bactericidal activities and stability to proteolysis. These findings support the conclusion that the function of the conserved salt bridge in Crp4 is not linked to bactericidal activity or proteolytic stability of the mature peptide.