Oral Immunotherapy With Human Secretory Immunoglobulin A Improves Survival in the Hamster Model of Clostridioides difficile Infection.

Coadministration of human secretory IgA (sIgA) together with subtherapeutic vancomycin enhanced survival in the Clostridioides difficile infection (CDI) hamster model. Vancomycin (5 or 10 mg/kg × 5 days) plus healthy donor plasma sIgA/monomeric IgA (TID × 21 days) or hyperimmune sIgA/monomeric IgA (BID × 13 days) enhanced survival. Survival was improved compared to vancomycin alone, P = .018 and .039 by log-rank Mantel-Cox, for healthy and hyperimmune sIgA, respectively. Passive immunization with sIgA (recombinant human secretory component plus IgA dimer/polymer from pooled human plasma) can be administered orally and prevents death in a partially treated CDI hamster model.

Induction of antitoxin responses in Clostridium-difficile-infected patients compared to healthy blood donors.

According to the literature Clostridium difficile antitoxins are present in up to 66% of humans. In a survey of ∼400 plasma samples from healthy blood donors we found that less than 6% were positive for anti-TcdA or anti-TcdB antitoxins. Using the same standard immunoassay protocol, we looked for IgG and IgA antitoxins in the blood and stool samples from 25 patients with C. difficile infection (CDI). Some patients with CDI had no antitoxin detected at all, while others had high levels of specific IgG- and IgA-antitoxins against both TcdA and TcdB in blood and IgA-anti-TcdA and -anti-TcdB antibodies in stool. Systemic responses to TcdB and mucosal responses to TcdA predominated. Among patients infected with the NAP1/027/BI strain, systemic IgG-anti-TcdB responses were particularly elevated. In contrast, patients infected with non-027 strains had more elevated mucosal IgA-anti-TcdA responses. Furthermore, high titer sera did not correlate with high neutralizing potential. We hypothesize that paradoxical killing of primed B-cells by antibody-mediated endosomal uptake of the Large Clostridial Toxins, TcdA and TcdB leads to clonal elimination of the fittest B-cells. If this hypothesis is confirmed, immune suppression rather than protective humoral immunity might be the consequence in some patients infected with toxigenic C. difficile.

Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

Autocatalytic cleavage of Clostridium difficile toxin B.

Clostridium difficile, the causative agent of nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis, possesses two main virulence factors: the large clostridial cytotoxins A and B. It has been proposed that toxin B is cleaved by a cytosolic factor of the eukaryotic target cell during its cellular uptake. Here we report that cleavage of not only toxin B, but also all other large clostridial cytotoxins, is an autocatalytic process dependent on host cytosolic inositolphosphate cofactors. A covalent inhibitor of aspartate proteases, 1,2-epoxy-3-(p-nitrophenoxy)propane, completely blocked toxin B function on cultured cells and was used to identify its catalytically active protease site. To our knowledge this is the first report on a bacterial toxin that uses eukaryotic signals for induced autoproteolysis to deliver its toxic domain into the cytosol of target cells. On the basis of our data, we present an integrated model for the uptake and inositolphosphate-induced activation of toxin B.

Treatment and Prevention of Recurrent Clostridium difficile Infection with Functionalized Bovine Antibody-Enriched Whey in a Hamster Primary Infection Model.

Toxin-induced Clostridium difficile infection (CDI) is a major disease characterized by severe diarrhea and high morbidity rates. The aim with this study was to develop an alternative drug for the treatment of CDI. Cows were repeatedly immunized to establish specific immunoglobulin G and A titers against toxins A (TcdA) and B (TcdB) and against C. difficile cells in mature milk or colostrum. The effect of three different concentrations of anti-C. difficile whey protein isolates (anti-CD-WPI) and the standard of care antibiotic vancomycin were investigated in an animal model of CD infected hamsters (6 groups, with 10 hamsters each). WPI obtained from the milk of exactly the same cows pre-immunization and a vehicle group served as negative controls. The survival of hamsters receiving anti-CD-WPI was 50, 80 and 100% compared to 10 and 0% for the control groups, respectively. Vancomycin suppressed the growth of C. difficile and thus protected the hamsters at the time of administration, but 90% of these hamsters nevertheless died shortly after discontinuation of treatment. In contrast, the surviving hamsters of the anti-CD-WPI groups survived the entire study period, although they were treated for only 75 h. The specific antibodies not only inactivated the toxins for initial suppression of CDI, but also provoked the inhibition of C. difficile growth after discontinuation, thus preventing recurrence. Oral administration of anti-CD-WPI is a functional therapy of CDI in infected hamsters for both primary treatment and prevention of recurrence. Thus, anti-CD-WPI could address the urgent unmet medical need for treating and preventing recurrent CDI in humans.

Revised nomenclature of Clostridium difficile toxins and associated genes.

Several different nomenclatures have been applied to the Clostridium difficile toxins and their associated genes. This paper summarizes the new nomenclature that has been agreed to by the research groups currently active in the field. The revised nomenclature includes C. difficile toxins and other related large clostridial toxins produced by Clostridium sordellii and Clostridium novyi, and corresponding toxin genes, as well as toxin production types of C. difficile strains.

Rac1 and PAK1 are upstream of IKK-epsilon and TBK-1 in the viral activation of interferon regulatory factor-3.

The anti-viral type I interferon (IFN) response is initiated by the immediate induction of IFN beta, which is mainly controlled by the IFN-regulatory factor-3 (IRF-3). The signaling pathways mediating viral IRF-3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF-3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFN beta promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF-3, we have identified the kinase p21-activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF-3 activators, I kappa B kinase- and TANK-binding kinase-1, establishing a first canonical virus-induced IRF-3 activating pathway.

Metabotropic glutamate receptors activate phospholipase D in astrocytes through a protein kinase C-dependent and Rho-independent pathway.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that mediate phospholipase D (PLD) activation in brain, but the mechanism underlying this response remains unclear. Here we used primary cultures of astrocytes as a cell model to explore the mechanism that links mGluRs to PLD. Glutamate activated both phospholipase C (PLC) and PLD with equal potency and this effect was mimicked by L-cysteinesulfinic acid, a putative neurotransmitter previously shown to activate mGluRs coupled to PLD, but not PLC, in adult brain. PLD activation by glutamate was dependent on Ca(2+) mobilization and fully blocked by both protein kinase C (PKC) inhibitors and PKC down-regulation, suggesting that PLD activation is secondary to PLC stimulation. Furthermore, brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation, partially inhibited the activation of PLD by glutamate. By contrast, pretreatment of astrocytes with Clostridium difficile toxin B, which inactivates small G proteins of the Rho family (Rho, Rac, and Cdc42), had no effect on PLD stimulation by glutamate. Taken together, these results indicate that PLD activation by mGluRs in astrocytes is dependent on PKC and small G proteins of the ARF family, but does not require Rho proteins.

Four distinct structural domains in Clostridium difficile toxin B visualized using SAXS.

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of approximately 275 A. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.

Expression and prognostic relevance of annexin A3 in prostate cancer.

OBJECTIVES: By differential quantitative protein expression, it has previously been shown that annexin A3 (ANXA3) expression is associated with prostate cancer. However little is known about the role and biology of ANXA3 in the human prostate. The aim of this study was to thoroughly analyze ANXA3 expression patterns and its potential as a prognostic marker in a large set of benign, preneoplastic, and neoplastic prostate tissue samples. METHODS: Immunohistochemistry-based ANXA3 protein expression was analyzed for 1589 prostate cancers as well as smaller subsets of benign epithelium and high-grade prostatic intraepithelial neoplasia (PIN) in a tissue microarray format. RESULTS: All samples of benign prostatic epithelium and PIN showed ANXA3 protein expression, with PIN lesions showing a decreased staining intensity compared with benign epithelium (p<0.0001). In cancer, ANXA3 protein expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT stage and Gleason score (p<0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor and enabled substratification of the large group of intermediate-risk patients (n=969) into high- and low-risk subgroups. CONCLUSIONS: ANXA3 represents a promising candidate tissue marker, and when combined with the standard prognostic parameters, is suggested to provide a more precise prediction of prognosis in the individual patient, therefore harboring the potential to contribute to future patient management.

Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells.

Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB(10463) between Leu(543) and Gly(544) and the naturally occurring variant TcdB(8864) between Leu(544) and Gly(545). The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB(10463)) and 62 761 Da (TcdB(8864))] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB(10463)) and 206 245 Da (TcdB(8864))] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.

The IStron CdISt1 of Clostridium difficile: molecular symbiosis of a group I intron and an insertion element.

The IStron CdISt1 was first discovered as an insertion into the tcdA gene of the clinical isolate C34. It combines structural and functional properties of a group I intron at its 5'-end with those of an insertion element at its 3'-end. Up to date four different types could be found, mainly differing in their IS-element portions. Contrasting classical group I introns, CdISt1 is always integrated in ORFs encoding bacterial protein. In case CdISt1 had only the IS-element function such insertion would inactivate the protein encoded by the host gene. It is only due to the self-splicing activity of the group I intron parts that CdISt1 integration does not abolish protein function. Both elements seem to exist in molecular symbiosis and CdISt1 could thus be a prototype of a novel class of genetic elements. Moreover, integration of the CdISt1 into the genome could be advantageous for the bacterium, a motor function for evolution of bacterial proteins is discussed. In clinical practice CdISt1 might well serve as a tool for epidemiological studies of C. difficile infections.

Clostridium difficile IStron CdISt1: discovery of a variant encoding two complete transposase-like proteins.

Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III. Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.

Activation of NF-kappaB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades.

Yersinia enterocolitica triggers activation of the nuclear factor (NF)-kappaB and production of the proinflammatory chemokine interleukin (IL)-8 in intestinal epithelial cells. This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells. Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho-GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF-kappaB by invasin. Invasin activated the mitogen activated protein kinases (MAPK) p38 and c-Jun N-terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK). The functional relevance of these pathways for invasin-mediated IL-8 expression was assessed by protein kinase inhibitors and dominant-negative kinase mutants. While NF-kappaB and JNK contribute to IL-8 transcription, p38 MAPK also acts through stabilization of IL-8 mRNA, as confirmed by quantitative RT-PCR and electrophoretic mobility shift assays. Transfection experiments with I-kappaB kinase (IKK)1 and IKK2 mutants indicate that the release of NF-kappaB from its cytoplasmic inhibitor I-kappaB and its translocation into the nucleus is mediated by these kinases. Our data identify Rac1 as a key intermediate in invasin-triggered IL-8 synthesis and demonstrate that maximum IL-8 induction involves several MAP kinase cascades.