NK cells are required for dendritic cell-based immunotherapy at the time of tumor challenge.

Increasing evidence suggests that NK cells act to promote effective T cell-based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria-dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.

Rapid interferon-gamma release from natural killer cells induced by a streptococcal commensal.

Interferon-gamma (IFN-γ) is a critical cytokine for the initiation of immune responses against a variety of infectious agents and malignancies. We found that a range of Gram-positive and Gram-negative bacteria stimulated the rapid release (<24 h) of IFN-γ from murine leukocytes. Using fluorescence activated cell sorting and cd1d(-/-) and rag1(-/-) mice, we determined that dendritic cells (DCs) and natural killer (NK) cells were primarily responsible for IFN-γ release by Streptococcus salivarius, a Gram-positive commensal, previously noted to possess potent interleukin-12 (IL-12)-inducing potential. IFN-γ release from NK cells required DC:NK membrane contact and IL-12/IL-18 expression, but was independent of lymphocyte function-associated antigen-1-mediated interactions. IFN-γ release in response to bacteria was maintained in mice deficient for Toll-like receptor (TLR)-2 and TLR-4, suggesting that bacteria activate antigen-presenting cells via multiple, redundant pathways. Together, our results suggest that Gram-positive bacteria may be useful in driving NK cell activation and T helper 1 polarization and have the potential for development as effective adjuvants.

Concentrated bovine milk whey active proteins facilitate osteogenesis through activation of the JNK-ATF4 pathway.

Concentrated fractions of low molecular weight whey proteins (1-30 kDa), that is concentrated bovine milk whey active proteins (CBP), have been found to enhance bone formation in both in vivo and clinical studies, but the underlying mechanisms are poorly understood. In this study, we found that CBP promoted osteoblastic differentiation in normal human osteoblasts, and determined the involvement of the c-jun NH2-terminal kinase (JNK)-activating transcription factor 4 (ATF4) pathway. We observed that alkaline phosphatase activity and mineralization were significantly induced by CBP treatment. In addition, mRNA expression of ATF4 was intensely elevated in CBP-treated osteoblasts, indicating that the late-phase events of differentiation were promoted. We found that CBP activated the phosphorylation of JNK and extracellular signal-regulated kinase (ERK). Furthermore, pathway analyses using the various signaling pathway-specific inhibitors revealed that JNK activation, but not ERK activation, is essential for CBP-induced mineralization and ATF4 expression. Our results indicate that the JNK-mediated ATF4 pathway is required for CBP-promotive osteogenesis.

Salivaricin G32, a Homolog of the Prototype Streptococcus pyogenes Nisin-Like Lantibiotic SA-FF22, Produced by the Commensal Species Streptococcus salivarius.

Salivaricin G32, a 2667 Da novel member of the SA-FF22 cluster of lantibiotics, has been purified and characterized from Streptococcus salivarius strain G32. The inhibitory peptide differs from the Streptococcus pyogenes-produced SA-FF22 in the absence of lysine in position 2. The salivaricin G32 locus was widely distributed in BLIS-producing S. salivarius, with 6 (23%) of 26 strains PCR-positive for the structural gene, slnA. As for most other lantibiotics produced by S. salivarius, the salivaricin G32 locus can be megaplasmid encoded. Another member of the SA-FF22 family was detected in two Streptococcus dysgalactiae of bovine origin, an observation supportive of widespread distribution of this lantibiotic within the genus Streptococcus. Since the inhibitory spectrum of salivaricin G32 includes Streptococcus pyogenes, its production by S. salivarius, either as a member of the normal oral microflora or as a commercial probiotic, could serve to enhance protection of the human host against S. pyogenes infection.

Identification of a haemolysin-like peptide with antibacterial activity using the draft genome sequence of Staphylococcus epidermidis strain A487.

Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery.

Identification of DysI, the immunity factor of the streptococcal bacteriocin dysgalacticin.

DysI is identified as the protein that confers specific immunity to dysgalacticin, a plasmid-encoded streptococcal bacteriocin. dysI is transcribed as part of the copG-repB-dysI replication-associated operon. DysI appears to function at the membrane level to prevent the inhibitory effects of dysgalacticin on glucose transport, membrane integrity, and intracellular ATP content.

Urinary soluble HLA-DR is a potential biomarker for acute renal transplant rejection.

BACKGROUND.: Urine is a potentially rich source of biomarkers for monitoring kidney dysfunction. In this study, we have investigated the potential of soluble human leukocyte antigen (sHLA)-DR in the urine for noninvasive monitoring of renal transplant patients. METHODS.: Urinary soluble HLA-DR levels were measured by sandwich enzyme-linked immunosorbent assay in 103 patients with renal diseases or after renal transplantation. sHLA-DR in urine was characterized by Western blotting and mass spectrometry. RESULTS.: Acute graft rejection was associated with a significantly elevated level of urinary sHLA-DR (P<0.0001), compared with recipients with stable graft function or healthy individuals. A receiver operating characteristic curve analysis showed the area under the curve to be 0.88 (P<0.001). At a selected threshold, the sensitivity was 80% and specificity was 98% for detection of acute renal transplant rejection. sHLA-DR was not exosomally associated and was of lower molecular weight compared with the HLA-DR expressed as heterodimer on the plasma membrane of antigen-presenting cells. CONCLUSIONS.: sHLA-DR excreted into urine is a promising indicator of renal transplant rejection.

Production of the Bsa lantibiotic by community-acquired Staphylococcus aureus strains.

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.

Mode of action of dysgalacticin: a large heat-labile bacteriocin.

OBJECTIVES: The mode of action of dysgalacticin, a large (21.5 kDa), heat-labile bacteriocin that is active against the human pathogen Streptococcus pyogenes, was investigated. METHODS: We used recombinant dysgalacticin to determine its mode of action against S. pyogenes. Antimicrobial activity of dysgalacticin was determined by MIC assays and viability counts. The extracellular pH of glucose-energized S. pyogenes cell suspensions was measured to determine the influence of dysgalacticin on glucose fermentation. To examine the effect of dysgalacticin on glucose transport, uptake of [14C]glucose and the non-metabolizable analogue [3H]2-deoxyglucose (2DG) was measured. Furthermore, the effect of dysgalacticin on membrane integrity, intracellular potassium concentration, membrane potential and [14C]serine uptake was determined. RESULTS: Dysgalacticin was bactericidal towards S. pyogenes and inhibited glucose fermentation by non-growing cell suspensions. Dysgalacticin blocked transport of both glucose and 2DG, indicating that dysgalacticin targets the phosphoenolpyruvate-dependent glucose- and mannose-phosphotransferase system (PTS) of S. pyogenes. This inhibitory activity was voltage-independent, and in addition to the inhibition of glucose transport, dysgalacticin increased the permeability of the cytoplasmic membrane mediating leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [14C]serine uptake, a membrane potential-dependent process in S. pyogenes. CONCLUSIONS: Taken together, these data indicate that dysgalacticin targets the glucose- and/or mannose-PTS as a receptor leading to inhibition of sugar uptake. As a result of this interaction, dysgalacticin perturbs membrane integrity leading to loss of intracellular K+ ions and dissipation of the membrane potential, ultimately leading to cell death.

Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor.

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not constitutive activity of B cells, but may be induced following cell: cell signaling.

Ubericin A, a class IIa bacteriocin produced by Streptococcus uberis.

Streptococcus uberis, a causal agent of bovine mastitis, produces ubericin A, a 5.3-kDa class IIa (pediocin-like) bacteriocin, which was purified and characterized. The uba locus comprises two overlapping genes: ubaA (ubericin A precursor peptide) and ubaI (putative immunity protein). Ubericin A is the first streptococcal class IIa bacteriocin to be characterized.

ef1097 and ypkK encode enterococcin V583 and corynicin JK, members of a new family of antimicrobial proteins (bacteriocins) with modular structure from Gram-positive bacteria.

Unlike the colicins, microcins and related peptide antibiotics, little is known about antibiotic proteins (M(r)>10,000) from Gram-positive bacteria, since only few examples have been described to date. In this study we used heterologous expression of recombinant proteins to access the 17 kDa antibiotic protein SA-M57 from Streptococcus pyogenes, along with two proteins of unknown function identified in publicly available databases: EF1097 from Enterococcus faecalis and YpkK from Corynebacterium jeikeium. Here we show that all three are antibiotic proteins with different spectra of antimicrobial activity that kill sensitive bacteria at nanomolar concentrations. In silico structure predictions indicate that although the three proteins share little sequence similarity, they may be composed of conserved secondary structural elements: a relatively unstructured, acidic N-terminal portion and a basic C-terminal portion characterized by two helical elements separated by a loop structure and stabilized by an essential disulphide. Expression of individual segments as well as protein chimaeras revealed that, at least in the case of YpkK, the C-terminal portion is responsible for the killing action of the protein, whereas the role of the N-terminal portion remains unclear. Both scnM57 and ef1097 appear to be widely distributed in Strep. pyogenes and Ent. faecalis (respectively), whereas ypkK is found only rarely amongst clinical isolates of C. jeikeium. Finally, we determined that the proteins kill sensitive bacteria without lysis, a feature that distinguishes them from known murolytic proteins.

Uberolysin: a novel cyclic bacteriocin produced by Streptococcus uberis.

Streptococcus uberis is commonly found in the environment and in association with various bovine body sites and is a major cause of bovine mastitis. Moreover, S. uberis is known to produce a variety of bacteriocin-like inhibitory substances, antimicrobial agents that generally inhibit closely related bacterial species. In this respect, S. uberis strain 42 has previously been shown to produce a novel nisin variant named nisin U. This paper reports that, in addition to nisin U, S. uberis strain 42 produces a second bacteriocin that induces the lysis of metabolically active, susceptible target bacteria and which has therefore been named uberolysin. Isolation of the native active antimicrobial agent revealed that uberolysin is a 7048 Da peptide that is refractory to sequence analysis by Edman degradation. Transposon mutagenesis was used to generate a uberolysin-negative mutant of S. uberis 42 and sequencing of DNA flanking the insertion site revealed, in addition to the structural gene (ublA), several open reading frames likely to be involved in post-translational modification, transport and producer self-protection (immunity), and possibly in regulation of the biosynthetic gene cluster. In addition, a pair of direct repeats that may be involved in bacteriocin acquisition were identified; indeed, ublA could be identified in 18 % of tested S. uberis strains. Enzymic hydrolysis of uberolysin was used to confirm that ublA does indeed encode the precursor of uberolysin, that an unusually short leader sequence of only six amino acids is cleaved during processing of the mature peptide and that uberolysin is post-translationally covalently modified to form a head-to-tail monocycle. Thus, uberolysin is a unique cyclic bacteriocin, belonging to the same family of bacteriocins as enterocin AS-48 and circularin A.

Circulating, soluble forms of major histocompatibility complex antigens are not exosome-associated.

In vitro studies have shown that soluble MHC (sMHC) released by cell lines is bound to nano-vesicles termed exosomes. It is thought that exosomes may represent the major reservoir of sMHC class I and II molecules in biological fluids. However, most studies have been confined to in vitro assays performed with cell lines. We show here that sMHC in the serum or plasma differs from exosome-bound sMHC in five ways: In contrast to exosome-associated sMHC, circulating sMHC is of low density, has a low apparent molecular mass (40-300 kDa) and is not detergent-labile. Moreover, the majority of MHC class II isoforms and MHC class I in blood are not physically linked and circulating HLA-DR is accessible to an antibody specific for the HLA-DR alpha-chain intracellular epitope, which is masked by its association with cellular or exosomal membranes. Finally, utilizing transcriptional activator of murine MHC class II (C2ta) promoter-mutant mice, we showed that the release of sMHC class II into the circulation is dependent on the C2ta pI promoter, but not pIII or pIV. This suggests that myeloid dendritic cells and/or macrophages, which preferentially use promoter pI of the C2ta gene, produce most of the sMHC class II found in the circulation.

Dysgalacticin: a novel, plasmid-encoded antimicrobial protein (bacteriocin) produced by Streptococcus dysgalactiae subsp. equisimilis.

Dysgalacticin is a novel bacteriocin produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 that has a narrow spectrum of antimicrobial activity directed primarily against the principal human streptococcal pathogen Streptococcus pyogenes. Unlike many previously described bacteriocins of Gram-positive bacteria, dysgalacticin is a heat-labile 21.5 kDa anionic protein that kills its target without inducing lysis. The N-terminal amino acid sequence of dysgalacticin [Asn-Glu-Thr-Asn-Asn-Phe-Ala-Glu-Thr-Gln-Lys-Glu-Ile-Thr-Thr-Asn-(Asn)-Glu-Ala] has no known homologue in publicly available sequence databases. The dysgalacticin structural gene, dysA, is located on the indigenous plasmid pW2580 of strain W2580 and encodes a 220 aa preprotein which is probably exported via a Sec-dependent transport system. Natural dysA variants containing conservative amino acid substitutions were also detected by sequence analyses of dysA elements from S. dysgalactiae strains displaying W2580-like inhibitory profiles. Production of recombinant dysgalacticin by Escherichia coli confirmed that this protein is solely responsible for the inhibitory activity exhibited by strain W2580. A combination of in silico secondary structure prediction and reductive alkylation was employed to demonstrate that dysgalacticin has a novel structure containing a disulphide bond essential for its biological activity. Moreover, dysgalacticin displays similarity in predicted secondary structure (but not primary amino acid sequence or inhibitory spectrum) with another plasmid-encoded streptococcal bacteriocin, streptococcin A-M57 from S. pyogenes, indicating that dysgalacticin represents a prototype of a new class of antimicrobial proteins.

Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis.

Streptococcus uberis is one of the principal causative agents of bovine mastitis. In this study, we report that S. uberis strain 42 produces a lantibiotic, nisin U, which is 78% identical (82% similar) to nisin A from Lactococcus lactis. The 15.6-kb nisin U locus comprises 11 open reading frames, similar in putative functionality but differing in arrangement from that of the nisin A biosynthetic cluster. The nisin U producer strain exhibits specific resistance (immunity) to nisin U and cross-resistance to nisin A, a finding consistent with the 55% sequence similarity of their respective immunity peptides. Homologues of the nisin U structural gene were identified in several additional S. uberis strains, and in each case cross-protective immunity was expressed to nisin A and to the other producers of nisin U and its variants. To our knowledge, this is the first report both of characterization of a bacteriocin by S. uberis, as well as of a member of the nisin family of peptides in a species other than L. lactis.

Bacteriocin (mutacin) production by Streptococcus mutans genome sequence reference strain UA159: elucidation of the antimicrobial repertoire by genetic dissection.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).

Comparative effects of the blue green algae Nodularia spumigena and a lysed extract on detoxification and antioxidant enzymes in the green lipped mussel (Perna viridis).

Nodularia spumigena periodically proliferates to cause toxic algal blooms with some aquatic animals enduring and consuming high densities of the blue green algae or toxic lysis. N. spumigena contains toxic compounds such as nodularin and lipopolysaccharides. This current work investigates physiological effects of exposure from bloom conditions of N. spumigena cells and a post-bloom lysis. Biochemical and antioxidative biomarkers were comparatively studied over an acute 3-day exposure. In general, a post-bloom N. spumigena lysis caused opposite physiological responses to bloom densities of N. spumigena. Specifically, increases in glutathione (GSH) and glutathione peroxidase (GPx) and decreases in glutathione S-transferase (GST) were observed from the N. spumigena lysis. In contrast, N. spumigena cell densities decreased GSH and increased GST and lipid peroxidation (LPO) in mussels. Findings also suggest that at different stages of a toxic bloom, exposure may result in toxic stress to specific organs in the mussel.

Identification of nlmTE, the locus encoding the ABC transport system required for export of nonlantibiotic mutacins in Streptococcus mutans.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic bacteriocin (mutacin) activity. In this study, we have combined bioinformatic and mutational analyses to identify the ABC transporter designated NlmTE, which is required for mutacin biogenesis in strain UA159 as well as in another mutacin producer, S. mutans N.