Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

Antigens of the type-three secretion system of Aeromonas salmonicida subsp. salmonicida prevent protective immunity in rainbow trout

Aeromonas salmonicida subsp. salmonicida is the etiologic agent of furunculosis, a frequent and significant disease of fisheries worldwide. The disease is largely controlled by commercial oil adjuvanted vaccines containing bacterins. However, the mechanisms leading to a protective immune response remain poorly understood. The type-three secretion system (T3SS) plays a central role in virulence of A. salmonicida subsp. salmonicida and thus may have an influence on the immune response of the host. The aim of this study was to evaluate the role of the T3SS antigens in mounting a protective immune response against furunculosis. Rainbow trout were intraperitoneally vaccinated in two independent experiments with bacterins prepared from a wild-type A. salmonicida strain and an isogenic strain carrying a deletion in the T3SS (ΔascV). Fish were challenged with the wt strain eight weeks after vaccination. In both trials, the survival rate of trout vaccinated with the ΔascV strain was significantly higher (23-28%) in comparison to the group vaccinated with the wt strain. High-throughput proteomics analysis of whole bacteria showed the ascV deletion in the mutant strain resulted in lower expression of all the components of the T3SS, several of which have a potential immunosuppressive activity. In a third experiment, fish were vaccinated with recombinant AcrV (homologous to the protective antigen LcrV of Yersinia) or S-layer protein VapA (control). AcrV vaccinated fish were not protected against a challenge while fish vaccinated with VapA were partially protected. The presence of T3SS proteins in the vaccine preparations decreased the level of protection against A. salmonicida infection and that AcrV was not a protective antigen. These results challenge the hypothesis that mounting specific antibodies against T3SS proteins should bring better protection to fish and demonstrate that further investigations are needed to better understand the mechanisms underlying effective immune responses against A. salmonicida infection.

Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.

Crystal chemistry and hydrogen bonding of rustumite Ca10(Si2O7)2(SiO4)(OH)2Cl2 with variable OH, Cl, F

Three samples of the skarn mineral rustumite Ca10(Si2O7)2(SiO4)(OH)2Cl2, space group C2/c, a ≈7.6, b ≈ 18.5, c ≈ 15.5 Å, β ≈ 104°, with variable OH, Cl, F content were investigated by electron microprobe, single-crystal X-ray structure refinements, and Raman spectroscopy. “Rust1LCl” is a low chlorine rustumite Ca10(Si2O7)2(SiO4)(OH1.88F0.12)(Cl1.28,OH0.72) from skarns associated with the Rize batholith near Ikizedere, Turkey. “Rust2F” is a F-bearing rustumite Ca10(Si2O7)2(SiO4)(OH1.13F0.87) (Cl1 96OH0.04) from xenoliths in ignimbrites of the Upper Chegem Caldera, Northern Caucasus, Russia. “Rust3LClF” represents a low-Cl, F-bearing rustumite Ca10(Si2O7)2(SiO4)0.87(H4O4)0.13(OH1.01F0.99) (Cl1.00 OH1.00) from altered merwinite skarns of the Birkhin massif, Baikal Lake area, Eastern Siberia, Russia. Rustumite from Birkhin massif is characterized by a significant hydrogarnet-like or fluorine substitution at the apices of the orthosilicate group, leading to specific atomic displacements. The crystal structures including hydrogen positions have been refined from single-crystal X-ray data to R1 = 0.0205 (Rust1_LCl), R1 = 0.0295 (Rust2_F), and R1 = 0.0243 (Rust3_LCl_F), respectively. Depletion in Cl and replacement by OH is associated with smaller unit-cell dimensions. The substitution of OH by F leads to shorter hydrogen bonds O-H⋯F instead of O-H⋯OH. Raman spectra for all samples have been measured and confirm slight strengthening of the hydrogen bonds with uptake of F.This study discusses the complex crystal chemistry of the skarn mineral rustumite and may provide a wider understanding of the chemical reactions related to contact metamorphism of limestones.

The role of zinc dynamics in growth hormone secretion

Human growth hormone (GH) causes a variety of physiological and metabolic effects in humans and plays a pivotal role in postnatal growth. In somatotroph cells of the anterior pituitary, GH is stored in concentrated forms in secretory granules to be rapidly released upon GH-releasing hormone stimulation. During the process of secretory granule biogenesis, self-association of GH occurs in the compartments of the early secretory pathway (endoplasmic reticulum and Golgi complex). Since this process is greatly facilitated by the presence of zinc ions, it is of importance to understand the potential role of zinc transporters that participate in the fine-tuning of zinc homeostasis and dynamics, particularly in the early secretory pathway. Thus, the role of zinc transporters in supplying the secretory pathway with the sufficient amount of zinc required for the biogenesis of GH-containing secretory granules is essential for normal secretion. This report, illustrated by a clinical case report on transient neonatal zinc deficiency, focuses on the role of zinc in GH storage in the secretory granules and highlights the role of specific zinc transporters in the early secretory pathway.

Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion.

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

Resolving Cl and SO4 Profiles in a Clay-Rich Rock Sequence

The chloride and sulfate concentration profiles in a 260 m thick clay-rich Mesozoic sediment sequence have been analyzed by various methods. Chloride data generally indicate a good consistency between different methods if anion exclusion is accounted for in leaching tests. For sulfate, however, there is an apparent inconsistency between leaching data and those obtained from the other methods, which points to the dissolution of a sulfur-bearing mineral. Traces of diagenetic gypsum seem to be a likely source, but other sulfur minerals cannot be ruled out.

Calibration of cosmogenic noble gas production in ordinary chondrites based on 36 Cl- 36 Ar ages. Part 1: Refined produced rates for cosmogenic 21 Ne and 38 Ar

We measured the concentrations and isotopic compositions of He, Ne, and Ar in bulk samples and metal separates of 14 ordinary chondrite falls with long exposure ages and high metamorphic grades. In addition, we measured concentrations of the cosmogenic radionuclides 10Be, 26Al, and 36Cl in metal separates and in the nonmagnetic fractions of the selected meteorites. Using cosmogenic 36Cl and 36Ar measured in the metal separates, we determined 36Cl-36Ar cosmic-ray exposure (CRE) ages, which are shielding-independent and therefore particularly reliable. Using the cosmogenic noble gases and radionuclides, we are able to decipher the CRE history for the studied objects. Based on the correlation 3He/21Ne versus 22Ne/21Ne, we demonstrate that, among the meteorites studied, only one suffered significant diffusive losses (about 35%). The data confirm that the linear correlation 3He/21Ne versus 22Ne/21Ne breaks down at high shielding. Using 36Cl-36Ar exposure ages and measured noble gas concentrations, we determine 21Ne and 38Ar production rates as a function of 22Ne/21Ne. The new data agree with recent model calculations for the relationship between 21Ne and 38Ar production rates and the 22Ne/21Ne ratio, which does not always provide unique shielding information. Based on the model calculations, we determine a new correlation line for 21Ne and 38Ar production rates as a function of the shielding indicator 22Ne/21Ne for H, L, and LL chondrites with preatmospheric radii less than about 65 cm. We also calculated the 10Be/21Ne and 26Al/21Ne production rate ratios for the investigated samples, which show good agreement with recent model calculations.

Role of Na/H exchange in insulin secretion by islet cells

PURPOSE OF REVIEW: Sodium/hydrogen exchangers (NHEs) are a large family of transport proteins catalyzing the exchange of cations for protons across lipid bilayer membranes. Several isoforms are expressed in β cells of the endocrine pancreas, including the recently discovered and poorly characterized isoform NHA2. This review will summarize advances in our understanding of the roles of NHEs in the regulation of insulin secretion in β cells. RECENT FINDINGS: Plasmalemmal full-length NHE1 defends β cells from intracellular acidification, but has no role in stimulus-secretion coupling and is not causally involved in glucose-induced alkalinization of the β cell. The function of a shorter NHE1 splice variant, which localizes to insulin-containing large dense core vesicles, remains currently unknown. In contrast, in-vitro and in-vivo studies indicate that the NHA2 isoform is required for insulin secretion and clathrin-mediated endocytosis in β cells. SUMMARY: Recent data highlight the importance of NHEs in the regulation of cellular pH, clathrin-mediated endocytosis and insulin secretion in β cells. Based on these studies, a pathophysiological role of NHEs in human disorders of the endocrine pancreas seems likely and should be investigated.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.