Zinc Sensing Receptor Signaling, Mediated by GPR39, Reduces Butyrate-Induced Cell Death in HT29 Colonocytes via Upregulation of Clusterin

Zinc enhances epithelial proliferation, protects the digestive epithelial layer and has profound antiulcerative and antidiarrheal roles in the colon. Despite the clinical significance of this ion, the mechanisms linking zinc to these cellular processes are poorly understood. We have previously identified an extracellular Zn2+ sensing G-protein coupled receptor (ZnR) that activates Ca2+ signaling in colonocytes, but its molecular identity as well as its effects on colonocytes' survival remained elusive. Here, we show that Zn2+, by activation of the ZnR, protects HT29 colonocytes from butyrate induced cell death. Silencing of the G-protein coupled receptor GPR39 expression abolished ZnR-dependent Ca2+ release and Zn2+-dependent survival of butyrate-treated colonocytes. Importantly, GPR39 also mediated ZnR-dependent upregulation of Na+/H+ exchange activity as this activity was found in native colon tissue but not in tissue obtained from GPR39 knock-out mice. Although ZnR-dependent upregulation of Na+/H+ exchange reduced the cellular acid load induced by butyrate, it did not rescue HT29 cells from butyrate induced cell death. ZnR/GPR39 activation however, increased the expression of the anti-apoptotic protein clusterin in butyrate-treated cells. Furthermore, silencing of clusterin abolished the Zn2+-dependent survival of HT29 cells. Altogether, our results demonstrate that extracellular Zn2+, acting through ZnR, regulates intracellular pH and clusterin expression thereby enhancing survival of HT29 colonocytes. Moreover, we identify GPR39 as the molecular moiety of ZnR in HT29 and native colonocytes

Substitution by Inosine at the 3′-Ultimate and Penultimate Positions of 16S rRNA Gene Universal Primers

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3′-ultimate or the 3′-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities. © 2010 Springer Science+Business Media, LLC

Microbial transcriptome profiling of black band disease in a Faviid coral during a seasonal disease peak

The etiology of black band disease (BBD), a persistent, globally distributed coral disease characterized by a dark microbial mat, is still unclear. A metatranscriptomics approach was used to unravel the roles of the major mat constituents in the disease process. By comparing the transcriptomes of the mat constituents with those of the surface microbiota of diseased and healthy corals, we showed a shift in bacterial composition and function in BBD-affected corals. mRNA reads of Cyanobacteria, Bacteroidetes and Firmicutes phyla were prominent in the BBD mat. Cyanobacterial adenosylhomocysteinase, involved in cyanotoxin production, was the most transcribed gene in the band consortium. Pathogenic and non-pathogenic forms of Vibrio spp., mainly transcribing the thiamine ABC transporter, were abundant and highly active in both the band and surface tissues. Desulfovibrio desulfuricans was the primary producer of sulfide in the band. Members of the Bacilli class expressed high levels of rhodanese, an enzyme responsible for cyanide and sulfide detoxification. These results offer a first look at the varied functions of the microbiota in the disease mat and surrounding coral surface and enabled us to develop an improved functional model for this disease.Published versio

Changes in the bacterial community associated with black band disease in a Red Sea coral, Favia sp., in relation to disease phases

© Inter-Research 2015, www.int-res.com. Changes of the black band disease (BBD)-associated microbial consortium on the surface of a Favia sp. coral colony were assessed in relation to the different disease phases. A number of highly active bacterial groups changed in numbers as the BBD disease signs changed. These included Gamma- and Epsilonproteobacteria, Bacteroidetes and Firmicutes groups. One cyanobacterium strain, BGP10-4ST (FJ210722), was constantly present in the disease interface and adjacent tissues of the affected corals, regardless of disease phase. The dynamics of the operational taxonomic units (OTUs) of this BBD-specific strain provide a marker regarding the disease phase. The disease's active phase is characterized by a wide dark band progressing along the tissue-skeleton interface and by numerous bacterial OTUs. Cyanobacterial OTUs decreased in numbers as the disease signs waned, perhaps opening a niche for additional microorganisms. Even when black band signs disappeared there was a consistent though low abundance of the BBD-specific cyanobacteria (BGP10-4ST), and the microbial community of the disease-skeleton interface remained surprisingly similar to the original band community. These results provide an indication that the persistence of even low numbers of this BBD-specific cyanobacterium in coral tissues during the non-active (or subclinical) state could facilitate reinitiation of BBD signs during the following summer. This may indicate that this bacterium is major constituent of the disease and that its persistence and ability to infiltrate the coral tissues may act to facilitate the assembly of the other BBD-specific groups of bacteria

The Possible Role of Cyanobacterial Filaments in Coral Black Band Disease Pathology

Black band disease (BBD), characterized by a black mat or line that migrates across a coral colony leaving behind it a bare skeleton, is a persistent disease affecting massive corals worldwide. Previous microscopic and molecular examination of this disease in faviid corals from the Gulf of Eilat revealed a number of possible pathogens with the most prominent being a cyanobacterium identified as Pseudoscillatoria coralii. We examined diseased coral colonies using histopathological and molecular methods in order to further assess the possible role of this cyanobacterium, its mode of entry, and pathological effects on the coral host tissues. Affected areas of colonies with BBD were sampled for examination using both light and transmission electron microscopies. Results showed that this dominant cyanobacterium was found on the coral surface, at the coral-skeletal interface, and invading the polyp tissues and gastrovascular cavity. Although tissues surrounding the invasive cyanobacterial filaments did not show gross morphological alterations, microscopic examination revealed that the coral cells surrounding the lesion were dissociated, necrotic, and highly vacuolated. No amoebocytes were evident in the mesoglea of affected tissues suggesting a possible repression of the coral immune response. Morphological and molecular similarity of the previously isolated BBD-associated cyanobacterium P. coralii to the current samples strengthens the premise that this species is involved in the disease in this coral. These results indicate that the cyanobacteria may play a pivotal role in this disease and that the mode of entry may be via ingestion, penetrating the coral via the gastrodermis, as well as through the skeletal-tissue interface. © 2013 Springer Science+Business Media New York

Richness and diversity in dust stormborne biomes at the Southeast Mediterranean

Dust storms include particulate matter that is transported over land and sea with biota that could impact downwind ecosystems. In addition to the physico-chemical compositions, organismal diversities of dust from two storm events in southern Israel, December 2012 (Ev12) and January 2013 (Ev13), were determined by pyro-sequencing using primers universal to 16S and 18S rRNA genes and compared. The bio-assemblages in the collected dust samples were affiliated with scores of different taxa. Distinct patterns of richness and diversity of the two events were influenced by the origins of the air masses: Ev13 was rich with reads affiliated to Betaproteobacteria and Embryophyta, consistent with a European origin. Ev12, originated in north-Africa, contained significantly more of the Actinobacteria and fungi, without conifers. The abundance of bacterial and eukaryotic reads demonstrates dissemination of biological material in dust that may impose health hazards of pathogens and allergens, and influence vegetation migration throughout the world.Published versio

Vibrio sp. as a potentially important member of the Black Band Disease (BBD) consortium in Favia sp. corals

Black Band Disease (BBD) is a well-described disease plaguing corals worldwide. It has been established that ecological and environmental stress factors contribute to the appearance and progression of the disease, believed to be caused by a diverse microbial consortium. We have identified and characterized Vibrio sp. associated with BBD in Eilat reef corals using both culture-dependent and -independent methods. Direct sampling using 16S rRNA gene clone libraries showed seasonal dynamics in the diversity of BBD-associated Vibrios. In the two sampling periods, BBD-associated Vibrio clones showed similarities to different groups: October samples were similar to known pathogens, while December samples were similar to general aquatic Vibrio sp. Cultured bacterial isolates of Vibrio sp. were highly homologous (≥99%) to previously documented BBD-associated bacteria from the Caribbean, Bahamas and Red Seas, and were similar to several known coral pathogens, such as Vibrio coralliilyticus. The proteolytic activity of Vibrio sp., as measured using casein- and azocasein-based assays, directly correlated with temperature elevation and peaked at 26-28 °C, with the microorganisms producing more proteases per bacterial cell or increasing the rate of proteolytic activity of the same proteases (potentially metalloproteases). This activity may promote coral tissue necrosis and aid in ensuing progression of the coral BBD. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved