Metabolically active bacteria in Lake Kinneret

Three staining methods were used to identify metabolically active bacteria in Lake Kinneret, northern Israel: CTC, DAPI staining followed by a propanol wash, and the Molecular Probes Live/Dead stain. Positive results from these methods purport to show, respectively, actively respiring bacteria (CTC+), cells with intact nucleoids (NuCC), and cells with intact membranes (MEM+). Concomitantly, bacterial metabolic activity was measured as electron transport system (ETS) flux, O₂ uptake, activities of peptidase, β-glucosidase and lipase, and rate of leucine incorporation in monthly samples taken for 2.5 yr at a pelagic lake station. Laboratory experiments followed changes during 22 or 40 h in the percentages of ‘active’ bacteria in GF/C-filtered lake water with or without substrate enrichment or antibiotic inhibitors of cell division, or with bacterivorous protists. In lake samples, each of the staining methods detected different aspects of cellular state or metabolic activity but all 3 indicated low percentages of ‘active’ bacteria relative to total bacterial abundance. CTC+ ranged from 1.0 to 27.3% (average 5.1%), NuCC from 1.4 to 42.9% (average 8.3%) and MEM+ from 1.0 to 29.9% (average 8.8%), with no clear seasonal or spatial patterns. No significant correlations were found between the proportions of ‘active’ bacteria in lake water as determined by these methods, although such correlations were observed in the laboratory experiments. Significant correlations were obtained between ETS and O₂ uptake, peptidase and β-glucosidase, and between leucine incorporation and peptidase. ETS was significantly correlated with CTC+ and NuCC cell abundance, but not with total bacteria (DAPI counts). In contrast, peptidase activity correlated with total bacterial counts. Results of time course experiments indicated that some bacteria which initially appear to be inactive can become active when stimulated by substrate addition, even though cell division is inhibited. Grazing by protists increased the percentage of active bacteria, at least during the active predator-prey phase. Our data support the hypothesis that in natural waters usually only a small fraction (probably <20%) of the entire bacterial assemblage is strongly active metabolically at any given time. This proportion may increase dramatically with localized substrate inputs. The concept of bacterial assemblages, heterogeneous not only in terms of phylotype, but also in terms of levels of metabolic activity will need to be considered in future aquatic ecosystem models.Keywords: Respiration, Lake Kinneret, Hydrolytic enzymes, Metabolically active bacteri

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (104–105M−1) and number of binding sites (106–107sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two disease