The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsC(Pcc) and RcsD(Pcc), and a response regulator RcsB(Pcc) as key components of this system, and an additional small lipoprotein RcsF(Pcc). This study indicates that inactivation of rcsC(Pcc), rcsD(Pcc) and rcsB(Pcc) enhances production of virulence factors with the highest effect detected for rcsB(Pcc). Interestingly, mutation of rcsF(Pcc) has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsB(Pcc) response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.     

In vitro potency,pharmacokinetic profiles and pharmacological activity of optimized anti-IL-21R antibodies in a mouse model of lupus

Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V_L CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas^lpr mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC_0-∞) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2–3-fold or 4–7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas^lpr mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.Key words: affinity maturation, phage display, monoclonal antibody, autoimmunity, lupus, IL-21, IL-21R, pharmacokinetics, anti-product antibodies     

History of anthropogenically mediated eutrophication of Lake Peipsi as revealed by the stratigraphy of fossil pigments and molecular size fractions of pore-water dissolved organic matter

We investigated stratigraphic changes in fossil pigments and the molecular structure of the UV-absorbing fraction of pore-water dissolved organic matter in a sedimentary record from Lake Peipsi (Estonia/Russia) temporally covering the 20th century. The aims of the study were to define the onset of eutrophication in the lake and to track its course. An attempt was also made to reconstruct lake conditions before the intensive nutrient loading began. Fossil pigment analysis indicated that the eutrophication of the lake started in the 1960s and accelerated in the 1970s. Sedimentary pigments also indicate a continuing tendency of the lake ecosystem towards eutrophy in the 1980s and 1990s. However, changes in the molecular size structure of pore-water dissolved organic matter indicated that the contribution of autochthonous matter to the organic pool of the lake ecosystem had already started to increase around the end of the 1930s. We conclude that this rise was generated by a coincidence of several anthropogenic and natural factors. The pore-water data also show that a slight relative reduction in the autochthonous organic matter took place in the 1990s. A discordance in the paleodata obtained for the beginning of the 20th century complicates clear conclusions about earlier conditions in the lake. On the one hand, the qualitative characteristics of pore-water dissolved organic matter and the low concentration of chlorophyll a indicate that the phytoplankton biomass was low in Lake Peipsi during that period. On the other hand, the concentrations of marker pigments of specific phytoplankton groups are high, comparable with the values in the recent sediments. Possible reasons for the high levels of these pigments in the early 1900s sediments, such as a shift in the preservation conditions of organic substances and their transport from the lake’s catchment, are discussed.     

Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS,IL-4, and IL-21, but not CD28, IL-7, and IL-15 responses

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4(+) T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Ralpha expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.     

AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production

Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239–246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.