Sensing properties of MtrB-MtrA of Corynebacterium glutamicum: a two-component system involved in the osmo- and chill stress response

Being an immobile Gram-positive soil bacterium, Corynebacterium glutamicum has to cope with dramatic changes of the environmental conditions like an altered osmolarity or temperature. It was recently shown, that the MtrB-MtrA two-component system of this bacterium mediates the expression regulation of osmo- and chill stress-related genes in response to an increased medium osmolarity or decreased environmental temperature. The response regulator of this system, MtrA, was shown to act as an activator of the genes proP, betP, and lcoP as well as a repressor of the mscL gene. These results led to the question, which physico-chemical signals are recognized by MtrB, in order to mediate the expression regulation of genes in response to changes of the environmental osmolarity and temperature. To avoid the high complexity of the cellular system, an in vitro system was established for the detailed analyses of the sensory properties of MtrB. Using E. coli liposomes enriched with MtrB, the basic characteristics of the bacterial two-component system could be detected. These included the autokinase activity of membrane-bound MtrB, the phosphoryl group transfer to soluble MtrA, and the MtrB-catalyzed dephosphorylation of MtrA-P. By use of the artificial membrane system the influence of a systematic variation of signals related to osmo- and chill stress conditions could be performed. The autokinase activity of MtrB turned out to be significantly stimulated by the presence of numerous osmolytes including sugars, amino acids, compatible solutes, and high molecular weight PEGs. In addition, the histidine kinase was shown to be strongly activated by exposure to low temperature. By fusion of proteoliposomes made from E. coli lipid with synthetic POPG, the sensory properties of MtrB furthermore turned out to significantly depend on the composition of the membrane surrounding. In order to detect the sensing domain of MtrB, various truncated derivatives of the protein were constructed. In contrast to most bacterial histidine kinases, which are thought to exhibit their sensory function within the extracytoplasmic loop, MtrB was shown to sense independently of this external domain. Taken together, the data of this work led to the model, that MtrB senses environmental hyperosmotic stress either as an increased osmolarity of the cytoplasm, or as altered membrane properties. Environmental chill conditions are probably sensed as an altered physical state of the membrane

Pseudomonas aeruginosa Increases Formation of Multidrug-Tolerant Persister Cells in Response to Quorum-Sensing Signaling Molecules▿ †

Bacterial persister cells constitute a small portion of a culture which is tolerant to killing by lethal doses of bactericidal antibiotics. These phenotypic variants are formed in numerous bacterial species, including those with clinical relevance like the opportunistic pathogen Pseudomonas aeruginosa. Although persisters are believed to contribute to difficulties in the treatment of many infectious diseases, the underlying mechanisms affecting persister formation are not well understood. Here we show that even though P. aeruginosa cultures have a significantly smaller fraction of multidrug-tolerant persister cells than cultures of Escherichia coli or Staphylococcus aureus, they can increase persister numbers in response to quorum-sensing-related signaling molecules. The phenazine pyocyanin (and the closely related molecule paraquat) and the acyl-homoserine lactone 3-OC12-HSL significantly increased the persister numbers in logarithmic P. aeruginosa PAO1 or PA14 cultures but not in E. coli or S. aureus cultures

In Vitro Analysis of the Two-Component System MtrB-MtrA from Corynebacterium glutamicum

The two-component system MtrBA is involved in the osmostress response of Corynebacterium glutamicum. MtrB was reconstituted in a functionally active form in liposomes and showed autophosphorylation and phosphatase activity. In proteoliposomes, MtrB activity was stimulated by monovalent cations used by many osmosensors for the detection of hypertonicity. Although MtrB was activated by monovalent cations, they lead in vitro to a general stabilization of histidine kinases and do not represent the stimulus for MtrB to sense hyperosmotic stress

Correction: Moeker, N.; et al. Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings. Antibodies 2017, 6, 15

The authors wish to make the following correction to the Conflict of Interests section in their published paper[...

Primary CD33-targeting CAR-NK cells for the treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is a malignant disorder derived from neoplastic myeloid progenitor cells characterized by abnormal proliferation and differentiation. Although novel therapeutics have recently been introduced, AML remains a therapeutic challenge with insufficient cure rates. In the last years, immune-directed therapies such as chimeric antigen receptor (CAR)-T cells were introduced, which showed outstanding clinical activity against B-cell malignancies including acute lymphoblastic leukemia (ALL). However, the application of CAR-T cells appears to be challenging due to the enormous molecular heterogeneity of the disease and potential long-term suppression of hematopoiesis. Here we report on the generation of CD33-targeted CAR-modified natural killer (NK) cells by transduction of blood-derived primary NK cells using baboon envelope pseudotyped lentiviral vectors (BaEV-LVs). Transduced cells displayed stable CAR-expression, unimpeded proliferation, and increased cytotoxic activity against CD33-positive OCI-AML2 and primary AML cells in vitro. Furthermore, CD33-CAR-NK cells strongly reduced leukemic burden and prevented bone marrow engraftment of leukemic cells in OCI-AML2 xenograft mouse models without observable side effects

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichia coli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress