A mycobacterial enzyme essential for cell division synergizes with resuscitation-promoting factor

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein-protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall-targeting beta-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein-protein interaction as one way of regulating its activity.     

SepF, a novel FtsZ-interacting protein required for a late step in cell division

Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.     

The role of cytokines in mycobacterial infection

Mycobacterial infections are a major cause of morbidity and mortality worldwide. The pathogenesis of infection and the mechanisms for the development of protective immunity are poorly known, but cytokines appear to play an important role in the modulation of the immune response. Evidence exists for the role of tumor necrosis factor (TNF-), granulocyte macrophage colony stimulating factor (GM-CSF) and interferon-gamma (IFN-) in the host defense against mycobacteria. In this article we discuss recent findings about the role of cytokines in leprosy, tuberculosis andMycobacterium avium infection, usingin vitro andin vivo human and murine data.Abbreviations M.TB Mycobacterium tuberculois - IFN- interferon gamma - TNF- tumor necrosis factor-alpha - LAM Lipoarabinomannam - IL-8 Interleukin-8 - IL-6 Interleukin-6 - IL-2 Interleukin-2 - CMI cell-mediated immunity - PPD purified protein derivative of tuberculin - MAC Mycobacterium avium complex - AIDS Acquired Immunodeficiency Syndrome - GM-CSF granulocyte macrophage colony stimulating factor - IL-10 Interleukin-10 - TGF transforming growth factor ₁     

The role of Toll-like receptors in immunity against mycobacterial infection

Recent work implicates Toll-like receptor (TLR) proteins as regulators of innate immune cell activation induced by Mycobacterium tuberculosis, which continues to ravage nearly one-third of the world's population. Novel insights into how TLR proteins may dictate the nature and extent of cellular immune responses against this pathogen will be discussed.     

The regulatory role of silicon in cell division

Several reports have suggested that silicon has an activating effect on cell proliferation. In order to test this hypothesis, both peripheral human lymphocytes and LDV/7 lymphoblast cells were cultured in the presence of a compound composed of monomethylsilanetriol (silanol), a soluble organic form of silicon, and serine. This molecule stimulates peripheral lymphocyte proliferation at an optimal concentration of 10 mg of silicon per liter of culture medium; in identical conditions, it inhibits the growth of lymphoblasto?d cells (p less than 0.001). Silanol-serine also inhibits the growth of PHA stimulated lymphocytes. The effect of silicon on cell growth has a negative correlation (p less than 0.001) with the mitotic activity of cultured cells: the more intense the latter, the stronger is the inhibitory effect of silanol-serine. This would suggest a regulatory role of this compound on the cell cycle.     

The essential role of phosphatidylglycerol in photosynthesis

Since the first identification of phosphatidylglycerol in Scenedesmus by Benson and Maruo in 1958, researchers have studied many biological functions of this phospholipid. Genetic, biochemical, and structural studies of photosynthetic organisms have revealed that phosphatidylglycerol is crucial to the photosynthetic transport of electrons, the development of chloroplasts, and tolerance to chilling. In this review, we summarize our present understanding of the biochemical and physiological functions of phosphatidylglycerol in cyanobacteria and higher plants. Submitted to the special issue in honor of Andrew A. Benson     

The role of cAMP in controlling yeast cell division

The studies on the cAMP-requiring mutants and their suppressors in the yeast, Saccharomyces cerevisiae, revealed that cAMP-dependent protein phosphorylation is involved in the G1 phase of the cell cycle, in conjugation, and in the post-meiotic stage of sporulation, and that inhibition of cAMP-dependent protein phosphorylation is required to induce meiotic division.     

The essential role of lipids in Alzheimer's disease

In the absence of efficient diagnostic and therapeutic tools, Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Although the precise cause of AD is still unknown, soluble β-amyloid (Aβ) oligomers are considered the proximate effectors of the synaptic injury and neuronal death occurring in the early stages of AD. Aβ oligomers may directly interact with the synaptic membrane, leading to impairment of synaptic functions and subsequent signalling pathways triggering neurodegeneration. Therefore, membrane structure and lipid status should be considered determinant factors in Aβ-oligomer-induced synaptic and cell injuries, and therefore AD progression. Numerous epidemiological studies have highlighted close relationships between AD incidence and dietary patterns. Among the nutritional factors involved, lipids significantly influence AD pathogenesis. It is likely that maintenance of adequate membrane lipid content could prevent the production of Aβ peptide as well as its deleterious effects upon its interaction with synaptic membrane, thereby protecting neurons from Aβ-induced neurodegeneration. As major constituents of neuronal lipids, n-3 polyunsaturated fatty acids are of particular interest in the prevention of AD valuable diet ingredients whose neuroprotective properties could be essential for designing preventive nutrition-based strategies. In this review, we discuss the functional relevance of neuronal membrane features with respect to susceptibility to Aβ oligomers and AD pathogenesis, as well as the prospective capacities of lipids to prevent or to delay the disease.     

An essential function of sphingolipids in yeast cell division

Ceramides are bioactive lipids and precursors to sphingolipids. They have been shown to take part in a wide variety of different physiological processes in eukaryotic organisms and are thought to be toxic at high concentrations. Ceramide is synthesized by condensation of the sphingoid base sphinganine and a fatty acyl CoA by ceramide synthases, a family of enzymes that differ in their specificity for the length of the acyl CoA substrate. We have engineered a yeast strain where the endogenous ceramide synthase has been replaced by one of the putative enzymes from cotton. As a result, the yeast strain produces C18 rather than C26 ceramides showing that the cotton protein is a bona fide ceramide synthase with specificity towards C18 acyl CoA. Strikingly, the accumulation of C18 ceramide is not toxic in Saccharomyces cerevisiae. This allows survival of the yeast after deletion of the normally essential AUR1 (inositol phosphorylceramide synthase) gene permitting us to address the essential roles of sphingolipids. Deletion of AUR1 allows cell growth, but leads to a defect in cytokinesis, which takes twice as long as in wild-type strains. Nuclear division and recruitment of septins is apparently not affected, but cytokinesis is delayed and cell separation is incomplete.     

Characterization of the essential cell division gene ftsL (yllD ) of Bacillus subtilis and its role in the assembly of the division apparatus

We have identified the Bacillus subtilis homologue of the essential cell division gene, ftsL , of Escherichia coli . Repression of ftsL in a strain engineered to carry a conditional promoter results in cell filamentation, with a near immediate arrest of cell division. The filaments show no sign of invagination, indicating that division is blocked at an early stage. FtsL is also shown to be required for septation during sporulation, and depletion of FtsL blocks the activation but not the synthesis of the prespore-specific sigma factor, σ^F. Immunofluorescence microscopy shows that depletion of FtsL has little or no effect on FtsZ ring formation, but the assembly of other division proteins, DivIB and DivIC, at the site of division is prevented. Repression of FtsL also results in a rapid loss of DivIC protein, indicating that DivIC stability is dependent on the presence of FtsL, in turn suggesting that FtsL is intrinsically unstable. The instability of one or more components of the division apparatus may be important for the cyclic assembly/disassembly of the division apparatus.     

The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.     

Restraining mycobacteria: role of granulomas in mycobacterial infections

The generation of prolonged immunity to Mycobacterium tuberculosis requires not only an antigen-specific IFN-gamma-producing T cell response, including both CD4 and CD8 T cells, but also the generation of protective granulomatous lesions, whereby the close apposition of activated T cells and macrophages acts to contain bacterial growth. The importance of the granulomatous lesion in controlling this immune response and in limiting both tissue damage and bacterial dissemination has been considered a secondary event but, as the present review illustrates, is no less important in surviving mycobacterial infection than an antigen-specific T-cell response. The formation of a protective granuloma involves the orchestrated production of a host of chemokines and cytokines, the upregulation of their receptors along with upregulation of addressins, selectins and integrins to coordinate the recruitment, migration and retention of cells to and within the granuloma. In the present review, the principal components of the protective response are outlined and the role of granuloma formation and maintenance in mediating prolonged containment of mycobacteria within the lung is addressed.     

Stem cell self-renewal: The role of asymmetric division

Asymmetric division occurs widely in different groups of organisms from single-celled to insects, mammals, and plants. The operation of asymmetrical division may differ widely in different organisms. In multicellular organisms, asymmetrical division is one of the essential features of stem cell biology. The data obtained assume one of the main biological functions of asymmetrical division to be maintenance of cell viability, beginning with stem cells. Cells continuously accumulate toxic inclusions, which are formed by damaged proteins which cannot be degraded by proteasomes. As a result of asymmetric division, these inclusions segregate into one of the daughter cells providing the ability of long-lived proliferation to another cell.     

The essential cell division protein FtsN contains a critical disulfide bond in a non‐essential domain

Disulfide bonds are found in many proteins associated with the cell wall of Escherichia coli, and for some of these proteins the disulfide bond is critical to their stability and function. One protein found to contain a disulfide bond is the essential cell division protein FtsN, but the importance of this bond to the protein's structural integrity is unclear. While it evidently plays a role in the proper folding of the SPOR domain of FtsN, this domain is non‐essential, suggesting that the disulfide bond might also be dispensable. However, we find that FtsN mutants lacking cysteines give rise to filamentous growth. Furthermore, FtsN protein levels in strains expressing these mutants were significantly lower than in a strain expressing the wild‐type allele, as were FtsN levels in strains incapable of making disulfide bonds (dsb^‐) exposed to anaerobic conditions. These results strongly suggest that FtsN lacking a disulfide bond is unstable, thereby making this disulfide critical for function. We have previously found that dsb^‐ strains fail to grow anaerobically, and the results presented here suggest that this growth defect may be due in part to misfolded FtsN. Thus, proper cell division in E. coli is dependent upon disulfide bond formation.