Ana Isabel Rico

Septum Enlightenment: Assembly of Bacterial Division Proteins

When observed under the microscope, cell division is just another dark event in the secret life of a bacterium: the cell grows up to a certain size, and then a constriction appears in its center that finally separates the cell into two daughters without any other perceptible changes. Upon a more

The order of the ring: assembly of Escherichia coli cell division components

Topological cues appear to override temporal events in the assembly of the Escherichia coli cell division ring. When a procedure that allows the recruitment of ring components based on their topological properties is used, a concerted mode of assembly of several components of the divisome, rather than a strict linear mode, is revealed. Three multimolecular complexes, the proto‐ring, the periplasmic connector and the peptidoglycan factory, show some degree of concertation for their assembly. In addition, back‐recruitment of all late proteins except FtsN into the division ring occurs even in the absence of proteins incorporated at earlier stages, i.e. FtsA or FtsQ.

Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein

We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide‐dependent manner, forming long corkscrew‐like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 β‐strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsAΔ1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two‐hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsAΔ1C are therefore incomplete and do not support division. The production of high levels of FtsAΔ1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12–S13 strands of domain 2B generates a protein (FtsAΔS12–13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild‐type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsAΔS12–13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid‐free cells. The S12–S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.

Immunostimulatory properties of the Leishmania infantum heat shock proteins HSP70 and HSP83

Emerging evidence indicates that the heat shock proteins (HSPs), a set of highly evolutionary conserved proteins, are playing essential roles in both normal processes of the immune system and specific immune responses. In a previous work, we demonstrated that the Leishmania infantum HSP70 possesses remarkable immunostimulatory properties. In the present work, we have extended the study to another HSP from this parasite, the HSP83. We show that this protein also has an adjuvant effect to an accompanying protein by stimulation of the humoral response when both proteins are fused and co-administered to BALBjc mice. The analysis of the IgG isotypes, IgG1 and IgG2a, indicated that the immunisations with the Leishmania HSPs, mainly the HSP70, potentiate a Thl-type response. It was found that the amino-terminal domain of the HSP70, the most evolutionary conserved region of the molecule, maintains the ability to stimulate the humoral response, whereas the carboxyl-terminal domain does not have a similar effect. Unexpectedly, we found that the L. infantum HSP70 and HSP83 recombinant proteins stimulated the proliferation of spleen cells from unprimed BALB/c mice. Remarkably, this proliferation was abolished either by thermal denaturing of the proteins or by using specific antibodies. The use of the T-cell inhibitor cyclosporin A in the splenocytes proliferation assays suggested that both T- and non-T-cells are stimulated by the Leishmania HSPs. These findings may be relevant for therapeutic and prophylactic applications.

In the Beginning, Escherichia coli Assembled the Proto-ring: An Initial Phase of Division

Cell division in Escherichia coli begins by assembling three proteins, FtsZ, FtsA, and ZipA, to form a proto-ring at midcell. These proteins nucleate an assembly of at least 35 components, the divisome. The structuring of FtsZ to form a ring and the processes that effect constriction have been explained by alternative but not mutually exclusive mechanisms. We discuss how FtsA and ZipA provide anchoring of the cytoplasmic FtsZ to the membrane and how a temporal sequence of alternative protein interactions may operate in the maturation and stability of the proto-ring. How the force needed for constriction is generated and how the proto-ring proteins relate to peptidoglycan synthesis remain as the main challenges for future research.

Role of Escherichia coli FtsN protein in the assembly and stability of the cell division ring.

Deprivation of FtsN, the last protein in the hierarchy of divisome assembly, causes the disassembly of other elements from the division ring, even extending to already assembled proto‐ring proteins. Therefore the stability and function of the divisome to produce rings active in septation is not guaranteed until FtsN is recruited. Disassembly follows an inverse sequential pathway relative to assembly. In the absence of FtsN, the frequencies of FtsN and FtsQ rings are affected similarly. Among the proto‐ring components, ZipA are more sensitive than FtsZ or FtsA rings. In contrast, removal of FtsZ leads to an almost simultaneous disappearance of the other elements from rings. Although restoration of FtsN allows for a quick reincorporation of ZipA into proto‐rings, the de novo joint assembly of the three components when FtsZ levels are restored to FtsZ‐deprived filaments is even faster. This suggests that the recruitment of ZipA into FtsZ‐FtsA incomplete proto‐rings may require first a period for the reversal of these partial assemblies.

Structural and Functional Model for Ionic (K+/Na+) and pH Dependence of GTPase Activity and Polymerization of FtsZ, the Prokaryotic Ortholog of Tubulin

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP-Mg(2+) and monovalent cations. The presence of a K(+) ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K(+). This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na(+) destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro.

The heat shock proteins, Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Abstract Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coli maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell–deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell–independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.

Roles of the essential protein FtsA in cell growth and division in Streptococcus pneumoniae

Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.