scholarly journals A bacterial dynamin-like protein confers a novel phage resistance strategy on the population level in Bacillus subtilis

2021 ◽  
Author(s):  
Lijun Guo ◽  
Laura Sattler ◽  
Peter Graumann ◽  
Marc Bramkamp
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Host Cell ◽  
Single Molecule ◽  
Membrane Integrity ◽  
Resistance Mechanism ◽  
Cell Lysis ◽  
Phage Infection ◽  
Host Cells ◽  
Protective Effects

Bacillus subtilis DynA is a member of the dynamin superfamily, involved in membrane remodeling processes. DynA was shown to catalyze full membrane fusion and it plays a role in membrane surveillance against antibiotics. We show here that DynA also provides a novel resistance mechanism against phage infection. Cells lacking DynA are efficiently lysed after phage infection and virus replication. DynA does not prevent phage infection and replication in individual cells, but significantly delays host cell lysis, thereby slowing down the release of phage progeny from the host cells. During the process, DynA forms large, almost immobile clusters on the cell membrane that seem to support membrane integrity. Single molecule tracking revealed a shift of freely diffusive molecules within the cytosol towards extended, confined motion at the cell membrane following phage induction. Thus, the bacterial dynamins are the first anti-phage system reported to delay host cell lysis and the last line of defense of a multilayered antiviral defense. DynA is therefore providing protective effects on the population, but not on single cell level.

scholarly journals Characterization of Stages of Hepatozoon americanum and of Parasitized Canine Host Cells

2005 ◽  
Vol 42 (6) ◽  
pp. 788-796 ◽  
Author(s):  
C. A. Cummings ◽  
R. J. Panciera ◽  
K. M. Kocan ◽  
J. S. Mathew ◽  
S. A. Ewing
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Striated Muscle ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Blood Leukocytes ◽  
Host Cell Membrane ◽  
Asexual Multiplication ◽  
Hepatozoon Americanum

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in “onion skin” cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a “tail” in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.

scholarly journals Visualization of the type III secretion mediated Salmonella–host cell interface using cryo-electron tomography

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Donghyun Park ◽  
Maria Lara-Tejero ◽  
M Neal Waxham ◽  
Wenwei Li ◽  
Bo Hu ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Protein Secretion ◽  
Electron Tomography ◽  
Host Cells ◽  
Target Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Cryo Electron Tomography ◽  
Cell Interface

Many important gram-negative bacterial pathogens use highly sophisticated type III protein secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

scholarly journals Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

2020 ◽  
Author(s):  
Marie Burghard-Schrod ◽  
Alexandra Kilb ◽  
Kai Krämer ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Metal Binding ◽  
Outer Cell ◽  
Dna Uptake ◽  
Cell Pole ◽  
Catalytic Function ◽  
C Terminus ◽  
Capture Mechanism

AbstractIn competent gram-negative and gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility within the periplasm as ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol.ImportanceBacteria can take up DNA from the environment and incorporate it into their chromosome in case similarity to the genome exists. This process of “natural competence” can result in the uptake of novel genetic information leading to horizontal gene transfer. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells with similar dynamics as DNA receptor ComEA, and thus takes a detour to get stored before uptake across the cell membrane into the cytosol by DNA permease ComEC. The latter assembles at a single cell pole, likely by a diffusion-capture mechanism, and requires its large C-terminus, including a conserved residue thought to confer nuclease function, for proper localization, function and mobility within the membrane.

Trypanosoma cruzi does not induce apoptosis in murine fibroblasts

Parasitology ◽  
1999 ◽  
Vol 118 (2) ◽  
pp. 167-175 ◽  
Author(s):  
R. K. CLARK ◽  
R. E. KUHN
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cell Apoptosis ◽  
Membrane Integrity ◽  
Host Cells ◽  
Mammalian Host ◽  
Host Cell Apoptosis ◽  
Intracellular Parasites ◽  
Murine Fibroblasts ◽  
Infected Cells

The intracellular cycle of Trypanosoma cruzi in mammalian host cells involves the differentiation of dividing amastigote forms into flagellated trypomastigote forms. The mechanism(s) regulating the growth and differentiation of the intracellular parasites is (are) not known. The number of parasites in infected cells can be several hundred and may be enough to induce apoptosis, a suicide-like death programme, generating products (e.g. nuclear proteins) that could function as signals to initiate the differentiation of amastigotes into trypomastigotes. Murine fibroblasts infected with T. cruzi were examined during a 5-day course of infection for evidence of apoptosis. However, characteristics of apoptosis, including degeneration of nuclear structure, condensation of chromatin, loss of plasma membrane integrity, or the cleavage of DNA into nucleosomal fragments, were not observed. Therefore, it is unlikely that products resulting from host cell apoptosis function to induce parasite differentiation. The possibility that T. cruzi might inhibit host cell apoptosis by increasing intracellular levels of Bcl-2, an endogenous inhibitor of apoptosis, was then investigated. Analysis of infected cells by flow cytometry did not demonstrate a significant amount of intracellular Bcl-2. This suggests that if the parasite is inhibiting host cell apoptosis, it is by a method that does not involve increasing levels of Bcl-2.

scholarly journals Cyclic di-GMP Signaling in Bacillus subtilis Is Governed by Direct Interactions of Diguanylate Cyclases and Cognate Receptors

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Sandra Kunz ◽  
Anke Tribensky ◽  
Wieland Steinchen ◽  
Luis Oviedo-Bocanegra ◽  
Patricia Bedrunka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Live Cells ◽  
Independent Manner ◽  
Single Molecule Tracking ◽  
Content Type ◽  
Diguanylate Cyclases

ABSTRACT Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro, which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro, showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient. IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis. Thus, local-pool signal transduction operates extremely efficiently and specifically.

scholarly journals Visualization of the type III secretion mediated Salmonella -host cell interface using cryo-electron tomography

2018 ◽  
Author(s):  
Jun Liu ◽  
Donghyun Park ◽  
Maria Lara-Tejero ◽  
Jorge E Galan ◽  
Wenwei Li ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Host Cell ◽  
Type Iii Secretion ◽  
Electron Tomography ◽  
Host Cells ◽  
Target Cells ◽  
Secretion Systems ◽  
Type Iii ◽  
Cryo Electron Tomography ◽  
Cell Interface

Many important gram-negative bacterial pathogens use highly sophisticated type III secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella -host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

scholarly journals A Cytoplasmic Antiholin Is Embedded In Frame with the Holin in a Lactobacillus fermentum Bacteriophage

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Tingting Guo ◽  
Yongping Xin ◽  
Chenchen Zhang ◽  
Jian Kong
Keyword(s):  
Amino Acid ◽  
Host Cell ◽  
Transmembrane Domain ◽  
Cell Lysis ◽  
Lactobacillus Fermentum ◽  
Host Cells ◽  
Amino Acid Residues ◽  
Content Type ◽  
C Terminus ◽  
Infection Cycles

ABSTRACT In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in Lactobacillus bacteriophages. A temperate bacteriophage of Lactobacillus fermentum , ϕPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The hyb5 gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in hyb5 , hyb5 157–465 and hyb5 209–328 , were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in trans with lyb5-hyb5 in Escherichia coli . Surprisingly, host cell lysis was delayed by Hyb5 157–465 but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5 157–465 acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5 157–465 , implying that the interaction between Hyb5 157–465 and Hyb5 occurs at the C terminus of the holin. In vivo and in vitro , Hyb5 157–465 and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5 157–465 and Hyb5. All the results suggest that Hyb5 157–465 is an antiholin of Hyb5 that is involved in lysis timing. IMPORTANCE Phage-encoded holins are considered to be the “molecular clock” of phage infection cycles. The interaction between a holin and its inhibitor antiholin precisely regulates the timing of lysis of the host cells. As a prominent biological group in dairy processes, phages of lactic acid bacteria (LAB) have been extensively genome sequenced. However, little is known about the antiholins of LAB phage holins and the holin-antiholin interactions. In this work, we identified an in-frame antiholin against the class III holin of Lactobacillus fermentum phage ϕPYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.

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