Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

Effect of Plasmid pSa and of Auxin on Attachment of Agrobacterium tumefaciens to Carrot Cells

When the plasmid pSa is introduced into Agrobacterium tumefaciens, its presence results in the suppression of bacterial virulence. A. tumefaciens(pSa) cells are virulent on Bryophyllum diagremontiana only when inoculated with auxin. A. tumefaciens(pSa) cells also bind to plant cells only in the presence of auxin. The effect of auxin is on the bacteria rather than on the plant cells, since the bacteria require auxin to bind to heat-killed carrot cells. Bacteria containing pSa and grown in the absence of auxin showed a lag in binding to carrot cells in auxin-containing medium. This lag was not seen during the binding of wild-type strains. Tetracycline inhibited the binding of A. tumefaciens(pSa) in auxin-containing medium, suggesting that bacterial protein synthesis is required for the auxin effect. No difference was seen in the size or ability to inhibit bacterial binding of lipopolysaccharides from bacteria containing or lacking pSa and grown with or without auxin. A. tumefaciens(pSa) cells grown in the absence of auxin lacked surface polypeptide(s) found in bacteria grown in the presence of auxin and in the wild-type bacteria, which do not contain pSa. Thus, the presence of certain polypeptides appears to be associated with the ability of the bacteria to bind to plant cells.     

Tumor induction by agrobacterium involves attachment of the bacterium to a site on the host plant cell wall

Cell wall preparations from primary bean leaves were found to inhibit tumor initiation by Agrobacterium tumefaciens strain B6 when inoculated with the bacteria on bean leaves. Membrane fractions from these same leaves were noninhibitory. The cell walls were effective when applied prior to or with bacteria, but application of cell walls about 15 minutes after bacteria did not affect the number of tumors initiated. Much of the inhibitory activity of the plant cell walls was eliminated by pretreatment with dead site-attaching bacteria or with lipopolysaccharide from these bacteria. Cells and lipopolysaccharide from non-site-attaching agrobacteria had no effect on the activity of the plant cell walls. About 30% inhibition of tumor initiation was obtained with plant cell walls at 50 μg/ml dry weight, and at 10 mg/ml dry weight about 70% inhibition was typical. Both early and late appearing tumors were affected by the cell walls, indicating that they do not exclusively affect tumors arising from either small or large wounds. These data show that plant cell walls but not membranes contain surfaces to which A. tumefaciens adheres and these exhibit the specificity typical of the host site to which virulent agrobacteria must attach to induce tumors. It is concluded that some portion of wound-exposed plant cell wall constitutes the host adherence site in Agrobacterium infections.     

Intercellular Correlations: Relation between DNA Synthesis and Cell Division in Early Stages of in Vitro Bud Neoformation

As well as showing the existence, during the first stages of in vitro bud neoformation, of cell populations in a tissue composed of a single cell layer (stem epidermis of Torenia fournieri Lind), some new physiological characteristics of mitosis are defined. Most of the cells which divide during organogenesis synthesize their DNA between 20 and 48 hours of culture. On an epidermal strip (10 × 2.5 millimeters composed of about 5,500 cells) 20% of the original cells enter the S-phase. The first division takes place at the 20-hour stage after the entry into the S-phase of a cell population of about 25 cells. Almost none of the cells of this population divide. The greatest percentage of divisions occurs in cells which synthesize DNA near the 48-hour stage. The relation [Formula: see text] has a value of about 25 at the beginning of cell division (20 hours) and falls to a value of about 1.4 for cells which synthesize DNA near the 48-hour stage. A hypothesis of the existence of a mitotic stimulant in the epidermis is put forward; this stimulant, at first weak, increases progressively.     

Octopine Accumulation Early in Crown Gall Development is Progressive

A purification of octopine from crown gall tissue was developed to quantitate conversion of precursor [³H]arginine into [³H]octopine. Plant wound tissue which was sterile or infected with an avirulent strain of Agrobacterium tumefaciens did not accumulate detectable quantities of octopine, consistent with opine synthesis not being induced by wounding or infection. Octopine was only recovered from tissue infected with virulent tumor-inducing strains of A. tumefaciens. In every case tested, the morphological appearance of tumors preceded the accumulation of octopine by at least 1 week, and in some instances 3 weeks. Thus, what was necessary and sufficient for the expression of plant hormones (auxin and cytokinin) required for tumor growth was not sufficient for the accumulation of octopine. The possible nature of the temporal difference in the expression of hormone autotrophy and octopine synthesis is discussed.     

Cell division after inhibition of chromosome replication in Escherichia coli

The residual cell divisions after thymine starvation of exponential cultures of TJK16, a thymine-requiring derivative of Escherichia coli B/r, were evaluated. The results indicate that under the conditions used (glucose minimal medium 37 °), (1) only cells that had terminated a round of replication divided; (2) once termination had occurred, thymine starvation and replication no longer affected the time of cell division; (3) synchronously terminating subpopulations of cells began to divide about 17 min after termination; after that time, the rate of division decreased exponentially. The results confirm the previously inferred asymmetric distribution of D-periods in an exponential population of E. coli bacteria and suggest that an event associated with termination of replication is required for cell division. The method of data evaluation presented can be used to determine the duration of the D-period and to find the parameter values (halflife and onset) of the stochastic phase of the D-period in exponential cultures, eliminating the need for synchronization procedures.     

Spontaneous Mitotic Recombination and Evidence for an X-Ray-Inducible System for the Repair of DNA Damage in DROSOPHILA MELANOGASTER

Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with normal division rates. The remaining 84% result from events generating cells with an average cell division rate one-third that of the surrounding cells; these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.     

Changing of the cell division axes drives vulva evolution in nematodes

Vulval epithelial tubes invaginate through concerted cell migration, ring formation, stacking of rings and intra-ring cell fusion in the nematodes Caenorhabditis elegans, Oscheius tipulae and Pristionchus pacificus. The number of rings forming the invaginations is invariantly seven, six, and eight, respectively. We hypothesize that each ring is formed from pairs of symmetrically positioned primordial vulval cells following three premises: If the final cell division is left-right, the daughters will fuse, migrate and form only one ring. If these cells do not divide, one ring will form. If the final division is anterior-posterior, two rings will form. We test the ring hypothesis and found coincidence between the patterns of vulva cell divisions and the number of rings for 12 species. We find heterochronic variations in the timing of division, migration and fusion of the vulval cells between species. We report a unique ring-independent pathway of vulva formation in Panagrellus redivivus. C. elegans lin-11(n389) mutation results in cell fate transformations including changes in the orientation of vulval cell division. lin-11 animals have an additional ring, as predicted by the ring hypothesis. We propose that the genetic pathway determining how vulval cells invaginate evolves through ring-dependent and ring-independent mechanisms.     

Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall

We compared the binding of Agrobacterium tumefaciens by freshly isolated root cap cells with susceptibility of plants to crown gall tumorigenesis. A high binding reaction was strongly correlated with susceptibility to tumorigenesis in a survey of the binding of strain B6 to cells from 48 species in 17 families. In reciprocal experiments with nine virulent A. tumefaciens strains, tumors developed in plant-bacteria combinations that gave a high binding response in the root cap cell assay. Binding was quantified by direct measurement of the number of bacteria bound to the periphery of individual cells. Root cap cells from six susceptible species bound significantly more bacteria than did cells from five resistant species.     

Pattern of cell division and wound vessel member differentiation in coleus pith explants

When pith parenchyma explants are taken from Coleus blumei plants and cultured on an agar medium containing sucrose and indoleacetic acid wound vessel members differentiate in 10 days. The time course of wound xylem appearance and an auxin requirement suggest that this uncomplicated system is responding in a manner comparable to wounded Coleus plants and cultured stem segments.

Histological examination and cell size comparisons confirm that parenchyma cells divide before differentiating. When colchicine is used to prevent mitosis no tracheary elements differentiate. Following the time course of this cytodifferentiation histologically shows that xylem differentiates from cells that are the products of several cell divisions.

     

Inhibitory Effects of a Pectin-Enriched Tomato Cell Wall Fraction on Agrobacterium tumefaciens Binding and Tumor Formation

A pectin-enriched soluble cell wall fraction (CWF) prepared from suspension cultured tomato cells inhibits binding of Agrobacterium tumefaciens to these cells. It was hypothesized that the CWF contains the plant surface binding site for A. tumefaciens (NT Neff, AN Binns 1985 Plant Physiol 77: 35-42). Experiments described here demonstrate that tomato CWF inhibited tumor formation on potato slices and Agrobacterium binding to intact tomato cells in a dose-dependent fashion. Boiling the fraction reduced both its binding and tumor inhibitory activities. Tumor inhibitory activity was titrated out by increased concentrations of bacterial inocula with no inhibition apparent at 1 × 10⁸ bacteria per milliliter. These results indicate that a tomato CWF is enriched for a putative A. tumefaciens binding site which may also be involved in tumor formation in potato.     

Agrobacterium tumefaciens Interaction with Suspension-Cultured Tomato Cells

Adherence of Agrobacterium tumefaciens to suspension-cultured tomato cells has been characterized using a quantitative binding assay. Saturable binding of radiolabeled A. tumefaciens to plant cells resulted in 100 to 300 bacteria bound per cell. Specificity of A. tumefaciens binding was also inferred from two additional results: (a) an initial incubation of plant cells with A. tumefaciens reduced subsequent binding of radiolabeled A. tumefaciens by 60% to 75%; (b) tomato cells bound less than three E. coli per cell. Protease treatment of plant cells had no effect on subsequent bacterial binding, but prior treatment of plant cells with pectinolytic enzymes increased binding 2- to 3-fold. Pectin-enriched and neutral polymer-enriched fractions were obtained from tomato cell walls. The soluble pectin-enriched fraction inhibited binding of bacteria to plant cells by 85% to 95%, whereas the neutral polymer fraction only partially inhibited binding. Preliminary characterization of the activity showed it is heat stable, partially inactivated by protease treatment, and substantially inactivated by acid hydrolysis.     

Transformation of Wall Deficient Cultured Tobacco Protoplasts by Agrobacterium tumefaciens

Attachment of virulent Agrobacterium tumefaciens to plant cells is required for transformation. To further study the components of the plant cell wall that may be involved in the attachment process, tobacco (Nicotiana tabacum L.) protoplasts were cultured in the presence of 2,6 dichlorobenzonitrile (DB), an inhibitor of cellulose biosynthesis, and then assayed for their ability to be transformed by Agrobacterium. The DB treated protoplasts were deficient in wall production. Nevertheless, they were transformable at high frequency by wild type Agrobacterium strains but not by mutant strains that lack the ability to bind to normal, walled cells. Small quantities of calcofluor white positive material present on DB treated cells were correlated with their competence to be transformed. Further, the plant:bacterial association that leads to transformation is shown to become stable within 5 hours after bacterial co-cultivation with either control or DB treated cells.     

Reactivation of the Cambium in Aesculus hippocastanum L.: A Transmission Electron Microscope Study

Changes taking place during cambial reactivation in Aesculushippocastanum have been studied using transmission electronmicroscopy. Cytoplasmic activity in the form of vesicle productionby dictyosomes and endoplasmic reticulum, and coated vesicleformation at the plasmalemma, was observed in samples collectedin mid-Feb. The first cell divisions occurred 1 month later,in cells to the phloem side of the cambium, and were of twotypes: penclinal divisions producing new phloem precursors,and oblique anticlinal divisions in phloem mother cells formedat the end of the previous growing season producing putativecompanion cell/sieve element pairs. The fusiform initial wasidentified as the cell adjacent to the boundary-layer of parenchymacells and was the last cell to divide, 2 weeks after the firstdivisions in phloem precursors. For the next 4 weeks phloemcells only were produced The first new differentiating xylemelements were formed in the middle of Apr., following a surgein the rate of cell division by the initial aRd its derivativexylem mother cells. These were a mixture of developing fibresand vessel elements. Some of the boundary-layer cells were converteddirectly to vessel elements without any division taking place,while others were derived from daughter cells of the fusiforminitial produced following its reactivation. Aesculus hippocastanum L., cambium, dormancy, reactivation     

Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs

The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) or autoclaved strains of B6 or B-48. Inhibition by B-48 or autoclaved B6 could still be obtained when these cells were added up to a maximum of 10 minutes after the addition of viable B6. Maximum inhibition occurred when these cells were added 10 minutes prior to the addition of B6. There was no inhibition observed when equal cell counts of B6 were added along with a Gram-positive bacterium or yeast cell, while inhibition was observed when these B6 cells were added simultaneously with other Gram-negative cells. These results suggest that a physical, specific bacterial attachment that occurs within 10 minutes is necessary for tumor formation on potato discs.     

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10⁸ bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41°C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.     

MACROMOLECULAR SYNTHESES RELATED TO THE REPRODUCTIVE CYST OF TETRAHYMENA PATULA

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.     

Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation

Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.     

Coordination of Division and Development Influences Complex Multicellular Behavior in Agrobacterium tumefaciens

The α-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse α-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJ homologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ΔpleC and ΔdivK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ΔpleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.     

Involvement of Carrot Cell Surface Proteins in Attachment of Agrobacterium tumefaciens

The initial step in tumor formation by Agrobacterium tumefaciens is the site-specific attachment of the bacteria to plant cells. A similar attachment to plant tissue culture cells has been observed. Binding to carrot suspension culture cells was not dependent on the presence of divalent cations and was not inhibited by the addition of mannose, α-methyl mannoside, galactose, arabinose, glucosamine, 2-deoxyglucose, or 0.25 molar NaCl to the culture medium. The ability of the carrot cells to bind A. tumefaciens was markedly reduced by elution of the cells with dilute detergent or CaCl₂ or by incubation of the cells with proteolytic enzymes. The carrot cells were not killed by these treatments and recovered the ability to bind A. tumefaciens within 3 to 6 hours. A. tumefaciens did not bind to carrot cells which had been induced to form embryos (AG Matthysse, RHG Gurlitz 1982 Physiol Plant Pathol 21: 381-387). A comparison of the peptides eluted from embryos and from uninduced cells using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there were several changes in extractable polypeptides after embryo induction. One or more of the polypeptides present before embryo induction and absent from embryos may be involved in the binding of A. tumefaciens to the carrot cell surface.