Antoinette Ryter

Procaryotic Cell Division with Respect to Wall and Membranes

(1971). Procaryotic Cell Division with Respect to Wall and Membranes. CRC Critical Reviews in Microbiology: Vol. 1, No. 1, pp. 29-72.

LOCALIZATION OF THE CHOLINERGIC RECEPTOR PROTEIN IN ELECTROPHORUS ELECTROPLAX 'BY HIGH RESOLUTION AUTORADIOGRAPHY

We first confirm that the [“HI o-toxin binds exclusively to the innervated (caudal) side of the eel electroplax and further show that the o-toxin binds both between the synapses and under the nerve terminals. However, taking into account the particular stereology of the cytoplasmic membrane, it appears that the density of a-toxin molecules bound per unit area of actual membrane surface is approx. 100 times larger under the synapses than outside the synapses. Absolute values of this density and of the total number of receptor sites per cell are given.

Localization of transcribing genes in the bacterial cell by means of high resolution autoradiography

The location of transcribing genes of the bacterial chromosome has been determined in Escherichia coli and in Bacillus subtilis by means of high resolution autoradiography. Statistical analysis of autoradiographs shows that the sites of RNA synthesis are located at the periphery of nuclear areas and far out into the cytoplasm. It also shows that a portion of the bacterial chromosome is spread out into the cytoplasm, and that the amount of this extra-nuclear DNA seems to be related to the growth rate. For bacteria in stationary phase or for those treated with puromycin, no extra-nuclear DNA is found. These results indicate that nuclear areas visible on thin sections of bacteria fixed under the Ryter-Kellenberger (1958) osmium tetroxide conditions contain only the inactive genes, and that the active genes are located in the cytoplasm around nuclear areas, forming probably the DNA-polysome complexes observed by Miller et al. (1970) .

The electron transparent zone in phagocytized Mycobacterium avium and other Mycobacteria Formation, persistence and role in bacterial survival

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages. Macrophages were also infected with bacteria killed with UV or gamma rays, H2O2, heat or glutaraldehyde. About 50% of bacilli killed with any of these treatments were found ETZ+ instead of 80-85% with live bacteria. Unlike live bacilli, for which the percentage of ETZ frequency remained stable throughout incubation time, ETZ frequency for killed bacilli decreased with time. ETZ assessment performed on M. tuberculosis H37 Rv for comparison showed that, despite a very low ETZ frequency (8-15%), the percentage of intact bacteria was identical to that observed with M. avium. In contrast, three rapidly growing non-pathogenic species (M. smegmatis, M. phlei and M. fallax) presented a low ETZ frequency after phagocytosis and were rapidly degraded. The process of ETZ formation and its role in bacterial survival are discussed.

Process of cellular division in Escherichia coli: Differentiation of growth zones in the sacculus

The murein sacculus is part of the bacterial cell envelope; its mechanical stability enables the cell to retain its shape. The growth pattern of this structure was studied in Escherichia coli wild type and in a series of temperature-sensitive mutants with defects in cell division, DNA initiation and DNA elongation. The murein of cells growing in rich medium under permissive and non-permissive conditions was specifically pulse-labelled with [ 3 H]diaminopimelic acid, and the distribution of label in isolated sacculi was analysed by high-resolution autoradiography. Two types of growth zones were found; one is assumed to be involved mainly in cell division, the other in cell elongation. Both can exist independently from each other as shown by the analysis of the autoradiographic grain patterns found in mutants.

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1β

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.

Intracellular differentiation of Leishmania amazonensis promastigotes to amastigotes: presence of megasomes, cysteine proteinase activity and susceptibility to leucine-methyl ester.

Intracellular differentiation of Leishmania promastigotes to amastigotes is a critical step in the establishment of infection. In this report three related features of mexicana subspecies amastigotes were used to follow the differentiation of the parasites within macrophages. Early after infection, (a) parasites did not contain ultrastructurally recognizable megasomes, (b) cysteine proteinase activity of parasite lysates was not detected in gelatin-containing acrylamide gels, and (c) parasites were essentially resistant to L-leucine-methyl ester (Leu-OMe). Typical megasomes were first identified on the 5th day, were more prevalent on day 7, and underwent swelling in macrophages exposed to Leu-OMe. Cysteine proteinase activity was first detected on day 3 and increased thereafter. Susceptibility to Leu-OMe of parasites studied in situ or isolated from infected macrophages increased with time of intracellular residence and by 7 days approached that of amastigotes isolated from mouse lesions. In contrast, parasites derived from either promastigotes or amastigotes were equally susceptible to another leishmanicidal compound, tryptophanamide (Trp-NH2). The results provide additional support for the involvement of megasomes and their cysteine proteinases in parasite killing by Leu-OMe, and highlight the slow pace of the intracellular differentiation of L. amazonensis promastigotes to amastigotes.

Megasomes as the targets of leucine methyl ester in Leishmania amazonensis amastigotes.

Certain L-amino acid esters, such as L-leucine methyl ester (Leu-OMe), can kill intracellular and isolated Leishmania amazonensis amastigotes. Killing appears to involve ester trapping and hydrolysis within an acidified parasite compartment (M. Rabinovitch and S. C. Alfieri, 1987, Brazilian Journal of Medical and Biological Research 20, 665-74). We show here by acid phosphatase light microscopic cytochemistry and by ultrastructural morphometry that megasomes, lysosome-like amastigote organelles, are the putative parasite targets of Leu-OMe. This conclusion is supported by the following observations. (a) Control amastigotes displayed a string of electron-dense, acid phosphatase-positive megasomes mostly located in the cellular poles opposite the flagellar pockets. Incubation of the amastigotes with Leu-OMe resulted in concentration-dependent swelling and fusion of the organelles as well as decreased electron density of the internal contents. These changes, which preceded parasite disruption, were followed by the progressive loss of parasite viability and the release of acid phosphatase activity into the medium. (b) Incubation of the amastigotes with L-isoleucine methyl ester, a non-leishmanicidal compound, induced only moderate fusion of the megasomes. (c) Pre-incubation of the parasites with the proteinase inhibitors antipain and chymostatin, previously shown to confer protection from Leu-OMe toxicity, nearly completely prevented the morphological changes of megasomes. (d) Exposure of amastigotes to tryptophanamide (Trp-NH2), the leishmanicidal activity of which is not reduced by antipain and chymostatin, did not result in swelling and fusion of the megasomes. This last finding suggests that different mechanisms underlie the destruction of amastigotes by Trp-NH2 and Leu-OMe.(ABSTRACT TRUNCATED AT 250 WORDS)

Leishmania mexicana: A cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.

Relationship between ultrastructure and specific functions of macrophages

The main function of the macrophages, which is to ingest and degrade any foreign molecules or particles penetrating the organism, appears in the development of the different structures implicated in endocytic activity. The macrophages high endocytic property first appears in its irregular shape and the large number of extensions of the cell membrane, allowing the rapid capture of extra-cellular material. Adhesion between macrophage cell surface and molecules or particles is greatly enhanced by the presence of varied kinds of receptors: lectin-like receptors which bind specific sugars or highly specific receptors such as Fc and C3b receptors, which increase phagocytosis of opsonized microbes. The microbicidal properties reside in part in the production of superoxide anions which result from the activity of a NAD(P)H oxidase. This enzyme is located in the plasma membrane. Its activity could be demonstrated with a cytochemical method, on the cell surface and along the phagosome membrane. It is, however, very weak in resident macrophages and increases after stimulation or activation. The second kind of bactericidal property corresponds to cationic proteins located in lysosomes. After fusion between lysosomes and phagosomes, they contribute to microbe killing by permeabilizing microbe envelopes. Lysosomes, which contain diverse acid hydrolases and are responsible for the degradation of ingested material, play a crucial role in macrophage endocytic activity. Their number increases in parallel with endocytic activity during macrophage differentiation and is particularly high after ingestion of degradable material. Contrary to polymorphonuclear leukocytes, macrophage is very poor in granules containing peroxidase. The latter, which are rather abundant in monocytes, disappear during macrophage maturation. They do not seem thus to be implicated in macrophage microbicidal activity. Endocytosis is accompanied by rapid and intense exchanges between the different membrane compartments of the cell (plasma membrane, pinosomes or phagosomes, endosomes, lysosomes, Golgi apparatus, etc.). These exchanges seem to occur by transitory fusions between vesicles coming from different compartments, rapidly followed by their recycling to their original compartment. This system of membrane shuttle has been clearly observed after formation of phagosomes or pinosomes in which the internalized plasma membrane is recycled back to the cell surface within a few minutes after their formation. This membrane traffic is especially intense in macrophages, the endocytic activity of which is very high, but it also exists in all cell types.(ABSTRACT TRUNCATED AT 400 WORDS)