Human intestinal tissue tropism of intimin epsilon O103 Escherichia coli

Human intestinal in vitro organ culture was used to assess the tissue tropism of human isolates of Escherichia coli O103:H2 and O103:H- that express intimin F. Both strains showed tropism for follicle associated epithelium and limited adhesion to other regions of the small and large intestine. This is similar to the tissue tropism shown by intimin gamma enterohaemorrhagic (EHEC) O157:H7, but distinct from that of intimin a enteropathogenic (EPEC) O127:H6. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserve

Exploitation of Other Social Amoebae by Dictyostelium caveatum

Dictyostelium amoebae faced with starvation trigger a developmental program during which many cells aggregate and form fruiting bodies that consist of a ball of spores held aloft by a thin stalk. This developmental strategy is open to several forms of exploitation, including the remarkable case of Dictyostelium caveatum, which, even when it constitutes 1/10(3) of the cells in an aggregate, can inhibit the development of the host and eventually devour it. We show that it accomplishes this feat by inhibiting a region of cells, called the tip, which organizes the development of the aggregate into a fruiting body. We use live-cell microscopy to define the D. caveatum developmental cycle and to show that D. caveatum amoebae have the capacity to ingest amoebae of other Dictyostelid species, but do not attack each other. The block in development induced by D. caveatum does not affect the expression of specific markers of prespore cell or prestalk cell differentiation, but does stop the coordinated cell movement leading to tip formation. The inhibition mechanism involves the constitutive secretion of a small molecule by D. caveatum and is reversible. Four Dictyostelid species were inhibited in their development, while D. caveatum is not inhibited by its own compound(s). D. caveatum has evolved a predation strategy to exploit other members of its genus, including mechanisms of developmental inhibition and specific phagocytosis

Figure 4

<p>A chemically induced block to development. A. Dialysis membranes of various pore sizes were filled with starving amoebae of <i>D. discoideum</i> (control) or <i>D. caveatum</i> at 10⁷/mL. Millipore filters, on which test populations of <i>D. discoideum</i> had been deposited, were placed on top of the dialysis membranes. The two populations were in chemical but not physical contact (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone-0000212-g005" target="_blank">Figure 5</a>). After 24 hours of development of the test populations, fruiting was complete in the case of the <i>D. discoideum</i>-filled membranes, partial in the case of the <i>D. caveatum</i>-filled 500 Da membrane. Development was blocked at the aggregation stage, in the case of <i>D. caveatum</i>-filled membranes of size greater than 1 kDa. Bar = 500 µm. A dialysate of a <i>D. caveatum</i>-filled membrane also inhibits development of <i>D. discoideum</i>. A dialysis membrane filled with <i>D. caveatum</i> was dialysed against 1.5 mL of SorC buffer for 24 hours. Test populations of <i>D. discoideum</i> on filters were then incubated with this dialysate or SorC (control). At 20 hours of development, the dialysate had inhibited <i>D. discoideum</i> development at the aggregate stage while control <i>D. discoideum</i> had reached the slug stage. The inhibition was a 4–6 hour lag at aggregation stage. Bar = 500 µm.</p

Figure 3

<p>In mixtures of 1/10³<i>D. caveatum</i>/<i>D. discoideum,</i> aggregation is blocked but cell-type differentiation occurs. Time in HH:MM. 00:00 corresponds to the beginning of recording, during the initiation of aggregation. Bar = 200 µm. A. In control developing <i>D. discoideum</i> populations, amoebae start to express CFP and YFP under the control of pre-spore or pre-stalk promoters after aggregation but before tip formation. The collective rotational motion of amoebae in aggregates is observed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s008" target="_blank">Movie S3A</a>). These markers are subsequently expressed in the pre-spore or pre-stalk regions of slugs and finally the stalk and spores of fruiting bodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s008" target="_blank">Movie S3A</a>). Two stages are shown here: late aggregation phase, before the fluorescent reporters are expressed; and tip formation, when the markers are already fully expressed and cells have sorted out to different regions of these aggregates. B. In 1/10³<i>D. caveatum</i>-infected <i>D. discoideum</i> populations, these markers are also expressed after aggregation. However there is no collective rotational motion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s009" target="_blank">Movie S3B</a>) and no tip formation. These markers persist until all <i>D. discoideum</i> amoebae are ingested and starving <i>D. caveatum</i> triggers its own development program. Here we show the late aggregation phase, before the fluorescent reporters are expressed; and the blocked aggregate phase, when markers are expressed but no tips form on aggregates.</p

Figure 1

<p>The phagocytosis of <i>D. discoideum</i> by <i>D. caveatum</i>. A. Time series of phagocytosis. A red-labeled <i>D. caveatum</i> amoeba (red arrow) phagocytoses a GFP-expressing <i>D. discoideum</i> amoeba (green arrow). Engulfment is complete within 2 minutes following contact. The prey cell is fragmented into several phagosomes within the same time frame (see GFP-positive fragments at 2.5 minutes following contact). After less than 10 minutes, phagocytosis is complete and the <i>D. caveatum</i> amoeba resumes migration until it comes into contact with another prey cell. Time is in minutes and seconds (MM:SS, see Movie <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s002" target="_blank">S1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s003" target="_blank">S1B</a>). Bar = 10 µm. B. Histological section of <i>D. caveatum</i> amoebae in an aggregate where the prey cells were initially at 1 <i>D. caveatum</i> for 1000 prey cells. <i>D. caveatum</i> amoebae are presumably the large darker cells showing a cytoplasmic basophilic straining (arrows), containing material resembling the majority of cells in the aggregate (fragments of <i>D. discoideum</i> cells). This section was prepared 16 hours after the beginning of development. Bar = 10 µm. C. Histological section of an aggregate in which nearly all <i>D. discoideum</i> amoebae have been consumed and only <i>D. caveatum</i> cells remain. These cells are packed in dense aggregates, and a few of them still contain remnants of <i>D. discoideum</i> amoebae. Dotted lines have been drawn around foci of aggregating <i>D. caveatum</i> within the cellular mass. This section corresponds to 36 hours after the beginning of development. Bar = 10 µm.</p

Range and specificity of inhibition and phagocytosis

<p>Four additional Dictyostelid species were tested according to the same protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone-0000212-g004" target="_blank">Figure 4A</a>. The table indicates the number of aggregates for each species as a percentage of the total number of developmental stages observed on filters after 24 hours of incubation (average (standard deviation)), as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone-0000212-g005" target="_blank">Figure 5</a>. In the controls, development at 24 h has passed the aggregate stage, while in the presence of <i>D. caveatum</i> (10⁸ cells/mL inside the dialysis membrane) development is blocked at this stage (compare first two columns). The ability of <i>D. caveatum</i> to phagocytose ameobae of these species, as assessed by live cell microscopy, is also indicated: all these Dictyostelid species are ingested by <i>D. caveatum</i> in the same manner as <i>D. discoideum</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s005" target="_blank">Movie S1D</a>).</p

Figure 2

<p>Time-lapse microscopy of the development of <i>D. caveatum</i>/<i>D. discoideum</i> mixtures. Cells of control GFP-expressing <i>D. discoideum</i> populations (A) and GFP-expressing <i>D. discoideum</i> populations containing 1/10³ Cell Tracker Red-labeled <i>D. caveatum</i> (B) were allowed to develop for 36 hours and continuously observed by time-lapse video microscopy. A. The control <i>D. discoideum</i> cultures undergo development from aggregation to fruiting (collective circular motion within aggregates is observed; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s006" target="_blank">Movie S2A</a>). Three stages are shown here: beginning of development (before aggregation), the tight aggregate stage, and the slug stage. Time is in HH:MM and 00:00 corresponds to the beginning of recording, during the initiation of aggregation. Bar = 200 µm. B. Aggregates infected with <i>D. caveatum</i> are blocked at the aggregate stage and no collective circular motion is observed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s007" target="_blank">Movie S2B</a>). Finally <i>D. caveatum</i> amoebae emerge as slugs and fruiting bodies when all of <i>D. discoideum</i> amoebae are consumed (and the GFP signal disappears).</p

Diagram accompanying Tables 1, 2 and S1.

<p>Diagram accompanying Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone-0000212-t001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone-0000212-t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone.0000212.s001" target="_blank">S1</a>.</p

Reversibility of the inhibition.

<p>Developing <i>D. discoideum</i> cells were incubated on filters on top of membranes containing <i>D. caveatum</i> or buffer (control) for 24 hours. Filters were then transferred to buffer-filled membranes and scored after 24, 48 and 72 hours. Each score represents the number of aggregates as a percentage of the total number of developmental stages observed at the time indicated (average (standard deviation)), as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000212#pone-0000212-g005" target="_blank">Figure 5</a>. The development inhibition is reversible: ∼75% of the <i>D. caveatum</i>-inhibited <i>D. discoideum</i> aggregates complete development to fruiting when rescued on SorC buffer for 72 hours.</p