\u3cem\u3eEscherichia coli\u3c/em\u3e Pathotypes Occupy Distinct Niches in the Mouse Intestine

Since the first step of the infection process is colonization of the host, it is important to understand how Escherichia coli pathogens successfully colonize the intestine. We previously showed that enterohemorrhagic O157:H7 strain E. coli EDL933 colonizes a niche in the streptomycin-treated mouse intestine that is distinct from that of human commensal strains, which explains how E. coli EDL933 overcomes colonization resistance imparted by some, but not all, commensal E. coli strains. Here we sought to determine if other E. coli pathogens use a similar strategy. We found that uropathogenic E. coli CFT073 and enteropathogenic E. coli E2348/69 occupy intestinal niches that are distinct from that of E. coli EDL933. In contrast, two enterohemorrhagic strains, E. coli EDL933 and E. coli Sakai, occupy the same niche, suggesting that strategies to prevent colonization by a given pathotype should be effective against other strains of the same pathotype. However, we found that a combination of commensal E. coli strains that can prevent colonization by E. coli EDL933 did not prevent colonization by E. coli CFT073 or E. coli E2348/69. Our results indicate that development of probiotics to target multiple E. coli pathotypes will be problematic, as the factors that govern niche occupation and hence stable colonization vary significantly among strains

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Role of catecholate siderophores in gram-negative bacterial colonization of the mouse gut

We investigated the importance of the production of catecholate siderophores, and the utilization of their iron (III) complexes, to colonization of the mouse intestinal tract by Escherichia coli. First, a ΔtonB strain was completely unable to colonize mice. Next, we compared wild type E. coli MG1655 to its derivatives carrying site-directed mutations of genes for enterobactin synthesis (ΔentA::Cm; strain CAT0), ferric catecholate transport (Δfiu, ΔfepA, Δcir, ΔfecA::Cm; CAT4), or both (Δfiu, ΔfepA, ΔfecA, Δcir, ΔentA::Cm; CAT40) during colonization of the mouse gut. Competitions between wild type and mutant strains over a 2-week period in vivo showed impairment of all the genetically engineered bacteria relative to MG1655. CAT0, CAT4 and CAT40 colonized mice 10[superscript 1]-, 10[superscript 5]-, and 10[superscript 2]-fold less efficiently, respectively, than MG1655. Unexpectedly, the additional inability of CAT40 to synthesize enterobactin resulted in a 1000-fold better colonization efficiency relative to CAT4. Analyses of gut mucus showed that CAT4 hyperexcreted enterobactin in vivo, effectively rendering the catecholate transport-deficient strain iron-starved. The results demonstrate that, contrary to prior reports, iron acquisition via catecholate siderophores plays a fundamental role in bacterial colonization of the murine intestinal tract

Summary of enterobactin production in vivo.

<p>The amount of FeEnt produced by the colonizing bacteria was standardized in relation to the amount of protein in the mucosal samples (CPM/A₂₈₀ nm). The plotted data therefore depicts the relative amount of enterobactin production by each of the strains on day 12 after inoculation. This experiment was performed only once, but each data point represents the mean values from pooled extracts of 5 animals. Despite the fact that the cell numbers of <i>E. coli</i> CAT4 were 4-logs lower than those of MG1655 at this time, we found more ⁵⁹FeEnt in the former strain's gut mucus. This finding, that CAT4 hyperexcretes enterobactin in vivo, corroborated previous findings <i>in vitro </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Cox1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Young1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Young2" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#pone.0050020-Seiffert1" target="_blank">[61]</a>. Even though they were present at 10,000-fold lower abundance (a finding that was substantiated by statistical analysis of three colonization experiments), CAT4 cells secreted as much or more enterobactin as wild type bacteria. This was validated by the background control, uncolonized mice treated with streptomycin, that established a baseline for the detection of FeEnt in the mice.</p

Colonization Competition between <i>E. coli</i> MG1655 and CAT0, CAT4 or CAT40.

<p>In each trial three streptomycin-treated mice were simultaneously fed with a mutant strain and wild type parent MG1655, which are both streptomycin resistant. If even a slight advantage exists between the two strains, the conditions of the large intestine select for the preferred strain, which dominates within a few days. If neither strain has an advantage, then the two strains co-colonize at almost equal levels. Fecal plate counts determined the relative colonizing abilities (the log difference in CFU/g of feces). Three-log differences or greater between the mutant and wild-type strain indicates in a major colonization defect. A 1.5 to 3 log difference shows a significant colonization defect, and a 1 to 1.5 log difference denotes a minor defect. Log differences less than 1 are not significant. In these experiments pairs of bacteria were orally inoculated into mice on day 0, and their presence in feces was monitored for 15 days. The plotted data represents the mean of two or more independent trials; error bars represent standard deviations of the means. <i>E. coli</i> MG1655 out-competed CAT0, CAT4 and CAT40 for colonization. Unexpectedly, CAT40 showed 1000-fold better persistence than CAT4, and maintained colonization at almost the same level as MG1655 for the first week.</p

SDS-PAGE of OM fractions.

<p>The prototypic <i>E. coli</i> strain MG1655 and its derivatives CAT4 (<i>Δfiu, ΔfepA, Δcir, ΔfecA::Cm</i>) and CAT40 (<i>Δfiu, ΔfepA, Δcir, ΔfecA, ΔentA::Cm</i>) were grown in LB broth, subcultured at 1% into iron-deficient MOPS minimal media at 37°C and grown to late log phase. The bacteria were collected by centrifugation, lysed in a French pressure cell and their OM fractions were purified, resolved by SDS-PAGE, and the gels were stained with coomassie blue R. Fiu, FepA and Cir are seen in MG1655 (Lane 1), but absent from CAT4 (lane 2) and CAT40 (lane 3). Molecular weight standards were included in lane 4. FecA, which is inducible by growth in the presence of citrate, is not visible in this experiment, but its absence was verified by PCR.</p

<i>E. coli</i> strains and plasmids.

<p><i>E. coli</i> strains and plasmids.</p

Enterobactin quantification in vivo.

<p>Mice were inoculated on day 0 and on day 12 caecal mucus from mice that were uncolonized or orally inoculated with <i>E. coli</i> strains MG1655, CAT4 or CAT40 was collected and diluted into 5 mM NaHPO₄, pH 6.9. Samples from five individual mice in each experimental group were consolidated, the solution was clarified by centrifugation, 100 uL of each supernatant was mixed with 10 µCi of ⁵⁹FeCl₃, the samples were incubated on ice for an hour and then chromatographed on Sephadex LH20 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050020#s4" target="_blank">Materials and Methods</a>). We chromatographed authentic FeEnt with the mucus as an internal marker, and determined and plotted the absorbances at 280 nm and 495 nm, and the radioactivity of each fraction. The black and red dashed lines show the absorbances of the eluted fractions at 280 nm and 495 nm, respectively: the blue line depicts their radioactivity.</p