Reactions of All <i>Escherichia coli</i> Lytic Transglycosylases with Bacterial Cell Wall

The reactions of all seven <i>Escherichia coli</i> lytic transglycosylases with purified bacterial sacculus are characterized in a quantitative manner. These reactions, which initiate recycling of the bacterial cell wall, exhibit significant redundancy in the activities of these enzymes along with some complementarity. These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall

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

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

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

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

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

A Chemical Biological Strategy to Facilitate Diabetic Wound Healing

A complication of diabetes is the inability of wounds to heal in diabetic patients. Diabetic wounds are refractory to healing due to the involvement of activated matrix metalloproteinases (MMPs), which remodel the tissue resulting in apoptosis. There are no readily available methods that identify active unregulated MMPs. With the use of a novel inhibitor-tethered resin that binds exclusively to the active forms of MMPs, coupled with proteomics, we quantified MMP-8 and MMP-9 in a mouse model of diabetic wounds. Topical treatment with a selective MMP-9 inhibitor led to acceleration of wound healing, re-epithelialization, and significantly attenuated apoptosis. In contrast, selective pharmacological inhibition of MMP-8 delayed wound healing, decreased re-epithelialization, and exhibited high apoptosis. The MMP-9 activity makes the wounds refractory to healing, whereas that of MMP-8 is beneficial. The treatment of diabetic wounds with a selective MMP-9 inhibitor holds great promise in providing heretofore-unavailable opportunities for intervention of this disease

Pathogenesis of strains in murine and Wax Moth models of infection.

<p>(A) CFUs of the Δ<i>frvA</i> and Δ<i>frvA</i> complemented strain enumerated from livers and spleens three days post infection. Error bars represent standard error of the mean and asterisks represent P<0.001 by the Student's t-test when compared to the wild-type and complement strains. (B) Pathogenesis of strains in the <i>Galleria mellonella</i> model of infection. Dotted line and cross indicates LT-50 (time in which 50% of insects had perished). (C) Pathogenesis of the Δ<i>lmo0642</i> mutant in the murine model of infection. Strains were inoculated into mice by the ip route and numbers were enumerated in the spleens at day three post-infection. Student t-test did not detect a significant difference between the wild-type and mutant strain.</p

Bacterial growth.

<p>The rates and extent of bacterial growth (A: EGD-e; C: Δ<i>lmo0641</i>; E:Δ<i>lmo0641</i>/pPL2<i>lmo0641</i>) were determined in iron-restricted MOPS-L media supplemented with Hb (panels <b>A</b>–<b>C</b>; open, gray and black symbols represent addition of 0.0, 0.02 and 2 uM Hb, respectively) or Hn (<b>D</b>, <b>E</b>; open, gray and black symbols represent addition of 0.0, 0.2 and 2 uM Hn, respectively), and in BHI broth (<b>F</b>). The bacteria were cultured in BHI broth overnight. In <b>A–E</b> they were then subcultured in MOPS-L to stationary phase, and at t = 0 subcultured again at 1% into MOPS-L containing different concentrations of Hb or Hn. In <b>F</b>, at t = 0 they were subcultured into BHI broth. The flasks were shaken at 37°C and absorbance at 600 nm (initially close to zero for all cultures) was monitored for 12–26 h (note different scales). Because of the slow growth of <i>L. monocytogenes</i> in iron-restricted minimal media, this graphic representation focuses on the comparison of the mutant strains at later times in the growth cycle.</p

Quantitative real-time PCR.

<p>Induction of <i>lmo0641</i> (<i>frvA</i>) transcription in Δ<i>fur</i> compared to the wild-type (black bar) and induction of gene transcriptions in Δ<i>frvA</i> compared to the wild-type (gray bars) in BHI. Up-regulated genes are represented by bars above the x-axis and the down-regulated gene (<i>fur</i>) is represented by the bar below the axis. Asterisks represent Fur-regulated genes. Error bars represent the mean ± SD of the relative change in gene expression of independent duplicate samples.</p