Succession in the Gut Microbiome following Antibiotic and Antibody Therapies for <em>Clostridium difficile</em>

<div><p>Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to <em>Clostridium difficile</em>. <em>C. difficile</em> infection is typically lethal in hamsters, but protection can be conferred with neutralizing antibodies against the A and B toxins. We compare treatment with neutralizing monoclonal antibodies (mAb) to treatment with vancomycin, which prolongs the lives of animals but ultimately fails to protect them from death. We carried out longitudinal deep sequencing analysis and found distinctive waves of succession associated with each form of treatment. Clindamycin sensitization prior to infection was associated with the temporary suppression of the previously dominant <em>Bacteroidales</em> and the fungus <em>Saccinobaculus</em> in favor of <em>Proteobacteria</em>. In mAb-treated animals, <em>C. difficile</em> proliferated before joining <em>Proteobacteria</em> in giving way to re-expanding <em>Bacteroidales</em> and the fungus <em>Wickerhamomyces</em>. However, the <em>Bacteroidales</em> lineages returning by day 7 were different from those that were present initially, and they persisted for the duration of the experiment. Animals treated with vancomycin showed a different set of late-stage lineages that were dominated by <em>Proteobacteria</em> as well as increased disparity between the tissue-associated and luminal cecal communities. The control animals showed no change in their gut microbiota. These data thus suggest different patterns of ecological succession following antibiotic treatment and <em>C. difficile</em> infection.</p> </div

PCoA analysis displaying the community structure through clindamycin treatment and recovery.

<p>The x-axis indicates time, the y-axis indicates the value of the first principal coordinate, a summary of the UniFrac matrix of distances between bacterial communities. The color code for the different groups is shown to the lower right. Each point represents the community of one animal on one day and the large colored dot on each day is the mean. Red dots- untreated control group; green triangles- UIg; teal squares- IIg; purple plus signs- IV. Vertical bar- range of two standard deviations from the mean.</p

Experimental design and pathology.

<p>(A) Hamsters were divided into seven groups, corresponding to one untreated control group (n = 3) and two clindamycin-treated arms with and without <i>C. difficile</i> challenge. Within each arm, hamsters were divided into three treatment groups- none, vancomycin, and antitoxin monoclonal antibody (n = 10 per treatment group). (B) Timeline of interventions. Clindamycin, <i>C. difficile</i>, vancomycin (green bar), and mAb (blue indicator) were given to the designated groups on the days indicated. (C) Gross pathology of Golden Syrian hamsters. Arrow indicates the cecum. [Left] Untreated control (UCtrl) animal on day 40; [Right] Infected, no treatment (I0) animal on day 2. The cecum of the infected, untreated hamster is enlarged, hyperemic, inflamed, and partially necrotic.</p

<i>C. difficile</i>-specific Taqman qPCR analysis of hamster feces and ceca.

<p>(A) A custom qPCR specific for <i>C. difficile</i> DNA was consistently negative for all tested samples from the untreated control group of hamsters. <i>C. difficile</i> DNA in the UIg group rises above the limit of detection by day 7 and remains elevated. The infected hamsters have a detectable spike by day 3 despite having no detectable <i>C. difficile</i> initially. The mAb-treated groups have near-complete survival to day 40 despite having a significant pathogen DNA burden as determined by PCR analysis. Vancomycin-treated animals have a lower spike by day 3 that falls below the limit of detection for most of the subsequent samples. (B) <i>C. difficile</i> 16S RNA copies were detected in luminal and cecal samples. Red bar- group mean 16S copies/ng DNA; gray dots- individual mean 16S copies/ng DNA. Limit of detection is 27 copies per reaction, or ca. 7.9×10³<i>C. difficile</i> genome equivalents per gram of dry weight feces assuming perfect recovery.</p

Taxonomic heatmap of 18S OTUs demonstrating microeukaryote succession in clindamycin-treated hamsters.

<p>Taxonomic heatmap comparing the three untreated control hamsters to five of the UIg individuals. The color code for the heat map is as in previous figures (more intense blue indicates a higher proportion).</p

Natural history of infection includes weight loss and high mortality rate.

<p>(A) Mean change in percent weight per group over the course of the experiment. Deceased hamsters were dropped from the plot. Red dot- mean weight as percent of initial; gray dot- individual weight as percent of initial; red bar- range within 2 standard deviations of the mean; gray dashed line- original weight. (B) Kaplan-Meier survival curve. Untreated control (UCtrl) hamsters survived the duration of the study. Both infected and uninfected untreated animals showed 100% mortality within six days. One animal died in both vancomycin-treated groups on day 2, while the rest of the animals survived between 11 and 16 days post infection, approximately one week after cessation of treatment. Two animals died in the uninfected, mAb-treated group, and one animal died in the infected, mAb-treated group about two weeks post infection. One more hamster from the infected, mAb-treated group died shortly before the end of the experiment and the rest survived until they were euthanized on day 40.</p

Taxonomic heatmap of fecal and cecal bacterial communities demonstrating large-scale community shift and multiple waves of succession following clindamycin administration.

<p>Heatmap showing relative abundance of taxa as a percent of total 16S rRNA tag reads. Each column represents the read-aggregated mean community for that treatment group and day, each row represents a taxon. The uninfected, clindamycin-naïve group is mostly stable and homogenous for the entirety of the experiment, while clindamycin induces a large-scale perturbation in the microbiota. The numbers of animals and the treatments are shown at the top (white plus (“+”) signs indicate dates of administration) and sample origin at the bottom. Proportion contributed by each taxon is shown by the color scale at the top right.</p

Divergent Bacteroidales present in the hamster gut at the beginning and end of the study.

<p>The 50 most abundant Bacteroidales OTUs from each group were identified at the beginning and the end of the study and marked–red indicates Bacteroidales OTUs predominant at the beginning, blue indicates the predominant Bacteroidales at the end of the study, grey indicates remaining, lower abundance Bacteroidales. The x-axis shows time, and the y-axis shows proportion over all bacterial lineages. OTU – color correspondence is constant for all panels.</p

Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

<div><p>New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC₅₀ less than 0.05 µg ml⁻¹. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.</p> </div

Thermodynamic parameters of PGT 121 antibodies and CD4 binding to gp120 and SOSIP.664 trimers measured by isothermal titration calorimetry.

%<p>Reported values are averages from at least two independent measurements and associated errors are approximately 10% of the average. Representative isotherms can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s006" target="_blank">Figs. S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s007" target="_blank">S7</a>.</p>*<p>The binding isotherms do not allow to accurately determining these binding parameters.</p>#<p>The change in Gibbs free energy (ΔG) was determined using the relationship: ΔG_binding = RTlnK_d<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342-deAzevedo1" target="_blank">[75]</a>.</p>&<p>The stoichiometry of binding (N) is directly affected by errors in protein concentration measurements, sample impurity and glycan heterogeneity on gp120.</p