Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding

DNA sequencing technologies provide unprecedented opportunities to analyze within-host evolution of microorganism populations. Often, within-host populations are analyzed via pooled sequencing of the population, which contains multiple individuals or "haplotypes." However, current next-generation sequencing instruments, in conjunction with single-molecule barcoded linked-reads, cannot distinguish long haplotypes directly. Computational reconstruction of haplotypes from pooled sequencing has been attempted in virology, bacterial genomics, metagenomics, and human genetics, using algorithms based on either cross-host genetic sharing or within-host genomic reads. Here, we describe PoolHapX, a flexible computational approach that integrates information from both genetic sharing and genomic sequencing. We demonstrated that PoolHapX outperforms state-of-the-art tools tailored to specific organismal systems, and is robust to within-host evolution. Importantly, together with barcoded linked-reads, PoolHapX can infer whole-chromosome-scale haplotypes from 50 pools each containing 12 different haplotypes. By analyzing real data, we uncovered dynamic variations in the evolutionary processes of within-patient HIV populations previously unobserved in single position-based analysis

Multiple Peptidoglycan Modification Networks Modulate Helicobacter pylori's Cell Shape, Motility, and Colonization Potential

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape

Functional Analysis of the Helicobacter pylori Flagellar Switch Proteins ▿ §

Helicobacter pylori uses flagellum-mediated chemotaxis to promote infection. Bacterial flagella change rotational direction by changing the state of the flagellar motor via a subcomplex referred to as the switch. Intriguingly, the H. pylori genome encodes four switch complex proteins, FliM, FliN, FliY, and FliG, instead of the more typical three of Escherichia coli or Bacillus subtilis. Our goal was to examine whether and how all four switch proteins participate in flagellation. Previous work determined that FliG was required for flagellation, and we extend those findings to show that all four switch proteins are necessary for normal numbers of flagellated cells. Furthermore, while fliY and fliN are partially redundant with each other, both are needed for wild-type levels of flagellation. We also report the isolation of an H. pylori strain containing an R54C substitution in fliM, resulting in bacteria that swim constantly and do not change direction. Along with data demonstrating that CheY-phosphate interacts with FliM, these findings suggest that FliM functions in H. pylori much as it does in other organisms

Identification of Helicobacter pylori Genes That Contribute to Stomach Colonization

Chronic infection of the human stomach by Helicobacter pylori leads to a variety of pathological sequelae, including peptic ulcer and gastric cancer, resulting in significant human morbidity and mortality. Several genes have been implicated in disease related to H. pylori infection, including the vacuolating cytotoxin and the cag pathogenicity island. Other factors important for the establishment and maintenance of infection include urease enzyme production, motility, iron uptake, and stress response. We utilized a C57BL/6 mouse infection model to query a collection of 2,400 transposon mutants in two different bacterial strain backgrounds for H. pylori genetic loci contributing to colonization of the stomach. Microarray-based tracking of transposon mutants allowed us to monitor the behavior of transposon insertions in 758 different gene loci. Of the loci measured, 223 (29%) had a predicted colonization defect. These included previously described H. pylori virulence genes, genes implicated in virulence in other pathogenic bacteria, and 81 hypothetical proteins. We have retested 10 previously uncharacterized candidate colonization gene loci by making independent null alleles and have confirmed their colonization phenotypes by using competition experiments and by determining the dose required for 50% infection. Of the genetic loci retested, 60% have strain-specific colonization defects, while 40% have phenotypes in both strain backgrounds for infection, highlighting the profound effect of H. pylori strain variation on the pathogenic potential of this organism

Assessment of the straight rod <i>H. pylori</i>'s colonization and pro-inflammatory potential.

<p>A) One week C57BL/6 mouse competition data compiled from three independent experiments. Data are plotted as a competitive index: [CFU/mL_MUT∶CFU/mL_{WT/Complement} stomach output]/[CFU/mL_MUT∶CFU/mL_{WT/Complement} inoculum] with each data point representing a single mouse. Black points indicate mice from which only one strain was recovered. Strains used: LSH100, LSH122, LSH124. B–D) Survival at low pH (B), in the presence of polymyxin B (C), or in high salt (D). Data comprise two independent experiments of four replicates per strain and condition (mean ± SD). Strains used: NSH57, LSH18. E) IL-8 production during infection of AGS gastric epithelial cells. Culture supernatants of triplicate wells were assayed for IL-8 using a commercial ELISA assay after infection at a multiplicity of infection of 10 (mean ± SD). Shown are data from one of three independent experiments with similar results. Strains used: NSH57, LSH13, LSH18.</p

Current understanding of muropeptide modification in <i>H. pylori</i>.

<p>This schematic shows peptide modification activities that can generate the muropeptides observed in the <i>H. pylori</i> sacculus. Known <i>H. pylori</i> proteins demonstrated (Csd3, Csd4) or predicted (Csd1, Csd2) to perform these activities are indicated. CPase, carboxypeptidase; EPase, endopeptidase.</p

<i>H. pylori</i> cell shape mutant morphologies and associated loci identified in a visual screen.

<p>The transposon insertion site and orientation (indicated by the spelling of the transposon's selectable marker, chloramphenicol acetyltransferase (<i>cat</i>)), is shown for each straight rod shape mutant identified in the screen. A) HPG27_353 (<i>csd4</i>) shape locus. B–E) Phase contrast (B, D) and transmission electron microscopy (TEM) (C, E) images of wild-type (B–C) and <i>csd4</i> mutant cells (D–E). F) HPG27_1195 (<i>csd5</i>) shape locus. G–H) Phase contrast (G) and TEM (H) images of <i>csd5</i> mutant cells. Strains used: NSH57, LSH18, LSH31, LSH36.</p

Functional analyses of Csd4 enzymatic activity and its role in shape determination.

<p>A) SDS-PAGE depicting steps in the purification of His-tagged <i>H. pylori</i> Csd4 protein from <i>E. coli</i> cells. Induced protein was purified using a Ni-NTA agarose column as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat.1002603.s001" target="_blank">Text S1</a>. WC, whole cell lysate; CL, cleared lysate; MW, molecular weight; FT, flow through. Positions of the 20 kDa and 50 kDa molecular weight markers are indicated. B) HPLC analysis of muropeptides released from purified <i>csd4</i> mutant (LSH122) PG treated with purified His-tagged Csd4 protein in the presence of Zn²⁺ or EDTA, or without protein. In the presence of Zn²⁺ but not EDTA, Csd4 trimmed the monomeric tripeptides to dipeptides, indicative of the protein having DL-carboxypeptidase activity. C–D) Muropeptides detected before and after incubation of Csd4 with purified disaccharide tripeptide (C) and disaccharide tetrapeptide substrates (D). Data indicate Csd4 cleaves tripeptide, but not tetrapeptide. E–F) Scatter plot arraying the wild-type, <i>csd4</i> deletion, <i>csd4</i> point, and <i>csd5</i> deletion mutant populations by length (x-axis, µm) and cell curvature (y-axis, arbitrary units). Each contour depicts the morphology of a single cell captured from a 1000× phase contrast image using CellTool software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat.1002603-Sycuro1" target="_blank">[13]</a>. The software algorithmically determines each cell's length along its two-dimensional central axis as well as the degree of cell body curvature (excluding the poles). 200–300 cells were analyzed for each strain. E) Smooth histograms displaying kernel density estimates of each strain's cell curvature (x-axis). Bootstrapped Kolmogorov–Smirnov statistical comparisons of population cell curvature distributions yielded p-values<0.001 for all pairwise comparisons with the exception of <i>csd4</i> vs. <i>csd4E222A</i>, p = 0.19. Strains used: NSH57, LSH18, LSH31, LSH146.</p

Motility of <i>H. pylori</i> cell shape mutants in soft agar and viscous polymer solutions.

<p>A, D) Motility phenotype of indicated strains in soft agar (mean halo diameter ± SD in 0.3% soft agar after four days). Data shown are from one experiment of 17–22 stabs/strain and are consistent with the findings from replicate experiments. Contours representative of each strain's average cell shape (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002603#ppat-1002603-g003" target="_blank">Figure 3</a> legend) are shown below panel D and are superimposed on a grid to highlight the slight differences in cell curvature that correlate with motility. p-values were generated using one-way ANOVA with the Bonferroni correction for multiple comparisons. B–C) Velocity of wild-type and the <i>csd4</i> mutant in broth containing porcine mucus (B) and methylcellulose (C). Data shown are the mean ± SD from measurements of 9–30 cells/strain/condition. No statistically significant differences between wild-type and the <i>csd4</i> mutant were observed in any condition (p>0.2, Student's t-test with equal variances). Strains used: A) LSH100, LSH122, LSH123; B–C) NSH57, LSH18; D) LSH100, LSH134, NSH152a, LSH146, NSH153a, NSH160a.</p