inGAP: an integrated next-generation genome analysis pipeline

Summary: We develop a novel mining pipeline, Integrative Next-generation Genome Analysis Pipeline (inGAP), guided by a Bayesian principle to detect single nucleotide polymorphisms (SNPs), insertion/deletions (indels) by comparing high-throughput pyrosequencing reads with a reference genome of related organisms. inGAP can be applied to the mapping of both Roche/454 and Illumina reads with no restriction of read length. Experiments on simulated and experimental data show that this pipeline can achieve overall 97% accuracy in SNP detection and 94% in the finding of indels. All the detected SNPs/indels can be further evaluated by a graphical editor in our pipeline. inGAP also provides functions of multiple genomes comparison and assistance of bacterial genome assembly

Microarray and Functional Analysis of Growth Phase-Dependent Gene Regulation in Bordetella bronchiseptica▿ †

Growth phase-dependent gene regulation has recently been demonstrated to occur in Bordetella pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived from a Bordetella bronchiseptica-like ancestor, we hypothesized that growth phase-dependent gene regulation would also occur in B. bronchiseptica. Microarray analysis revealed and quantitative real-time PCR (qRT-PCR) confirmed that growth phase-dependent gene regulation occurs in B. bronchiseptica, resulting in prominent temporal shifts in global gene expression. Two virulence phenotypes associated with these gene expression changes were tested. We found that growth-dependent increases in expression of some type III secretion system (TTSS) genes led to a growth phase-dependent increase in a TTSS-dependent function, cytotoxicity. Although the transcription of genes encoding adhesins previously shown to mediate adherence was decreased in late-log and stationary phases, we found that the adherence of B. bronchiseptica did not decrease in these later phases of growth. Microarray analysis revealed and qRT-PCR confirmed that growth phase-dependent gene regulation occurred in both Bvg+ and Bvg− phase-locked mutants, indicating that growth phase-dependent gene regulation in B. bronchiseptica can function independently from the BvgAS regulatory system

Effect of TLR4 stimulation on the rapid antibody-mediated clearance of <i>B. parapertussis</i>.

<p>(A) Groups of wild type (C3H/HEOuJ) and TLR4-deficient (C3H/HEJ) mice were inoculated with <i>B. parapertussis</i> (Bpp) or Bpp and heat-killed <i>B. bronchiseptica</i> (HK Bb) and given I.P. injections of naïve serum (NS) or immune serum (IS). Groups of mice were also inoculated with Bpp and (B) Bb LPS (BbLPS) or (C) <i>E. coli</i> LPS (EcLPS) and treated with naïve or immune serum. Bacterial numbers were quantified 3 days later and are expressed as the Log₁₀ mean+/−SD. Asterisks denote P-values<0.05.</p

<i>In vivo</i> growth of <i>B. bronchiseptica</i> on <i>B. parapertussis</i> upon a co-inoculation.

<p>(A) Groups of C57BL/6 mice were inoculated with <i>B. parapertussis</i> (Bpp), <i>B. bronchiseptica</i> (Bb), or both bacteria (Bpp+Bb) and sacrificed 0.5, 1, 2, or 3 days later to quantify neutrophils in the BAL fluid. Groups of mice were also sacrificed at these times to quantify (B) Bb numbers and (C) Bpp numbers in the lungs. Neutrophil numbers are expressed as the mean+/−SD and bacterial numbers are expressed as the Log₁₀ mean+/−SD. Asterisks represent P-values<0.05 when comparing <i>B. parapertussis</i>-infected mice to co-infected mice.</p

Evidence for Horizontal Gene Transfer of Two Antigenically Distinct O Antigens in Bordetella bronchiseptica▿ †

Host immunity is a major driving force of antigenic diversity, resulting in pathogens that can evade immunity induced by closely related strains. Here we show that two Bordetella bronchiseptica strains, RB50 and 1289, express two antigenically distinct O-antigen serotypes (O1 and O2, respectively). When 18 additional B. bronchiseptica strains were serotyped, all were found to express either the O1 or O2 serotype. Comparative genomic hybridization and PCR screening showed that the expression of either the O1 or O2 serotype correlated with the strain containing either the classical or alternative O-antigen locus, respectively. Multilocus sequence typing analysis of 49 B. bronchiseptica strains was used to build a phylogenetic tree, which revealed that the two O-antigen loci did not associate with a particular lineage, evidence that these loci are horizontally transferred between B. bronchiseptica strains. From experiments using mice vaccinated with purified lipopolysaccharide from strain RB50 (O1), 1289 (O2), or RB50Δwbm (O antigen deficient), our data indicate that these O antigens do not confer cross-protection in vivo. The lack of cross-immunity between O-antigen serotypes appears to contribute to inefficient antibody-mediated clearance between strains. Together, these data are consistent with the idea that the O-antigen loci of B. bronchiseptica are horizontally transferred between strains and encode antigenically distinct serotypes, resulting in inefficient cross-immunity

Replacement of Adenylate Cyclase Toxin in a Lineage of Bordetella bronchiseptica▿ §

Bordetella bronchiseptica is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of B. bronchiseptica strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that cyaA, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the cya locus, which encodes, activates, and secretes CyaA, was replaced by an operon (ptp) predicted to encode peptide transport proteins. Other B. bronchiseptica strains from the same phylogenetic lineage as that of strain 253 also lacked the cya locus, contained the ptp genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the ptp genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of B. bronchiseptica