Allelic Variation on Murine Chromosome 11 Modifies Host Inflammatory Responses and Resistance to Bacillus anthracis

Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36–74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT

A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection

Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated buclgenes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates

Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of “inactivated” Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy

Metabolic and regulatory genes in the MSHR668 genome that were not present in either K96243 or 1106a.

<p>*MSHR668 has one or more additional genes for this function.</p><p>#candidate chokepoint.</p><p>NA: function too general or no pathway associated.</p><p>Metabolic and regulatory genes in the MSHR668 genome that were not present in either K96243 or 1106a.</p

Interrogation of the <i>Burkholderia pseudomallei</i> Genome to Address Differential Virulence among Isolates

<div><p>Infection by the Gram-negative pathogen <i>Burkholderia pseudomallei</i> results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of <i>B. pseudomallei</i> is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number of <i>B. pseudomallei</i> genomes that are being sequenced and compared.</p><p>Here, we compared three <i>B. pseudomallei</i> genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of <i>B. pseudomallei</i> infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three <i>B. pseudomallei</i> isolates. While this work contributes to our understanding of <i>B. pseudomallei</i> genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to <i>B. pseudomallei</i> metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of <i>B. pseudomallei</i> on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what constitutes a fully virulent <i>Burkholderia</i> isolate may provide for better diagnostic and medical countermeasure strategies.</p></div

Venn diagram illustrating the numbers of CDS shared by <i>B. pseudomallei</i> strains K96243, MSHR668 and 1106a, determined by a bidirectional best BLAST hits analysis.

<p>The number of CDS unique to each genome in each pairwise comparison and the number of putative paralogs are shown. The total number of CDS present in each genome is given below the genome name.</p

General genome features.

<p>General genome features.</p