Microbial Community Composition Impacts Pathogen Iron Availability during Polymicrobial Infection

Iron is an essential nutrient for bacterial pathogenesis, but in the host, iron is tightly sequestered, limiting its availability for bacterial growth. Although this is an important arm of host immunity, most studies examine how bacteria respond to iron restriction in laboratory rather than host settings, where the microbiome can potentially alter pathogen strategies for acquiring iron. One of the most important transcriptional regulators controlling bacterial iron homeostasis is Fur. Here we used a combination of RNA-seq and chromatin immunoprecipitation (ChIP)-seq to characterize the iron-restricted and Fur regulons of the biofilm-forming opportunistic pathogen Aggregatibacter actinomycetemcomitans. We discovered that iron restriction and Fur regulate 4% and 3.5% of the genome, respectively. While most genes in these regulons were related to iron uptake and metabolism, we found that Fur also directly regulates the biofilm-dispersing enzyme Dispersin B, allowing A. actinomycetemcomitans to escape from iron-scarce environments. We then leveraged these datasets to assess the availability of iron to A. actinomycetemcomitans in its primary infection sites, abscesses and the oral cavity. We found that A. actinomycetemcomitans is not restricted for iron in a murine abscess mono-infection, but becomes restricted for iron upon co-infection with the oral commensal Streptococcus gordonii. Furthermore, in the transition from health to disease in human gum infection, A. actinomycetemcomitans also becomes restricted for iron. These results suggest that host iron availability is heterogeneous and dependent on the infecting bacterial community

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans. One of this bacterium\u27s key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field\u27s lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising \u3e1,500 unique insertions and used a sequencing-based approach to define each mutant\u27s position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen

Co-infecting microbes dramatically alter pathogen gene essentiality during polymicrobial infection

Identifying genes required by pathogens during infection is critical for antimicrobial development. Here, we used a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data to determine the role of infection site and co-infecting microbes on the in vivo ‘essential’ genome of Staphylococcus aureus. We discovered that co-infection of murine surgical wounds with Pseudomonas aeruginosa results in conversion of ~25% of the in vivo S. aureus mono-culture essential genes to non-essential. Furthermore, 182 S. aureus genes are uniquely essential during co-infection. These “Community Dependent Essential” (CoDE) genes illustrate the importance of studying pathogen gene essentiality in polymicrobial communities

Localization of Synuclein Protein in Mouse Auditory Tissue

From the Washington University Senior Honors Thesis Abstracts (WUSHTA), Volume 2, Spring 2010. Published by the Office of Undergraduate Research. Henry Biggs, Director, Office of Undergraduate Research / Associate Dean, College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator / Assistant Dean in the College of Arts & Sciences; E. Holly Tasker, Editor. Mentor: Brian Faddi

Localization of Synuclein Protein in Mouse Auditory Tissue

Mentor: Brian Faddis From the Washington University Undergraduate Research Digest: WUURD, Volume 6, Issue 1, Fall 2010. Published by the Office of Undergraduate Research. Henry Biggs, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator, Co-editor, and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor

The biogeography of polymicrobial infection

Bacteria usually cause human infections as multispecies communities. These communities often spatially organize into surface-attached structures known as biofilms. Within biofilms, bacteria interact by exchanging metabolites and competing for nutrients, such as carbon and iron sources. These interactions can result in synergy, or enhanced bacterial persistence. Despite its clinical relevance, we lack approaches for understanding synergy. One of the most prevalent polymicrobial infections is periodontitis, or severe gum inflammation. This condition leads to tooth loss, and the associated bacteria also cause life-threatening abscesses. The most abundant bacteria in the oral cavity are streptococci. Streptococci release high amounts of lactate and peroxide as waste. Previously we showed that Streptococcus gordonii (Sg) enhances the persistence of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) in murine abscesses. Aa prefers lactate as a carbon source, and cross-feeding by Aa on lactate made by Sg is critical for synergy in abscesses. Unclear from these studies was how Aa simultaneously tolerates peroxide, an antimicrobial, in the abscess. Furthermore, Aa can only catabolize lactate if oxygen is available, but abscesses are generally considered anaerobic. Through 3D spatial analysis of abscesses, I showed that Aa senses peroxide to localize to a 4-13 μm distance from Sg, where it can presumably cross-feed on lactate but avoid peroxide. I then applied high-throughput genomic approaches to study Aa synergy with Sg. Through transposon mutant fitness profiling (Tn-seq) on Aa in abscesses, I showed that Sg enhances oxygen availability, shifting Aa from a low- to high-energy metabolism where it can cross-feed on lactate. Through RNA-seq and chromatin immunoprecipitation followed by sequencing (ChIP-seq) on the Aa Ferric uptake regulator (Fur), I showed that in mono-abscesses Aa can access iron, an essential nutrient often sequestered by the host, but in co-culture abscesses Sg reduces iron availability. Furthermore, I showed that in human oral infections, the shift to disease also reduces Aa iron availability. Together, these studies reveal novel interactions that underlie synergy in model and human polymicrobial infections.Microbiolog

Iron and Fur regulate Dispersin B.

<p>(A) Structure of the <i>dspB</i> promoter. Gray, OxyR box; orange, Fur box; underlined, -35 and -10 regions; +1, transcriptional start site; bold, start codon. (B) <i>dspB</i> transcription in colony biofilms was measured using a <i>dspB</i> promoter-<i>lacZ</i> transcriptional fusion. Left panel: blue, <i>A</i>. <i>actinomycetemcomitans</i> strain 624; red, <i>A</i>. <i>actinomycetemcomitans</i> strain VT1169. Right panel: blue, <i>A</i>. <i>actinomycetemcomitans</i> strain 624 wild type (wt); red, <i>A</i>. <i>actinomycetemcomitans</i> strain 624 <i>Δfur</i> (<i>Δfur</i>). Chelator is 250 μM 2,2’-dipyridyl, and Fe is 250 μM FeSO₄. Y axis is fold change (FC) in <i>dspB</i> expression relative to no chelator (-chelator) and no FeSO₄ (-Fe) addition. Error bars represent standard deviation (n = 3). (C) <i>dspB</i> mRNA levels in colony biofilms was measured using reverse transcriptase PCR in iron-replete (+Fe) and iron-restricted (-Fe) conditions. <i>clpX</i> serves as a control that is not regulated by iron or Fur. Wild type (wt), <i>Δfur</i> (<i>Δfur</i>), <i>Δfur</i> + <i>fur-vsv-g</i> (<i>Δfur</i> genetically complemented with VSV-G tagged Fur). (D) Biofilm dispersal assay. A second, higher ring biofilm (indicated by arrow) indicates dispersal. The purple stain is crystal violet. Chelator is 250 μM 2,2’-dipyridyl; -oxygen is anaerobic growth; +oxygen is aerobic growth.</p

<i>A</i>. <i>actinomycetemcomitans</i> is iron-restricted in murine abscess co-infection.

<p>(A) Principal component analysis of the 93 genes regulated by iron. Each dot is a single replicate. Legend: Fe+, biofilm on rich media; Fe-, biofilm on iron-chelated media; mono, abscess mono-infection; co, abscess co-infection with <i>S</i>. <i>gordonii</i>. Axes: Percentages are the amount of variation captured by each principal component. (B) Correlation analysis of the 93 genes regulated by iron. Spearman’s rank correlation was determined by comparing Fe+ and Fe- <i>in vitro</i> biofilms to <i>A</i>. <i>actinomycetemcomitans</i> gene expression in mono-infection (mono vs. Fe+ and Fe-) or co-infection with <i>S</i>. <i>gordonii</i> (co vs. Fe+ and Fe-). Error bars represent standard deviation (n = 4–6 pairwise comparisons). Significance was determined using a 2-tailed t test.</p

The <i>A</i>. <i>actinomycetemcomitans</i> iron-restricted regulon.

<p>Cellular processes differentially expressed by iron restriction. Shaded numbers above each process indicate fold change. 1.5–2.0 fold, light shade; >2.0–4.0 fold, medium shade; >4.0 fold, dark shade. Octagon, ferritin; Q, quinone; R, respiratory reductase; TMAO, trimethylamine N-oxide; TMA, trimethylamine; PFL, pyruvate formate lyase; FHL, formate hydrogen lyase; Afu and Afe, characterized transporters; Hg, hemoglobin; Tf, transferrin; Cys, cysteine; G3P, glycerol-3-phosphate; hairpin, sRNA; TRX, thioredoxin; DspB, Dispersin B.</p

<i>A</i>. <i>actinomycetemcomitans</i> is not iron-restricted in murine abscess mono-infection.

<p>(A) Principal component analysis of the 93 genes regulated by iron. Each dot is a single replicate. Legend: Fe+, biofilm on rich media; Fe-, biofilm on iron-chelated media; abscess, wild-type abscess infection. Axes: Percentages are the amount of variation captured by each principal component. (B) Correlation analysis of the 93 genes regulated by iron. Spearman’s rank correlation was determined by comparing gene expression in wild-type <i>A</i>. <i>actinomycetemcomitans</i> abscess infection to Fe+ and Fe- <i>in vitro</i> biofilms. Error bars represent standard deviation (n = 6 pairwise comparisons). Significance was determined using a 2-tailed t test. (C) Survival of the wild type (wt) and <i>Δfur</i> mutant in abscesses. Each dot is a single abscess (n = 2 biological replicates). Significance was determined using a Mann-Whitney U test. Y axis represents colony forming units (CFU) per abscess after 3 days post-infection. (D) Venn diagram showing the overlap between the <i>in vitro</i> and <i>in vivo</i> ChIP-seq results.</p