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

Abstract

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's 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's 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 >1,500 unique insertions and used a sequencing-based approach to define each mutant's 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.IMPORTANCE Millions suffer from gum disease, which often is caused by Aggregatibacter actinomycetemcomitans, a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for A. actinomycetemcomitans by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause A. actinomycetemcomitans to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen.

Keywords: Aggregatibacter; Tn-seq; antibiotics; biofilms; essential genome.

FIG 1

The A. actinomycetemcomitans essential genome. (A) Density plots of the fold change in mutant abundance compared to expected value in VT1169 (left) and 624 (right). Essential genes (red) have significantly fewer mutants than expected (gray). (B) Scatter plot of the log₂ fold change (FC) in mutant abundance compared to expected in VT1169 (x axis) versus 624 (y axis). Each point corresponds to an ortholog. Gray, not essential in either strain; red, essential in both strains; blue, essential in VT1169 only; purple, essential in 624 only.

FIG 2

The A. actinomycetemcomitans essential metabolic network. Dots and lines represent compounds and reactions, respectively. Blue, pathways essential in both strains; green, pathways essential in VT1169 only; red, pathways essential in 624 only. The shaded regions indicate specific metabolites or metabolic pathways. PRPP, phosphoribosyl pyrophosphate; CoA, coenzyme A.

FIG 3

Validation of the A. actinomycetemcomitans ordered library. P, plate; W, well; Site, transposon insertion site in the VT1169 genome; Product, gene product nearest the transposon insertion; PTS, phosphotransferase; Ltx, leukotoxin. Each box under the Sanger and PCR headings indicates a replicate. Green, positive Sanger/PCR result; red, negative Sanger/PCR result.

FIG 4

A. actinomycetemcomitans processes affected by subinhibitory erythromycin. The horizontal gray lines represent the outer (top line) and inner (bottom line) membranes. LtxA, leukotoxin; e⁻, electron; DMSO, dimethyl sulfoxide; fum, fumarate; Gln, glutamine; Glu, glutamate; poly-GlcNAc, poly-N-acetylglucosamine. R, B, and D represent RNases.

FIG 5

A. actinomycetemcomitans factors that mediate antibiotic-induced attachment. Growth rates (A) and attachment levels (B) were measured for the wild type (wt) and mutants in half-MIC (+) erythromycin or none (−). y axis in panel A, doublings per hour; y axis in panel B, absorbance (A₆₂₀) of crystal violet bound to biofilm; blue, mutants that decreased in abundance after erythromycin exposure; orange, mutants that increased in abundance after erythromycin exposure; purple, defective for antibiotic-induced attachment; ND, not detected. Data labels underneath indicate each mutant's disrupted gene product and coordinates in the ordered library. poly-GlcNAc, poly-N-acetylglucosamine; Ltx, leukotoxin. Error bars represent standard deviations (n = 3 to 4 for growth rate; n = 11 to 16 for attachment). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student's t test).