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 Ͼ1,500 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 Tnseq-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 O ne of the longest-studied human microbiomes is the "animalcules" (bacteria), first observed by van Leeuwenhoek (1), that inhabit the oral cavity. Oral bacteria readily attach to tooth surfaces and develop into spatially organized, multispecies biofilms (2). These communities also persist beneath the gum line in the subgingival pocket, where they are kept in check by host immune cells (3). Though normally benign, subgingival communities can accumulate opportunistic oral p...

Genomewide Identification of Essential Genes and Fitness Determinants of Streptococcus mutans UA159

“…Genes encoding products related to translation, lipid metabolism, and cell wall biogenesis were enriched among essential genes (COGs J, I, and M) ( Fig. 2 ), consistent with essential genomes identified in other bacterial species ( 23 , 27 ). Genes associated with signal transduction, transcription, general functions, or of unknown function were depleted (COGs K, R, S, and T), suggesting that these functions are less important for growth on blood agar.…”

“…Conversely, 100 to 300 noncore genes were estimated previously to differ between any two strains of S. mutans ( 29 ). When comparing the essential genomes of two strains of the same species, differences in essential gene content and metabolic pathways have been reported ( 26 , 27 ). Clearly, accessory gene content is important and could alter gene-gene networks in such a way that genes that are essential in one strain become functionally redundant in another strain.…”

Aggregatibacter, a Low Abundance Pathobiont That Influences Biogeography, Microbial Dysbiosis, and Host Defense Capabilities in Periodontitis: The History of a Bug, and Localization of Disease

“…This ltx gene deletion resulted in a complete lack of attachment in vivo and reduced binding to hydroxyapatite in vitro, resulting from reduced expression of genes in the tad operon [55]. This linkage between ltx genes and flp genes was confirmed in another model, which showed that, when A. actinomycetemcomitans was stressed, four genes were linked: flp, ltx, pgA, and tfox [56].…”

Transcriptomic Analysis of Aggregatibacter actinomycetemcomitans Core and Accessory Genes in Different Growth Conditions

“…In this study, the core genes were identified by comparative genomics and may or may not be the same as essential genes that require experimental confirmation. A previous study utilizing rapid transposon mutant sequencing (Tn-Seq) had established the presence of essential genomes in two divergent A. actinomycetemcomitans strains, VT1169 and 624 strains [24]. Notably, 307 of the 319 essential genes matched to our core gene pool.…”

“…To determine pathways affected by the differentially expressed genes, we used the KEGG Mapper Search and Color Pathway tool as previously described [24,49] using the locus tag of each differentially expressed genes obtained from each media. A comparison of the pathways affected were then attempted.…”