A Commensal Bacterium Promotes Virulence of an Opportunistic Pathogen via Cross-Respiration

Stacy, Apollo; Fleming, Derek; Lamont, Richard J.; Rumbaugh, Kendra P.; Whiteley, Marvin

doi:10.1128/mbio.00782-16

Cited by 78 publications

(87 citation statements)

References 38 publications

(59 reference statements)

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“…We also used the same mutagenesis procedure, conjugation with Escherichia coli on rich, undefined medium (tryptic soy agar supplemented with yeast extract [TSAYE]). Of note, we performed conjugations under both aerobic and anaerobic conditions, reasoning that this would increase mutant diversity since many genes in A. actinomycetemcomitans are required specifically under high or low oxygen levels (33). After generating the mutant pools, we defined the location and abundance of the insertions via Tn-seq.…”

Section: Resultsmentioning

confidence: 99%

“…AcrAB can export not only antibiotics but also host substrates, such as bile and steroids, and so in many pathogens AcrAB acts as a virulence factor (54). Although AcrAB is not essential for A. actinomycetemcomitans survival in abscesses (33), it may play an important role in the oral cavity.…”

Section: Discussionmentioning

confidence: 99%

“…The location and frequency of transposon insertions were determined largely as described previously (33,83). See Tables S1 and S2 for details and a summary of the analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The essential genome of each strain of A. actinomycetemcomitans was determined largely as described previously (41,48) using the following two replicates per strain: (i) Tn-seq on an aliquot of the mutant pool before amplification and (ii) Tn-seq on an aliquot of the mutant pool after amplification (see "A. actinomycetemcomitans mutant pools"). After correcting for polymerase slippage as described previously (33,87), the countif function in Microsoft Excel was used to filter for insertions present in both replicates (see Table S1). Local smoothing (LOESS) was then used as described previously (41,48) to correct for how multifork replication can inflate the abundance of insertions close to the origin of replication (see Table S2).…”

Section: Methodsmentioning

confidence: 99%

“…Software/language versions were fqgrep v0.4.3 (https://github.com/indraniel/fqgrep), cutadapt v1.12 (85), python v2.7.9, bowtie2 v2.2.5 (86), and R v3.3.1. Differential mutant abundance was tested at the site and gene level as described previously (33,83) with minor modifications. Read counts for the control and antibiotic treatments were merged by insertion site using the full_join() dplyr function in R. Read counts for sites present in one sample but not the other were set to 1.…”

Section: Ordered Library Validationmentioning

confidence: 99%

See 4 more Smart Citations

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

Narayanan

Ramsey

Stacy

et al. 2017

Appl Environ Microbiol

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…The location and frequency of transposon insertions were determined largely as described previously (33,83). See Tables S1 and S2 for details and a summary of the analysis.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ordered Library Validationmentioning

confidence: 99%

See 3 more Smart Citations

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

Narayanan

Ramsey

Stacy

et al. 2017

Appl Environ Microbiol

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Evaluation of microbiome in primary and permanent dentition in grade C periodontitis in young individuals

Koo,

Fernandes,

et al. 2024

Journal of Periodontology

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BackgroundThe aim of the present study was to evaluate the subgingival microbiome in patients with grade C molar‐incisor pattern periodontitis (C‐MIP) affecting the primary or permanent dentitions.MethodsDNA was isolated from subgingival biofilm samples from diseased and healthy sites from 45 C‐MIP patients and subjected to phylogenetic microarray analysis. C‐MIP sites were compared between children affected in the primary to those affected in the permanent dentitions. Within‐subject differences between C‐MIP‐affected sites and dentition‐matched healthy sites were also evaluated.ResultsC‐MIP sites of subjects affected in the primary dentition showed partially overlapping but distinct microbial communities from C‐MIP permanent dentition sites (p < 0.05). Differences were due to increased levels in primary C‐MIP sites of certain species of the genera Capnocytophaga and Leptotrichia, while C‐MIP permanent dentition sites showed higher prevalence of Filifactor alocis. Aggregatibacter actinomycetemcomitans (Aa) was among species seen in high prevalence and levels in both primary and permanent C‐MIP sites. Moreover, both permanent and primary C‐MIP sites showed distinct microbial communities when compared to dentition‐matched healthy sites in the same subject (p < 0.01).ConclusionsPrimary and permanent teeth with C‐MIP showed a dysbiotic microbiome, with children affected in the primary dentition showing a distinct profile from those affected in the permanent dentition. However, Aa was enriched in both primary and permanent diseased sites, confirming that this microorganism is implicated in C‐MIP in both dentitions.

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Querying Legionella Genomes Using Transposition-Sequencing

Hardy

Charpentier²

2019

Methods in Molecular Biology

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A Commensal Bacterium Promotes Virulence of an Opportunistic Pathogen via Cross-Respiration

Cited by 78 publications

References 38 publications

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

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

Evaluation of microbiome in primary and permanent dentition in grade C periodontitis in young individuals

Querying Legionella Genomes Using Transposition-Sequencing

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