Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in
            <i>Actinobacillus actinomycetemcomitans</i>
            and
            <i>Actinobacillus pleuropneumoniae</i>
            Biofilms

Kaplan, Jeffrey B.; Velliyagounder, Kabilan; Ragunath, Chandran; Rohde, Holger; Mack, Dietrich; Knobloch, J; Ramasubbu, Narayanan

doi:10.1128/jb.186.24.8213-8220.2004

Cited by 268 publications

(283 citation statements)

References 40 publications

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“…As we anticipated, a number of these mutants were disrupted for genes associated with A. actinomycetemcomitans biofilm formation, including 3 genes in the tight adherence (tad) locus, involved in assembling Flp pili (17), and 1 gene in the pga locus, involved in synthesizing the biofilm polysaccharide poly-N-acetylglucosamine (60) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

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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%

“…5B) encoded products known to assist in A. actinomycetemcomitans biofilm formationFlp pili (tadZ [17]) and poly-GlcNAc (pgaC [60]) (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

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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“…[8]). EMs consist of proteins, extracellular DNA (eDNA) and polysaccharides, such as poly-N-acetylglucosamine (PGA), which is the major polysaccharide in A. actinomycetemcomitans biofilms [9]. However, A. actinomycetemcomitans requires eDNA for cohesive biofilm formation, efficient adhesion to surfaces [10,11], and leukotoxin attachment to the bacterial membrane [12].…”

Section: Introductionmentioning

confidence: 99%

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

et al. 2018

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Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43 nM in static settings and 2.4 μM in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested β-lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.

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“…(One of the ends of the incoming DNA can have as little as 78 bp of homology.) The use of plasmid DNA to get allelic exchange has been helpful for site-directed mutagenesis of chromosomal genes (see, e.g., Kaplan et al, 2004;Fine et al, 2005;Rhodes et al, 2005;Perez et al, 2006). Transformation with undigested replicative plasmid DNA can yield A. actinomycetemcomitans strains that carry an extrachromosomal plasmid.…”

Section: Discussionmentioning

confidence: 99%

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Bhattacharjee

Fine

Figurski

2007

Gene

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AbstracttfoX (sxy) is a regulatory gene needed to turn on competence genes. Aggregatibacter (Actinobacillus) actinomycetemcomitans has a tfoX gene that is important for transformation. We cloned this gene on an IncQ plasmid downstream of the inducible tac promoter. When this plasmid was resident in cells of A. actinomycetemcomitans and tfoX was induced, the cells became competent for transformation. Several strains of A. actinomycetemcomitans, including different serotypes, as well as rough (adherent) and isogenic smooth (nonadherent) forms were tested. Only our two serotype f strains failed to be transformed. With the other strains, we could easily get transformants with extrachromosomal plasmid DNA when closed circular, replicative plasmid carrying an uptake signal sequence (USS) was used. When a replicative plasmid carrying a USS and cloned DNA from the chromosome of A. actinomycetemcomitans was linearized by digestion with a restriction endonuclease or when genomic DNA was used directly, the outcome was allelic exchange. To facilitate allelic exchange, we constructed a suicide plasmid (pMB78) that does not replicate in A. actinomycetemcomitans and carries a region with two inverted copies of a USS. This vector gave allelic exchange in the presence of cloned and induced tfoX easily and without digestion. Using transposon insertions in cloned katA DNA, we found that as little as 78 bp of homology at one of the ends was sufficient for that end to participate in allelic exchange. The cloning and induction of tfoX makes it possible to transform nearly any strain of A. actinomycetemcomitans, and allelic exchange has proven to be important for site-directed mutagenesis.

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Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms

Cited by 268 publications

References 40 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

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

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