Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development

Moule, Madeleine G.; Hemsley, Claudia; Seet, Qihui; Guerra-Assunção, José Afonso; Lim, Jiali; Sarkar‐Tyson, Mitali; Clark, Taane G.; Tan, Patrick; Titball, Richard W.; Cuccui, Jon; Wren, Brendan W.

doi:10.1128/mbio.00926-13

Cited by 73 publications

(101 citation statements)

References 34 publications

(57 reference statements)

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“…Essential genomes are the full set of genes required for the viability of an organism, and they can be leveraged to discover novel drug targets (43,44). Here, we used a Monte Carlo approach (41) to define the essential genome in two strains of the periodontal pathogen A. actinomycetemcomitans, VT1169 and 624.…”

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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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: 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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“…For example, the relative lack of mutants in a particular genetic element in a high-density transposon library can indicate the essentiality of that element. Previous studies have used several criteria to determine the immutable regions of a bacterial genome from Tn-seq data in the absence of control conditions, including the prevalence of transposon insertions detected per genetic element or the probability of encountering a DNA segment with no insertions given that segment's length (15)(16)(17). However, these methods do not always account for two types of information available in Tn-seq data: (i) the abundance of each transposon mutant and (ii) the variability observed in Tn-seq biological replicates.…”

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confidence: 99%

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

Turner

Wessel

Palmer

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

368

491

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Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.

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“…bpsl1444 shows similarity to the glycotransferase gene waaG. This is notable because many of the other waa genes, which are involved in the biosynthesis and construction of the core sugar of the B. pseudomallei LPS, were identified as essential genes (28). It would be of interest in future experiments to determine if bpsl1444 plays a role in virulence due to being important to the structural integrity of the bacterium or if this phenotype is due to compromised LPS.…”

Section: Discussionmentioning

confidence: 99%

“…This technique has been applied to large bacterial libraries to identify every essential gene within the genome and to identify new in vivo virulence factors (27,28). It can also be retrospectively applied to previously screened STM libraries to identify the insertion sites and phenotypes of mutants that were previously overlooked due to technical constraints, allowing the identification of new virulence factors without undertaking further animal experiments (29).…”

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confidence: 99%

Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

et al. 2016

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dBurkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates. Burkholderia pseudomallei is a Gram-negative, motile saprophytic bacterium that is the causative agent of melioidosis. This emerging human pathogen is endemic to the soil and water of tropical areas, including Thailand, Singapore, and northern Australia, and can cause infection through contact with broken skin or through ingestion or inhalation of the bacterium (1). The resulting disease can manifest as a localized skin ulcer or can progress to a systemic infection that is associated with mortality rates as high as 50% in some regions of endemicity (2). There is currently no licensed vaccine against B. pseudomallei available, and it is highly resistant to most antibiotics, severely limiting treatment options (3). Due to the virulent nature of the pathogen, potential for aerosol transmission, and lack of therapeutic options, B. pseudomallei is listed as a Tier 1 bioterrorism threat by the Centers for Disease Control and Prevention (4).B. pseudomallei is a facultative intracellular pathogen capable of invading and replicating within both epithelial cells and macrophages (5). While B. pseudomallei is capable of extracellular growth and survival and is highly resistant to complement-mediated killing in human sera, intracellular growth is essential for virulence (2, 6). When B. pseudomallei enters the host cell, either through phagocytosis or by inducing its own uptake into nonphagocytic cells, it is able to escape from the phagosome or endocyt...

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Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development

Cited by 73 publications

References 34 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

Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

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