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

Shields, Robert C.; Zeng, Lin; Culp, David J.; Burne, Robert A.

doi:10.1128/msphere.00031-18

Cited by 52 publications

(96 citation statements)

References 70 publications

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“…Deletion of rmlB and rmlC in S. mutans resulted in a phenotype similar as previously observed for an rmlD deletion mutant (van der Beek et al, 2015), underscoring that inhibition of the dTDP-rhamnose biosynthesis pathway severely impacts bacterial viability. Indeed, the rmlABCD genes are essential for S. mutans in the competitive environment of a mutant transposon library (Shields et al, 2018), similar to GAS and GCS (Le Breton et al, 2013;Charbonneau et al, 2017). We are however able to construct rml deletion mutants in S. mutans in isolation in contrast to similar attempts in GAS (van Sorge et al, 2014).…”

Section: Discussionmentioning

confidence: 88%

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Beek

Zorzoli

Çanak

et al. 2019

Molecular Microbiology

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Summary Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus ; GAS), Streptococcus mutans and Mycobacterium tuberculosis . Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans , we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC 50 of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.

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Section: Discussionmentioning

confidence: 88%

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Beek

Zorzoli

Çanak

et al. 2019

Molecular Microbiology

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“…Although some loci have no COG assigned, 714 genes were assigned a COG functional category (Table S2). Each COG category was plotted against its “COG Enrichment Index”, which is calculated as the percentage of non-neutral genes in each COG category divided by the percent of the whole genome with that COG (51). A subset of COGs, particularly cell cycle control, translation, lipid and coenzyme metabolism, and cell wall biogenesis were associated with non-neutral genes at a frequency significantly higher than expected based on their genomic representation (Fig S2A).…”

Section: Resultsmentioning

confidence: 99%

“…To determine if COGs were enriched in certain groups of genes (such as conditionally depleted genes), a COG enrichment index was calculated as in (51). The COG Enrichment Index is the percentage of the genes of a certain category (essential genes or CD genes) assigned to a specific COG divided by the percentage of genes in that COG in the entire genome.…”

Section: Methodsmentioning

confidence: 99%

“…A) Non-neutral genes (defined by EL-ARTIST as either regional or underrepresented) by Clusters of Orthologous Groups (COG) classification. COG enrichment index (displayed as log2 enrichment) is calculated as defined in (51) as the percentage of the CD genes assigned to a specific COG divided by the percentage of genes in that COG in the entire genome. A two-tailed Fisher’s exact test with a Bonferroni correction was used to test the null hypothesis that enrichment is independent of TIS status.…”

Section: Supplementary Figure Legendsmentioning

confidence: 99%

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Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

Warr

Hubbard

Múnera

et al. 2019

Preprint

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31 Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne 32 pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to 33 identify genes required for EHEC and commensal E. coli K-12 growth in vitro and for 34 EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci 35 contribute to EHEC's but not to K-12's growth in vitro, suggesting that gene acquisition 36 during EHEC evolution has heightened the pathogen's reliance on certain metabolic 37 processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC 38 colonization of the rabbit colon, which complicated identification of EHEC genes 39 facilitating growth in vivo. Both a refined version of an existing analytic framework as 40 well as PCA-based analysis were used to compensate for the effects of the infection 41 bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion 42 apparatus is required for growth in vivo and revealed that only a few effectors are critical 43 for in vivo fitness. Numerous mutants not previously associated with EHEC 44 survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative 45 in vivo fitness factors were validated. Some were found to contribute to efficient type-46 three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote 47 EHEC resistance to host-derived stresses encountered in vivo. cvpA, which is also 48 required for intestinal growth of several other enteric pathogens, proved to be required 49 for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt 50 deoxycholate. Collectively, our findings provide a comprehensive framework for 51 understanding EHEC growth in the intestine. 523 53 Author Summary 54 Enterohemorrhagic E. coli (EHEC) are important food-borne pathogens that infect the 55 colon. We created a highly saturated EHEC transposon library and used transposon 56 insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo 57 in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit 58 model of intestinal colonization, and refined two analytic approaches to facilitate 59 rigorous identification of new EHEC genes that promote fitness in vivo. Besides the 60 known type III secretion system, more than 200 additional genes were found to 61 contribute to EHEC survival and/or growth within the intestine. The requirement for 62 some of these new in vivo fitness factors was confirmed, and their contributions to 63 infection were investigated. This set of genes should be of considerable value for future 64 studies elucidating the processes that enable the pathogen to proliferate in vivo and for 65 design of new therapeutics. 66 4 67 Introduction 68 Enterohemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen that 69 causes gastrointestinal (GI) infections worldwide. EHEC is a non-invasive pathogen that 70 colonizes the human colon and gives rise to sporadi...

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“…We compared the in silico deletions to two experimental gene deletion studies in S. mutans UA159: a transposon mutagenesis sequencing (Tn-seq) experiment (47) and a screen of an ordered array of single gene deletion strains (48). The Tn-seq study used a mariner-family transposon to generate random insertions across the UA159 genome (47). The transposon/genomic DNA junctions were amplified and sequenced to quantify fitness after growth in a defined media (FMC).…”

Section: Gene Deletion Simulations Match Experimental Datamentioning

confidence: 99%

Metabolic modeling ofStreptococcus mutansreveals complex nutrient requirements of an oral pathogen

Jijakli

Jensen

2018

Preprint

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Streptococcus mutans is a Gram positive bacterium that thrives under acidic conditions and is a primary cause of tooth decay (dental caries). To better understand the metabolism of S. mutans on a systematic level, we manually constructed a genomescale metabolic model of the S. mutans type strain UA159. The model, called iSMU, contains 656 reactions involving 514 metabolites and the products of 488 genes.We interrogated S. mutans' nutrient requirements using model simulations and nutrient removal experiments in defined media. The iSMU model matched experimental results in greater than 90% of the conditions tested. We also simulated effects of single gene deletions. The model's predictions agreed with 78.1% and 84.4% of the gene essentiality predictions from two experimental datasets. Our manually curated model is more accurate than S. mutans models generated from automated reconstruction pipelines. We believe the iSMU model is an important resource for understanding how metabolism enables the cariogenicity of S. mutans.

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

Cited by 52 publications

References 70 publications

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

Metabolic modeling ofStreptococcus mutansreveals complex nutrient requirements of an oral pathogen

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