Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening

Matano, Leigh M.; Morris, Heidi G.; Wood, B.M.; Meredith, Timothy C.; Walker, Suzanne

doi:10.1016/j.bmc.2016.08.003

Cited by 25 publications

(32 citation statements)

References 62 publications

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“…S. aureus evasion from human neutrophils and colonization and virulence in mouse models were dependent on the D-alanine modification (30,31). In an effort to stem the tide of antibiotic-resistant S. aureus infections, the DLT pathway has been suggested as a target for small molecule inhibitors, and some have already been identified (33,34).…”

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confidence: 99%

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

Wood

Maria²,

Matano

et al. 2018

Journal of Biological Chemistry

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Modifications to the Gram-positive bacterial cell wall play important roles in antibiotic resistance and pathogenesis, but the pathway for the D-alanylation of teichoic acids (DLT pathway), a ubiquitous modification, is poorly understood. The D-alanylation machinery includes two membrane proteins of unclear function, DltB and DltD, which are somehow involved in transfer of D-alanine from a carrier protein inside the cell to teichoic acids on the cell surface. Here, we probed the role of DltD in the human pathogen Staphylococcus aureus using both cell-based and biochemical assays. We first exploited a known synthetic lethal interaction to establish the essentiality of each gene in the DLT pathway for D-alanylation of lipoteichoic acid (LTA) and confirmed this by directly detecting radiolabeled D-Ala-LTA both in cells and in vesicles prepared from mutant strains of S. aureus. We developed a partial reconstitution of the pathway by using cell-derived vesicles containing DltB, but no other components of the D-alanylation pathway, and showed that D-alanylation of previously formed lipoteichoic acid in the DltB vesicles requires the presence of purified and reconstituted DltA, DltC, and DltD, but not of the LTA synthase LtaS. Finally, based on the activity of DltD mutants in cells and in our reconstituted system, we determined that Ser-70 and His-361 are essential for D-alanylation activity, and we propose that DltD uses a catalytic dyad to transfer D-alanine to LTA. In summary, we have developed a suite of assays for investigating the bacterial DLT pathway and uncovered a role for DltD in LTA D-alanylation.

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confidence: 99%

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

Wood

Maria²,

Matano

et al. 2018

Journal of Biological Chemistry

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“…1B, Fig. 2A) (49, 51). Using spot titre assays, we found here that Δ ltaA strains, like Δ ugtP strains, are susceptible to both DltB inhibitors (Fig.…”

Section: Resultsmentioning

confidence: 95%

The length of lipoteichoic acid polymers controlsStaphylococcus aureuscell size and envelope integrity

Hesser¹,

Matano²,

Vickery³

et al. 2020

Preprint

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24The opportunistic pathogen Staphylococcus aureus is protected by a cell envelope 25 that is crucial for viability. In addition to peptidoglycan, lipoteichoic acid (LTA) is an 26 especially important component of the S. aureus cell envelope. LTA is an anionic polymer 27 anchored to a glycolipid in the outer leaflet of the cell membrane. It was known that 28 deleting the gene for UgtP, the enzyme that makes this glycolipid anchor, causes cell 29 growth and division defects. In Bacillus subtilis, growth abnormalities from the loss of ugtP 30 have been attributed to the absence of the encoded protein, not to loss of its enzymatic 31 activity. Here, we show that growth defects in S. aureus ugtP deletion mutants are due to 32 the long, abnormal LTA polymer that is produced when the glycolipid anchor is missing 33 from the outer leaflet of the membrane. Dysregulated cell growth leads to defective cell 34 division, and these phenotypes are corrected by mutations in the LTA polymerase, ltaS, 35 that reduce polymer length. We also show that S. aureus mutants with long LTA are 36 sensitized to cell wall hydrolases, beta-lactam antibiotics, and compounds that target 37 other cell envelope pathways. We conclude that control of LTA polymer length is 38 important for S. aureus physiology and promotes survival under stressful conditions, 39including antibiotic stress. 40 41 IMPORTANCE 42Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of 43 community-and hospital-acquired infections and is responsible for a large fraction of 44 deaths caused by antibiotic-resistant bacteria. S. aureus is surrounded by a complex cell 45 envelope that protects it from antimicrobial compounds and other stresses. Here we show 46 3 that controlling the length of an essential cell envelope polymer, lipoteichoic acid, is critical 47 for controlling S. aureus cell size and cell envelope integrity. We also show that genes 48 involved in LTA length regulation are required for resistance to beta-lactam antibiotics in 49MRSA. The proteins encoded by these genes may be targets for combination therapy 50 with an appropriate beta-lactam. 51 52 129 RESULTS 130 ugtP and ltaA mutants are larger than wild type cells and have cell division 131 defects 132A previous study in S. aureus reported that ugtP cells are larger than wild type 133 cells and have other morphological defects (32), but ltaA mutant cells have not been 134 examined in detail. We compared the morphology of otherwise isogenic wild type, ltaA, 135 and ugtP strains by transmission electron microscopy (TEM) and quantified cell size 136 using brightfield and epifluorescence microscopy after staining with a membrane dye. 137 8. Weidenmaier C, Peschel A, Kempf VA, Lucindo N, Yeaman MR, Bayer AS. 2005. DltABCD-and 595 MprF-mediated cell envelope modifications of Staphylococcus aureus confer resistance to 596 platelet microbicidal proteins and contribute to virulence in a rabbit endocarditis model. Infect 597 Immun 73:8033-8.598 9.

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“…As an alternative, pathway-directed whole cell screening approaches, as have been used to discover small molecule inhibitors of teichoic acid biosynthesis in S . aureus , may provide advantages including the ability to screen in the relevant organism, a simple assay format, the presence of biological activity, expectation of on-target activity, and the ability to target multiple enzymes simultaneously [ 89 – 92 ].…”

Section: Discussionmentioning

confidence: 99%

A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in Acinetobacter baumannii

et al. 2018

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Acinetobacter baumannii ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes lpxA, lpxC or lpxD. Although LOS itself is not essential for growth of A. baumannii ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.

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Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening

Cited by 25 publications

References 62 publications

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

The length of lipoteichoic acid polymers controlsStaphylococcus aureuscell size and envelope integrity

A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in Acinetobacter baumannii

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