MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB47255 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR6589710_1.fastq.gz | Fastqsanger.gz | |||
| ERR6589710_2.fastq.gz | Fastqsanger.gz | |||
| ERR6589711_1.fastq.gz | Fastqsanger.gz | |||
| ERR6589711_2.fastq.gz | Fastqsanger.gz | |||
| ERR6589712_1.fastq.gz | Fastqsanger.gz |
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Project description:Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to activate PG biosynthesis and further enhanced the intrinsic cephalosporin resistance of L. monocytogenes. This latter effect required the RodA3/PBP B3 transglycosylase/transpeptidase pair. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during standard laboratory growth conditions and intracellular growth in macrophages. These findings have important implications for the understanding of PG biosynthesis regulation and β-lactam resistance of L. monocytogenes and related Gram-positive bacteria.
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Project description:PrkA controls peptidoglycan biosynthesis through the essential phosphorylation of ReoM
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Project description:Identifying antibody escape mutations using the evolutionary capacity of trVLP-ASV
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Project description:CD11b+ CD11c- cells were isolated by cell sorting from the spleens of NOD and NOD.Idd3 mice. Cells were then either unstimulated or stimulated with peptidoglycan (5ug/ml) for 4 hours at 37 degrees prior to RNA extraction. Peptidoglycan was chosen as a stimulus due to its know ability to stimulate CD11b+ cells through Toll-like receptor 2 and our previous data showing biological differences between NOD and NOD.Idd3-derived CD11b+CD11c- cells after stimulation with peptidoglycan. CD11b+ cells from NOD and NOD.Idd3 were either unstimulated or stimulated with peptidoglycan(PGN) for 4 hours at 37 degerees prior to RNA extraction.
2010-09-28 | E-GEOD-24350 | biostudies-arrayexpress
Project description:Streptococcus pneumoniae is a human commensal bacterium that causes serious diseases. Peptidoglycan (PG) is the critical component of bacteria that maintains cell shape, size, chaining, and turgor resistance. Because of its surface exposure and eubacterial-specific mechanism of biosynthesis, PG biosynthesis remains an outstanding target for discovery of new antibiotics to resistant “superbugs,” like S. pneumoniae. Pneumococcal PG biosynthesis is carried out by a balance of septal and peripheral (side-wall-like) PG synthesis mediated by bPBP2x and bPBP2b, respectively, that emanates from midcell regions. We selected for additional suppressor mutations, besides mltG, that eliminate the requirement for essential bPBP2b in peripheral PG synthesis. We found that mutations that inactivate a putative RNA binding protein, designated KhpA, suppressed the requirement for some, but not all of the essential proteins required for peripheral PG synthesis. KhpA was used as bait in co-IP experiments to demonstrate interactions with a second RNA binding protein, designated KhpB, in cells. Mutations in khpA or khpB phenocopy each other and result in slower growth rates, smaller cell size with normal aspect ratios, and induction of cell wall stress responses. RIP-Seq analyses confirmed KhpAB as RNA binding protein in cells. Another suppressor of Δpbp2b implicated induction of a cell division operon as a way to bypass the requirement for bPBP2b. We show that increased expression of this cell division operon occurs in ΔkhpAB mutants and that this induction is necessary and sufficient to account for suppression of Δpbp2b. Comparisons of relative transcript amounts and protein levels indicate that induction of this cell division operon in ΔkhpAB mutants occurs primarily at the post-transcriptional level. Together, our results show that KhpAB, which are virulence factors in S. pneumoniae and are conserved in other Gram-positive pathogens, acts as a new class of RNA binding protein, with properties analogous to Hfq in Gram-negative bacteria.
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Project description:Using transcriptomics, we studied the transcriptional response of Vibrio cholerae to 10 min of exogenously supplied peptidoglycan at 300 µg/mL.
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2010-09-28 | GSE24350 | GEO
Project description:(Manuscript abstract) "Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently."
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