scholarly journals Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium

2021 ◽  
Author(s):  
Gregory S. Canfield ◽  
Anushila Chatterjee ◽  
Juliel Espinosa ◽  
Mihnea R. Mangalea ◽  
Emma K. Sheriff ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Phage Therapy ◽  
Selective Pressure ◽  
Phage Resistance ◽  
Resistance Mutations ◽  
Penicillin Binding Proteins ◽  
Molecular Events ◽  
Wall Teichoic Acids ◽  
Arginine Aspartate

Enterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-aspartate-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could serve as effective antimicrobials for the treatment of E. faecium infections.

scholarly journals Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium

2020 ◽  
Author(s):  
Gregory S. Canfield ◽  
Anushila Chatterjee ◽  
Mihnea R. Mangalea ◽  
Emma K. Sheriff ◽  
Micah Keidan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Phage Therapy ◽  
Selective Pressure ◽  
Phage Resistance ◽  
Resistance Mutations ◽  
Penicillin Binding Proteins ◽  
Molecular Events ◽  
Wall Teichoic Acids ◽  
Arginine Aspartate

AbstractEnterococcus faecium, a commensal of the human intestine, has emerged as a hospital-adapted, multi-drug resistant (MDR) pathogen. Bacteriophages (phages), natural predators of bacteria, have regained attention as therapeutics to stem the rise of MDR bacteria. Despite their potential to curtail MDR E. faecium infections, the molecular events governing E. faecium-phage interactions remain largely unknown. Such interactions are important to delineate because phage selective pressure imposed on E. faecium will undoubtedly result in phage resistance phenotypes that could threaten the efficacy of phage therapy. In an effort to understand the emergence of phage resistance in E. faecium, three newly isolated lytic phages were used to demonstrate that E. faecium phage resistance is conferred through an array of cell wall-associated molecules, including secreted antigen A (SagA), enterococcal polysaccharide antigen (Epa), wall teichoic acids, capsule, and an arginine-arginine-aspartate (RDD) protein of unknown function. We find that capsule and Epa are important for robust phage adsorption and that phage resistance mutations in sagA, epaR, and epaX enhance E. faecium susceptibility to ceftriaxone, an antibiotic normally ineffective due to its low affinity for enterococcal penicillin binding proteins. Consistent with these findings, we provide evidence that phages potently synergize with cell wall (ceftriaxone and ampicillin) and membrane-acting (daptomycin) antimicrobials to slow or completely inhibit the growth of E. faecium. Our work demonstrates that the evolution of phage resistance comes with fitness defects resulting in drug sensitization and that lytic phages could potentially serve as antimicrobial adjuvants in treating E. faecium infections.

scholarly journals Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

2019 ◽  
Author(s):  
Anushila Chatterjee ◽  
Cydney N. Johnson ◽  
Phat Luong ◽  
Karthik Hullahalli ◽  
Sara W. McBride ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Phage Therapy ◽  
Selective Pressure ◽  
Acquired Resistance ◽  
Productive Infection ◽  
Phage Resistance ◽  
Intestinal Colonization ◽  
Intestinal Dysbiosis ◽  
Loss Of Resistance ◽  
Mutant Strains

AbstractEnterococcus faecalisis a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multi-drug resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism againstE. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain specific genes. We discovered that exposure to phage selective pressure favors mutation in non-conservedepagenes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance,epamutant strains exhibited a loss of resistance to the cell wall targeting antibiotics, vancomycin and daptomycin. Finally, we show that anE. faecalis epamutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin preventing their overgrowth during antibiotic treatment.

Cell wall teichoic acids of Bacillus licheniformis VKM B-511T, Bacillus pumilus VKM B-508T, and other strains previously assigned to Bacillus pumilus

Microbiology ◽  
2012 ◽  
Vol 81 (4) ◽  
pp. 425-434 ◽  
Author(s):  
G. M. Streshinskaya ◽  
A. S. Shashkov ◽  
Yu. I. Kozlova ◽  
E. M. Tul’skaya ◽  
E. B. Kudryashova ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Bacillus Pumilus ◽  
Teichoic Acids ◽  
Wall Teichoic Acids

New structures and composition of cell wall teichoic acids from Nocardiopsis synnemataformans, Nocardiopsis halotolerans, Nocardiopsis composta and Nocardiopsis metallicus: a chemotaxonomic value

2014 ◽  
Vol 106 (6) ◽  
pp. 1105-1117 ◽  
Author(s):  
Elena M. Tul’skaya ◽  
Alexander S. Shashkov ◽  
Galina M. Streshinskaya ◽  
Natalia V. Potekhina ◽  
Ludmila I. Evtushenko
Keyword(s):  
Cell Wall ◽  
Teichoic Acids ◽  
Wall Teichoic Acids

scholarly journals Neisseria gonorrhoeaePBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
Ryan E. Schaub ◽  
Krizia M. Perez-Medina ◽  
Kathleen T. Hackett ◽  
Daniel L. Garcia ◽  
Joseph P. Dillard
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
Inflammatory Response ◽  
Neisseria Gonorrhoeae ◽  
Wild Type ◽  
Cross Link ◽  
Content Type ◽  
Lytic Transglycosylases ◽  
Penicillin Binding Proteins ◽  
Proinflammatory Molecules

ABSTRACTNeisseria gonorrhoeaereleases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, >40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in eitherdacB, encoding PBP3, orpbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation indacBcaused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminald-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of bothdacBandpbpGeliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by thedacB pbpGmutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Rapid in vivo development of resistance to daptomycin in vancomycin-resistant Enterococcus faecium due to genomic rearrangements

2021 ◽  
Author(s):  
Sarah Mollerup ◽  
Christine Elmeskov ◽  
Heidi Gumpert ◽  
Mette Pinholt ◽  
Tobias Steen Sejersen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Chromosomal Rearrangements ◽  
Resistance Mechanisms ◽  
Resistant Isolate ◽  
Wall Structure ◽  
Whole Genome ◽  
Cyclic Lipopeptide ◽  
Vancomycin Resistant

AbstractBackgroundDaptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here we analysed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 40 days of daptomycin and linezolid combination therapy.MethodsThe two isogenic VREfm isolates (daptomycin-susceptible and daptomycin-resistant) were analysed using whole genome sequencing with Illumina and Nanopore.ResultsWhole genome comparative analysis identified the loss of a 46.5 kb fragment and duplication of a 29.7 kb fragment in the daptomycin-resistant isolate, with many implicated genes involved in cell wall synthesis. Two plasmids of the daptomycin-susceptible isolate were also found integrated in the chromosome of the resistant isolate. One nonsynonymous SNP in the rpoC gene was identified in the daptomycin-resistant isolate.ConclusionsDaptomycin resistance developed through chromosomal rearrangements leading to altered cell wall structure. Such novel types of resistance mechanisms can only be identified by comparing closed genomes of isogenic isolates.

scholarly journals Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Volker Winstel ◽  
Patricia Sanchez-Carballo ◽  
Otto Holst ◽  
Guoqing Xia ◽  
Andreas Peschel
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biosynthesis Pathway ◽  
Glycerol Phosphate ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Wall Teichoic Acids

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

scholarly journals FmhA and FmhC of Staphylococcus aureus incorporate serine residues into peptidoglycan cross-bridges

2020 ◽  
Vol 295 (39) ◽  
pp. 13664-13676 ◽  
Author(s):  
Stephanie Willing ◽  
Emma Dyer ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Proteins ◽  
Penicillin Binding Proteins ◽  
Daughter Cells ◽  
Cross Bridge ◽  
Resistant Strains ◽  
The Cross ◽  
Cross Bridges ◽  
Adjacent Wall

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In Staphylococcus aureus, pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of S. aureus. FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.

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