scholarly journals A new class of cell wall-recycling L,D-carboxypeptidase determines β-lactam susceptibility and morphogenesis in Acinetobacter baumannii

2021 ◽  
Author(s):  
Yunfei Dai ◽  
Victor Pinedo ◽  
Amy Y Tang ◽  
Felipe Cava ◽  
Edward Geisinger
Keyword(s):  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Cell Envelope ◽  
Membrane Lipid ◽  
Model Organisms ◽  
Complex Cell ◽  
Intrinsic Resistance ◽  
New Class ◽  
Hospital Acquired ◽  
Cell Wall Recycling

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for cell elongation and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell-wall-modifying L,D-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic L,D-carboxypeptidase (LDC) that provides a critical step in cell-wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, elongation, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including L,D-crosslink formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on cell envelope physiology. We thus reveal a novel class of cell-wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria.

scholarly journals A New Class of Cell Wall-Recycling l , d -Carboxypeptidase Determines β-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii

mBio ◽  
2021 ◽  
Author(s):  
Yunfei Dai ◽  
Victor Pinedo ◽  
Amy Y. Tang ◽  
Felipe Cava ◽  
Edward Geisinger
Keyword(s):  
Immune Response ◽  
Cell Wall ◽  
Enzymatic Activity ◽  
Acinetobacter Baumannii ◽  
Drug Resistant ◽  
Content Type ◽  
New Class ◽  
Recycling Pathway ◽  
And Control ◽  
Cell Wall Recycling

To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l , d -carboxypeptidase (LDC). Acinetobacter baumannii , an “urgent-threat” pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog.

scholarly journals Lon Protease Has Multifaceted Biological Functions inAcinetobacter baumannii

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Carly Ching ◽  
Brendan Yang ◽  
Chineme Onwubueke ◽  
David Lazinski ◽  
Andrew Camilli ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Developmental Process ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Lon Protease ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Biofilm Development ◽  
Hospital Acquired

ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) ofA. baumanniican withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process inA. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease inA. baumanniiaffects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing keyA. baumanniiprocesses to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumanniiis an opportunistic pathogen and is a leading cause of hospital-acquired infections.A. baumanniiis difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation inA. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon inA. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology ofA. baumannii, which will permit us to find effective ways to eliminate the bacterium.

scholarly journals Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

2020 ◽  
Author(s):  
Edward Geisinger ◽  
Nadav J. Mortman ◽  
Yunfei Dai ◽  
Murat Cokol ◽  
Sapna Syal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Cell Envelope ◽  
Opportunistic Pathogen ◽  
Drug Action ◽  
Antibiotic Development ◽  
Cell Wall Assembly ◽  
Combination Strategies ◽  
High Doses ◽  
Wall Assembly

AbstractAcinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of β-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

scholarly journals CraA, a Major Facilitator Superfamily Efflux Pump Associated with Chloramphenicol Resistance in Acinetobacter baumannii

2009 ◽  
Vol 53 (9) ◽  
pp. 4013-4014 ◽  
Author(s):  
I. Roca ◽  
S. Marti ◽  
P. Espinal ◽  
P. Martínez ◽  
I. Gibert ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Acinetobacter Baumannii ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Intrinsic Resistance ◽  
Chloramphenicol Resistance ◽  
Hospital Acquired Infections ◽  
Major Facilitator ◽  
Hospital Acquired

ABSTRACT Acinetobacter baumannii has been increasingly associated with hospital-acquired infections, and the presence of multidrug resistance strains is of great concern to clinicians. A. baumannii is thought to possess a great deal of intrinsic resistance to several antimicrobial agents, including chloramphenicol, although the mechanisms involved in such resistance are not well understood. In this work, we have identified a major facilitator superfamily efflux pump present in most A. baumannii strains, displaying strong substrate specificity toward chloramphenicol.

scholarly journals Oxidative Stress Enhances Cephalosporin Resistance of Enterococcus faecalis through Activation of a Two-Component Signaling System

2014 ◽  
Vol 59 (1) ◽  
pp. 159-169 ◽  
Author(s):  
Dušanka Djorić ◽  
Christopher J. Kristich
Keyword(s):  
Oxidative Stress ◽  
Enterococcus Faecalis ◽  
Cell Envelope ◽  
Signaling System ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Prior Therapy ◽  
Two Component ◽  
Cephalosporin Resistance ◽  
Hospital Acquired

ABSTRACTEnterococcus faecalisis a low-GC Gram-positive bacterium, a normal resident of the gastrointestinal (GI) tract, and an important hospital-acquired pathogen. An important risk factor for hospital-acquired enterococcal infections is prior therapy with broad-spectrum cephalosporins, antibiotics that impair cell wall biosynthesis by inhibiting peptidoglycan cross-linking. Enterococci are intrinsically resistant to cephalosporins; however, environmental factors that modulate cephalosporin resistance have not been described. While searching for the genetic determinants of cephalosporin resistance inE. faecalis, we unexpectedly discovered that oxidative stress, whether from external sources or derived from endogenous metabolism, drives enhanced intrinsic resistance to cephalosporins. A particular source of oxidative stress, H2O2, activates signaling through the CroR-CroS two-component signaling system, a known determinant of cephalosporin resistance inE. faecalis. We find that CroR-CroS is required for adaptation to H2O2stress and that H2O2potentiates the activities of cephalosporins againstE. faecaliswhen the CroR-CroS signaling system is nonfunctional. Rather than directly detecting H2O2, our data suggest that the CroR-CroS system responds to cell envelope damage caused by H2O2exposure in order to promote cell envelope repair and enhanced cephalosporin resistance.

scholarly journals DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

2018 ◽  
Author(s):  
Emily S. Melzer ◽  
Caralyn E. Sein ◽  
James J. Chambers ◽  
M. Sloan Siegrist
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
De Novo ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Population Level ◽  
Model Organisms ◽  
Bacterial Cells ◽  
Peptidoglycan Synthesis ◽  
Mycobacterial Cell

AbstractIn many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. InCaulobacter crescentusandEscherichia coli, MreB is also required to sculpt this morphologyde novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or sub-polar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself.Mycobacterium smegmatisrendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms,M. smegmatisalso establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

scholarly journals Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

2019 ◽  
Vol 201 (10) ◽  
Author(s):  
Benjamin D. Labbe ◽  
Cherisse L. Hall ◽  
Stephanie L. Kellogg ◽  
Yao Chen ◽  
Olivia Koehn ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Kinase Activity ◽  
Cell Envelope ◽  
Activation Loop ◽  
Intrinsic Resistance ◽  
Content Type

ABSTRACTTransmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP]andSer/Thr-associated) domains are ubiquitous amongActinobacteriaandFirmicutesspecies. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylationin vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood.Enterococcus faecalisis a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. InE. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreKin vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their functionin vivoandin vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activityin vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreKin vivoupon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activityin vivoto promote adaptation to cell wall stresses.IMPORTANCETransmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous amongActinobacteriaandFirmicutesspecies and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase ofEnterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistancein vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

scholarly journals Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Edward Geisinger ◽  
Nadav J. Mortman ◽  
Yunfei Dai ◽  
Murat Cokol ◽  
Sapna Syal ◽  
...  
Keyword(s):  
Cell Wall ◽  
Acinetobacter Baumannii ◽  
Antibiotic Susceptibility ◽  
Cell Envelope ◽  
Specific Cell ◽  
Cell Wall Assembly ◽  
Transposon Insertion ◽  
Wall Assembly ◽  
Transposon Insertion Sequencing ◽  
Susceptibility Profiles

AbstractA unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

scholarly journals Bacillus subtilis σVConfers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and d-Alanylation of Teichoic Acids

2011 ◽  
Vol 193 (22) ◽  
pp. 6223-6232 ◽  
Author(s):  
Veronica Guariglia-Oropeza ◽  
John D. Helmann
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Double Mutant ◽  
Cell Envelope ◽  
Intrinsic Resistance ◽  
Triple Mutant ◽  
Cell Wall Modification ◽  
Content Type ◽  
Teichoic Acids ◽  
Σ Factor

The seven extracytoplasmic function (ECF) sigma (σ) factors ofBacillus subtilisare broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σVas including at least 20 operons, many of which are also regulated by σM, σX, or σW. The σVregulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance ofB. subtilisto lysozyme. Strains with null mutations in eithersigVor all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σVin the Δ7ECF background restores lysozyme resistance, whereas induction of σM, σX, or σWdoes not. Lysozyme resistance results from the ability of σVto activate the transcription of two operons: the autoregulatedsigV-rsiV-oatA-yrhKoperon anddltABCDE. Genetic analyses reveal thatoatAanddltare largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas anoatA dltAdouble mutant is as sensitive as asigVnull strain. Moreover, thesigV oatA dltAtriple mutant is no more sensitive than theoatA dltAdouble mutant, indicating that there are no other σV-dependent genes necessary for lysozyme resistance. Thus, we suggest that σVconfers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA andd-alanylation of teichoic acids by DltABCDE.

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

1994 ◽  
Vol 52 ◽  
pp. 276-277
Author(s):  
Mary Beth Downs ◽  
Wilson Ribot ◽  
Joseph W. Farchaus
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Cell Envelope ◽  
Single Species ◽  
Supporting Cell ◽  
Surface Layers ◽  
Rabbit Igg ◽  
In Vitro Assembly ◽  
Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

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