scholarly journals In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

2013 ◽  
Vol 57 (9) ◽  
pp. 4470-4480 ◽  
Author(s):  
Min Jung Kwun ◽  
Gabriela Novotna ◽  
Andrew R. Hesketh ◽  
Lionel Hill ◽  
Hee-Jeon Hong
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Transcriptional Response ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Expression Of Genes ◽  
Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

scholarly journals Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

2015 ◽  
Vol 36 (6) ◽  
pp. 913-922 ◽  
Author(s):  
Nallani Vijay Kumar ◽  
Jianbo Yang ◽  
Jitesh K. Pillai ◽  
Swati Rawat ◽  
Carlos Solano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcriptional Response ◽  
Transcriptional Regulator ◽  
Yeast Protein ◽  
Content Type ◽  
Arsenic Tolerance ◽  
Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

scholarly journals Substrate Inhibition of VanA by d-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

2016 ◽  
Vol 60 (8) ◽  
pp. 4930-4939 ◽  
Author(s):  
Lizah T. van der Aart ◽  
Nicole Lemmens ◽  
Willem J. van Wamel ◽  
Gilles P. van Wezel
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Substrate Inhibition ◽  
Model Organism ◽  
Vancomycin Resistance ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
Lipid Ii ◽  
Vancomycin Resistant

ABSTRACTThe increasing resistance of clinical pathogens against the glycopeptide antibiotic vancomycin, a last-resort drug against infections with Gram-positive pathogens, is a major problem in the nosocomial environment. Vancomycin inhibits peptidoglycan synthesis by binding to thed-Ala–d-Ala terminal dipeptide moiety of the cell wall precursor lipid II. Plasmid-transferable resistance is conferred by modification of the terminal dipeptide into the vancomycin-insensitive variantd-Ala–d-Lac, which is produced by VanA. Here we show that exogenousd-Ala competes withd-Lac as a substrate for VanA, increasing the ratio of wild-type to mutant dipeptide, an effect that was augmented by several orders of magnitude in the absence of thed-Ala–d-Ala peptidase VanX. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that high concentrations ofd-Ala led to the production of a significant amount of wild-type cell wall precursors, whilevanX-null mutants produced primarily wild-type precursors. This enhanced the efficacy of vancomycin in the vancomycin-resistant model organismStreptomyces coelicolor, and the susceptibility of vancomycin-resistant clinical isolates ofEnterococcus faecium(VRE) increased by up to 100-fold. The enhanced vancomycin sensitivity ofS. coelicolorcells correlated directly to increased binding of the antibiotic to the cell wall. Our work offers new perspectives for the treatment of diseases associated with vancomycin-resistant pathogens and for the development of drugs that target vancomycin resistance.

scholarly journals Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

2014 ◽  
Vol 58 (3) ◽  
pp. 1738-1743 ◽  
Author(s):  
Russell E. Lewis ◽  
Guangling Liao ◽  
Katherine Young ◽  
Cameron Douglas ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Activator Protein 1 ◽  
Chitin Synthesis ◽  
Synthesis Inhibitor ◽  
Content Type

ABSTRACTAntifungal exposure can elicit immunological effects that contribute to activityin vivo, but this activity is rarely screenedin vitroin a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates ofCandida albicansandAspergillus fumigatusthat conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the β-glucan catalytic subunit of glucan synthase,FKS1, increased macrophage NF-κB/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis inC. albicansor when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.

scholarly journals Mycobacterium tuberculosis Resists Stress by Regulating PE19 Expression

2015 ◽  
Vol 84 (3) ◽  
pp. 735-746 ◽  
Author(s):  
Pavithra Ramakrishnan ◽  
Alisha M. Aagesen ◽  
John D. McKinney ◽  
Anna D. Tischler
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Transcriptional Profiling ◽  
Constitutive Expression ◽  
System Component ◽  
Uptake System ◽  
Aberrant Expression ◽  
Content Type

Mycobacterium tuberculosisrequires the phosphate-sensing signal transduction system Pst/SenX3-RegX3 to resist host immune responses. A ΔpstA1mutant lacking a Pst phosphate uptake system component is hypersensitive to diverse stress conditionsin vitroand is attenuatedin vivodue to constitutive expression of the phosphate starvation-responsive RegX3 regulon. Transcriptional profiling of the ΔpstA1mutant revealed aberrant expression of certainpeandppegenes. PE and PPE proteins, defined by conserved N-terminal domains containing Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs, account for a substantial fraction of theM. tuberculosisgenome coding capacity, but their functions are largely uncharacterized. Because some PE and PPE proteins localize to the cell wall, we hypothesized that overexpression of these proteins sensitizesM. tuberculosisto stress by altering cell wall integrity. To test this idea, we deletedpeandppegenes that were overexpressed by ΔpstA1bacteria. Deletion of a singlepegene,pe19, suppressed hypersensitivity of the ΔpstA1mutant to both detergent and reactive oxygen species. Ethidium bromide uptake assays revealed increased envelope permeability of the ΔpstA1mutant that was dependent on PE19. The replication defect of the ΔpstA1mutant in NOS2−/−mice was partially reversed by deletion ofpe19, suggesting that increased membrane permeability due to PE19 overexpression sensitizesM. tuberculosisto host immunity. Our data indicate that PE19, which comprises only a 99-amino-acid PE domain, has a unique role in the permeability of theM. tuberculosisenvelope that is regulated to resist stresses encountered in the host.

scholarly journals The Level of AdpA Directly Affects Expression of Developmental Genes in Streptomyces coelicolor

2011 ◽  
Vol 193 (22) ◽  
pp. 6358-6365 ◽  
Author(s):  
Marcin Wolański ◽  
Rafał Donczew ◽  
Agnieszka Kois-Ostrowska ◽  
Paweł Masiewicz ◽  
Dagmara Jakimowicz ◽  
...  
Keyword(s):  
Early Stage ◽  
Response Regulator ◽  
Streptomyces Coelicolor ◽  
Streptomyces Griseus ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Content Type ◽  
Protein Functions

AdpA is a key regulator of morphological differentiation inStreptomyces. In contrast toStreptomyces griseus, relatively little is known about AdpA protein functions inStreptomyces coelicolor. Here, we report for the first time the translation accumulation profile of theS. coelicoloradpA(adpASc) gene; the level ofS. coelicolorAdpA (AdpASc) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpAScspecifically binds theadpAScpromoter regionin vitroandin vivo, suggesting that its expression is autoregulated; surprisingly, in contrast toS. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpAScon the expression of several genes whose products play key roles in the differentiation ofS. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpAScprotein on the expression of the analyzed genes presumably results mainly from different affinities of AdpAScprotein to individual promoters.

scholarly journals The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Regina Selb ◽  
Christian Derntl ◽  
Reinhard Klein ◽  
Beatrix Alte ◽  
Christoph Hofbauer ◽  
...  
Keyword(s):  
Life Cycle ◽  
Constitutive Expression ◽  
Viral Proteins ◽  
Inducible Promoter ◽  
Mutant Virus ◽  
Viral Gene ◽  
Tail Fiber ◽  
Content Type ◽  
Lysogenic Strain

ABSTRACT In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea. By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadii. IMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii. Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

Expression of genes for B7-H3 and other T cell ligands by nasal epithelial cells during differentiation and activation

2004 ◽  
Vol 287 (1) ◽  
pp. L217-L225 ◽  
Author(s):  
Bahman Saatian ◽  
Xiao-Ying Yu ◽  
Andrew P. Lane ◽  
Thanh Doyle ◽  
Vincenzo Casolaro ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Constitutive Expression ◽  
Human Leukocyte ◽  
Costimulatory Molecules ◽  
Human Respiratory Tract ◽  
Brush Biopsy ◽  
Expression Of Genes ◽  
Mucociliary Differentiation

Epithelial cells of the human respiratory tract express human leukocyte antigen (HLA) and the costimulatory molecules B7-1 and B7-2. Little is known, however, about the constitutive expression of genes encoding for the more recently identified members of the B7 homolog family of costimulatory molecules or about the influence of cellular differentiation and cytokines on their activity or on that of HLA or B7-1 and B7-2. Human nasal epithelial (HNE) cells were grown at the air-liquid interface (ALI) for 2 or 21 days to model in vivo conditions. Expression of genes for HLA-B and HLA-DR1 increased during mucociliary differentiation during this period and became more similar to HNE cells obtained fresh by brush biopsy from nasal turbinates. Gene transcripts for B7-H3 and B7-H2 were abundantly expressed in cells cultured at the ALI, but neither their activities nor that of B7-2 was significantly altered during differentiation. IFN-γ and TNF-α upregulated mRNA encoding for both HLA molecules, but not for the B7 molecules. This study describes, for the first time, the expression of B7-H3 and B7-H2 by HNE cells and thus expands the range of potential costimulatory signals through which these cells may interact with activated mucosal T lymphocytes. In addition, the results suggest that the extent of mucociliary differentiation of cultured cells may influence this capability.

scholarly journals Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Shichun Lun ◽  
David Miranda ◽  
Andre Kubler ◽  
Haidan Guo ◽  
Mariama C. Maiga ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Synthetic Lethality ◽  
Multidrug Resistant ◽  
Cell Wall Biosynthesis ◽  
Drug Resistant ◽  
Content Type ◽  
Innate Resistance ◽  
Extensively Drug Resistant

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. IMPORTANCE The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of β-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to β-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with β-lactams in the mouse model.

scholarly journals An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
L. Zhang ◽  
K. Ramijan ◽  
V. J. Carrión ◽  
L. T. van der Aart ◽  
J. Willemse ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sequence Similarity ◽  
Streptomyces Coelicolor ◽  
Genomic Analysis ◽  
Environmental Harm ◽  
Cell Wall Synthesis ◽  
Precursor Molecule ◽  
Content Type ◽  
Lipid Ii ◽  
A Cell

ABSTRACT The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces. CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis. IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.

scholarly journals The C Terminus of Substrates Is Critical but Not Sufficient for Their Degradation by the Pseudomonas aeruginosa CtpA Protease

2020 ◽  
Vol 202 (16) ◽  
Author(s):  
Sammi Chung ◽  
Andrew J. Darwin
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Membrane ◽  
Content Type ◽  
C Terminus ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Carboxyl Terminal

ABSTRACT Bacterial carboxyl-terminal processing proteases (CTPs) are widely conserved and have been linked to important processes, including signal transduction, cell wall metabolism, and virulence. However, the features that target proteins for CTP-dependent cleavage are unclear. Studies of the Escherichia coli CTP Prc suggested that it cleaves proteins with nonpolar and/or structurally unconstrained C termini, but it is not clear if this applies broadly. Pseudomonas aeruginosa has a divergent CTP, CtpA, which is required for virulence. CtpA works in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. In this study, we investigated if the C termini of two nonhomologous CtpA substrates are important for their degradation. We determined that these substrates have extended C termini compared to those of their closest E. coli homologs. Removing 7 amino acids from these extensions was sufficient to reduce their degradation by CtpA both in vivo and in vitro. Degradation of one truncated substrate was restored by adding the C terminus from the other but not by adding an unrelated sequence. However, modification of the C termini of nonsubstrates, by adding the C-terminal amino acids from a substrate, did not cause their degradation by CtpA. Therefore, the C termini of CtpA substrates are required but not sufficient for their efficient degradation. Although C-terminal truncated substrates were protected from degradation, they still associated with the LbcA-CtpA complex in vivo. Therefore, degradation of a protein by CtpA requires a C terminus-independent interaction with the LbcA-CtpA complex, followed by C terminus-dependent degradation, perhaps because CtpA normally initiates cleavage at a C-terminal site. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life, but exactly how they work is poorly understood, including how they recognize substrates. Bacterial CTPs have been associated with virulence, including CtpA of Pseudomonas aeruginosa, which works in complex with the outer membrane lipoprotein LbcA to degrade potentially dangerous peptidoglycan hydrolases. We report an important advance by revealing that efficient degradation by CtpA requires at least two separable phenomena and that one of them depends on information encoded in the substrate C terminus. A C terminus-independent association with the LbcA-CtpA complex is followed by C terminus-dependent cleavage by CtpA. Increased understanding of how CTPs target proteins is significant, due to their links to virulence, peptidoglycan remodeling, and other important processes.

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