scholarly journals Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Simon-Ulysse Vallet ◽  
Lykke Haastrup Hansen ◽  
Freja Cecillie Bistrup ◽  
Signe Aagaard Laursen ◽  
Julien Bortoli Chapalay ◽  
...  
Keyword(s):  
Cell Wall ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Wall Stress ◽  
Two Component System ◽  
Content Type ◽  
Division Plane ◽  
Cell Wall Stress ◽  
Two Component ◽  
Weak Points

ABSTRACT Rod-shaped bacteria frequently localize proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the roles of polar factors in responses to extracellular stimuli have been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal resistance of cell envelope-directed antibiotics, including vancomycin which does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin sensitivity. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to the wild type under some, but not all, cell wall stress conditions that this strain was sensitized to, in particular cefixime and detergent exposure. Together, these results indicate that TipN contributes to cell envelope stress resistance in addition to its roles in intracellular development, and its loss influences signaling through the ChvIG two-component system which has been co-opted as a sensor of cell wall stress in Caulobacter. IMPORTANCE Maintenance of an intact cell envelope is essential for free-living bacteria to protect themselves against their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and reform the cell envelope at the division plane as the cells divide. We have found that TipN, a factor required for correct division and cell pole development in Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilization of these weak points of the cell envelope as well as its other roles inside the cell.

scholarly journals Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

2019 ◽  
Author(s):  
Simon-Ulysse Vallet ◽  
Lykke Haastrup Hansen ◽  
Freja Cecillie Bistrup ◽  
Julien Bortoli Chapalay ◽  
Marc Chambon ◽  
...  
Keyword(s):  
Cell Wall ◽  
Caulobacter Crescentus ◽  
Cell Envelope ◽  
Wall Stress ◽  
Two Component System ◽  
Component System ◽  
Division Plane ◽  
Cell Wall Stress ◽  
Two Component ◽  
Weak Points

AbstractRod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.Author summaryMaintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.

scholarly journals Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Stephanie L. Kellogg ◽  
Jaime L. Little ◽  
Jessica S. Hoff ◽  
Christopher J. Kristich
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecium ◽  
Wall Stress ◽  
Signaling System ◽  
Beta Lactam ◽  
Two Component System ◽  
Component System ◽  
Content Type ◽  
Cell Wall Stress ◽  
Two Component

ABSTRACT Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis. Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis. Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium. Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci.

scholarly journals Identification of Genes in the σ22 Regulon of Pseudomonas aeruginosa Required for Cell Envelope Homeostasis in Either the Planktonic or the Sessile Mode of Growth

mBio ◽  
2012 ◽  
Vol 3 (3) ◽  
Author(s):  
Lynn F. Wood ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Stress Response ◽  
Sigma Factor ◽  
Cell Envelope ◽  
Wall Stress ◽  
Content Type ◽  
Response System ◽  
Cell Wall Stress ◽  
Stress Response System

ABSTRACTThePseudomonas aeruginosaextracytoplasmic functioning (ECF) sigma factor σ22is encoded byalgT/algUand is inhibited by anti-sigma factor MucA. σ22was originally discovered for its essential role in the expression of the exopolysaccharide alginate by mucoid strains associated with chronic pulmonary infection. However, σ22is now known to also have a large regulon associated with the response to cell wall stress. Our recent transcriptome analysis identified 293 open reading frames (ORFs) in the σ22stress stimulon that include genes for outer envelope biogenesis and remodeling, although most of the genes have undefined functions. To better understand the σ22-dependent stress response, mutants affected in 27 genes of the σ22stimulon were examined and expression was studied withlacZfusions. Mutants constructed in the 27 genes showed no major change in response to cell wall-acting antibiotics or growth at elevated temperatures nor in alginate production. The mutants were examined for their effects on the expression of the σ22-dependent promoter of the alginate biosynthetic operon (PalgD) as a measure of σ22derepression from MucA. By testing PalgDexpression under both planktonic and sessile growth conditions, 11 genes were found to play a role in the stress response that activates σ22. Some mutations caused an increase or a decrease in the response to cell wall stress. Interestingly, mutations in 7 of the 11 genes caused constitutive PalgDexpression under nonstressed conditions and thus showed that these genes are involved in maintaining envelope homeostasis. Mutations in PA0062 and PA1324 showed constitutive PalgDexpression during both the planktonic and the sessile modes of growth. However, the PA5178 mutation caused constitutive PalgDexpression only during planktonic growth. In contrast, mutations in PA2717, PA0567, PA3040, and PA0920 caused constitutive PalgDexpression only in the sessile/biofilm mode of growth. This provides evidence that the σ22stimulon for cell envelope homeostasis overlaps with biofilm control mechanisms.IMPORTANCEDuring chronic lung infections, such as in cystic fibrosis patients,Pseudomonas aeruginosaproduces the exopolysaccharide alginate and forms biofilms that shield the organisms from the immune response and increase resistance to antibiotics. Activation of alginate genes is under the control of an extracytoplasmic stress response system that releases an alternative sigma factor (σ22) in response to cell wall stress and then activates expression of a large regulon. In this study, a mutant analysis of 27 members of the regulon showed that 11 play a role in envelope homeostasis and affect the stress response system itself. Interestingly, some genes demonstrate effects only in either the planktonic (free-swimming) or the sessile (biofilm) mode of growth, which leads to persistence and antibiotic tolerance. The studies presented here provide an important initial step in dissecting the mechanisms that regulate a critical signal transduction pathway that impactsP. aeruginosapathogenesis.

scholarly journals Epigallocatechin Gallate Induces Upregulation of the Two-Component VraSR System by Evoking a Cell Wall Stress Response in Staphylococcus aureus

2012 ◽  
Vol 78 (22) ◽  
pp. 7954-7959 ◽  
Author(s):  
Oren Levinger ◽  
Tamar Bikels-Goshen ◽  
Elad Landau ◽  
Merav Fichman ◽  
Roni Shapira
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Epigallocatechin Gallate ◽  
Wall Stress ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type ◽  
Cell Wall Stress ◽  
Two Component ◽  
A Cell

ABSTRACTWe previously found that a short exposure ofStaphylococcus aureusto subinhibitory (SI) doses of epigallocatechin gallate (EGCG) results in increased cell wall thickness, adaptation, and enhanced tolerance to cell-wall-targeted antibiotics. In this study, the response to EGCG ofsigBandvraSRtranscription factor mutants was characterized. We show that in contrast to the results observed for wild-type (WT) strains, anS. aureus315vraSRnull mutant exposed to SI doses of EGCG did not exhibit increased tolerance to EGCG and oxacillin. A diminished increase in tolerance to ampicillin (from 16-fold to 4-fold) and no change in the magnitude of resistance to vancomycin were observed. Preexposure to EGCG enhanced the tolerance of wild-type andsigBnull mutant cells to lysostaphin, but this enhancement was much weaker in thevraSRnull mutant. Marked upregulation (about 60-fold) ofvraRand upregulation of the peptidoglycan biosynthesis-associated genesmurA,murF, andpbp2(2-, 5-, and 6-fold, respectively) in response to SI doses of EGCG were determined by quantitative reverse transcription-PCR (qRT-PCR). EGCG also induced the promoter ofsas016(encoding a cell wall stress protein of unknown function which is not induced invraSRnull mutants) in a concentration-dependent manner, showing kinetics comparable to those of cell-wall-targeting antibiotics. Taken together, our results suggest that the two-component VraSR system is involved in modulating the cell response to SI doses of EGCG.

scholarly journals The Transcription Factor SomA Synchronously Regulates Biofilm Formation and Cell Wall Homeostasis in Aspergillus fumigatus

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Yuan Chen ◽  
Francois Le Mauff ◽  
Yan Wang ◽  
Ruiyang Lu ◽  
Donald C. Sheppard ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Wall Stress ◽  
Fungal Cell ◽  
Glucan Synthase ◽  
Content Type ◽  
Cell Wall Stress ◽  
Chitin Synthases

ABSTRACT Polysaccharides are key components of both the fungal cell wall and biofilm matrix. Despite having distinct assembly and regulation pathways, matrix exopolysaccharide and cell wall polysaccharides share common substrates and intermediates in their biosynthetic pathways. It is not clear, however, if the biosynthetic pathways governing the production of these polysaccharides are cooperatively regulated. Here, we demonstrate that cell wall stress promotes production of the exopolysaccharide galactosaminogalactan (GAG)-depend biofilm formation in the major fungal pathogen of humans Aspergillus fumigatus and that the transcription factor SomA plays a crucial role in mediating this process. A core set of SomA target genes were identified by transcriptome sequencing and chromatin immunoprecipitation coupled to sequencing (ChIP-Seq). We identified a novel SomA-binding site in the promoter regions of GAG biosynthetic genes agd3 and ega3, as well as its regulators medA and stuA. Strikingly, this SomA-binding site was also found in the upstream regions of genes encoding the cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Thus, SomA plays a direct regulation of both GAG and cell wall polysaccharide biosynthesis. Consistent with these findings, SomA is required for the maintenance of normal cell wall architecture and compositions in addition to its function in biofilm development. Moreover, SomA was found to globally regulate glucose uptake and utilization, as well as amino sugar and nucleotide sugar metabolism, which provides precursors for polysaccharide synthesis. Collectively, our work provides insight into fungal adaptive mechanisms in response to cell wall stress where biofilm formation and cell wall homeostasis were synchronously regulated. IMPORTANCE The cell wall is essential for fungal viability and is absent from human hosts; thus, drugs disrupting cell wall biosynthesis have gained more attention. Caspofungin is a member of a new class of clinically approved echinocandin drugs to treat invasive aspergillosis by blocking β-1,3-glucan synthase, thus damaging the fungal cell wall. Here, we demonstrate that caspofungin and other cell wall stressors can induce galactosaminogalactan (GAG)-dependent biofilm formation in the human pathogen Aspergillus fumigatus. We further identified SomA as a master transcription factor playing a dual role in both biofilm formation and cell wall homeostasis. SomA plays this dual role by direct binding to a conserved motif upstream of GAG biosynthetic genes and genes involved in cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Collectively, these findings reveal a transcriptional control pathway that integrates biofilm formation and cell wall homeostasis and suggest SomA as an attractive target for antifungal drug development.

scholarly journals Reciprocal Regulation of Resistance-Nodulation-Division Efflux Systems and the Cpx Two-Component System in Vibrio cholerae

2014 ◽  
Vol 82 (7) ◽  
pp. 2980-2991 ◽  
Author(s):  
Dawn L. Taylor ◽  
X. Renee Bina ◽  
Leyla Slamti ◽  
Matthew K. Waldor ◽  
James E. Bina
Keyword(s):  
Antimicrobial Resistance ◽  
Vibrio Cholerae ◽  
Cell Envelope ◽  
Two Component System ◽  
Reciprocal Regulation ◽  
Component System ◽  
Content Type ◽  
Expression Of Genes ◽  
Two Component ◽  
Resistance Nodulation Division

ABSTRACTThe Cpx two-component regulatory system has been shown inEscherichia colito alleviate stress caused by misfolded cell envelope proteins. TheVibrio choleraeCpx system was previously found to respond to cues distinct from those in theE. colisystem, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by theV. choleraeCpx system. Our observations suggest that the activation of theV. choleraeCpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopiccpxRexpression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting thatvexABandvexGHand thecpxP-cpxRAsystem are reciprocally regulated. We speculate that the reciprocal regulation of theV. choleraeRND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems.

scholarly journals The Posttranslocational Chaperone Lipoprotein PrsA Is Involved in both Glycopeptide and Oxacillin Resistance in Staphylococcus aureus

2012 ◽  
Vol 56 (7) ◽  
pp. 3629-3640 ◽  
Author(s):  
Ambre Jousselin ◽  
Adriana Renzoni ◽  
Diego O. Andrey ◽  
Antoinette Monod ◽  
Daniel P. Lew ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Wall Stress ◽  
Growth Conditions ◽  
Transcriptional Analysis ◽  
Pharmacological Intervention ◽  
Content Type ◽  
Cell Wall Stress ◽  
Oxacillin Resistance ◽  
Mrsa Strains

ABSTRACTUnderstanding in detail the factors which permitStaphylococcus aureusto counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistantS. aureus(MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolylcis/transisomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays thatprsAis directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption ofprsAis tolerated byS. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption ofprsAleads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals thatprsAandvraRare coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediateS. aureus(GISA) strains compared to their susceptible parents. Collectively, our results point to a role forprsAas a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding whyprsAis a key element of the VraRS-mediated cell wall stress response.

scholarly journals Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2470-2484 ◽  
Author(s):  
Eric Botella ◽  
Sebastian Hübner ◽  
Karsten Hokamp ◽  
Annette Hansen ◽  
Paola Bisicchia ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Expression Profiles ◽  
Phosphate Limitation ◽  
Envelope Gene ◽  
Two Component System ◽  
Cell Wall Metabolism ◽  
Component System ◽  
Two Component

The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (ΔphoPR) cells.

scholarly journals Mutational Analyses of Open Reading Frames within thevraSROperon and Their Roles in the Cell Wall Stress Response ofStaphylococcus aureus

2011 ◽  
Vol 55 (4) ◽  
pp. 1391-1402 ◽  
Author(s):  
N. McCallum ◽  
P. Stutzmann Meier ◽  
R. Heusser ◽  
B. Berger-Bächi
Keyword(s):  
Signal Transduction ◽  
Cell Wall ◽  
Cell Envelope ◽  
Wall Stress ◽  
Integral Membrane Protein ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Cell Wall Stress ◽  
Type Strains

ABSTRACTThe exposure ofStaphylococcus aureusto a broad range of cell wall-damaging agents triggers the induction of a cell wall stress stimulon (CWSS) controlled by the VraSR two-component system. ThevraSRgenes form part of the four-cistron autoregulatory operonorf1-yvqF-vraS-vraR. The markerless inactivation of each of the genes within this operon revealed thatorf1played no observable role in CWSS induction and had no influence on resistance phenotypes for any of the cell envelope stress-inducing agents tested. The remaining three genes were all essential for the induction of the CWSS, and mutants showed various degrees of increased susceptibility to cell wall-active antibiotics. Therefore, the role of YvqF inS. aureusappears to be opposite that in other Gram-positive bacteria, where YvqF homologs have all been shown to inhibit signal transduction. This role, as an activator rather than repressor of signal transduction, corresponds well with resistance phenotypes of ΔYvqF mutants, which were similar to those of ΔVraR mutants in which CWSS induction also was completely abolished. Resistance profiles of ΔVraS mutants differed phenotypically from those of ΔYvqF and ΔVraR mutants on many non-ß-lactam antibiotics. ΔVraS mutants still became more susceptible than wild-type strains at low antibiotic concentrations, but they retained larger subpopulations that were able to grow on higher antibiotic concentrations than ΔYvqF and ΔVraR mutants. Subpopulations of ΔVraS mutants could grow on even higher glycopeptide concentrations than wild-type strains. The expression of a highly sensitive CWSS-luciferase reporter gene fusion was up to 2.6-fold higher in a ΔVraS than a ΔVraR mutant, which could be linked to differences in their respective antibiotic resistance phenotypes. Bacterial two-hybrid analysis indicated that the integral membrane protein YvqF interacted directly with VraS but not VraR, suggesting that it plays an essential role in sensing the as-yet unknown trigger of CWSS induction.

scholarly journals Phosphoric Metabolites Link Phosphate Import and Polysaccharide Biosynthesis for Candida albicans Cell Wall Maintenance

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Ning-Ning Liu ◽  
Maikel Acosta-Zaldívar ◽  
Wanjun Qi ◽  
Joann Diray-Arce ◽  
Louise A. Walker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Stress Resistance ◽  
Wall Stress ◽  
Phosphate Starvation ◽  
Nucleotide Sugar ◽  
Content Type ◽  
Cell Wall Stress ◽  
Chitin Synthases ◽  
Nucleotide Sugars

ABSTRACT The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans. IMPORTANCE Candida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans. Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

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