scholarly journals VimA-Dependent Modulation of Acetyl Coenzyme A Levels and Lipid A Biosynthesis Can Alter Virulence in Porphyromonas gingivalis

2011 ◽  
Vol 80 (2) ◽  
pp. 550-564 ◽  
Author(s):  
A. Wilson Aruni ◽  
J. Lee ◽  
D. Osbourne ◽  
Y. Dou ◽  
F. Roy ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Genetic Background ◽  
Lipid A ◽  
Coenzyme A ◽  
Signal Sequence ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Defective Mutant ◽  
Associated Proteins ◽  
Branched Chain

ABSTRACTThePorphyromonas gingivalisVimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA,P. gingivalisFLL406 carrying an additionalvimAgene and avimA-defective mutant in a differentP. gingivalisgenetic background were evaluated. ThevimA-defective mutant (FLL451) in theP. gingivalisATCC 33277 genetic background showed a phenotype similar to that of thevimA-defective mutant (FLL92) in theP. gingivalisW83 genetic background. In contrast to the wild type, gingipain activity was increased inP. gingivalisFLL406, avimAchimeric strain.P. gingivalisFLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated withP. gingivalisFLL92 showed a decrease in invasion, in contrast toP. gingivalisFLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in thevimA-defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting.

scholarly journals Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

2014 ◽  
Vol 83 (3) ◽  
pp. 1019-1029 ◽  
Author(s):  
Julienne C. Kaiser ◽  
Sameha Omer ◽  
Jessica R. Sheldon ◽  
Ian Welch ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Defined Medium ◽  
Infection Model ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth ofStaphylococcus aureusbut also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment.S. aureusencodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all threebrnQparalogs so as to characterize their substrate specificities and their roles in growthin vitroandin vivo. We demonstrated that in the community-associated, methicillin-resistantS. aureus(CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that abrnQ2mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression ofbrnQ1. In a murine infection model, thebrnQ1mutant was attenuated, but in contrast,brnQ2mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhancedin vivoscavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA.

scholarly journals Acid Stress-Mediated Metabolic Shift in Lactobacillus sanfranciscensis LSCE1

2011 ◽  
Vol 77 (8) ◽  
pp. 2656-2666 ◽  
Author(s):  
Diana I. Serrazanetti ◽  
Maurice Ndagijimana ◽  
Sylvain L. Sado-Kamdem ◽  
Aldo Corsetti ◽  
Rudi F. Vogel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Acid Stress ◽  
Redox Balance ◽  
Gas Chromatography Mass Spectrometry ◽  
Metabolic Shift ◽  
Content Type ◽  
Lactobacillus Sanfranciscensis ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTLactobacillus sanfranciscensisLSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth inL. sanfranciscensisin restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.

scholarly journals Free Lipid A Isolated from Porphyromonas gingivalis Lipopolysaccharide Is Contaminated with Phosphorylated Dihydroceramide Lipids: Recovery in Diseased Dental Samples

2011 ◽  
Vol 80 (2) ◽  
pp. 860-874 ◽  
Author(s):  
Frank C. Nichols ◽  
Bekim Bajrami ◽  
Robert B. Clark ◽  
William Housley ◽  
Xudong Yao
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Alveolar Bone ◽  
Ionization Mass ◽  
Subgingival Plaque ◽  
Content Type ◽  
Free Lipid ◽  
The Matrix ◽  
Tlr2 Activation ◽  
Porphyromonas Gingivalis Lipopolysaccharide

ABSTRACTRecent reports indicate thatPorphyromonas gingivalismediates alveolar bone loss or osteoclast modulation through engagement of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopolysaccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides ofP. gingivalishave been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because conflicting evidence has been reported concerning the capacity ofP. gingivalisLPS or lipid A to engage TLR2 versus TLR4. In the present study, we first preparedP. gingivalisLPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphorylated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray-MS methods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived fromP. gingivalisare contaminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingival plaque do not contain free lipid A constituents ofP. gingivalisbut contain substantial amounts of phosphorylated dihydroceramide lipids. Therefore, the free lipid A ofP. gingivalisis not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A ofP. gingivaliscould actually be mediated by phosphorylated dihydroceramides.

scholarly journals Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Jose Solbiati ◽  
Ana Duran-Pinedo ◽  
Fernanda Godoy Rocha ◽  
Frank C. Gibson ◽  
Jorge Frias-Lopez
Keyword(s):  
Chronic Inflammation ◽  
Porphyromonas Gingivalis ◽  
Clinical Findings ◽  
Bacterial Virulence ◽  
Oral Microbiome ◽  
Periodontal Pathogens ◽  
Content Type ◽  
Virulence Potential ◽  
Associated Proteins ◽  
Polymicrobial Disease

Porphyromonas gingivalis is a key pathogen of periodontitis, a polymicrobial disease characterized by a chronic inflammation that destroys the tissues supporting the teeth. Thus, understanding the virulence potential of P. gingivalis is essential to maintaining a healthy oral microbiome. In nonoral organisms, CRISPR-Cas systems have been shown to modulate a variety of microbial processes, including protection from exogenous nucleic acids, and, more recently, have been implicated in bacterial virulence. Previously, our clinical findings identified activation of the CRISPR-Cas system in patient samples at the transition to disease; however, the mechanism of contribution to disease remained unknown. The importance of the present study resides in that it is becoming increasingly clear that CRISPR-associated proteins have broader functions than initially thought and that those functions now include their role in the virulence of periodontal pathogens. Studying a P. gingivalis cas3 mutant, we demonstrate that at least one of the CRISPR-Cas systems is involved in the regulation of virulence during infection.

scholarly journals Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
C. D. Shaw ◽  
H. Imamura ◽  
T. Downing ◽  
G. Blackburn ◽  
G. D. Westrop ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Wild Type ◽  
Genetic Changes ◽  
Content Type ◽  
Novel Treatments ◽  
Branched Chain Amino Acid ◽  
Amastigote Stage ◽  
Branched Chain ◽  
Intracellular Amastigote ◽  
Selection Of

ABSTRACT Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 μM at the promastigote stage and 32 and 195 μM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr. In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

scholarly journals Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, inPorphyromonas gingivalis

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Sumita Jain ◽  
Ana M. Chang ◽  
Manjot Singh ◽  
Jeffrey S. McLean ◽  
Stephen R. Coats ◽  
...  
Keyword(s):  
Immune Response ◽  
Porphyromonas Gingivalis ◽  
Innate Immune Response ◽  
Lipid A ◽  
Innate Immune ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Gene Encoding ◽  
Content Type ◽  
Chronic Inflammatory Condition

ABSTRACTRemoval of one acyl chain from bacterial lipid A by deacylase activity is a mechanism used by many pathogenic bacteria to evade the host's Toll-like receptor 4 (TLR4)-mediated innate immune response. InPorphyromonas gingivalis, a periodontal pathogen, lipid A deacylase activity converts a majority of the initially synthesized penta-acylated lipid A, a TLR4 agonist, to tetra-acylated structures, which effectively evade TLR4 sensing by being either inert or antagonistic at TLR4. In this paper, we report successful identification of the gene that encodes theP. gingivalislipid A deacylase enzyme. This gene, PGN_1123 inP. gingivalis33277, is highly conserved withinP. gingivalis, and putative orthologs are phylogenetically restricted to theBacteroidetesphylum. Lipid A of ΔPGN_1123 mutants is penta-acylated and devoid of tetra-acylated structures, and the mutant strain provokes a strong TLR4-mediated proinflammatory response, in contrast to the negligible response elicited by wild-typeP. gingivalis. Heterologous expression of PGN_1123 inBacteroides thetaiotaomicronpromoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCEPeriodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population.Porphyromonas gingivalisis a bacterium closely associated with periodontitis, although how and if it is a cause for the disease are not known. It has a formidable capacity to dampen the host's innate immune response, enabling its persistence in diseased sites and triggering microbial dysbiosis in animal models of infection.P. gingivalisis particularly adept at evading the host's TLR4-mediated innate immune response by modifying the structure of lipid A, the TLR4 ligand. In this paper, we report identification of the gene encoding lipid A deacylase, a key enzyme that modifies lipid A to TLR4-evasive structures.

scholarly journals A Novel Class of Lipoprotein Lipase-Sensitive Molecules Mediates Toll-Like Receptor 2 Activation by Porphyromonas gingivalis

2013 ◽  
Vol 81 (4) ◽  
pp. 1277-1286 ◽  
Author(s):  
Sumita Jain ◽  
Stephen R. Coats ◽  
Ana M. Chang ◽  
Richard P. Darveau
Keyword(s):  
Lipoprotein Lipase ◽  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Bacterial Cells ◽  
Toll Like Receptor ◽  
Two Phase ◽  
Content Type ◽  
Tlr2 Ligand ◽  
Toll Like Receptor 2 ◽  
Major Factors

ABSTRACTInfection by the chronic periodontitis-associated pathogenPorphyromonas gingivalisactivates a Toll-like receptor 2 (TLR2) response that triggers inflammation in the host but also promotes bacterial persistence. Our aim was to define ligands on the surfaces of intactP. gingivaliscells that determine its ability to activate TLR2. Molecules previously reported as TLR2 agonists include lipopolysaccharide (LPS), fimbriae, the lipoprotein PG1828, and phosphoceramides. We demonstrate that these molecules do not comprise the major factors responsible for stimulating TLR2 by whole bacterial cells. First,P. gingivalismutants devoid of the reported protein agonists, PG1828 and fimbriae, activate TLR2 as strongly as the wild type. Second, two-phase extraction of whole bacteria resulted in a preponderance of TLR2 agonist activity partitioning to the hydrophilic phase, demonstrating that phosphoceramides are not a major TLR2 ligand. Third, analysis of LPS revealed that TLR2 activation is independent of lipid A structural variants. Instead, activation of TLR2 and TLR2/TLR1 by LPS is in large part due to copurifying molecules that are sensitive to the action of the enzyme lipoprotein lipase. Strikingly, intactP. gingivalisbacterial cells treated with lipoprotein lipase were attenuated in their ability to activate TLR2. We propose that a novel class of molecules comprised by lipoproteins constitutes the major determinants that confer toP. gingivalisthe ability to stimulate TLR2 signaling.

scholarly journals Porphyromonas gingivalis Lipid A Phosphatase Activity Is Critical for Colonization and Increasing the Commensal Load in the Rabbit Ligature Model

2013 ◽  
Vol 82 (2) ◽  
pp. 650-659 ◽  
Author(s):  
Camille Zenobia ◽  
Hatice Hasturk ◽  
Daniel Nguyen ◽  
Thomas E. Van Dyke ◽  
Alpdogan Kantarci ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Lipid A ◽  
Cellular Responses ◽  
Structural Heterogeneity ◽  
Microbial Load ◽  
Wild Type ◽  
Microbial Composition ◽  
Content Type ◽  
Mutant Strains ◽  
Repeated Applications

ABSTRACTPeriodontitis is a disease of polymicrobial etiology characterized by inflammation, degradation of host tissue, and bone that irreversibly destroys the supporting apparatus of teeth.Porphyromonas gingivaliscontains lipid A with structural heterogeneity that has been postulated to contribute to the initiation of dysbiosis in oral communities by modulating the host response, thereby creating a permissive environment for its growth. We examined twoP. gingivalislipid A phosphatase mutants which contain different “locked” lipid A structures that induce different host cellular responses for their ability to induce dysbiosis and periodontitis in rabbits. Lipopolysaccharide (LPS) preparations obtained from these strains were also examined. After repeated applications of all strains and their respective LPS preparations,P. gingivaliswild type, but not the lipid A mutants, had a significant impact on both the oral commensal microbial load and composition. In contrast, in rabbits exposed to the mutant strains or the LPS preparations, the microbial load did not increase, and yet significant changes in the oral microbial composition were observed. All strains and their respective LPS preparations induced periodontitis. Therefore, the ability to alter the lipid A composition in response to environmental conditions by lipid A phosphatases is required for both colonization of the rabbit and increases in the microbial load. Furthermore, the data demonstrate that multiple dysbiotic oral microbial communities can elicit periodontitis.

scholarly journals Role of Acetyltransferase PG1842 in Gingipain Biogenesis inPorphyromonas gingivalis

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Arunima Mishra ◽  
Francis Roy ◽  
Yuetan Dou ◽  
Kangling Zhang ◽  
Hui Tang ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Porphyromonas Gingivalis ◽  
Late Onset ◽  
Etiologic Agent ◽  
Host Immune System ◽  
Content Type ◽  
Isogenic Mutant ◽  
Defective Mutant ◽  
Lysine Residues ◽  
Catalytically Active

ABSTRACTPorphyromonas gingivalis, the major etiologic agent in adult periodontitis, produces large amounts of proteases that are important for its survival and pathogenesis. The activation/maturation of gingipains, the major proteases, inP. gingivalisinvolves a complex network of processes which are not yet fully understood. VimA, a putative acetyltransferase and virulence-modulating protein inP. gingivalis, is known to be involved in gingipain biogenesis.P. gingivalisFLL92, avimA-defective isogenic mutant (vimA::ermF-ermAM) showed late-onset gingipain activity at stationary phase, indicating the likelihood of a complementary functional VimA homolog in that growth phase. This study aimed to identify a functional homolog(s) that may activate the gingipains in the absence of VimA at stationary phase. A bioinformatics analysis showed five putative GCN5-relatedN-acetyltransferases (GNAT) encoded in theP. gingivalisgenome that are structurally related to VimA. Allelic exchange mutagenesis was used to make deletion mutants for these acetyltransferases in theP. gingivalisvimA-defective mutant FLL102 (ΔvimA::ermF) genetic background. One of the mutants, designatedP. gingivalisFLL126 (ΔvimA-ΔPG1842), did not show any late-onset gingipain activity at stationary phase compared to that of the parent strainP. gingivalisFLL102. A Western blot analysis of stationary-phase extracellular fractions with antigingipain antibodies showed immunoreactive bands that were similar in size to those for the progingipain species present only in the ΔvimA-ΔPG1842isogenic mutant. Both recombinant VimA and PG1842 proteins acetylated Y230, K247, and K248 residues in the pro-RgpB substrate. Collectively, these findings indicate that PG1842 may play a significant role in the activation/maturation of gingipains inP. gingivalis.IMPORTANCEGingipain proteases are key virulence factors secreted byPorphyromonas gingivalisthat cause periodontal tissue damage and the degradation of the host immune system proteins. Gingipains are translated as an inactive zymogen to restrict intracellular proteolytic activity before secretion. Posttranslational processing converts the inactive proenzyme to a catalytically active protease. Gingipain biogenesis, including its secretion and activation, is a complex process which is still not fully understood. One recent study identified acetylated lysine residues in the three gingipains RgpA, RgpB, and Kgp, thus indicating a role for acetylation in gingipain biogenesis. Here, we show that the acetyltransferases VimA and PG1842 can acetylate the pro-RgpB gingipain species. These findings further indicate that acetylation is a potential mechanism in the gingipain activation/maturation pathway inP. gingivalis.

scholarly journals Growth Inhibition of Pathogenic Bacteria by Sulfonylurea Herbicides

2012 ◽  
Vol 57 (3) ◽  
pp. 1513-1517 ◽  
Author(s):  
Jason F. Kreisberg ◽  
Nicholas T. Ong ◽  
Aishwarya Krishna ◽  
Thomas L. Joseph ◽  
Jing Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Targets ◽  
Burkholderia Pseudomallei ◽  
Pathogenic Bacteria ◽  
Acetohydroxyacid Synthase ◽  
Sulfonylurea Herbicides ◽  
Biosynthetic Enzyme ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTEmerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, includingBurkholderia pseudomallei,Pseudomonas aeruginosa, andAcinetobacter baumannii. Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy.

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