Gene Encoding Research Articles

Erratum for Kieffer et al., "mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin".

Volume 63, no. 9, e00965-19, 2019, [https://doi.org/10.1128/AAC.00965-19][1]. Discussion section, 5th paragraph: The primer sequences were incorrect in the originally published article and should read as follows. MCR-9F (5’-GGT GAA AGT TAT TCC GCT GG-3’) and MCR-9R (5’-TCG CGG TCA GGA TTA TAG

VvWRKY13 from Vitis vinifera negatively modulates salinity tolerance

WRKY transcription factors participate in the salt stress responses of various plant species, but the underlying functional mechanisms are not well understood, particularly in grape. In this study, the role of VvWRKY13 in the salt stress response was characterized and the underlying physiological and molecular mechanisms were explored. The results show that VvWRKY13 expression was induced by salt stress, and the VvWRKY13 transcript level in the salt stress-sensitive cultivar was significantly higher than that in the resistant cultivars. Overexpression of VvWRKY13 in Arabidopsis significantly inhibited the growth of transgenic plants under salt stress, indicating that VvWRKY13 has a negative effect on the salt stress response. The photosynthetic rate, contents of proline and soluble sugars, activities of superoxide dismutase (SOD) and catalase (CAT), as well as transcriptional levels of proline and soluble sugar metabolic genes, such as P5CS1, SS1, SS2 and G6PDH, as well as SOD and CAT encoding genes CAT1, CAT2, and Cu/Zn-SOD, all decreased under salt stress, whereas hydrogen peroxide and oxygen-free radical contents increased in transgenic Arabidopsis plants compared with those of wild-type plants. VvWRKY13 suppressed upregulation of SnRK2.3, ABF1, ABF2, ABF3, RD29B, RD29A, and RAB18 induced by salt stress, but showed no significant effect on the transcript levels of SnRK2.2, SnRK2.6, or RD22, indicating that VvWRKY13 affects salt stress tolerance via a specific abscisic acid signaling pathway. VvWRKY13, isolated from grapevine, negatively regulate salt stress tolerance by depressing photosynthetic capacity, inhibiting salt-induced accumulation of osmolytes and up-regulation in antioxidant activity as well as expression of related genes. VvWRKY13 may affects salt stress tolerance by specific ABA signalling.

The Metallochaperone Encoding Gene hypA Is Widely Distributed among Pathogenic Aeromonas spp. and Its Expression Is Increased under Acidic pH and within Macrophages.

Metallochaperones are essential proteins that insert metal ions or metal cofactors into specific enzymes, that after maturation will become metalloenzymes. One of the most studied metallochaperones is the nickel-binding protein HypA, involved in the maturation of nickel-dependent hydrogenases and ureases. HypA was previously described in the human pathogens Escherichia coli and Helicobacter pylori and was considered a key virulence factor in the latter. However, nothing is known about this metallochaperone in the species of the emerging pathogen genus Aeromonas. These bacteria are native inhabitants of aquatic environments, often associated with cases of diarrhea and wound infections. In this study, we performed an in silico study of the hypA gene on 36 Aeromonas species genomes, which showed the presence of the gene in 69.4% (25/36) of the Aeromonas genomes. The similarity of Aeromonas HypA proteins with the H. pylori orthologous protein ranged from 21−23%, while with that of E. coli it was 41−45%. However, despite this low percentage, Aeromonas HypA displays the conserved characteristic metal-binding domains found in the other pathogens. The transcriptional analysis enabled the determination of hypA expression levels under acidic and alkaline conditions and after macrophage phagocytosis. The transcriptional regulation of hypA was found to be pH-dependent, showing upregulation at acidic pH. A higher upregulation occurred after macrophage infection. This is the first study that provided evidence that the HypA metallochaperone in Aeromonas might play a role in acid tolerance and in the defense against macrophages.

Genetic diversity and biofilm formation analysis of Staphylococcus aureus causing urinary tract infections in Tehran, Iran.

Over the past decades, prevalence of biofilm-forming Staphylococcus aureus strains has significantly increased in urinary tract infections. The aim of this study was to investigate prevalence of biofilm forming and adhesion encoding genes and to analyze distribution of different agr and spa types in S. aureus isolates. In the present study, 75 S. aureus isolates obtained from patients with urinary tract infections were examined for susceptibility to antimicrobial agents. Adhesion, biofilm, and spa encoding genes were detected by PCR screening; agr types were determined using multiplex PCR. Among the 75 isolates, 72% were biofilm producers and 28% were non-biofilm producers. Notably, the ability to produce biofilm was higher among MRSA strains ompared to MSSA strains. The most prevalent biofilm forming gene was icaD (77.3%), followed by icaA (76%), icaB (57.3%) and icaC (50.7%). Adhesion genes clfA, clfB, fnbB, can, fnbA, ebp and bap were detected in 94.7%, 92%, 68%, 64%, 64%, 60% and 5.3% of the isolates, respectively. The spa types t426 and t7789 were found among the non-MDR isolates. It was found that t790, t084, t7789 and t325 spa types were biofilm producers, while t426 and t1339 spa types were non-biofilm producers. Biofilm encoding genes icaD and spa type t790 and agr type III were the most prevalent factors among MDR biofilm producer isolates. The study emphasized that identification of genes and characterization of molecular types involved in biofilm formation should be considered.

Phylogeny Characterization of Seb Gene Encoding Enterotoxins in Staphylococcus aureus Isolated from Raw Milk and Cheese

Food poisoning due to the bacteria is a big global problem in economically and human's health. This problem refers to an illness which is due to infection or the toxin exists in nature and the food that use. Milk is considered a nutritious food because it contains proteins and vitamins. The aim of this study is to detect and phylogeny characterization of staphylococcal enterotoxin B gene (Seb). A total of 200 milk and cheese samples were screened. One hundred ten isolates of Staphylococcus aureus pre-confirmed using selective and differential media with biochemical tests. Genomic DNA was extracted from the isolates and the SEB gene detects using conventional PCR with specific primers. Three staphylococcus aureus isolates were found to be positive for Seb gene using PCR and confirmed by sequencing. Sequence homology showed variety range of identity starting from (100% to 38%). Phylogenetic tree analyses show that samples (6 and 5) are correlated with S. epidermidis. This study discovered that isolates (A6-RLQ and A5-RLQ) are significantly clustered in a group with non- human pathogen Staphylococcus agnetis.

Identification and Characterization of Two New S-Adenosylmethionine-Dependent Methyltransferase Encoding Genes Suggested Their Involvement in Stipe Elongation of Flammulina velutipes

Two new SAM-dependent methyltransferase encoding genes (fvsmt1 and fvsmt2) were identified from the genome of Flammulina velutipes. In order to make a comprehensive characterization of both genes, we performed in silico analysis of both genes and used qRT-PCR to reveal their expression patterns during the development of F. velutipes. There are 4 and 6 exons with total length of 693 and 978 bp in fvsmt2 and fvsmt1, respectively. The deduced proteins, i.e., FVSMT1 and FVSMT2 contained 325 and 230 amino acids with molecular weight 36297 and 24894 Da, respectively. Both proteins contained a SAM-dependent catalytic domain with signature motifs (I, p-I, II, and III) defining the SAM fold. SAM-dependent catalytic domain is located either in the middle or at the N-terminal of FVSMT2 and FVSMT1, respectively. Alignment and phylogenic analysis showed that FVSMT1 is a homolog to a protein–arginine omega-N-methyltransferase, while FVSMT2 is of cinnamoyl CoA O-methyltransferase type and predicted subcellular locations of these proteins are mitochondria and cytoplasm, respectively. qRT-PCR showed that fvsmt1 and fvsmt2 expression was regulated in different developmental stages. The maximum expression levels of fvsmt1 and fvsmt2 were observed in stipe elongation, while no difference was found in mycelium and pileus. These results positively demonstrate that both the methyltransferase encoding genes are involved in the stipe elongation of F. velutipes.

Molecular Cloning and Functional Identification of a Squalene Synthase Encoding Gene from Alfalfa (Medicago sativa L.).

The quality of alfalfa, a main legume forage worldwide, is of great importance for the dairy industry and is affected by the content of triterpene saponins. These natural terpenoid products of triterpene aglycones are catalyzed by squalene synthase (SQS), a highly conserved enzyme present in eukaryotes. However, there is scare information on alfalfa SQS. Here, an open reading frame (ORF) of SQS was cloned from alfalfa. Sequence analysis showed MsSQS had the same exon/intron composition and shared high homology with its orthologs. Bioinformatic analysis revealed the deduced MsSQS had two transmembrane domains. When transiently expressed, GFP-MsSQS fusion protein was localized on the plasma membrane of onion epidermal cells. Removal of the C-terminal transmembrane domain of MsSQS improved solubility in Escherichia coli. MsSQS was preferably expressed in roots, followed by leaves and stems. MeJA treatment induced MsSQS expression and increased the content of total saponins. Overexpression of MsSQS in alfalfa led to the accumulation of total saponins, suggesting a correlation between MsSQS expression level with saponins content. Therefore, MsSQS is a canonical squalene synthase and contributes to saponin synthesis in alfalfa. This study provides a key candidate gene for genetic manipulation of the synthesis of triterpene saponins, which impact both plant and animal health.

Competition between Escherichia coli Populations with and without Plasmids Carrying a Gene Encoding Extended-Spectrum Beta-Lactamase in the Broiler Chicken Gut.

Extended-spectrum-beta-lactamase (ESBL)/AmpC-producing Escherichia coli strains are widely found in E. coli isolates from broiler feces, largely due to the presence of the blaCTX-M-1 gene on IncI1 plasmids. Plasmid carriage is theorized to cause fitness loss and thus should decrease under conditions of reduced antibiotic use. However, in vitro studies showed plasmid carriage to increase in the absence of antimicrobials, due to plasmid conjugation. We investigated whether this translates to increased levels of plasmid in the gastrointestinal tracts of chickens, where conjugation rates may be different and subtle differences in growth rates may have a larger impact on colonization. Eight groups of five chickens were orally inoculated at 4 days of age with a 0.5-ml volume containing 106 CFU/ml E. coli cells, of which 0%, 0.1%, 10%, or 100% carried the IncI1 plasmid with the gene blaCTX-M-1 At 13 time points during 41 days, fecal samples were taken from each chicken. E. coli strains with and without plasmids were quantified. Trends in E. coli subpopulations were analyzed using generalized linear mixed models, and population dynamics were studied by fitting to a mechanistic model. Trends in E. coli subpopulations were different between groups rather than between individual chickens, suggesting substantial levels of E. coli exchange between chickens in a group. The IncI1 plasmid carrying blaCTX-M-1 was transferred with conjugation coefficients at levels higher than those observed in vitro Across groups, the plasmids disappeared or were established independently of the initial fraction of plasmid-carrying E. coli, but no major increase occurred as observed in vitro Differences in growth rates were observed, but competitive exclusion of plasmid-carrying variants was counteracted by conjugation.IMPORTANCE Bacteria that produce extended-spectrum beta-lactamases are resistant to an important class of antimicrobials in human and veterinary medicine. Reduction in antibiotic use is expected to decrease the prevalence of resistance. However, resistance genes often lie on plasmids which can be copied and transferred to other bacteria by conjugation, so in vitro resistance was observed to increase in the absence of antimicrobials. We sought to determine whether this also occurs in the chicken gut and if competitive exclusion by similar E. coli variants without the resistance occurred. We studied the excretion of E. coli carrying IncI1 plasmids with the blaCTX-M-1 resistance gene in small groups of broiler chickens, after inoculating the chickens with E. coli suspensions containing different fractions of plasmid-carrying cells. Our results showed little variation between chickens within groups but large differences between groups that were independent of the ratio of variants with and without the plasmid and with persistence or extinction of the plasmid. However, there was no major plasmid increase as observed in vitro We conclude that in vivo studies with sufficient independent replications are important for intervention studies on plasmid-mediated antimicrobial resistance.

GNPDA2 Gene Affects Adipogenesis and Alters the Transcriptome Profile of Human Adipose-Derived Mesenchymal Stem Cells.

Background Genome-wide association studies have found an obesity-related single-nucleotide polymorphism rs10938397 near the glucosamine-6-phosphate deaminase 2 gene (GNPDA2) encoding, an enzyme that catalyzes the deamination of the glucosamine-6-phosphate involved in the hexosamine signaling pathway, but the molecular mechanisms underlying the missing link between GNPDA2 and obesity remain elusive. Methods As obesity is accompanied by an increase in the size and the number of adipocytes, the present study investigates the possible mechanism of the GNPDA2 in adipogenesis using GeneChip® Human Transcriptome Array 2.0 in human adipose-derived mesenchymal stem cells. Results We found that overexpression of GNPDA2 enhanced accumulation of lipid droplets, and knocking down the gene decreased accumulation of lipid droplets. GO term enrichment analysis indicated that most differentially expressed genes (DEGs) affected by deficiency of GNPDA2 have functions to lipid and glucose metabolism. Further KEGG enrichment analysis showed that the greatest proportion of DEGs are involved in thermogenesis, peroxisome proliferator-activated receptor (PPAR) signaling pathway, carbon metabolism, and fatty acid metabolism including fatty acid degradation, elongation, and biosynthesis. Conclusion These findings suggest that GNPDA2 may be a critical gene for lipid and glucose metabolism, and the expression level of GNPDA2 alters the transcriptome profile of human adipose-derived mesenchymal stem cells.

HPLC analysis on citrinin and lovastatin and their genes expression of two selected Monascus purpureus strains

A physiological and genetic study was conducted to profile two selected of Monascus purpureus strains based on lovastatin produce which was analyzed by HPLC and its gene expression. After completed on producing angkak, a fermented rice product, by each M. purpureus strains at 25°C, HPLC analysis on the yield of citrinin and lovastatin was conducted. The expression of citrinin or lovastatin encoding gene of the two strains was analyzed by using reverse transverse (RT)-PCR of its RNA. Each RNA was extracted from mycelial masses by using Trizol (Invitrogen) kit. The primers used for RT-PCR were pksCT and LovB for citrinin and lovastatin encoding gene respectively. The results showed that the two strains were different physiologically and genetically. M. purpureus Skw2 was high potency as a good producer of lovastatin (38.5 mg/g). Unfortunately, this strain still produced citrinin although at low concentration (13.03 ppm). While M. purpureus Serasi was not good producer of lovastatin (7.4 mg/g). However, based on safety concern on health, M. purpureus Serasi was potential for future use for its benefits application because of its negative result based on the citrinin analysis result. Therefore, enhancement of lovastatin produce would be of one of the interests for further research of this Monascus strain.

Intestine-specific activity of insulin signaling pathway in response to microgravity stress in Caenorhabditis elegans

Insulin signaling pathway is conserved and important during the control of biological processes in organisms. In this study, using the simulated microgravity system in Caenorhabditis elegans, we examined the response of insulin signaling pathway to simulated microgravity. After simulated microgravity treatment for 24-h, we observed the significant decrease in expressions of daf-2 encoding an insulin receptor and two genes (age-1 and akt-1) encoding kinase proteins, and increase in expression of daf-16 encoding a FOXO transcriptional factor. Simulated microgravity also induced an obvious translocation and accumulation of DAF-16::GFP signals in the nucleus. Mutation of daf-2, age-1, or akt-1 caused the resistance to toxicity of simulated microgravity, and daf-16 mutation caused the susceptibility to toxicity of simulated microgravity, indicating the protective response of insulin signaling to simulated microgravity. RNA interference (RNAi) knockdown of daf-16 could suppress the resistance of daf-2, age-1, or akt-1 mutant nematodes to toxicity of simulated microgravity, implying the involvement of signaling cascade of DAF-2-AGE-1-AKT-1-DAF-16 in regulating the response to simulated microgravity. Tissue-specific activity analysis demonstrated that the insulin signaling pathway only acted in the intestine to regulate the response to simulated microgravity. Moreover, in the intestine, insulin signaling functioned synergistically with p38 mitogen-activated protein kinase (MAPK) signaling to regulate the response to simulated microgravity. Therefore, our results suggest the crucial role of intestinal insulin signaling pathway in response to simulate microgravity in nematodes.

Detection of Ampicillin Resistance Encoding Gene of Escherichia coli from Chickens in Bandung and Purwakarta

The purpose of this study was to test the resistance and to detect antibiotic resistence encoding gene in E. coli bacteria from chickens in Bandung and Purwakarta livestock. 18 E. coli isolates were tested for antibiotics resistance using the disk diffusion method. Isolates that were categorized as resistant and intermediate to antibiotics, then polymerase chain reaction was utilized to detect the resistent coding gene. The test results showed that all E. coli isolates from chickens in Bandung and Purwakarta were resistant to ampicillin (100%). E. coli isolates were still sensitive to chloramphenicol (11.1%) and gentamicin (22.2%). The gene encoding for ampC resistance from the test were in the amount of 77.7%. Sensitivity test results and detection of resistance coding gene showed that almost all isolates were resistant to ampicillin antibiotics and E. coli isolates were still sensitive to chlorampenicol and gentamicin.

Identification of Mutations in the mrdA Gene Encoding PBP2 That Reduce Carbapenem and Diazabicyclooctane Susceptibility of Escherichia coli Clinical Isolates with Mutations in ftsI (PBP3) and Which Carry blaNDM-1.

Penicillin-binding proteins (PBPs) are essential for bacterial cell wall biosynthesis, and several are clinically validated antibacterial targets of β-lactam antibiotics. We identified mutations in the mrdA gene encoding the PBP2 protein in two Escherichia coliblaNDM-1 clinical isolates that reduce susceptibility to carbapenems and to the intrinsic antibacterial activity of a diazabicyclooctane (DBO) PBP2 and β-lactamase inhibitor. These mutations coexisted with previously described mutations in ftsI (encoding PBP3) that reduce susceptibility to monobactams, penicillins, and cephalosporins. Clinical exposure to β-lactams is driving the emergence of multifactorial resistance that may impact the therapeutic usefulness of existing antibacterials and novel compounds that target PBPs.IMPORTANCE Emerging antibacterial resistance is a consequence of the continued use of our current antibacterial therapies, and it is limiting their utility, especially for infections caused by multidrug-resistant isolates. β-Lactams have enjoyed extensive clinical success, but their broad usage is linked to perhaps the most extensive and progressive example of resistance development for any antibacterial scaffold. In Gram-negative pathogens, this largely involves constant evolution of new β-lactamases able to degrade successive generations of this scaffold. In addition, more recently, alterations in the targets of these compounds, penicillin-binding proteins (PBPs), are being described in clinical isolates, which often also have multiple β-lactamases. This study underscores the multifactorial nature of β-lactam resistance by uncovering alterations of PBP2 that reduce susceptibility to carbapenems in E. coli clinical isolates that also have alterations of PBP3 and express the NDM-1 β-lactamase. The changes in PBP2 also reduced susceptibility to the intrinsic antibacterial activity of some diazabicyclooctane (DBO) compounds that can target PBP2. This may have implications for the development and use of the members of this relatively newer scaffold that are inhibitors of PBP2 in addition to their inhibition of serine-β-lactamases.

Isolation, Molecular Identification and Verification of Gene Encoding Bacterial Keratinase from Crocodile (Crocodylus porosus) Feces

Keratinase is a group of protease enzymes which acts to degrade keratin. Keratin is a fiberous protein difficult to be degraded because of hydrogen and disulfide bonds. The purpose of this study was to isolate and to identify keratinase coding genes using molecular approach. The object of this study was crocodile feces from Asam Kumbang crocodile farm. Bacterial isolation was done by using feather agar (FA) and was screened in skim milk agar. Keratinolytic bacteria isolates were identified molecularly with 16S rDNA specific markers. Detection of keratinase gene was done by using keratinase-specific primers through primer3 application (version 0.4.0). The result showed that isolate FB3 are potential to produce keratinase. Isolate FB3 showed a clear zone in FA and SMA. FB3 was 100% similar to Aeromonas hydrophila. Amlification using kerD gene specific primer showed that FB3 was a novel bacteria possessing kerD gene with a fragment length of 750 bp. This result provided a new database of kerD gene.

PCR Amplification and In-Silico Analysis of Putative Boiling Stable Protein Encoding Genes from Invasive Alien Plant Lantana camara

Lantana camara, a noxious weed flourishing worldwide is one of the world’s 10 worst weed causing biological pollution. In this study, we amplified and sequenced stress responsive boiling stable protein encoding genes from leaves of L. camara. We also describe in-silico characterization and molecular modeling of LCDHY hydrophilic protein through homology search, secondary structure prediction, active site prediction and 3D structure analysis. Clustal W analysis showed the presence of conserved amino acid sequence. 3D structure was predicted using Swiss-model server and homology modeling. Structure was refined by energy minimization and stereo-chemical qualities validated with PROCHECK, VADAR, QMEAN servers. Active sites were predicted form target protein structure. NEST analysis revealed structural motifs in functionally important regions of protein structure. Based on gene ontology (GO) terms in ProFunc server, putative functions of modeled LCDHY protein were predicted.

The ion channels and transporters gene expression profile indicates a shift in excitability and metabolisms during malignant progression of Follicular Lymphoma

The definition of the gene expression profile of genes encoding Ion Channels and Transporters (ICT-GEP) represents a novel and attracting aspect in cancer. We determined the ICT-GEP of Follicular Lymphoma (FL), and compared it with that of the more aggressive Diffuse Large B Cell Lymphoma (DLBCL). cDNA microarray data were collected both from patients enrolled for this study, and from public datasets. In FL the ICT-GEP indicated the overexpression of both the K+ channel encoding gene KCNN4, and SLC2A1, which encodes the Glut1 glucose transporter. SLC2A1 turned out to represent the hub of a functional network, connecting channels and transporters in FL. Relapsed FL patients were characterised by 38 differentially expressed ICT genes, among which ATP9A, SLC2A1 and KCNN4 were under-expressed, indicating a down-regulation of both excitability and glycolysis. A completely different profile of K+ channel encoding genes emerged in DLBCL accompanied by the over-expression of the fatty acid transporter-encoding gene SLC27A1 as well as of the metabolism regulator NCoR1. This indicates a change in excitability and a shift towards an oxidative metabolism in DLBCL. Overall, the ICT-GEP may contribute to identifying novel lymphoma biomarkers related to excitability and metabolic pathways, with particular relevance for drug resistant, relapsed FL.

Molecular and functional characterization of odorant-binding protein genes in Holotrichia oblita Faldermann

Holotrichia oblita is one of the nastiest pests in China. In present research, four full-length cDNA encoding of HoblOBP genes were cloned and sequenced from H. oblita. The mRNA of HoblOBPs were predominantly expressed in antenna. The recombinant HoblOBPs proteins were obtained for fluorescence binding assays. Four of HoblOBPs could mediate the response of H. oblita to organic fertilizers-derived attractants, including HoblOBP5 binding to skatole; HoblOBP8 binding to p-cresol, indole and skatole; HoblOBP9 binding to indole and 4-allylanisole; and HoblOBP24 binding to p-cresol, indole and 4-ethylphenol. Further, RNA interference demonstrated that transcripts of HoblOBP5, 8, 9, and 24 decreased in a time-dependent manner after dsRNA-injection. Knockdown of HoblOBP5, 8, 9, and 24 by injection of dsRNA successfully interfered with behavioral responses towards the target compounds in beetles. Our results showed that HoblOBP5, HoblOBP8, HoblOBP9 and HoblOBP24 are essential in mediating the approach behavior of H. oblita.

Bacteriological and Molecular Studies on Virulence Encoding Genes in Escherichia coli Isolated From Diseased Ducks

Avian colibacillosis is considered a major bacterial disease in the poultry industry worldwide and one of the most common avian diseases that communicable to human. A prevalence study on APEC was carried out on 56 cloacal swabs isolated from 17 weeks old ducks suffering from diarrhea. The isolates were cultured onto MacConkey and EMB agar for isolation and identification of E. coli followed by serotyping then PCR for the detection of virulence encoding genes (iss, fimH, eaeA, tsh, ibeA, stx1and stx2) and their phylogenetic group. The collected data showed that, out of 56 isolates 12 isolates were typical APEC commonly belong to7 different serogroups and having at least two virulence genes. The data demonstrated that, iss and fimH encoding genes were present in all the examined serogroups (100%). In contrast stx1 and eaeA were absent in all isolates, while tsh and ibeA were variable (63.63%, 27.27%), respectively. The presented phylogenetic groups were A and B1 in percentage of 27.27% and 72.72%, respectively. The use of phylogenetic groups makes a revolution in the identification of APEC which enhanced our knowledge for APEC pathogenicity and helping pave the road for the application of the suitable preventive and curative measures in order to reduce the economic drawbacks of avian colibacillosis in large-scale farms.

Identification of Biofilm Encoding Genes (agg) in the Escherichia coli Isolates by Multiplex-PCR Method

Identification of Biofilm Encoding Genes (agg) in the Escherichia coli Isolates by Multiplex-PCR Method

Identification of PGN_1123 as the Gene Encoding Lipid A Deacylase, an Enzyme Required for Toll-Like Receptor 4 Evasion, in Porphyromonas gingivalis.

Removal 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. In Porphyromonas 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 the P. gingivalis lipid A deacylase enzyme. This gene, PGN_1123 in P. gingivalis 33277, is highly conserved within P. gingivalis, and putative orthologs are phylogenetically restricted to the Bacteroidetes phylum. 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-type P. gingivalis Heterologous expression of PGN_1123 in Bacteroides thetaiotaomicron promoted lipid A deacylation, confirming that PGN_1123 encodes the lipid A deacylase enzyme.IMPORTANCE Periodontitis, commonly referred to as gum disease, is a chronic inflammatory condition that affects a large proportion of the population. Porphyromonas gingivalis is 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. gingivalis is 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.