scholarly journals EptC of Campylobacter jejuni Mediates Phenotypes Involved in Host Interactions and Virulence

2012 ◽  
Vol 81 (2) ◽  
pp. 430-440 ◽  
Author(s):  
Thomas W. Cullen ◽  
John P. O'Brien ◽  
David R. Hendrixson ◽  
David K. Giles ◽  
Rhonda I. Hobb ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Lipid A ◽  
Inner Core ◽  
Complex Surface ◽  
Surface Structures ◽  
Avian Host ◽  
Toll Like Receptor ◽  
Core Oligosaccharide ◽  
Gene Encoding ◽  
Content Type

ABSTRACTCampylobacter jejuniis a natural commensal of the avian intestinal tract. However, the bacterium is also the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Like many bacterial pathogens,C. jejuniassembles complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work inC. jejuniidentified a gene encoding a novel phosphoethanolamine (pEtN) transferase, EptC (Cj0256), that plays a promiscuous role in modifying the flagellar rod protein, FlgG; the lipid A domain of lipooligosaccharide (LOS); and severalN-linked glycans. In this work, we report that EptC catalyzes the addition of pEtN to the first heptose sugar of the inner core oligosaccharide of LOS, a fourth enzymatic target. We also examine the role pEtN modification plays in circumventing detection and/or killing by host defenses. Specifically, we show that modification ofC. jejunilipid A with pEtN results in increased recognition by the human Toll-like receptor 4–myeloid differentiation factor 2 (hTLR4-MD2) complex, along with providing resistance to relevant mammalian and avian antimicrobial peptides (i.e., defensins). We also confirm the inability of aberrant forms of LOS to activate Toll-like receptor 2 (TLR2). Most exciting, we demonstrate that strains lackingeptCshow decreased commensal colonization of chick ceca and reduced colonization of BALB/cByJ mice compared to wild-type strains. Our results indicate that modification of surface structures with pEtN by EptC is key to its ability to promote commensalism in an avian host and to survive in the mammalian gastrointestinal environment.

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 Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

2016 ◽  
Vol 82 (9) ◽  
pp. 2738-2750 ◽  
Author(s):  
Ryan Simkovsky ◽  
Emily E. Effner ◽  
Maria José Iglesias-Sánchez ◽  
Susan S. Golden
Keyword(s):  
Biomass Production ◽  
Lipid A ◽  
Synechococcus Elongatus ◽  
Cyanobacterial Blooms ◽  
Core Oligosaccharide ◽  
Diverse Range ◽  
Content Type ◽  
O Antigen ◽  
Population Structures ◽  
Pcc 7942

ABSTRACTIn natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacteriumSynechococcus elongatusPCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678–16683, 2012,http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide ofS. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains.

scholarly journals Lack of Lipid A Pyrophosphorylation and FunctionallptAReduces Inflammation by Neisseria Commensals

2012 ◽  
Vol 80 (11) ◽  
pp. 4014-4026 ◽  
Author(s):  
Constance M. John ◽  
Mingfeng Liu ◽  
Nancy J. Phillips ◽  
Zhijie Yang ◽  
Courtney R. Funk ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Lipid A ◽  
Polymyxin B ◽  
Toll Like Receptor ◽  
Inflammatory Signaling ◽  
Toll Like Receptor 4 ◽  
Bioinformatics Analyses ◽  
Monocytic Cells ◽  
Content Type ◽  
High Level

ABSTRACTThe interaction of the immune system withNeisseriacommensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenicNeisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded bylptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensalNeisseriaspecies confirmed thatlptAwas absent in 15 of 17 strains examined but was present inN. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lackinglptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensalNeisseriastrains that lackedlptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of twoN. lactamicacommensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcallptAdeletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensalNeisseriastrains by reducing the inflammatory potential of LOS.

scholarly journals Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Eugenia Faber ◽  
Eugenia Gripp ◽  
Sven Maurischat ◽  
Bernd Kaspers ◽  
Karsten Tedin ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Campylobacter Jejuni ◽  
Effector Proteins ◽  
Host Responses ◽  
Toll Like Receptor ◽  
Cross Tolerance ◽  
Content Type ◽  
Gene Coding ◽  
Cecal Microbiota ◽  
Toll Like Receptor 5

ABSTRACT Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins. The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins.

scholarly journals Relative Contributions of Lipooligosaccharide Inner and Outer Core Modifications to Nontypeable Haemophilus influenzae Pathogenesis

2013 ◽  
Vol 81 (11) ◽  
pp. 4100-4111 ◽  
Author(s):  
Pau Morey ◽  
Cristina Viadas ◽  
Begoña Euba ◽  
Derek W. Hood ◽  
Montserrat Barberán ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Outer Core ◽  
Nontypeable Haemophilus Influenzae ◽  
Core Oligosaccharide ◽  
Biofilm Growth ◽  
Content Type ◽  
Relative Contribution ◽  
Self Aggregation

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show thatopsX,lgtF,lpsA,lic1, andlic2Acontribute to bacterial resistance to AMP;lic1is related to NTHi self-aggregation;lgtF,lic1, andsiaBare involved in biofilm growth;opsXandlgtFparticipate in epithelial infection; andopsX,lgtF, andlpsAcontribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.

scholarly journals Roles of 3-Deoxy-d-manno-2-Octulosonic Acid Transferase from Moraxella catarrhalis in Lipooligosaccharide Biosynthesis and Virulence

2005 ◽  
Vol 73 (7) ◽  
pp. 4222-4230 ◽  
Author(s):  
Daxin Peng ◽  
Biswa P. Choudhury ◽  
Ronald S. Petralia ◽  
Russell W. Carlson ◽  
Xin-Xing Gu
Keyword(s):  
Moraxella Catarrhalis ◽  
Lipid A ◽  
Therapeutic Interventions ◽  
Core Oligosaccharide ◽  
Gene Encoding ◽  
Oligosaccharide Moiety ◽  
Normal Human ◽  
Human Infections

ABSTRACT Lipooligosaccharide (LOS), a major outer membrane component of Moraxella catarrhalis, is a possible virulence factor in the pathogenesis of human infections caused by the organism. However, information about the roles of the oligosaccharide chain from LOS in bacterial infection remains limited. Here, a kdtA gene encoding 3-deoxy-d-manno-2-octulosonic acid (Kdo) transferase, which is responsible for adding Kdo residues to the lipid A portion of the LOS, was identified by transposon mutagenesis and construction of an isogenic kdtA mutant in strain O35E. The resulting O35EkdtA mutant produced only lipid A without any core oligosaccharide, and it was viable. Physicochemical and biological analysis revealed that the mutant was susceptible to hydrophobic reagents and a hydrophilic glycopeptide and was sensitive to bactericidal activity of normal human serum. Importantly, the mutant showed decreased toxicity by the Limulus amebocyte lysate assay, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. These data suggest that the oligosaccharide moiety of the LOS is important for the biological activity of the LOS and the virulence capability of the bacteria in vitro and in vivo. This study may bring new insights into novel vaccines or therapeutic interventions against M. catarrhalis infections.

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 Campylobacter jejuni Disrupts Protective Toll-Like Receptor 9 Signaling in Colonic Epithelial Cells and Increases the Severity of Dextran Sulfate Sodium-Induced Colitis in Mice

2012 ◽  
Vol 80 (4) ◽  
pp. 1563-1571 ◽  
Author(s):  
Jennifer R. O'Hara ◽  
Troy D. Feener ◽  
Carrie D. Fischer ◽  
Andre G. Buret
Keyword(s):  
Campylobacter Jejuni ◽  
Dextran Sulfate ◽  
Dextran Sulfate Sodium ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Toll Like Receptor ◽  
Intestinal Epithelial Barrier ◽  
Content Type ◽  
Tlr9 Agonist ◽  
Dss Colitis

ABSTRACTInflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation associated with a dysregulated immune response to commensal bacteria in susceptible individuals. The relapse of IBD may occur following an infection withCampylobacter jejuni. Apical epithelial Toll-like receptor 9 (TLR9) activation by bacterial DNA is reported to maintain colonic homeostasis. We investigated whether a priorC. jejuniinfection disrupts epithelial TLR9 signaling and increases the severity of disease in a model of mild dextran sulfate sodium (DSS) colitis in mice. In a further attempt to identify mechanisms, T84 monolayers were treated withC. jejunifollowed by a TLR9 agonist. Transepithelial resistance (TER) and dextran flux across confluent monolayers were monitored. Immunohistochemistry, Western blotting, and flow cytometry were used to examine TLR9 expression. Mice colonized byC. jejunilacked any detectable pathology; however, in response to low levels of DSS, mice previously exposed toC. jejuniexhibited significantly reduced weight gain and increased occult blood and histological damage scores. Infected mice treated with DSS also demonstrated a significant reduction in levels of the anti-inflammatory cytokine interleukin-25.In vitrostudies indicated that apical application of a TLR9 agonist enhances intestinal epithelial barrier function and that this response is lost inC. jejuni-infected monolayers. Furthermore, infected cells secreted significantly more CXCL8 following the basolateral application of a TLR9 agonist. Surface TLR9 expression was reduced inC. jejuni-infected monolayers subsequently exposed to a TLR9 agonist. In conclusion, infection byC. jejunidisrupts TLR9-induced reinforcement of the intestinal epithelial barrier, and colonization byC. jejuniincreases the severity of mild DSS colitis.

scholarly journals Interaction of Copper Toxicity and Oxidative Stress in Campylobacter jejuni

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Susan P. Gardner ◽  
Jonathan W. Olson
Keyword(s):  
Oxidative Stress ◽  
Campylobacter Jejuni ◽  
Copper Toxicity ◽  
Foodborne Pathogen ◽  
Growth Media ◽  
Avian Host ◽  
Copper Binding ◽  
Copper Transporter ◽  
Content Type ◽  
Metabolic Requirement

ABSTRACT Copper is both a required micronutrient and a source of toxicity in most organisms, including Campylobacter jejuni. Two proteins expressed in C. jejuni (termed CopA and CueO) have been shown to be a copper transporter and multicopper oxidase, respectively. We have isolated strains with mutations in these genes, and here we report that they were more susceptible to both the addition of copper in the growth media and to induced oxidative stress. Both mutant strains were defective in colonization of an avian host, and copper in the feed exacerbated the colonization deficiency. Overexpression of a cytoplasmic peptide derived from the normally periplasmic copper-binding region of CueO also caused copper intolerance compared to nonexpressing strains or strains expressing the non-copper-binding versions of the peptide. Taken together, the results indicate that copper toxicity in C. jejuni is due to a failure to effectively sequester cytoplasmic copper, resulting in an increase in copper-mediated oxidative damage. IMPORTANCE Copper is a required micronutrient for most aerobic organisms, but it is universally toxic at elevated levels. These organisms use homeostatic mechanisms that allow for cells to acquire enough of the element to sustain metabolic requirements while ensuring that lethal levels cannot build up in the cell. Campylobacter jejuni is an important foodborne pathogen that typically makes its way into the food chain through contaminated poultry. C. jejuni has a metabolic requirement for copper and encodes a copper detoxification system. In the course of studying this system, we have learned that it is important for avian colonization. We have also gained insight into how copper exerts its toxic effects in C. jejuni by promoting oxidative stress.

scholarly journals mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Nicolas Kieffer ◽  
Guilhem Royer ◽  
Jean-Winoc Decousser ◽  
Anne-Sophie Bourrel ◽  
Mattia Palmieri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Acquired Resistance ◽  
Regulatory System ◽  
Amino Acid Identity ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
A Minor

ABSTRACT The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae. Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9. Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

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