Rongsui Gao

Diversified mcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota

ABSTRACT Colistin is an ultimate line of refuge against multidrug-resistant Gram-negative pathogens. Very recently, the emergence of plasmid-mediated mcr-1 colistin resistance has become a great challenge to global public health, raising the possibility that dissemination of the mcr-1 gene is underestimated and diversified. Here, we report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients. Structural and functional analyses determined that the colistin resistance is conferred purely by the single mcr-1 gene. Genetic and sequence mapping revealed that mcr-1-harbouring plasmid reservoirs are present in diversity. Together, the data represent the first evidence of diversity in mcr-1-harbouring plasmid reservoirs of human gut microbiota. IMPORTANCE The plasmid-mediated mobile colistin resistance gene (mcr-1) challenged greatly the conventional idea mentioned above that colistin is an ultimate line of refuge against lethal infections by multidrug-resistant Gram-negative pathogens. It is a possibility that diversified dissemination of the mcr-1 gene might be greatly underestimated. We report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients and functionally define the colistin resistance conferred purely by the single mcr-1 gene. Genetic and sequence mapping revealed unexpected diversity among the mcr-1-harbouring plasmid reservoirs of human gut microbiota. The plasmid-mediated mobile colistin resistance gene (mcr-1) challenged greatly the conventional idea mentioned above that colistin is an ultimate line of refuge against lethal infections by multidrug-resistant Gram-negative pathogens. It is a possibility that diversified dissemination of the mcr-1 gene might be greatly underestimated. We report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients and functionally define the colistin resistance conferred purely by the single mcr-1 gene. Genetic and sequence mapping revealed unexpected diversity among the mcr-1-harbouring plasmid reservoirs of human gut microbiota.

Dissemination and Mechanism for the MCR-1 Colistin Resistance

Polymyxins are the last line of defense against lethal infections caused by multidrug resistant Gram-negative pathogens. Very recently, the use of polymyxins has been greatly challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1). However, the mechanistic aspects of the MCR-1 colistin resistance are still poorly understood. Here we report the comparative genomics of two new mcr-1-harbouring plasmids isolated from the human gut microbiota, highlighting the diversity in plasmid transfer of the mcr-1 gene. Further genetic dissection delineated that both the trans-membrane region and a substrate-binding motif are required for the MCR-1-mediated colistin resistance. The soluble form of the membrane protein MCR-1 was successfully prepared and verified. Phylogenetic analyses revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase in Paenibacili, a known producer of polymyxins. The fact that the plasmid-borne MCR-1 is placed in a subclade neighboring the chromosome-encoded colistin-resistant Neisseria LptA (EptA) potentially implies parallel evolutionary paths for the two genes. In conclusion, our finding provids a first glimpse of mechanism for the MCR-1-mediated colistin resistance.

Deciphering MCR-2 Colistin Resistance

ABSTRACT Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2. Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus. Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1. Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4′ position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens. IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2. IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Mycobacterium smegmatis BioQ defines a new regulatory network for biotin metabolism.

Biotin (vitamin H), the sulfur‐containing enzyme cofactor, is an essential micronutrient for three domains of life. Given the fact that biotin is an energetically expensive molecule whose de novo biosynthesis demands 20 ATP equivalents each, it is reasonable that bacteria have evolved diversified mechanisms in various microorganisms to tightly control biotin metabolism. Unlike the Escherichia coli BirA, the prototypical bi‐functional version of biotin protein ligase (BPL) in that it acts as a repressor for biotin biosynthesis pathway, the BirA protein of Mycobacterium smegmatis (M. smegmatis), a closely relative of the tuberculosis‐causing pathogen, Mycobacterium tuberculosis, lacked the DNA‐binding activity. It raised a possibility that an alternative new regulator might be present to compensate the loss of regulatory function. Here we report that this is the case. Genomic context analyses of M. smegmatis detected a newly identified BioQ homolog classified into the TetR family of transcription factor and its recognizable palindromes. The M. smegmatis BioQ protein was overexpressed and purified to homogeneity. Size‐exclusion chromatography combined with chemical cross‐linking studies demonstrated that the BioQ protein had a propensity to dimerize. The promoters of bioFD and bioQ/B were mapped using 5′‐RACE. Electrophoretic mobility shift assays revealed that BioQ binds specifically to the promoter regions of bioFD and bioQ/B. Further DNase I foot‐printing elucidated the BioQ‐binding palindromes. Site‐directed mutagenesis suggested the important residues critical for BioQ/DNA binding. The isogenic mutant of bioQ (ΔbioQ) was generated using the approach of homologous recombination. The in vivo data from the real‐time qPCR combined with the lacZ transcriptional fusion experiments proved that removal of bioQ gave significant increment with expression of bio operons. Also, expression of bio operons were repressed by exogenous addition of biotin, and this repression seemed to depend on the presence of BioQ protein. Thereby, we believed that M. smegmatis BioQ is not only a negative auto‐regulator but also a repressor for bioFD and bioB operons involved in the biotin biosynthesis pathway. Collectively, this finding defined the two‐protein paradigm of BirA and BioQ, representing a new mechanism for bacterial biotin metabolism.

Unexpected complexity of multidrug resistance in the mcr-1-harbouring Escherichia coli.

The discovery that the mobile colistin resistance gene (mcr-1) is encoded by plasmids and is prevalent in food animals and human beings worldwide (Hasman et al., 2015; Liu et al., 2015) has challenged greatly our traditional idea that polymyxin (consisting of two isoforms：polymyxin B and polymyxin E (colistin)) acts as an ultimate line of refuge in the clinical treatment against the severe infections by the multidrug-resistant Gram-negative pathogens (Nation et al., 2015; Paterson and Harris, 2015). To make matters worse, the mcr-1 gene was recently found to be co-localized with other drug resistance genes in the plasmid pKH457-3-BE with an IncP backbone from a bovine isolate in Belgium (Surbi Malhotra-Kumar, 2016), which is far different from the pig-isolated plasmid, pHNSHP45 with an IncI2 backbone (Liu et al., 2015). It raised the possibility that super-bugs with pan-drug resistance might be emerging. Given the fact that (1) genomic sequences of the mcr-1-harbouring plasmids are extremely limited right now, and (2) co-occurrence of MCR-1-mediated colistin resistance with other multidrug resistance remains unclear, we screened a collection of antibiotic-resistant isolates (no., 102) from swine tissues in China. In particular, only 6 of the 16 colistin-resistant isolates (namely WH01, WH02, …, WH16) are verified to be mcr-1-positive Escherichia coli (Figure 1A, 1B and S1). They are namely WH03, WH07, WH09, WH12, WH13, and WH15. In contrast, the majority of the isolates we checked is mcr-1-negative, but remains appreciable level of the colistin resistance (not shown), implying the possibility that some mystical machiner-ies/vectors claim for this antibiotic resistance. Subsequently, we tested the sensitivity of the six mcr-1-positive E. coli isolates to a dozen of various antibiotics (Figure 1C and S1). The antibiotics (15 in total) we used here are categorized into eight groups 1: β-lactams antibiotics including ampicillin (AMP), cefotaxime (CTX); 2: quinolone antibiotic such as ciprofloxacin (CIP), norfloxacin (NOR) and levofloxacin (LEV); 3: tetracycline antibiotics (tetracycline (TET) and doxycycline (DOX)); 4: aminoglycoside antibiotics like amikacin (AMK), gentamycin (GEN) and kana-mycin (KAN); 5: amino alcohol antibiotic, chloramphenicol (CHL); 6: sulfonamide antibiotic, trimethoprim (TMP); 7: nitrofuran antibiotic, macrodantin (NFT); and 8: cationic polypeptide antibiotic, colistin (COL)). To our surprise, the unexpected complexity of the multi-drug resistance was observed in the mcr-1-harbouring isolates. First, the mcr-1-poitive isolate WH13 exhibited the mostly-broad-spectrum antibiotic resistance in that it can be tolerant with nearly all the 15 antibiotics with an exception of amikacin (Figure 1C). Second to the WH13, the mcr-1-carrying …

Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

ABSTRACTThe Escherichia colifadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ–proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensisfabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensisfabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of ShewanellafabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.

Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of the fad Regulon

ABSTRACT Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The β-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon. IMPORTANCE The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.

A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein

Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.

Structural and Functional Characterization of the FadR Regulatory Protein from Vibrio alginolyticus

The structure of Vibrio cholerae FadR (VcFadR) complexed with the ligand oleoyl-CoA suggests an additional ligand-binding site. However, the fatty acid metabolism and its regulation is poorly addressed in Vibrio alginolyticus, a species closely-related to V. cholerae. Here, we show crystal structures of V. alginolyticus FadR (ValFadR) alone and its complex with the palmitoyl-CoA, a long-chain fatty acyl ligand different from the oleoyl-CoA occupied by VcFadR. Structural comparison indicates that both VcFadR and ValFadR consistently have an additional ligand-binding site (called site 2), which leads to more dramatic conformational-change of DNA-binding domain than that of the E. coli FadR (EcFadR). Isothermal titration calorimetry (ITC) analyses defines that the ligand-binding pattern of ValFadR (2:1) is distinct from that of EcFadR (1:1). Together with surface plasmon resonance (SPR), electrophoresis mobility shift assay (EMSA) demonstrates that ValFadR binds fabA, an important gene of unsaturated fatty acid (UFA) synthesis. The removal of fadR from V. cholerae attenuates fabA transcription and results in the unbalance of UFA/SFA incorporated into membrane phospholipids. Genetic complementation of the mutant version of fadR (Δ42, 136-177) lacking site 2 cannot restore the defective phenotypes of ΔfadR while the wild-type fadR gene and addition of exogenous oleate can restore them. Mice experiments reveals that VcFadR and its site 2 have roles in bacterial colonizing. Together, the results might represent an additional example that illustrates the Vibrio FadR-mediated lipid regulation and its role in pathogenesis.

Crystal structure and acetylation of BioQ suggests a novel regulatory switch for biotin biosynthesis in Mycobacterium smegmatis

Biotin (vitamin B7), a sulfur‐containing fatty acid derivative, is a nutritional virulence factor in certain mycobacterial species. Tight regulation of biotin biosynthesis is important because production of biotin is an energetically expensive process requiring 15–20 equivalents of ATP. The Escherichia coli bifunctional BirA is a prototypical biotin regulatory system. In contrast, mycobacterial BirA is an unusual biotin protein ligase without DNA‐binding domain. Recently, we established a novel two‐protein paradigm of BioQ–BirA. However, structural and molecular mechanism for BioQ is poorly understood. Here, we report crystal structure of the M. smegmatis BioQ at 1.9 Å resolution. Structure‐guided functional mapping defined a seven residues‐requiring motif for DNA‐binding activity. Western blot and MALDI‐TOF MS allowed us to unexpectedly discover that the K47 acetylation activates crosstalking of BioQ to its cognate DNA. More intriguingly, excess of biotin augments the acetylation status of BioQ in M. smegmatis. It seems likely that BioQ acetylation proceeds via a non‐enzymatic mechanism. Mutation of this acetylation site K47 in BioQ significantly impairs its regulatory role in vivo. This explains in part (if not all) why BioQ has no detectable requirement of the presumable bio‐5’‐AMP effecter, which is a well‐known ligand for the paradigm E. coli BirA regulator system. Unlike the scenario seen with E. coli carrying a single biotinylated protein, AccB, genome‐wide search and Streptavidin blot revealed that no less than seven proteins require the rare post‐translational modification, biotinylation in M. smegmatis, validating its physiological demand for biotin at relatively high level. Taken together, our finding defines a novel biotin regulatory machinery by BioQ, posing a possibility that development of new antibiotics targets biotin, the limited nutritional virulence factor in certain pathogenic mycobacterial species.