Contribution Of Amino Acid Substitution Research Articles

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Evolutionary changes between pre- and post-vaccine South African group A G2P[4] rotavirus strains, 2003-2017.

The transient upsurge of G2P[4] group A rotavirus (RVA) after Rotarix vaccine introduction in several countries has been a matter of concern. To gain insight into the diversity and evolution of G2P[4] strains in South Africa pre- and post-RVA vaccination introduction, whole-genome sequencing was performed for RVA positive faecal specimens collected between 2003 and 2017 and samples previously sequenced were obtained from GenBank (n=103; 56 pre- and 47 post-vaccine). Pre-vaccine G2 sequences predominantly clustered within sub-lineage IVa-1. In contrast, post-vaccine G2 sequences clustered mainly within sub-lineage IVa-3, whereby a radical amino acid (AA) substitution, S15F, was observed between the two sub-lineages. Pre-vaccine P[4] sequences predominantly segregated within sub-lineage IVa while post-vaccine sequences clustered mostly within sub-lineage IVb, with a radical AA substitution R162G. Both S15F and R162G occurred outside recognised antigenic sites. The AA residue at position 15 is found within the signal sequence domain of Viral Protein 7 (VP7) involved in translocation of VP7 into endoplasmic reticulum during infection process. The 162 AA residue lies within the hemagglutination domain of Viral Protein 4 (VP4) engaged in interaction with sialic acid-containing structure during attachment to the target cell. Free energy change analysis on VP7 indicated accumulation of stable point mutations in both antigenic and non-antigenic regions. The segregation of South African G2P[4] strains into pre- and post-vaccination sub-lineages is likely due to erstwhile hypothesized stepwise lineage/sub-lineage evolution of G2P[4] strains rather than RVA vaccine introduction. Our findings reinforce the need for continuous whole-genome RVA surveillance and investigation of contribution of AA substitutions in understanding the dynamic G2P[4] epidemiology.

The Molecular Determinants of Thermoadaptation:Methanococcalesas a Case Study

Previous reports have shown that environmental temperature impacts proteome evolution in Bacteria and Archaea. However, it is unknown whether thermoadaptation mainly occurs via the sequential accumulation of substitutions, massive horizontal gene transfers, or both. Measuring the real contribution of amino acid substitution to thermoadaptation is challenging, because of confounding environmental and genetic factors (e.g., pH, salinity, genomic G + C content) that also affect proteome evolution. Here, using Methanococcales, a major archaeal lineage, as a study model, we show that optimal growth temperature is the major factor affecting variations in amino acid frequencies of proteomes. By combining phylogenomic and ancestral sequence reconstruction approaches, we disclose a sequential substitutional scheme in which lysine plays a central role by fine tuning the pool of arginine, serine, threonine, glutamine, and asparagine, whose frequencies are strongly correlated with optimal growth temperature. Finally, we show that colonization to new thermal niches is not associated with high amounts of horizontal gene transfers. Altogether, although the acquisition of a few key proteins through horizontal gene transfer may have favored thermoadaptation in Methanococcales, our findings support sequential amino acid substitutions as the main factor driving thermoadaptation.

Using epidemics to map H3 equine influenza virus determinants of antigenicity

Equine influenza is a major cause of respiratory infections in horses and causes widespread epidemics, despite the availability of commercial vaccines. Antigenic drift within the haemagglutinin (HA) glycoprotein is thought to play a part in vaccination breakdown. Here, we carried out a detailed investigation of the 1989 UK outbreak, using reverse genetics and site-directed mutagenesis, to determine the individual contribution of amino acid substitutions within HA. Mutations at positions 159, 189 and 227 all altered antigenicity, as measured by haemagglutination-inhibition assays. We also compared HA sequences for epidemic and vaccine strains from four epidemics and found that at least 8 amino acid differences were present, affecting multiple antigenic sites. Substitutions within antigenic site B and at least one other were associated with each outbreak, we also identified changes in loop regions close to antigenic sites that have not previously been highlighted for human H3 influenza viruses.

Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae

Amino acid substitutions at position 89 or 91 in GyrA of fluoroquinolone-resistant Mycobacterium leprae clinical isolates have been reported. In contrast, those at position 94 in M. tuberculosis, equivalent to position 95 in M. leprae, have been identified most frequently. To verify the possible contribution of amino acid substitutions at position 95 in M. leprae to fluoroquinolone resistance, we conducted an in vitro assay using wild-type and mutant recombinant DNA gyrases. Fluoroquinolone-mediated supercoiling activity inhibition assay and DNA cleavage assay revealed the potent contribution of an amino acid substitution of Asp to Gly or Asn at position 95 to fluoroquinolone resistance. These results suggested the possible future emergence of quinolone-resistant M. leprae isolates with these amino acid substitutions and the usefulness of detecting these mutations for the rapid identification of fluoroquinolone resistance in leprosy.

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme.

To elucidate correlative relationships between structural change and thermodynamic stability in proteins, a series of mutant human lysozymes modified at two buried positions (Ile56 and Ile59) were examined. Their thermodynamic parameters of denaturation and crystal structures were studied by calorimetry and X-ray crystallography. The mutants at positions 56 and 59 exhibited different responses to a series of amino acid substitutions. The changes in stability due to substitutions showed a linear correlation with changes in hydrophobicity of substituted residues, having different slopes at each mutation site. However, the stability of each mutant was found to be represented by a unique equation involving physical properties calculated from mutant structures. By fitting present and previous stability data for mutant human lysozymes substituted at various positions to the equation, the magnitudes of the hydrophobicity of a carbon atom and the hydrophobicity of nitrogen and neutral oxygen atoms were found to be 0.178 and -0.013 kJ/mol.A(2), respectively. It was also found that the contribution of a hydrogen bond with a length of 3.0 A to protein stability was 5.1 kJ/mol and the entropy loss of newly introduction of a water molecules was 7.8 kJ/mol.

Maturation of the immune response to (4-hydroxy-3-nitrophenyl)-acetyl (NP) haptens in C57BL/6 mice

Changes with time in specificity and affinity of anti-NP antibodies in C57BL/6 mice after immunization with NP 22-chicken gamma-globulin (CGG) were studied by comparing the abilities of the antibodies to bind to NP 3-bovine serum albumin (NP 3-BSA) at pH 5 and 8. Early anti-NP antibodies (on day 14 after immunization) bound to NP 3-BSA at pH 8, but not pH 5. This pH-dependence of binding was explained in terms of the low affinity of the antibody to the phenolic form of NP on the basis of results of fluorescence quenching titration of a monoclonal anti-NP antibody that showed similar specificity to that of the early anti-NP antibodies. Since NP on the CGG molecule ionized with an apparent p K of about 7.4, more than half the NP should be in the unionized (phenolic) form under the immunization conditions. However, early anti-NP antibodies bound preferentially to the ionized (phenolate) form of NP, which was a minor form at neutral pH, whereas later anti-NP antibodies showed ability to bind to both the phenolate and phenolic forms of NP. This change in specificity with time was observed on immunization with T cell-dependent (TD) antigens such as NP-CGG and NP keyhole limpet hemocyanin (KLH), but not with a T cell-independent (TI) antigen such as NP-Ficoll. The heavy (H) chains from the two monoclonal antibodies 3G6 and 3C6, which bound to the phenolate form and both the phenolate and phenolic forms, respectively, were recombined with λ1 chains (L 3G6 and L 3C6) from these antibodies as well as a λ 1 chain (L HOPC-1) with the amino acid sequence of the germline. Ability to bind to the phenolate form of NP was recovered in all the reconstituted IgGs, while ability to bind to both the phenolate and phenolic form of NP was observed only with IgG reconstituted from H 3 c6 and L 3 c6 . These results suggest that the specificity corresponding to early anti-NP antibodies were generated even by λ 1 chains of a germline sequence, but that of late anti-NP antibodies was expressed only by an appropriate pair of H and L chains. The contribution of amino acid substitution by somatic point mutation to the change of specificity with time was discussed.