Emergence of a KPC-90 Variant that Confers Resistance to Ceftazidime-Avibactam in an ST463 Carbapenem-Resistant Pseudomonas aeruginosa Strain

For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance. CZA resistance was mediated by a 2 amino acid insertion outside the KPC omega-loop region in CRPA.

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/α-ketoglutarate-dependent oxygenase SptF

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

Structure and Fc-Effector Function of Rhesusized Variants of Human Anti-HIV-1 IgG1s

Passive transfer of monoclonal antibodies (mAbs) of human origin into Non-Human Primates (NHPs), especially those which function predominantly by a Fc-effector mechanism, requires an a priori preparation step, in which the human mAb is reengineered to an equivalent NHP IgG subclass. This can be achieved by changing both the Fc and Fab sequence while simultaneously maintaining the epitope specificity of the parent antibody. This Ab reengineering process, referred to as rhesusization, can be challenging because the simple grafting of the complementarity determining regions (CDRs) into an NHP IgG subclass may impact the functionality of the mAb. Here we describe the successful rhesusization of a set of human mAbs targeting HIV-1 envelope (Env) epitopes involved in potent Fc-effector function against the virus. This set includes a mAb targeting a linear gp120 V1V2 epitope isolated from a RV144 vaccinee, a gp120 conformational epitope within the Cluster A region isolated from a RV305 vaccinated individual, and a linear gp41 epitope within the immunodominant Cys-loop region commonly targeted by most HIV-1 infected individuals. Structural analyses confirm that the rhesusized variants bind their respective Env antigens with almost identical specificity preserving epitope footprints and most antigen-Fab atomic contacts with constant regions folded as in control RM IgG1s. In addition, functional analyses confirm preservation of the Fc effector function of the rhesusized mAbs including the ability to mediate Antibody Dependent Cell-mediated Cytotoxicity (ADCC) and antibody dependent cellular phagocytosis by monocytes (ADCP) and neutrophils (ADNP) with potencies comparable to native macaque antibodies of similar specificity. While the antibodies chosen here are relevant for the examination of the correlates of protection in HIV-1 vaccine trials, the methods used are generally applicable to antibodies for other purposes.

Uncovering the Role of PhzC as DAHP Synthase in Shikimate Pathway of Pseudomonas chlororaphis HT66

DAHP synthase catalyzes the first step in the shikimate pathway, deriving the biosynthesis of aromatic amino acids (Trp, Phe and Tyr), phenazine-1-carboxamide, folic acid, and ubiquinone in Pseudomonas chlororaphis. In this study, we identified and characterized one DAHP synthase encoding gene phzC, which differs from the reported DAHP synthase encoding genes aroF, aroG and aroH in E. coli. PhzC accounts for approximately 90% of the total DAHP synthase activities in P. chlororaphis HT66 and plays the most critical role in four DAHP synthases in the shikimate pathway. Inactivation of phzC resulted in the reduction of PCN production by more than 90%, while the absence of genes aroF, aroG and aroH reduced PCN yield by less than 15%, and the production of PCN was restored after the complementation of gene phzC. Moreover, the results showed that phzC in P. chlororaphis HT66 is not sensitive to feedback inhibition. This study demonstrated that gene phzC is essential for PCN biosynthesis. The expression level of both phzC and phzE genes are not inhibited in feedback by PCN production due to the absence of a loop region required for allosteric control reaction. This study highlighted the importance of PhzC and applying P. chlororaphis for shikimate pathway-derived high-value biological production.

Decreased mitochondrial D-loop region methylation mediates an increase in mitochondrial DNA copy number in CADASIL

Background Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a typical neurodegenerative disease associated with mitochondrial dysfunction. Methylation of the D-loop region and mitochondrial DNA copy number (mtDNAcn) play a critical role in the maintenance of mitochondrial function. However, the association between D-loop region methylation, mtDNAcn and CADASIL remains unclear. Methods Overall, 162 individuals were recruited, including 66 CADASIL patients and 96 age- and sex-matched controls. After extracting genomic DNA from the peripheral white blood cells, levels of D-loop methylation and mtDNAcn were assessed using MethylTarget sequencing and real-time PCR, respectively. Results We observed increased mtDNAcn and decreased D-loop methylation levels in CADASIL patients compared to the control group, regardless of gender stratification. Besides, we found a negative correlation between D-loop methylation levels and mtDNAcn. Mediation effect analysis shows that the proportion of the association between mtDNAcn and CADASIL that is mediated by D-loop methylation is 11.6% (95% CI 5.6, 22.6). After gender stratification, the proportions of such associations that are mediated by D-loop methylation in males and females were 7.2% (95% CI 2.4, 19.8) and 22.0% (95% CI 7.4, 50.1), respectively. Conclusion Decreased methylation of the D-loop region mediates increased mtDNAcn in CADASIL, which may be caused by a compensatory mechanism of mitochondrial dysfunction in patients with CADASIL.

Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.

A PRELIMINARY COMPARISON OF MITOCHONDRIAL D-LOOP REGION OF FUNAAB ALPHA AND NIGERIAN INDIGENOUS CHICKENS

Nigerian indigenous chickens possess immunity from endemic diseases and have a better survival rate than commercial hybrid strains under local production conditions. FUNAAB Alpha chicken was developed by improving Nigerian indigenous chickens through crossbreeding and selection. This study compared the mitochondrial d-loop of FUNAAB Alpha and Nigerian indigenous chickens to check likely genetic erosion and loss of diversity in development of FUNAAB Alpha breed. Blood samples were collected from Nigerian indigenous (n=23) and FUNAAB Alpha (n=20) chickens sampled from farms and houses in Ogun state, Nigeria. The Hypervariable 1 (HV1) of the mitochondrial d-loop region was amplified and sequenced. Single nucleotide polymorphisms present in HV1 of chickens were identified using Clustal W. Genetic diversity of the region was determined using DnaSp v5 while selective forces acting on the chickens were predicted using HyPhy software implemented inside MEGA 6 software. Phylogenetic relationship among FUNAAB Alpha, Nigerian indigenous and other chicken breeds was determined using MEGA 6 software. Five polymorphisms were identified in FUNAAB Alpha chickens while twelve were identified in Nigerian indigenous chickens. All the polymorphisms identified in FUNAAB Alpha chickens were also observed in Nigerian indigenous chickens while seven polymorphisms were unique to Nigerian indigenous chickens. Higher diversity indices were observed in Nigerian indigenous chickens (number of haplotype: 4; haplotype diversity: 0.743±0.012; nucleotide diversity: 0.014±0.0013 and average number of nucleotide differences: 4.332) compared with FUNAAB Alpha chickens (number of haplotype: 2; haplotype diversity: 0.485±0.001; nucleotide diversity: 0.008±0.0001 and average number of nucleotide differences: 2.424). Positive selective forces were acting on FUNAAB Alpha chickens while negative selective forces were acting on Nigerian indigenous chickens. Phylogenetic analysis revealed that FUNAAB Alpha chickens clustered with Nigerian indigenous and South American chickens. It can be concluded that there was likely genetic erosion and loss of diversity in development of FUNAAB Alpha breed. Breeding programmes aimed at improvement of genetic diversity and reduction of genetic erosion should be applied in subsequent improvement of FUNAAB Alpha chickens.

Primer design of D-loop region for wild population genetics of Rusa timorensis in Indonesia

Rusa timorensis (Javan deer) is endemic wildlife in Indonesia and is estimated at less than 10.000 individuals with continuously declining populations due to habitat loss and illegal hunting in the wild. This declining low population indicates a greater risk of extinction. Unfortunately, the genetic information of the wild Javan deer population for conservation management strategies still lacks data due to challenging sampling in the wild. Most recent studies were analysing the breeding populations outside Indonesia. Here, we propose the primer design of the D-loop genetic marker to determine the genetic population of wild Javan deer. We used metadata analysis of genetic sequences and new samples from five wild populations to design the specific primer of the D-loop region of the wild Javan deer in Indonesia. We used software, i.e.., Primer3 to design the primers, BLAST for specificity and Oligo Analyzer™ Tool for efficiency of the primer. The Annealing temperature optimisation started with pre-denaturation at 94 °C followed by 35 cycles of denaturation at 95°C; 51-56°C annealing for each one degree’s different per PCR treatment; and 72°C extensions. We successfully designed a specific primer (RL-3.1a) to amplify 235 bp of the D-loop region at 52°C annealing’s temperature.

Extensive Drug-Resistant Salmonella enterica Isolated From Poultry and Humans: Prevalence and Molecular Determinants Behind the Co-resistance to Ciprofloxacin and Tigecycline

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella. The tet(A) and tet(X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt.