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Corynebacterium amycolatum is a nonlipophilic coryneform which is increasingly being recognized as a relevant human and animal pathogen showing multidrug resistance to commonly used antibiotics. However, little is known about the molecular mechanisms involved in transition from colonization to the MDR invasive phenotype in clinical isolates. In this study, we performed a comprehensive pan-genomic analysis of C. amycolatum, including 26 isolates from different countries. We obtained the novel genome sequences of 8 of them, which are multidrug resistant clinical isolates from Spain and Tunisia. They were analyzed together with other 18 complete or draft C. amycolatum genomes retrieved from GenBank. The species C. amycolatum presented an open pan-genome (α = 0.854905), with 3,280 gene families, being 1,690 (51.52%) in the core genome, 1,121 related to accessory genes (34.17%), and 469 related to unique genes (14.29%). Although some classic corynebacterial virulence factors are absent in the species C. amycolatum, we did identify genes associated with immune evasion, toxin, and antiphagocytosis among the predicted putative virulence factors. Additionally, we found genomic evidence for extensive acquisition of antimicrobial resistance genes through genomic islands.

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Corynebacterium striatum, a common constituent of the human skin microbiome, is now considered an emerging multidrug-resistant pathogen of immunocompromised and chronically ill patients. However, little is known about the molecular mechanisms in the transition from colonization to the multidrug-resistant (MDR) invasive phenotype in clinical isolates. This study performed a comprehensive pan-genomic analysis of C. striatum, including isolates from "normal skin microbiome" and from MDR infections, to gain insights into genetic factors contributing to pathogenicity and multidrug resistance in this species. For this, three novel genome sequences were obtained from clinical isolates of C. striatum of patients from Brazil, and other 24 complete or draft C. striatum genomes were retrieved from GenBank, including the ATCC6940 isolate from the Human Microbiome Project. Analysis of C. striatum strains demonstrated the presence of an open pan-genome (α = 0.852803) containing 3816 gene families, including 15 antimicrobial resistance (AMR) genes and 32 putative virulence factors. The core and accessory genomes included 1297 and 1307 genes, respectively. The identified AMR genes are primarily associated with resistance to aminoglycosides and tetracyclines. Of these, 66.6% are present in genomic islands, and four AMR genes, including aac(6')-ib7, are located in a class 1-integron. In conclusion, our data indicated that C. striatum possesses genomic characteristics favorable to the invasive phenotype, with high genomic plasticity, a robust genetic arsenal for iron acquisition, and important virulence determinants and AMR genes present in mobile genetic elements.

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Biochemical, serological, and molecular methods have been developed for the laboratory diagnosis of diseases caused by C. pseudotuberculosis (CP), but the identification of the pathogen and biovars differentiation may be time-consuming, expensive, and confusing compared with other bacteria. This study aimed to evaluate MALDI Biotyper and Overall Genome Relatedness Index (OGRI) analysis to optimize the identification and differentiation of biovars of C. pseudotuberculosis. Out of 230 strains isolated from several hosts and countries, 202 (87.8%) were precisely classified using MALDI Biotyper and the BioNumerics platform. The classification accuracies for the Ovis and Equi biovars were 80 (88.75%) and 82 (92.68%), respectively. When analyzing a sampling of these strains by Average Nucleotide Identity based on BLAST and TETRA analyses using genomic sequence data, it was possible to differentiate 100% of the strains in Equi and Ovis. Our data show that MALDI Biotyper and OGRI analysis help identify C. pseudotuberculosis at the species and biovar levels.

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Common strategies to improve recombinant protein production in Escherichia coli often involve the test and optimization of several different variables, when using traditional expression vectors that are commercially available. Now, modern synthetic biology-based strategies allow for extensive modifications of these traditional vectors, or even construction of entirely new modular vectors, so as to permit tunable production of the recombinant proteins of interest. Herein, we describe the engineering of a new expression operating unit (EOU; 938 bp) for producing recombinant proteins in E. coli, through the combinatorial assembly of standardized and well-characterized genetic elements required for transcription and translation (promoter, operator site, RBS, junction RBS-CDS, cloning module, transcriptional terminator). We also constructed a novel T7 promoter variant with increased transcriptional activity (1.7-fold higher), when compared to the canonical wild type T7 promoter sequence. This new EOU yielded an improved production of the reporter protein superfolder GFP (sfGFP) in E. coli BL21(DE3) (relative fluorescence units/RFU = 70.62 ± 1.62 A U.) when compared to a high-producing control expression vector (plasmid BBa_I746909; RFU = 59.68 ± 1.82 A U.). The yields of purified soluble recombinant sfGFP were also higher when using the new EOU (188 mg L-1 culture vs. 108 mg L-1 in the control) and it performed similarly well when inserted into different plasmid backbones (pOPT1.0/AmpR and pOPT2.0/CmR).

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Toxocariasis is an infection caused by the round worms Toxocara canis and Toxocara cati. It occurs worldwide though it is more prevalent in developing countries. For the diagnosis of toxocariasis, the most used method is the indirect enzyme-linked immunosorbent assay (indirect ELISA), based on the detection of specific antibodies using the excreted/secreted products from T. canis larvae (TES) as antigens, but it cross-reacts with several helminth infections. For this reason, there is a need to investigate species-specific immunoreactive proteins, which can be used for the development of a more sensitive and specific diagnosis. This study aims to investigate immunoreactive protein candidates to be used for the development of a more sensitive and specific diagnosis of Toxocara spp. infection in humans. We have used immunoblotting and mass spectrometry to select four Toxocara canis immunoreactive proteins that were recombinantly expressed in bacteria and evaluated as potential new diagnostic antigens (rMUC3, rTES 26, rTES32 and rCTL4). The recognition of these recombinant proteins by total serum IgG and IgG4 was assayed using the purified proteins in an isolated manner or in combination. The IgG ELISAs performed with individual recombinant antigens reached values of sensitivity and specificity that ranged from 91.7% to 97.3% and 94.0% to 97.9%, respectively. Among the analyses, the IgG4 immunoassay was proven to be more effective, revealing a sensitivity that ranged from 88.8% to 98.3% and a specificity of 97.8%-97.9%. The IgG4 ELISA was shown to be more effective and presented no cross-reactivity when using combinations of the rTES 26 and rCTL4 recombinant proteins. The combination of these two molecules achieved 100% sensitivity and specificity. The use of only two recombinant proteins can contribute to improve the current panorama of toxocariasis immunodiagnosis for, with a better optimization and reduced cost.

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Blomia tropicalis and Dermatophagoides pteronyssinus play an important role in triggering allergy. Glycycometus malaysiensis causes IgE reaction in sensitive people, but is rarely reported in domestic dust, because it is morphologically similar to B. tropicalis making the identification of these species difficult. The identification of mites is mostly based on morphology, a time-consuming and ambiguous approach. Herein, we describe a multiplex polymerase chain reaction (mPCR) assay based on ribosomal DNA capable to identify mixed cultures of B. tropicalis, D. pteronyssinus and G. malaysiensis, and/or to identify these species from environmental dust. For this, the internal transcribed spacer 2 (ITS2) regions, flanked by partial sequences of the 5.8S and 28S genes, were PCR-amplified, cloned and sequenced. The sequences obtained were aligned with co-specific sequences available in the GenBank database for primer design and phylogenetic studies. Three pairs of primers were chosen to compose the mPCR assay, which was used to verify the frequency of different mites in house dust samples (n = 20) from homes of Salvador, Brazil. Blomia tropicalis was the most frequent, found in 95% of the samples, followed by G. malaysiensis (70%) and D. pteronyssinus (60%). Besides reporting for the first time the occurrence of G. malaysiensis in Brazil, our results confirm the good resolution of the ITS2 region for mite identification. Furthermore, the mPCR assay proved to be a fast and reliable tool for identifying these mites in mixed cultures and could be applied in future epidemiological studies, and for quality control of mite extract production for general use.

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BACKGROUND: Allergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant. METHODS: Herein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions. RESULTS: Four main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy. CONCLUSIONS: When produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001). GENERAL SIGNIFICANCE: This study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.

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Recombinant proteins are generally fused with solubility enhancer tags to improve the folding and solubility of the target protein of interest. However, the fusion protein strategy usually requires expensive proteases to perform in vitro proteolysis and additional chromatographic steps to obtain tag-free recombinant proteins. Expression systems based on intracellular processing of solubility tags in Escherichia coli, through co-expression of a site-specific protease, simplify the recombinant protein purification process, and promote the screening of molecules that fail to remain soluble after tag removal. High yields of soluble target proteins have already been achieved using these protease co-expression systems. Herein, we review approaches for controlled intracellular processing systems tailored to produce soluble untagged proteins in E. coli. We discuss the different genetic systems available for intracellular processing of recombinant proteins regarding system design features, advantages, and limitations of the various strategies.

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Corynebacterium spp. are Gram-positive rods that are recognized to cause opportunistic diseases under certain predisposing clinical conditions. Some species have been described in urinary tract infections. In this report we document a new episode of urinary tract infection caused by Corynebacterium phoceense and describe the whole-genome sequencing, phenotypic characteristics and mass spectra obtained by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on genome identification and DNA-to-DNA hybridization, we can assume that our strain is the second isolate of C. phoceense to be described in a urine sample. No other infectious diseases have been reported to be associated with this species.

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Varroa destructor is an ectoparasite mite that attacks bees leading to colony disorders worldwide. microRNAs (miRNAs) are key molecules used by eukaryotes to post-transcriptional control of gene expression. Nevertheless, still lack information aboutV. destructor miRNAs and its regulatory networks. Here, we used an integrative strategy to characterize the miRNAs in the V. destructor mite. We identified 310 precursors that give rise to 500 mature miRNAs, which 257 are likely mite-specific elements. miRNAs showed canonical length ranging between 18 and 25 nucleotides and 5' uracil preference. Top 10 elements concentrated over 80% of total miRNA expression, with bantam alone representing ~50%. We also detected non-templated bases in precursor-derived small RNAs, indicative of miRNA post-transcriptional regulatory mechanisms. Finally, we note that conserved miRNAs control similar processes in different organisms, suggesting a conservative role. Altogether, our findings contribute to the better understanding of the mite biology that can assist future studies on varroosis control.

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