Heterologous expression and purification of calmodulin from Plasmodium knowlesi using codon-optimized synthetic gene

Aims@#Calmodulin (CaM) is vital for the survival of Plasmodium knowlesi, a simian malaria parasite that infects both macaques and humans in Southeast Asia. To advance antimalarial drugs development targeting this protein, it is imperative to produce ample quantities of pure CaM for further research. Hence, this study aims to establish a robust strategy for the heterologous expression and purification of CaM from Plasmodium knowlesi (Pk-CaM). @*Methodology and results@#First, we optimised the gene sequence of Pk-CaM for Escherichia coli expression, chemically synthesised it and integrated it into the pET28a plasmid. The optimised gene displayed a 45.15% GC content and a 0.81 codon-adaptation index, making it highly compatible with E. coli. Pk-CaM expression was assessed under various conditions, with the best results achieved at a post-induction temperature of 20 °C for 16 h, yielding a fully soluble protein. Subsequently, we purified the protein using Ni2+-NTA affinity chromatography and size-exclusion chromatography (SEC), obtaining 15 mg from 1 L of culture. The folding properties of purified Pk-CaM were analysed using far-UV circular dichroism (CD) spectroscopy, revealing a predominance of helical structures, both with and without Ca2+ ions. Binding to Ca2+ ions induced structural changes, increasing the helical content compared to when Ca2+ ions were absent. @*Conclusion, significance and impact of study@#The optimal conditions for the recombinant expression and purification of Pk-CaM in a correctly folded and functional form were successfully established in this study. This achievement provides a solid foundation for conducting further comprehensive research in the pursuit of novel antimalarial drugs.

Inhibition properties of collagen hydrolysates against FKBP35 of Plasmodium knowlesi

Aim@#FK506-binding protein 35 from Plasmodium knowlesi (Pk-FKBP35), a member of peptidyl-prolyl cis-trans isomerase (PPIase), is considered a viable target for the development of the novel antimalarial drug targeting zoonotic malaria in Malaysia. While FK506 effectively inhibits this protein, this drug is not applicable due to its immunosuppressive effects. This study aims to assess the inhibitory potential of different collagen hydrolysates (CH) against Pk-FKBP35, as FK506 replacers.@*Methodology and results@#Recombinant full-length Pk-FKBP35 was initially over-expressed using Escherichia coli (BL21) host cells and subsequently purified via affinity chromatography coupled with size-exclusion chromatography. In this study, four distinct CH were employed, originating from bovine, bone broth, fish and swine. The results revealed that all CH inhibited PPIase catalytic activity of Pk-FKBP35 with IC50 values 1.63 mg/mL (bovine CH), 2.97 mg/mL (fish CH), 33.01 mg/mL (swine CH) and 13.91 mg/mL (bone broth CH), which were much higher than that of FK506. Furthermore, these CHs retained the ability of Pk-FKBP35 to inhibit calcineurin phosphatase activity, yet not as extreme as FK506. @*Conclusion, significance and impact of study@#The inhibition is predicted due to the presence of proline-rich peptides in CH, which were able to block the substrate binding cavity of Pk-FKBP35. This study suggested that CH might have no serious immunosuppressant effect and is promising for further harnessing for antimalarial compounds

Transposable elements: Uniqueness and applications

@#The study of transposable genetic elements, a cornerstone of molecular genetics, offers profound insights into the dynamic nature of genetic material. This exploration encompasses various types found in bacteria, including insertion sequences, composite and non-composite transposons. These elements are instrumental in shaping bacterial genomes by facilitating the movement of genetic information, driving evolution and contributing to genetic diversity. Understanding the mechanisms of transposition is essential for unravelling the intricate processes governing genetic rearrangements. Replicative and conservative transposition mechanisms, exemplified by the Tn3 family and phage Mu, illustrate the remarkable adaptability of these systems in reshaping genomes. However, it is the transposon Tn5 that steals the spotlight as a versatile molecular genetics tool. Tn5's transposition mechanism, characterized by precise control over gene expression, translational regulation, protein localization and the induction of conditional mutations, empowers researchers to dissect gene regulation intricacies with unprecedented accuracy. Transposable genetic elements, epitomized by Tn5, are indispensable instruments in molecular genetics. They allow researchers to navigate the intricate landscape of genetics, exploring gene regulation, protein function, and genetic diversity with unparalleled precision. These elements continue to be at the forefront of molecular genetics research, driving innovations that deepen our understanding of the fundamental mechanisms governing life's genetic code.

Biofilm formation of serotype 19 Streptococcus pneumoniae clinical isolates in relation to clinical isolate source, pH and Fe(III) supplementation

Aims@#Streptococcus pneumoniae is one the world’s foremost bacterial pathogens that cause massive global mortality and morbidity in young children and immunocompromised adults especially in developing countries. Biofilms have been increasingly recognized as an important prerequisite to disease. Individual S. pneumoniae strains differ markedly in their virulence phenotypes, but genetic heterogeneity has complicated attempts to identify any association between a given clonal lineage and propensity to cause a particular disease type. This study investigated serotype 19 S. pneumoniae from blood and ear isolates for biofilm formation capacity in relation to isolate source, pH and ferric oxide [Fe(III)] supplementation.@*Methodology and results@#Viable count and density biofilm assays, microscopy and multi locus sequence typing (MLST) were applied to investigate biofilm formation capacity and genetic diversity of serotype 19 S. pneumoniae from blood and ear isolates. Generally, blood isolates were observed to produce more biofilms at both pH 7.4 and 6.8 compared to the ear isolates. The supplementation of Fe(III) was also found to support biofilm growth. Upon MLST typing of the isolates, marked differences in biofilm formation within the same sequence types (ST) of ST199 and ST177 was observed. This strongly indicated that strains within the same sequence type show differences in biofilm formation capacity.@*Conclusion, significance and impact of study@#This study showed that despite belonging to the same serotype, serotype 19, S. pneumoniae blood and ear isolates showed high diversity in biofilm formation ability in relation to pH and Fe(III) supplementation. Additionally, pneumococcal isolates from sequence types ST199 and ST177 also gave rise to differences in biofilm formation ability within the same sequence type (ST). The diversity of biofilm formation within serotype 19 seen in this study is significant to further inform of vaccination strategies against pneumococcal infections, in that due to variations in biofilm formation capacity within the same ST. It is possible that within serotype 19 may show variable vaccination or drug treatment responses. This also indicates that the current treatment strategy which employs specific serotype selection as for PCV14 and PCV7 pneumococcal vaccines may not produce the desired therapeutic results.