Lessons from a Single Amino Acid Substitution: Anticancer and Antibacterial Properties of Two Phospholipase A2-Derived Peptides

The membrane-active nature of phospholipase A2-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA2 toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A2 isoforms, is again demonstrated as a valuable source of therapeutic peptides.

Creation of photocyclic vertebrate rhodopsin by single amino acid substitution

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photo-convert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, the molecular property of vertebrate rhodopsin successfully converted to the photocyclic and photoreversible properties by a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by the regulation of the recovery from the active state to the original dark state.

Hemoglobin Genotypes Modulate Inflammatory Response to Plasmodium Infection

In 2018, 228 million cases and 405,000 malaria-associated deaths were reported worldwide with a majority being in Africa. A wide range of factors, including parasitemia, host immunity, inflammatory responses to infection, and host hemoglobin genotype, mediate the severity of malaria. Among the hemoglobinopathies, hemoglobin S (HbS) is caused by a single amino acid substitution of Glutamic Acid replaced by Valine at the sixth position of the beta-globin chain (E6V). Hemoglobin C (HbC) on the other hand, involves a single amino acid substitution of Glutamic Acid by a Lysine (E6K), which has received the most attention. These substitutions alter the stability of Hb leading to wide-ranging hematological disorders. The homozygous state of hemoglobin S (HbSS) results in sickle cell anemia (SCA) whereas the heterozygous state (HbAS) results in sickle cell trait (SCT). Both mutations are reported to mediate the reduction in the severity and fatality of Plasmodium falciparum malaria. The mechanism underlying this protection is poorly understood. Since both malaria and sickle cell disease (SCD) are associated with the destruction of erythrocytes and widespread systemic inflammation, identifying which inflammatory factor(s) mediate susceptibility of individuals with different hemoglobin genotypes to Plasmodium infection could result in the discovery of new predictive markers and interventions against malaria or SCD severity. We hypothesized that hemoglobin genotypes modulate the inflammatory response to Plasmodium infection. We conducted a cross-sectional study in Ghana, West Africa, between 2014 and 2019 to ascertain the relationships between blood inflammatory cytokines, Plasmodium infection, and hemoglobin genotype. A total of 923 volunteers were enrolled in the study. A total of 74, age and sex-matched subjects were identified with various genotypes including HbAS, HbAC, HbSS, HbSC, HbCC, or HbAA. Complete blood counts and serum inflammatory cytokine expression levels were assessed. The results indicate that differential expression of CXCL10, TNF-α, CCL2, IL-8, and IL-6 were tightly linked to hemoglobin genotype and severity of Plasmodium infection and that these cytokine levels may be predictive for susceptibility to severe malaria or SCD severity.

Genotypic Variation in Trichomonas vaginalis Detected in South African Pregnant Women

Background. Trichomonas vaginalis is the causative agent of trichomoniasis. The genetic characterisation of T. vaginalis isolates reveals significant genetic diversity in this organism. Data on the prevalence of different genotypes of T. vaginalis in South African populations is lacking. This study investigated the diversity of T. vaginalis in a pregnant population in South Africa. Methods. In this study, 362 pregnant women from the King Edward VIII Hospital in Durban, South Africa, provided vaginal swabs to be tested for the presence of T. vaginalis. T. vaginalis was detected using the TaqMan assay using commercially available primers and probes specific for this protozoan (Pr04646256_s1). The actin gene from T. vaginalis was amplified with gene-specific primers. The actin amplicons were digested with HindII, MseI, and RsaI, and the banding patterns were compared across the three digests for assignment of genotypes. Phylogenetic analysis was conducted using MEGA. Results. The prevalence of T. vaginalis in the study population was 12.9% (47/362). Genotype G was the most frequent genotype in our study population. Genotypes H and I were detected in one sample each. According to the multiple sequence alignments and phylogenetic analysis, a level of diversity was observed across and within genotypes. Four different single-nucleotide changes in the actin gene were detected. Sample TV358 (H genotype) contained a single amino acid substitution from glutamine to lysine. Sample TV184 (G genotype) contained a single amino acid substitution from glutamic acid to arginine. Sample TV357 (G genotype) contained two amino acid substitutions, arginine to leucine and glycine to aspartic acid. Conclusion. Three different genotypes were observed in the pregnant population. Diversity was observed across and within genotypes. The observed diversity can be challenging for future vaccine design and development of antigen-based rapid diagnostic tests for trichomoniasis.