Enzymatic and antibacterial activity of the recombinant endolysin PVP-SE1gp146 expressed in Hansenula polymorpha.

Antibiotic resistance, a major global concern, highlights the need for discovering alternative therapies. Recently, endolysins have garnered attention as antibacterial tools with a lower resistance development rate compared to conventional antibiotics, and their production in various expression hosts holds significance. Given its generally recognized as safe (GRAS) status and other advantages, Hansenula polymorpha offers a promising host for endolysin production. PVP-SE1gp146 originates from the Salmonella Enteritidis-specific phage PVP-SE1, which has been previously characterized. We inserted the PVP-SE1gp146 coding gene into the H. polymorpha expression vector pHIPX4. The resulting recombinant, pHIPX4-PVP-SE1gp146, was then introduced into H. polymorpha NCYC495 to facilitate the production of the endolysin PVP-SE1gp146. The expression level of the PVP-SE1gp146 protein was assessed, and it was determined to be approximately 43 mg/l of yeast culture medium. The enzymatic (muralytic) activity of this endolysin was also evaluated, corresponding to the version produced by the E. coli Bl21 strain. The endolysin exhibited admissible antibacterial activity against several gram-negative species, including P. aeruginosa, E. coli, and A. baumannii, while showing an almost negligible impact on K. pneumoniae. Endolysin production within GRAS-approved hosts holds potential for combating antibiotic-resistant bacteria. Challenges involve optimizing concentrations, targeting gram-negative species and improving attachment to bacterial cell walls. Addressing these issues requires dedicated research in endolysin engineering and a comprehensive evaluation of their production in diverse expression hosts.

Modified Phenol/Chloroform-Free DNA Isolation from Yeast and Other Fungi by Non-Enzymatic Salting Out Method.

The most commonly used fungal DNA extraction techniques require enzymatic digestion or hazardous chemicals. Here, we describe a quick non-enzymatic salt-out method that we developed for isolating high-quality DNA from various fungal species. DNA was extracted from four species of typical fungi, including Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, and Aspergillus flavus. Results that we obtained from agarose gel electrophoresis and spectrophotometry verified the integrity, high quality, and purity of the extracted DNAs. Also, PCR amplification and restriction enzyme digestion confirmed the efficiency of the extracted DNAs in molecular biology applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Yeast and fungal cultivation for DNA extraction Basic Protocol 2: Modified salting out DNA extraction protocol Support Protocol 1: PCR amplification of extracted genomes Support Protocol 2: Digestion with restriction enzyme.

Association between rs1049174 NKG2D gene polymorphism and idiopathic recurrent spontaneous abortion in Iranian women: a case-control study.

Natural killer group 2, member D (NKG2D) is one of the best known activating receptors of NK cells, which recognises its ligand on altered or stressed cells and activates NK cells to kill them. In this study, the single nucleotide polymorphism of the NKG2D gene for rs1049174 mutation was compared in 140 women with recurrent spontaneous abortion (RSA) and 175 control women with at least one successful pregnancy and without any known pregnancy loss. The findings just revealed that GG genotype and G allele were significantly higher in the case group compared with the control group (p < .001). Our results regarding decreased risk of RSA in C allele (OR = 0.438; 95%CI = 0.310-0.619; p < .001), and GC genotype (OR = 0.492; 95%CI = 0.214-0.574; p < .001) compared with G allele and GG genotype respectively. This study demonstrated a significant association between NKG2D gene polymorphism (rs1049174 G/C) and the risk of RSA in Iranian women.Impact statementWhat is already known on this subject? According to previous investigations, maternal immune responses may affect the foetus, causing recurrent spontaneous abortion (RSA). The main cause of RSA has not yet been detected in nearly 50% of the cases.What do the results of this study add? The results showed that the frequency of G allele and C allele were significantly different in the case group and control group.What are the implications of these findings for clinical practice and/or further research? The results suggest a protective function of C allele because it significantly decreased the risk of RSA compared to G allele. It improves inhibition of NK cells and probably participates in maintaining pregnancy in fertile controls; whereas, G allele is related to a slight inhibition of NK cells, probably leading to increase effectiveness of NK activation and undesirable inflammation, which consequently causes foetal rejection.

Evaluating cytotoxic effects of recombinant fragaceatoxin C pore forming toxin against AML cell lines.

OBJECTIVES: Current therapeutic strategies for cancer are associated with side effects and lack of specificity in treatments. Biological therapies including monoclonal antibodies and immune effectors have been the subject of multiple research projects. Pore-forming proteins may become the other biological strategy to overcome the problems associated with current treatments. But detailed mechanisms of their action on target membranes remained to be elucidated. We aimed to study the cytotoxic effects of recombinant form of fragaceatoxin C on AML cell lines HL-60 and KG-1. MATERIALS AND METHODS: We cloned the FraC gene in pET-28a (+) bacterial expression vector and the expressed recombinant FraC protein was purified by affinity chromatography. Then, cytotoxic effects of the recombinant protein were examined on two AML cell lines, HL-60 and KG-1. Effects of serum and calcium ion were explored by hemolysis assay in more details. RESULTS: Our results showed that the recombinant C-terminal polyhistidine-tagged FraC protein has potent cytotoxic effects on both AML cell lines, with IC50=5.6, and 4.6 µg.ml-1 for HL-60 and KG-1 cells, respectively. Serum showed dose-dependent and also time-dependent inhibitory effects on the hemolytic and cytotoxic activities of the FraC protein. Pre-incubation of the toxin with different concentrations of calcium ion also inhibited hemolytic activity of FraC toxin. CONCLUSION: Results of the present study showed that FraC has potential anti-tumor effects. By detailed investigation of the inhibition mechanism of serum and calcium effects in the future, it can be possible to design target sites for clinical applications of the toxin.

Interleukin-17 A and F gene polymorphisms affect the risk of tuberculosis: An updated meta-analysis.

BACKGROUND: Cytokines are fundamental elements in mediating and stimulating the immune response against tuberculosis (TB). Growing evidence indicated that polymorphisms in the interleukin-17 (IL-17) A and F genes are implicated in TB. OBJECTIVES: This meta-analysis was aimed to re-evaluate and update the relationship between IL-17A rs2275913 G/A and IL17F rs763780 T/C polymorphisms and TB risk. METHODS: Using inclusive searches of the PubMed, MEDLINE, EMBASE, Web of Science and Elsevier Science Direct, we identified outcome data from all articles estimating the association between IL-17 A and F polymorphisms and TB risk. RESULTS: A total of 15 studies comprising 7130 patients and 7540 controls were included. Our pooled analysis demonstrated that the IL-17A rs2275913 G/A SNP was not associated with the risk of TB in overall, or in Asians and Caucasians, but it conferred resistance to TB in Latin Americans using allele (OR=0.53), codominant (OR=0.53 and 0.38), dominant (OR=0.49) and recessive (OR=0.46) inheritance models. For IL-17F rs763780 T/C, the pooled evidence indicated that this variation was a risk factor for TB in allele (C vs T) and dominant (TC+CC vs TT) models in overall (OR of 1.35) and among Asians (OR=1.40), but not in Caucasians. CONCLUSION: In summary, our meta-analysis suggested that the IL-17A rs2275913 was a protective factor against TB, but -17F rs763780 T/C was a risk factor for TB.

Peginterferon Alfa-2a/Ribavirin treatment efficacy in chronic hepatitis C patients is related to natural killer group 2D gene rs1049174 GC polymorphism.

Natural killer group 2D (NKG2D), as an activating receptor, plays pivotal role in viral infectious diseases. Several single nucleotide polymorphisms (SNPs) in the NKG2D gene have characterized that the rs1049174G/C SNP of NKG2D is in the spotlight of notice because of its role in activating of human T cells. This study aimed to investigate rs1049174G/C genetic polymorphism in Chronic Hepatitis C (CHC) patients. The study compromised 107 CHC patients with genotype 1a and 1b. All recruited patients were under treatment with Peginterferon Alfa-2a/Ribavirin according to standard protocol. After completing treatment, 67 patients showed sustained virologic response (SVR) and the rest of patients did not respond to the treatment and considered as non-responder (NR). Genotyping of NKG2D rs1049174G/C SNP was performed using PCR-RFLP method in SVR and NR patients. The NKG2D rs1049174 genotypes frequency for GG, GC and CC were 45, 41 and 14 % respectively. Genotypes distribution were significantly different between SVR and NR groups (p = 0.005). So that the patients with the homozygous GG genotype demonstrated a higher response to Peginterferon Alfa-2a/Ribavirin therapy against HCV infection (OR = 6.0, 95 %CI 1.71-21.08, p = 0.005). In conclusion, the rs1049174 GG genotype of NKG2D receptor is an effective factor in successfully treatment of CHC patients by Peginterferon Alfa-2a/Ribavirin.

Relationship between γ-interferon gene polymorphisms and susceptibility to brucellosis infection.

Interferon-gamma (IFN-γ) is a pro-inflammatory cytokine that plays a pivotal role in the defense mechanism against Brucella infection. It was hypothesized that the IFN-γ in (+874 A/T in intron 1) TT and +5644 T/A, TT genotypes, which are reportedly associated with high IFN production, are associated with susceptibility to brucellosis in Iranian subjects. Genotyping of these IFN-γ variants by an allele-specific polymerase chain reaction method was performed in 281 subjects, comprising 153 patients with active brucellosis and 128 healthy controls. It was found that the +874 minor allele (A) and homozygote genotype (AA) were significantly more frequently present in brucellosis patients than in controls (OR = 2.588; 95% CI, 1.313-5.104; P = 0.006 for the AA genotype; OR = 1.575; 95% CI, 1.124-2.216; P = 0.010 for the A allele). However, the allelic and genotypic distribution of the IFN-γ polymorphism at position UTR5644 A>T did not differ significantly between patients and controls (P > 0.05). The distribution of haplotypes in this study suggests that the T/A haplotype (+874/UTR5644), which was present more frequently in controls than in patients, may protect subjects against Brucella infection. It is suggested that IFN-γ +874 AA genotype and A allele are risk factors for developing brucellosis infection in Iranian subjects.

The association of interleukin-18 promoter polymorphisms and serum levels with duodenal ulcer, and their correlations with bacterial CagA and VacA virulence factors.

BACKGROUND: We analyzed the impact of interleukin (IL)-18 promoter polymorphisms on IL-18 serum levels in Helicobacter pylori-infected duodenal ulcer (DU) patients and healthy asymptomatic (AS) carriers. We also aimed to determine the association of the H. pylori virulence factors CagA and VacA antibodies with serum concentrations of IL-18 in order to elucidate any correlation between them. METHODS: Three groups of patients were enrolled: DU patients (67 individuals), AS carriers (48 individuals), and H. pylori-negative subjects (26 individuals). Serum concentrations of IL-18 were determined by ELISA. Patient sera were tested by Western blot method to determine the presence of serum antibodies to bacterial CagA and VacA. Genotyping of IL-18 promoter polymorphisms at positions - 137G/C and - 607C/A were performed by allele-specific primer PCR protocol. RESULTS: Our study revealed that serum IL-18 levels are positively influenced by CagA-positive H. pylori strains, so that maximum levels of IL-18 were detected in DU patients with the CagA(+) phenotype, regardless of the presence of the anti-VacA antibody. Regarding IL-18 promoter polymorphisms, the AA genotype and A allele at position - 607C/A were found to be significantly lower in DU patients than in AS carriers and H. pylori-negative subjects (p = 0.032 and 0.043, respectively). CONCLUSIONS: The IL-18 - 607C variant was associated with higher levels of serum IL-18 and an increased risk of DU. Moreover, our findings indicated that serum concentrations of IL-18 were influenced by CagA factor, irrespective of the VacA status, suggesting that high levels of IL-18 in CagA-positive subjects predisposes to susceptibility to DU.

Circulating levels of interleukin (IL)-12 and IL-13 in Helicobacter pylori-infected patients, and their associations with bacterial CagA and VacA virulence factors.

OBJECTIVE: The aim of this study was to determine the association of the Helicobacter pylori virulence factors, cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) antibodies, with serum levels of interleukin (IL)-12 and IL-13 in H. pylori-infected duodenal ulcer (DU) patients and H. pylori-infected asymptomatic (AS) carriers in order to elucidate any correlation between them. METHODS: A total of 67 DU patients, 48 AS individuals, and 26 healthy H. pylori-negative subjects were enrolled in this study. Serum concentrations of IL-12 and IL-13 were determined by enzyme-linked immunosorbent assay (ELISA) method. Patient sera were tested by Western blot method to determine the presence of serum antibodies to bacterial virulence antigens p120 (CagA) and p95 (VacA). Serum concentrations of IL-12 and IL-13 were compared in 9 groups, including 4 AS phenotypes (CagA⁺VacA⁺, CagA⁺VacA⁻, CagA⁻VacA⁺, CagA⁻VacA⁻), 4 DU phenotypes (CagA⁺VacA⁺, CagA⁺VacA⁻, CagA⁻VacA⁺, CagA⁻VacA⁻), and 1 control group. RESULTS: The results revealed that DU patients positive for CagA, independent of the anti-VacA antibody status, showed drastically elevated levels of IL-12 (251 ± 43 pg/ml) when compared with the other groups (p = 0.0001). No significant difference was found between groups regarding levels of IL-13 (p > 0.05). CONCLUSIONS: Our findings indicate that in the DU group, the serum concentrations of IL-12 but not of IL-13 were influenced by bacterial CagA, independent of the VacA status, suggesting that high IL-12 levels may contribute to susceptibility to DU in CagA-positive individuals. These findings could possibly be considered to improve the predictive or prognostic values of inflammatory cytokines for DU, and also to design possible novel therapeutic approaches.