Deletion of the Candida albicans PIR32 Results in Increased Virulence, Stress Response, and Upregulation of Cell Wall Chitin Deposition

Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.     

Knocking down Israa,the Zmiz1 intron-nested gene,unveils interrelated T cell activation functions in mouse

We previously reported Israa (immune-system-released activating agent), a novel gene nested in intron 6 of the mouse Zmiz1 gene. Zmiz1 is involved in several functions such as fertility and T cell development and its knockout leads to non-viable embryos. We also reported ISRAA's expression in lymphoid organs, particularly in the thymus CD³⁺ T cells during all developmental stages. In addition, we showed that ISRAA is a binding partner of Fyn and Elf-1 and regulates the expression of T cell activation-related genes in vitro. In this paper, we report the generation and characterization of an Israa^?/? constitutive knockout mouse. The histological study shows that Israa^?/? mice exhibit thymus and spleen hyperplasia. Israa^?/? derived T cells showed increased proliferation compared to the wild-type mice T cells. Moreover, gene expression analysis revealed a set of differentially expressed genes in the knockout and wild-type animals during thymus development (mostly genes of T cell activation pathways). Immunological phenotyping of the thymocytes and splenocytes of Israa^?/- showed no difference with those of the wild-type. Moreover, we observed that knocking out the Zmiz1 intron embedded Israa gene does not affect mice fertility, thus does not disturb this Zmiz1 function. The characterization of the Israa^?/- mouse confirms the role ISRAA plays in the expression regulation of genes involved in T cell activation established in vitro. Taken together, our findings point toward a potential functional interrelation between the intron nested Israa gene and the Zmiz1 host gene in regulating T cell activation. This constitutively Israa^?/? mice can be a good model to study T cell activation and to investigate the relationship between host and intron-nested genes.     

The seroprevalence of SARS-CoV-2 IgG antibodies among asymptomatic blood donors in Saudi Arabia

BackgroundIn late 2019, cases of severe pneumonia with unidentified etiology began to emerge in Wuhan, China, before progressively spreading first nationally and then globally.The current study sought to investigate the seroprevalence of immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among blood donors in Al-Madinah, Saudi Arabia. To our knowledge, this is the first study in Saudi Arabia to screen blood donors who were not known to be previously infected with SARS-CoV-2.MethodsThis study was a cross-sectional study to assess individuals who donated blood to the central blood bank in Al-Madinah between mid-May and mid-July 2020. An enzyme-linked immunosorbent assay (ELISA) was designed and established to detect antibodies directed against the SARS-CoV-2 spike protein in serum samples. A total of 1,212 healthy blood donors participated in this study. The donors were males and met the requirements for blood donation during the COVID-19 pandemic period in Saudi Arabia.ResultsThe SARS-CoV-2 seroprevalence among blood donors in Al-Madinah was 19.31% (n = 234/1212; 95% confidence interval: 17.12%–21.64%). No statistically significant difference was identified in seropositivity according to age. However, significant differences (p < 0.001) were identified according to ABO blood groups, with those with type A blood presenting the highest rate of seropositivity (29.18%) compared with the other blood groups (12.65% for type B, 16.36% for type AB, and 15.11% for type O).ConclusionA high prevalence of SARS-CoV-2 antibodies was detected among blood donors in Al-Madinah, which indicated a high level of exposure to the virus within the population. This further suggested that as high as one-fifth of the population may have acquired innate immunity against the virus.     

Association of interleukin-6 polymorphisms with obesity and metabolic alterations in young Saudi population

Rising levels of obesity are a global problem that is being exported from affluent to developing nations through the gradual “westernization of lifestyle”. Population of Saudi Arabia is going through a nutrition transition where customary and traditional food is being replaced by fast food high in fat, sugar and salt. Interleukin-6 (IL-6) is a central player in the regulation of inflammation, haematopoiesis, immune response and host defense mechanisms. During the last decade, an accumulating amount of data suggested a pivotal role for IL-6 in metabolic processes, thus fortifying the picture of IL-6 as a multifaceted, pleiotropic cytokine. The Objective is to investigate the relationship between IL-6 (rs1554606) polymorphism and the risk of obesity in young Saudi population. Totally 204 Saudi young obese subjects were involved in this study. Genotyping of IL-6 was performed by the real-time polymerase chain reaction technology, using the Taq Man 5′-allele discrimination assay. IL-6 (rs1554606) AA versus AG (p < 0.01) and AA versus GG (p < 0.01) shows significant difference between Male and female group in genotypic as well as allelic distribution differ significantly, while AG versus GG did not differ significantly (p > 0.5). We have observed significant effects for Genotyping, LDL, CHOL, AST, ALP, BILIT, BMI at 5 % (0.05) significance level in the study population. Our results shown that IL-6 polymorphism have significantly differ in both male and females subjects. We have observed that some evidence of interactions of the IL-6 polymorphism and have shown statistical significant association with elevated BMI, Lipid profile and total bilurubin in the study subjects.     

Association between HLA Variations and Chronic Hepatitis B Virus Infection in Saudi Arabian Patients

Hepatitis B virus (HBV) infection is a leading cause of liver diseases including cirrhosis and hepatocellular carcinoma. Human leukocyte antigens (HLAs) play an important role in the regulation of immune response against infectious organisms, including HBV. Recently, several genome-wide association (GWAS) studies have shown that genetic variations in HLA genes influence disease progression in HBV infection. The aim of this study was to investigate the role of HLA genetic polymorphisms and their possible role in HBV infection in Saudi Arabian patients. Variations in HLA genes were screened in 1672 subjects who were divided according to their clinical status into six categories as follows; clearance group, inactive carriers, active carriers, cirrhosis, hepatocellular carcinoma (HCC) patients and uninfected healthy controls. Three single nucleotide polymorphisms (SNPs) belonged to HLA-DQ region (rs2856718, rs7453920 and rs9275572) and two SNPs belonged to HLA-DP (rs3077 and rs9277535) were studied. The SNPs were genotyped by PCR-based DNA sequencing (rs2856718) and allele specific TaqMan genotyping assays (rs3077, rs7453920, rs9277535 and rs9275572). The results showed that rs2856718, rs3077, rs9277535 and rs9275572 were associated with HBV infection (p = 0.0003, OR = 1.351, CI = 1.147–1.591; p = 0.041, OR = 1.20, CI = 1.007–1.43; p = 0.045, OR = 1.198, CI = 1.004–1.43 and p = 0.0018, OR = 0.776, CI = 0.662–0.910, respectively). However, allele frequency of rs2856718, rs7453920 and rs9275572 were found more in chronically infected patients when compared to clearance group infection (p = 0.0001, OR = 1.462, CI = 1.204–1.776; p = 0.0178, OR = 1.267, CI = 1.042–1.540 and p = 0.010, OR = 0.776, CI = 0.639–0.942, respectively). No association was found when polymorphisms in HLA genes were compared in active carriers versus cirrhosis/HCC patients. In conclusion, these results suggest that variations in HLA genes could affect susceptibility to and clearance of HBV infection in Saudi Arabian patients.     

The H50Q Mutation Enhances α-Synuclein Aggregation,Secretion, and Toxicity

Over the last two decades, the identification of missense mutations in the α-synuclein (α-Syn) gene SNCA in families with inherited Parkinson disease (PD) has reinforced the central role of α-Syn in PD pathogenesis. Recently, a new missense mutation (H50Q) in α-Syn was described in patients with a familial form of PD and dementia. Here we investigated the effects of this novel mutation on the biophysical properties of α-Syn and the consequences for its cellular function. We found that the H50Q mutation affected neither the structure of free or membrane-bound α-Syn monomer, its interaction with metals, nor its capacity to be phosphorylated in vitro. However, compared with the wild-type (WT) protein, the H50Q mutation accelerated α-Syn fibrillization in vitro. In cell-based models, H50Q mutation did not affect α-Syn subcellular localization or its ability to be phosphorylated by PLK2 and GRK6. Interestingly, H50Q increased α-Syn secretion from SHSY5Y cells into culture medium and induced more mitochondrial fragmentation in hippocampal neurons. Although the transient overexpression of WT or H50Q did not induce toxicity, both species induced significant cell death when added to the culture medium of hippocampal neurons. Strikingly, H50Q exhibited more toxicity, suggesting that the H50Q-related enhancement of α-Syn aggregation and secretion may play a role in the extracellular toxicity of this mutant. Together, our results provide novel insight into the mechanism by which this newly described PD-associated mutation may contribute to the pathogenesis of PD and related disorders.     

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.A unique lipid composition and lipid-protein interactions appear to exist at the transient membrane domain that defines the division site in bacterial cells (1). Using the cardiolipin (CL)⁴-specific fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found CL-enriched membrane domains located at cell poles and near potential division sites in Escherichia coli (2). Subsequently others reported similar CL domains in Bacillus subtilis (3) and Pseudomonas putida (4). In addition, cell pole and division site enrichment in CL in E. coli was confirmed by lipid analysis of minicells spontaneously budded off from the cell poles of a ΔminCDE mutant (5). We suggested that formation of CL domains at cell pole/division sites plays an important role in selection and recognition of the division site by amphitropic cell cycle and cell division proteins, such as DnaA (initiation of DNA replication at oriC), MinD (a part of MinCDE system preventing positioning of the divisome at cell poles in E. coli), and FtsA (bacterial actin, which is a linker protein for cytoskeletal protein FtsZ (bacterial tubulin), responsible for targeting the Z-ring to the mid-cell membrane domain). They interact directly with membrane phospholipids through specific amphipathic motifs enriched in basic amino acids, which confers the preference for anionic lipids (for references see Ref. 1). In E. coli the ATP-bound form of MinD recruits an inhibitor of Z-ring formation, MinC, to the membrane, whereas the topological regulator, MinE, induces hydrolysis of ATP bound to MinD resulting in release of MinD, and consequently MinC, from the membrane into the cytoplasm. As a result, all three proteins oscillate between the cell poles maintaining the maximum concentration of the inhibitor MinC at the cell poles and its minimum concentration at the cell center. Pole-to-pole oscillation of Min proteins occurs by dynamic redistribution of the proteins within a helical oligomeric structure that winds around the cell (for recent review and references see Ref. 6).Our previous study of a mutant lacking phosphatidylethanolamine (PE) and containing highly elevated levels of phosphatidylglycerol (PG) and CL demonstrated a strong inhibition of cell division and aggregation of MinD and FtsZ/FtsA proteins at domains enriched in CL (7, 8). To further investigate the role of lipids in the process of cell division, we chose an E. coli mutant with an opposite extreme in phospholipid composition to PE-lacking mutants, namely a ΔpgsA mutant (pgsA encodes phosphatidylglycerol phosphate synthase, which catalyzes the committed step to PG and CL synthesis (9)). This mutant is devoid of PG and CL (contribute ∼20 mole % of phospholipids in wild type) and contains higher levels of PE (∼90 mole % versus 80 mole % in wild type) (10, 11). Interestingly, the ΔpgsA null mutant accumulates elevated amounts of the phospholipid precursors, phosphatidic acid (PA) (∼4 mole %) and CDP-diacylglycerol (∼3 mole %), which are also anionic lipids that were proposed to fulfill the structural and functional roles of PG and CL (10, 11). These results suggest that a minimum of 5–10% anionic lipid is required to support viability. Another minor anionic phospholipid, N-acylphosphatidylethanolamine was suggested to be present in wild-type E. coli (12), which, if proven true, might be also elevated in this mutant. Finally, if anionic lipids are essential for cell division, then we would expect these normally minor lipids to segregate into similar anionic lipid domains, as does CL.In this report we identified N-acyl-PE in E. coli and, along with PA, its enrichment in polar/septal membrane domains of the ΔpgsA mutant UE54 (11) lacking PG and CL. Thus E. coli has a mechanism for preferential segregation of anionic phospholipids to the polar/septal regions where several amphitropic proteins, which show preference for interaction with anionic phospholipids in vitro, are functionally located.     

DNA sequence recognition by an imidazole-containing isopropyl-substituted thiazole polyamide (thiazotropsin B)

We have used DNA footprinting and fluorescence melting experiments to study the sequence specific binding of an imidazole-containing isopropyl-substituted thiazole polyamide (thiazotropsin B) to DNA. While the parent compound (thiazotropsin A) binds to the hexanucleotide sequence ACTAGT, changing one of the N-methylpyrrole groups to N-methylimidazole changes the preferred binding sequence to (A/T)CGCG(T/A). Experiments with DNA fragments that contain variants of this sequence suggest that the ligand can also bind, with lower affinity, to sequences which differ from this by 1bp in any position.     

Maternal and developmental toxicity of Bisphenol-A in SWR/J mice

Bisphenol-A (BPA), an organic compound with two phenol functional groups, is a widely used industrial plasticizer with known estrogenic properties. It is used in the manufacture of epoxy resins and polycarbonate plastics. This study was designed to evaluate and assess the possible toxicity arising from the oral administration of BPA to pregnant mice. Pregnant SWR/J mice (15 mice/group) were administrated oral doses of BPA (125, 250 and 500 mg/kg/day) over the course of five-day intervals during gestation (D1-5, D6-10 and D11-15), while control groups received only corn oil. The results indicated that BPA was associated with a reduction in the body weight of the pregnant mice from around 2–3 days after administration until the end of gestation. The greatest effects were evident when the BPA was given during the later stages of pregnancy, and with higher doses. They also showed marked reduction in food intake and, to a lesser extent, in water intake. Furthermore, doses of BPA induced a reduction in implantation sites, lower foetal body weight and increased mortality rates. Abortion and foetal resorption rates were not affected by BPA administration, however. The above findings were concluded by discussing the possible mechanisms involved in producing these effects.