Exploring the mechanisms by which camel lactoferrin can kill Salmonella enterica serovar typhimurium and Shigella sonnei.

There is a continuously increasing pressure associated with the appearance of Salmonella enterica Serovar typhimurium (S. typhimurium) and Shigella sonnei (S. sonnei) that have developed pathogenic multiple antibiotic resistance and the cost of cure and control of these enterobacteriaceae infections increases annually. The current report for first time demonstrated the distinguished antimicrobial action of camel lactoferrin (cLf) obtained from the milk of different clans of camel in Saudi Arabia against S. typhimurium and S. sonnei. These cLf subtypes showed comparable antimicrobial potential when tested against the two bacterial strains but were superior to either bovine (bLf) or human lactoferrin (hLf). The synergism between lactoferrins and antibiotics concerning their antibacterial efficacies against the two bacterial strains was evident. Exploring mechanisms by which camel lactoferrin can kill S. typhimurium and S. sonnei revealed that cLf affects bacterial protein profile. Besides, it interacts with bacterial lipopolysaccharides (LPS) and numerous membrane proteins of S. typhimurium and S. sonnei, with each bacterial strain possessing distinctive binding membrane proteins for lactoferrin. Furthermore, as evidenced by electron microscopy analysis, cLf induces extracellular and intracellular morphological changes in the test bacterial strains when used alone or in combination treatment with antibiotics. Lactoferrin and antibiotics combination strongly disrupts the integrity of the bacterial cells and their membranes. Therefore, cLf can kill S. typhimurium and S. sonnei by four different mechanisms, such as iron chelation, affecting some bacterial proteins, binding to bacterial LPS and membrane proteins, and impairing the integrity of the bacterial cells and their membranes.

Natural resources to control COVID-19: could lactoferrin amend SARS-CoV-2 infectivity?

The world population is still facing the second wave of the COVID-19 pandemic. Such a challenge requires complicated tools to control, namely vaccines, effective cures, and complementary agents. Here we present one candidate for the role of an effective cure and/or complementary agent: lactoferrin. It is the cross-talking mediator between many organs/cellular systems in the body. It serves as a physiological, immunological, and anti-microbial barrier, and acts as a regulator molecule. Furthermore, lactoferrin has receptors on most tissues cells, and is a rich source for bioactive peptides, particularly in the digestive system. In the past months, in vitro and in vivo evidence has accumulated regarding lactoferrin's ability to control SARS-CoV-2 infectivity in different indicated scenarios. Also, lactoferrin or whey milk (of human or other mammal's origin) is a cheap, easily available, and safe agent, the use of which can produce promising results. Pharmaceutical and/or food supplementary formulas of lactoferrin could be particularly effective in controlling the gastrointestinal COVID-19-associated symptoms and could limit the fecal-oral viral infection transmission, through mechanisms that mimic that of norovirus infection control by lactoferrin via induction of intestinal innate immunity. This natural avenue may be effective not only in symptomatic patients, but could also be more helpful in asymptomatic patients as a main or adjuvant treatment.

Comparative Analysis of Milk Fat Globular Membrane (MFGM) Proteome between Saudi Arabia Camelus dromedary Safra and Wadha Breeds.

Camel milk is traditionally known to have medicinal properties and many potential health benefits. Natural milk contains many soluble proteins and nanoparticles, such as a milk fat globule membrane (MFGM), a three-layered membrane covering of milk fat globule mainly composed of proteins and lipids, which plays an important role in human health. MFGM proteins account for 1%-4% of total milk proteins, and their nutritive value and distribution depends on the different breeds. The differential composition of these membrane proteins among different camel breeds has not been explored. The current study, therefore, aimed to quantitatively analyze and compare the MFGM proteome between the milk produced by the two most common Saudi camel breeds, Camelus dromedarius: Safra and Wadha. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analysis revealed a total of 44 MFGM proteins that were identified with a significant difference in abundance (p ≤ 0.05; fold change ≥ 1.5) between the two breeds. Thirty-one proteins were up-regulated and 13 proteins were down-regulated in the Safra breed compared to the Wadha breed. The proteins identified with an increased abundance included α-lactalbumin, lactadherin, and annexin a8, whereas the down-regulated proteins included butyrophilin subfamily 1 member a1, lactotransferrin, and vinculin. The differentially abundant proteins were analyzed by the UNIPROT system and gene ontology (GO) to reveal their associations with known biological functions and pathways. Enzyme-linked immunosorbent assay (ELISA) confirmed the 2D-DIGE findings of butyrophilin (BTN) and α-lactalbumin (α-LA) levels obtained from Safra and Wadha breeds.

Disorder in milk proteins: adipophilin and TIP47, important constituents of the milk fat globule membrane.

Milk fat globules (MFGs), which are secreted by the epithelial cells of the lactating mammary glands, account for the most of the nutritional value of milk. They are enveloped by the milk fat globule membrane (MFGM), a complex structure consisting of three phospholipid membrane monolayers and containing various lipids. Depending on the origin of milk, specific proteins accounts for 5-70% of the MFGM mass. Proteome of MFGMs includes hundreds of proteins, with nine major components being adipophilin, butyrophilin, cluster of differentiation 36, fatty acid binding protein, lactadherin, mucin 1, mucin 15, tail-interacting protein 47 (TIP47), and xanthine oxidoreductase. Two of the MFGM components, adipophilin and TIP47, belong to the five-member perilipin family of lipid droplet proteins. Adipophilin is involved in the formation of cytoplasmic lipid droplets and secretion of MFGs. This protein is also related to the formation of other lipid droplets that exist in most cell types, playing an important role in the transport of lipids from ER to the surface of lipid droplets. TIP47 acts as a cytoplasmic sorting factor for mannose 6-phosphate receptors and is recruited to the MFGM. Therefore, both adipophilin and TIP47 are moonlighting proteins, each possessing several unrelated functions. This review focuses on the main functions and specific structural features of adipophilin and TIP47, analyzes similarities and differences of these proteins among different species, and describes these proteins in the context of other members of the perilipin family.Communicated by Ramaswamy H. Sarma.

Bacteriostatic and Bactericidal Activities of Camel Lactoferrins Against Salmonella enterica Serovar Typhi.

Lactoferrin is an iron-binding glycoprotein present in various secretions (e.g., milk, tears, saliva, pancreatic juice), which performs multiple functions, with one of them being the antimicrobial defense. Purified camel lactoferrins (cLfs) from different Saudi camel clans, as well as human and bovine lactoferrins (hLf or bLf) were tested as antimicrobial agents against Salmonella enterica serovar Typhi (S. Typhi). All cLfs showed superior antibacterial potentials relative to hLf or bLf, while there was no noticeable difference in the antimicrobial capabilities between the cLfs from different camel clans. We observed synergy between the inhibitory activities of Lfs and antibiotics against bacterial growth. Expression of numerous bacterial proteins was affected by the treatment with Lf and its combinations, giving insight into the molecular mechanisms of the Lf action. Furthermore, several bacterial proteins were shown to interact with cLf-biotin. Scanning and transmission electron microscopy revealed the presence of obvious extracellular and intracellular changes after S. Typhi treatment by antibiotic (carbenicillin) or cLf alone, and in combination. The effects of antibiotics and Lf were synergistic, supporting the potential of the use of Lf-antibiotic combinations.

Publisher Correction: Genomic analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Synergistic Killing of Pathogenic Escherichia coli Using Camel Lactoferrin from Different Saudi Camel Clans and Various Antibiotics.

Current study aimed to analyze the synergistic killing of pathogenic Escherichia coli using camel lactoferrin from different Saudi camel clans and various antibiotics. Methods: using multiple microbiological and protein analysis techniques, the results were shown that the purified camel lactoferrins (cLfs) from different Saudi camel have strong antimicrobial potentials against two strains of E. coli. Although all cLfs were superior relative to human or bovine lactoferrins (hLf or bLf), there was no noticeable difference in the antimicrobial potentials of cLfs from different camel clans. The effects of antibiotics and cLfs were synergistic, indicating the superiority of using cLf-antibiotic combinations against E. coli growth. Since these combinations possessed distinguished synergy profiles, it is likely that they can be used to enhance the low efficacy of antibiotics, as well as to control the problems associated with bacterial resistance. Furthermore, these combinations can reduce the cost of cure of bacterial infections, especially in the developing countries. The analysis of the molecular mechanisms of lactoferrin action revealed that expression of several E. coli proteins was affected by the treatment with these antibacterial factors. Several proteins of different molecular weights interacting with cLf-biotin were found. Scanning and transmission electron microscopy analysis revealed the presence of noticeable morphological changes associated with the treatment of E. coli strains by antibiotic carbenicillin or cLf alone, and in combination. Camel lactoferrin has superior potential killing of E. coli over bovine and human lactoferrin, and this potential can be further synergistically enhanced of cLF is combined with antibiotics.

Genomic analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East.

Tuberculosis (TB) represents a significant challenge to public health authorities, especially with the emergence of drug-resistant (DR) and multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis. We sought to examine the genomic variations among recently isolated strains of M. tuberculosis in two closely related countries with different population demography in the Middle East. Clinical isolates of M. tuberculosis from both Egypt and Saudi Arabia were subjected to phenotypic and genotypic analysis on gene and genome-wide levels. Isolates with MDR phenotypes were highly prevalent in Egypt (up to 35%) despite its relatively stable population structure (sympatric pattern). MDR-TB isolates were not identified in the isolates from Saudi Arabia despite its active guest worker program (allopatric pattern). However, tuberculosis isolates from Saudi Arabia, where lineage 4 was more prevalent (>65%), showed more diversity than isolates from Egypt, where lineage 3 was the most prevalent (>75%). Phylogenetic and molecular dating analyses indicated that lineages from Egypt were recently diverged (~78 years), whereas those from Saudi Arabia were diverged by over 200 years. Interestingly, DR isolates did not appear to cluster together or spread more widely than drug-sensitive isolates, suggesting poor treatment as the main cause for emergence of drug resistance rather than more virulence or more capacity to persist.

Immunogenicity comparison of lactoferrin purified from Saudi Arabia camel clans milk.

Secretory lactoferrins play a crucial rolls at mucosal surfaces as not only antimicrobial molecules in primate as well as human, but as physiological protein. Its multiple functions extended to be one of immunogen could elicited autoimmune disorders. Purified camel lactoferrin (cLfs) from different Saudi camel clans were shown to be a potent immunogen when injected into rabbit. Four rabbit were subcutaneously immunized with different camel clans lactoferrin/Freunds adjuvant. Anti-cLfs potency titration was reach 1:32000 and did not significantly differences between different cLfs. The cross-reactivity level of different anti-Lfs were highly significant, specially between cLfs and bLf/hLf.

Correction to: Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels.

The original version of this article contained mistakes in author names and affiliations. The last names of the authors Salah Korim, Amro Samra, and Hussein A. Amhedar were misspelled. The corrected spelling is Saleh A. Alkarim, Amr A. El-Hanafy, and Hussein A. Almehdar. The correct list of author names and affiliations are published with this erratum.

Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels.

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR-RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype.

Disorder in Milk Proteins: Lactadherin Multifunctionality and Structure.

Milk fat globule membrane (MFGM) is one of the milk components that is produced by the lactating mammary glands and released to the milk in the form of vesicles. MFGM surrounds milk fat globule secreted by the milk producing cells and has a complex structure containing various lipids (e.g., triacylglycerides, phospholipids, and cholesterol), proteins and other macromolecules. Among the proteinaceous components of MFGM is lactadherin, also known as milk fat globule-EGF factor 8 protein (MFG-E8). Being one of the main proteins present in MFGM, lactadherin is related to milk secretion, has antimicrobial and antiviral effects, and plays important roles in the immune defense as one of the immune system molecules. Furthermore, lactadherin belongs to the family of secreted extracellular matrix proteins, and clearly can be considered as a multifunctional (or moonlighting) glycoprotein involved in regulation of many biological and physiological processes, such as angiogenesis, atherosclerosis, haemostasis, phagocytosis, and tissue remodeling. This review focuses on the similarities and differences of lactadherin among different species and describes the main functions of this protein, as well as its structure.

Prediction of Disordered Regions and Their Roles in the Anti-Pathogenic and Immunomodulatory Functions of Butyrophilins.

Butyrophilins (BTNs) are a group of the moonlighting proteins, some members of which are secreted in milk. They constitute a large family of structurally similar type 1 transmembrane proteins from the immunoglobulin superfamily. Although the founding member of this family is related to lactation, participating in the secretion, formation and stabilization of milk fat globules, it may also have a cell surface receptor function. Generally, the BTN family members are known to modulate co-stimulatory responses, T cell selection, differentiation, and cell fate determination. Polymorphism of these genes was shown to be associated with the pathology of several human diseases. Despite their biological significance, structural information on human butyrophilins is rather limited. Based on their remarkable multifunctionality, butyrophilins seem to belong to the category of moonlighting proteins, which are known to contain intrinsically disordered protein regions (IDPRs). However, the disorder status of human BTNs was not systematically investigated as of yet. The goal of this study is to fill this gap and to evaluate peculiarities of intrinsic disorder predisposition of the members of human BTN family, and to find if they have IDPRs that can be attributed to the multifunctionality of these important proteins.

Divergent Anticancer Activity of Free and Formulated Camel Milk α-Lactalbumin.

Alpha-lactalbumin (α-LA), a small milk calcium-binding globular protein, is known to possess noticeable anticancer activity, which is determined by the ability of this protein to form complexes with oleic acid (OA). To date, in addition to human and bovine α-LA, the ability to form such anti-tumor complexes with OA was described for goat and camel α-LA. Although the mechanisms of the anticancer activity of human and bovine α-LA are already well-studied, little is currently known about the anticancer action of this camel protein. The goal of this study was to fill this gap and to analyze the anticancer and pro-apoptotic activities of camel α-LA in its free form (α-cLA) and as an OA-containing complex (OA-α-cLA) using four human cancer cell lines, including Caco-2 colon cancer cells, PC-3 prostate cancer cells, HepG-2 hepatoma cells, and MCF-7 breast cancer cells as targets. The anti-tumor activities of OA-α-cLA and α-cLA were analyzed using MTT test, annexin/PI staining, cell cycle analysis, nuclear staining, and tyrosine kinase (TK) inhibition methods. We show here that the OA-α-cLA complex does not affect normal cells but has noticeable anti-cancer activity, especially against MCF-7 cells, thus boosting the anticancer activity of α-cLA and improving the selectivity of OA. The OA-α-cLA complex mediated cancer cell death via selective induction of apoptosis and cell-cycle arrest at lower IC50 than that of free α-cLA by more than two folds. However, OA induced apoptosis at higher extent than OA-α-cLA and α-cLA. OA also caused unselective apoptosis-dependent cell death in both normal and cancer cells to a similar degree. The apoptosis and cell-cycle arresting effect of OA-α-cLA may be attributed to the TK inhibition activity of OA. Therefore, OA-α-cLA serves as efficient anticancer complex with two functional components, α-cLA and OA, possessing different activities. This study declared the effectiveness of OA-α-cLA complex as a promising entity with anticancer activity, and these formulated OA-camel protein complexes constitute an auspicious approach for cancer remedy, particularly for breast cancer.

Reduction in CD16/CD56 and CD16/CD3/CD56 Natural Killer Cells in Coronary Artery Disease.

BACKGROUND: Natural killer (NK) cells are the potential modulators of inflammatory reactions that exert several unique biological effects and could lead to future adverse events of coronary artery disease (CAD). HYPOTHESIS: The purpose of this study was to find out the possible association of modulation in NK cell, TNK cells, T cells, B cells, and tumor necrosis factor alpha (TNF-α) in CAD patients and various forms of myocardial infarction. METHODS: The present study included total 190 subjects (98 confirmed CAD patients both men and women and 92 healthy control individuals). Serum concentration of TNF-α was measured by ELISA method. For the measurement of various immune cells, viz., NK cell, TNK cells, T cells, and B cells, flow-cytometric analysis was performed. RESULTS: A significant reduction by 15% (P < 0.001) in CD16/CD56 NK cells was observed in CAD patients. Moreover, non-ST segment elevation myocardial infarction (NSTEMI), ST segment elevation myocardial infarction (STEMI), unstable angina (UA), and combined UA + NSTEMI group also showed a significant decline in NK cells compared with control individuals. CD16/CD56/CD3 TNK cells showed a significant reduction in CAD, NSTEMI, STEMI, and UA categories. However, UA + NSTEMI group did not show any significant change in TNK cells. On the other hand, the level of TNF-α was found to be significantly elevated in CAD, STEMI, and UA groups. NSTEMI and combined UA + NSTEMI group did not show any significant change in TNF-α level. CONCLUSION: Current study provides an insight toward the association of immune cells and inflammation with CAD.

Assessment of IL-18 Serum Level and Its Promoter Polymorphisms in the Saudi Coronary Artery Disease (CAD) Patients.

The purpose of the current study was to find out the possible changes polymorphic site at the promoter region of IL-18 gene in Saudi CAD patients. We have also measured serum IL-18 level to find out, the likely association between its level and polymorphic site. The present study included total 197 subjects (98 confirmed CAD patients both men and women and 99 healthy control individuals). Serum concentration of IL-18 was measured by enzyme linked immuno-sorbent assay. For SNPs analysis, sanger method of DNA sequencing was followed. We observed variable numbers of SNPs at -137 C/G, -607 A/C, and -656 T/G promoter sites in our studied samples. However, the observed changes in the number of SNP hotspots were found to be non-significant compared with control. IL-18 level was found to be significantly (P < 0.001) elevated in CAD patients compared with control individuals. The highest rise of around 36% (P < 0.001) in IL-18 level was recorded in unstable angina (UA) patients. Moreover, the group belonging to UA and non-ST segment elevation myocardial infarction (NSTEMI) showed only 6% rise. On the basis of our result, inflammation seems to have a role in the pathogenesis of CAD but not leading to the significant changes at the genetic level. J. Cell. Biochem. 118: 1849-1854, 2017. © 2017 Wiley Periodicals, Inc.

In Vitro Exploration of the Anti-HCV Potential of the Synthetic Spacer Peptides Derived from Human, Bovine, and Camel Lactoferrins.

BACKGROUND: Chronic liver disease is often associated with the infection by hepatitis C virus (HCV), which is an enveloped RNA virus belonging to the Flaviviridae family. Many studies found that milk proteins, such as lactoferrin, might have profound antiviral activity against HCV. Various secretory fluids ranging from milk, to tears, saliva, and nasal secretion, and to bile and pancreatic juice, as well as neutrophils, mucosal surfaces, and blood contain a widely spread multifunctional glycoprotein, lactoferrin (Lf), structure of which can be depicted as two homologous domains connected by the short spacer peptide. OBJECTIVE: This study aimed to understand the effectiveness of the synthetic peptides cLfsp, bLfsp, hLfsp1, and hLfsp2 corresponding to the spacer peptides of camel, bovine, and human Lfs, respectively, against HCV in in vitro settings. METHOD: We used RT-nested PCR to evaluate the antiviral activity of the synthesized spacer peptides against HCV infectivity in PBMC and HepG2 cells looking at their neutralization, protection, and intracellular treatment potentials. RESULTS AND CONCLUSION: We show that direct interaction of hLfsp1, hLfsp2, and bLfsp with viral particles is able to neutralize the HCV entry into HepG2 cells (with hLfsp2 being more potent neutralizer than hLfsp1 and bLfsp), whereas cLfsp does not show any neutralizing potential. Therefore, our analysis revealed that different spacer peptides are characterized by different antiviral potentials and use different mechanisms for antiviral protection.

Assessment of genetic diversity in IL-6 and RANTES promoters and their level in Saudi coronary artery disease patients.

BACKGROUND: The present study consisted of a total of 200 subjects (100 confirmed coronary artery disease (CAD) patients), both men and women, and 100 healthy control individuals. METHODS: Serum concentration of IL-6 and RANTES were measured by enzyme-linked immunosorbent assay kit. For SNPs analysis, sanger method of DNA sequencing was followed. RESULTS: We observed variable numbers of SNP sites at -174 G/C, -572 G/C, and -597 G/A in IL-6 and -28 C/G and -109 C/T in RANTES promoters in CAD patients compared with control individuals. However, the observed changes in the number of SNPs were found to be non-significant compared with control individuals. The IL-6 level was found to be significantly (P<.001) elevated in CAD patients compared with control. Moreover, RANTES serum level did not show any significant change in CAD patients. CONCLUSION: Based on our result, it is quite clear that inflammation has a role in the pathogenesis of CAD but does not lead to significant changes at the genetic level in our population. As far as our knowledge goes, this is the first report that shows the genetic diversity in IL-6 and RANTES promoters and their respective levels in Saudi CAD patients.

Elevated Concentration of Defensins in Hepatitis C Virus-Infected Patients.

Hepatitis C virus (HCV) is the major etiological agent of human non-A and non-B hepatitis, affecting around 180 million people worldwide. Defensins, small cysteine-rich cationic peptides, are shown to have potent antibacterial, antiviral, and antifungal properties. Defensins can be found in both normal and microbial infected patients, at variable concentrations. Notably, viral infections are often associated with elevated concentrations of defensins. The current study aimed to estimate the concentrations of total, α-, and ß-defensins in serum taken from normal and HCV-infected patients. 12 healthy (noninfected) and 34 HCV-infected patients were enrolled. Standardized immunoassay kits were used to obtain serum concentrations of defensins. The obtained results were calibrated against kit standard reagents. Total defensin concentrations in HCV-infected patients were significantly higher (2- to 105-fold) compared to healthy individuals. The concentrations of α-defensins were also significantly elevated in the HCV-infected patients (31-1398 ng/50 µL). However, concentrations of ß-defensins ranged from 44.5 ng/50 µL to 1056 ng/50 µL. The results did not reveal differences in serum defensin concentration between male and female HCV-infected patients. A-defensin concentration of ≥250 ng/50 µL was found to contain more ß-defensins than total defensins and α-defensins. This study concludes, for the first time, that serum defensin levels are elevated in HCV-infected patients.