Lactoferrin: Role in iron homeostasis and host defense against microbial infection

The transferrin family of non-heme iron binding glycoproteins are believed to play a central role in iron metabolism and have been implicated in iron transport, cellular iron delivery and control of the level of free iron in external secretions. Lactoferrin (LF) is a member of this family that is widely localized in external fluids including milk and mucosal secretions, in addition to being a prominent component of the secondary granules of neutrophils. Although structurally related to transferrin, LF appears to have a broader functional role mediated by both iron dependent and iron independent mechanisms. In this review, we will focus on our current understanding on the role of LF in regulating iron homeostasis and its role in host protection against microbial infection at the mucosal surface. In addition, recent insights obtained from analyzing the phenotypic consequences of LF ablation in lactoferrin knockout mice (LFKO), which challenge the long held dogma that LF is required for intestinal iron absorption in the neonate, are summarized.     

重组猪乳铁蛋白N端的高效表达及抑菌活性检测

为获得表达猪乳铁蛋白基因的重组菌株,并检测其表达的重组猪乳铁蛋白抑菌活性,应用RT-PCR方法从泌乳3d后母猪乳腺组织中扩增了猪乳铁蛋白N端1077bp的PLF-N基因片段,与GenBank上发表的4株猪乳铁蛋白基因序列相比,核苷酸同源性均达到99%以上。为了得到高表达量的PLF-N基因,以扩增的PLF-N片段为参考模板,经过密码子优化,全基因合成了编码猪乳铁蛋白N端的基因PLF-NS。将其定向插入到原核表达载体pET-30b中,转化大肠杆菌BL21,获得了表达PLF-NS的重组菌pET-PLF-NS/BL21;经IPTG诱导,并对表达条件进行优化,以及通过SDS-PAGE和Western blotting分析均表明猪乳铁蛋白得到了正确表达,其产物分子量约为42kDa,最优表达条件下蛋白表达量占菌体总蛋白的32%,表达产物以包涵体形式存在。包涵体经裂解、纯化、复性处理后纯度达到98%。用琼脂孔穴扩散抑菌法检测表明重组猪乳铁蛋白具有明显的抑菌作用。表明通过基因优化对表达量低的基因进行改造使之高效表达,是一种提高表达效率的有效手段。     

九香虫抗菌肽CcAMP1的分离纯化和抗菌活性检测

【目的】从药用昆虫九香虫 Coridius chinensis 中分离纯化抗菌肽,为进一步开发九香虫抗菌肽资源及深入挖掘九香虫的药用功能奠定基础。【方法】用大肠杆菌Escherichia coli 和金黄色葡萄球菌 Staphylococcus aureus 混合物作诱导源刺激九香虫产生抗菌肽,对血淋巴进行提取、凝胶过滤层析、固相萃取及反相色谱纯化,活性组分经质谱测定。对分离得到的这种抗菌肽进行人工合成,并进行抗菌活性检测。【结果】本研究获得一种九香虫抗菌肽CcAMP1,由17个氨基酸残基组成,分子量为1 997.37 u,带1个正电荷,表面有5个疏水氨基酸。对人工合成的CcAMP1进行抗菌活性检测表明,该抗菌肽与九香虫血淋巴一样对金黄色葡萄球菌等革兰氏阳性菌和大肠杆菌等革兰氏阴性菌都有较好的抗菌活性,且对革兰氏阴性菌的抗菌活性更强。【结论】从九香虫中分离得到具有较强抗菌活性的阳离子抗菌肽CcAMP1,有较大的开发利用价值。     

Comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin

AIMS: To compare amidation and acylation of lactoferrin (LF) from bovine milk, as a means of enhancing its antimicrobial and antiviral properties. METHODS AND RESULTS: LF was chemically modified by amidation with a 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) in the presence of ammonium ions or by acylation with either succinic or acetic anhydride. In the test systems used, amidation substantially enhanced the activity of LF against Pseudomonas fluorescens in comparison with native LF. However, increasing the net negative charge of LF by acylation had no effect on the activity of LF against P. fluorescens, and abrogated the antimicrobial activity of LF against Bacillus subtilis and Saccharomyces cerevisiae. Increasing the net negative charges of LF by acylation eliminated its antimicrobial and antiviral effects against poliovirus and feline calicivirus (nonenveloped viruses). CONCLUSIONS: The addition of positive charges to LF via amidation enhanced antimicrobial properties in contrast to increasing the negative charges by acylation, which abolished both the antimicrobial and antiviral properties of LF. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of charge alteration of LF determined in this study provides a basis for further development of LF formulations with enhanced antimicrobial effectiveness for use in food process hygiene, veterinary and health-care applications.     

银杏内生菌的分离及其抑菌活性筛选

从健康的银杏（Ginkgo biloba）茎和叶片中分离内生菌,结果从银杏叶和茎上共分离到内生真菌20株,其中9株来自银杏茎部,11株来自银杏叶部;内生放线菌23株,其中15株来自于银杏茎部,8株来自于银杏叶部;内生细菌15株,其中8株来自于银杏茎部,7株来自于银杏叶部。以金黄色葡萄球菌（Staphylococcus aureus）、枯草芽胞杆菌（Bacillus subtilis）、大肠埃希菌（Escherichia coli）、黑曲霉（Aspergillus niger）、酿酒酵母（Saccharomyces cerevisiae）作为指示菌,采用双层平板法对内生菌进行抑菌活性筛选,结果表明：20株内生真菌中有12株出现了抑菌活性,有抑菌活性菌株的比例为60.0%;23株内生放线菌中,仅3株出现了抑菌活性,有抑菌活性菌株的比例为13.0%;15株内生细菌中,有4株出现了抑菌活性,有抑菌活性菌株的比例为26.7%。     

Lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins that is abundantly expressed and secreted from glandular epithelial cells. In secretions, such as milk and fluids of the intestinal tract, lactoferrin is an important component of the first line of host defence. During the inflammatory process, lactoferrin, a prominent component of the secondary granules of neutrophils (PMNs), is released in infected tissues and in blood and then it is rapidly cleared by the liver. In addition to the antimicrobial properties of lactoferrin, a set of studies has focused on its ability to modulate the inflammatory process and the overall immune response. Though many in vitro and in vivo studies report clear regulation of the immune response and protective effect against infection and septic shock by lactoferrin, elucidation of all the cellular and molecular mechanisms of action is far from being achieved. At the cellular level, lactoferrin modulates the migration, maturation and function of immune cells. At the molecular level and in addition to iron binding, interactions of lactoferrin with a plethora of compounds, either soluble or membrane molecules, account for its modulatory properties. This paper reviews our current understanding of the cellular and molecular mechanisms that explain the regulatory properties of lactoferrin in host defence.     

乳铁蛋白与骨质疏松症

本文主要阐述了乳铁蛋白对造骨细胞、破骨细胞及半胱氨酸蛋白酶的作用,由于其对造骨细胞有促进作用及对破骨细胞和半胱氨酸蛋白酶（如K、L）的抑制作用,因此乳铁蛋白是治疗骨质疏松症的一种潜在的治疗手段。     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

Factors contributing to the potency of antimicrobial cationic peptides from the N-terminal region of human lactoferrin

This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.     

Mycoplasma pneumoniae infection induces a neutrophil-derived antimicrobial peptide, cathelin-related antimicrobial peptide

In innate immunity, cationic antimicrobial peptides including cathelin-related antimicrobial peptide (CRAMP) are known to play critical roles in protecting the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between CRAMP and mycoplasmas. In the present study, the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in bronchoalveolar lavage fluid (BALF) of M. pneumoniae-infected mice was examined. CRAMP at 10-20 μg/mL reduced the growth of two strains of M. pneumoniae by 100 to 1000-fold. The amount of CRAMP in the BALF of M. pneumoniae-infected mice was 20～25 ng/mL by ELISA. The presence of mature CRAMP in BALF was observed by Western blotting. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm by immunofluorescence. Furthermore, the addition of M. pneumoniae resulted in the release of a large amount of CRAMP from neutrophils induced by thioglycolate. These results suggest that CRAMP from neutrophils may play an important role in protection against M. pneumoniae infection.     

人乳铁蛋白基因在烟草中的表达及抗细菌与病毒的能力

从人血液中性白细胞中分离细胞总ＲＮＡ,用ｏｌｉｇｏ（ｄＴ） 为引物进行ｃＤＮＡ 第１ 条链合成,再用乳铁蛋白ｃＤＮＡ 的特异性引物进行聚合酶链反应（ＰＣＲ） ,分两段合成并拼成完整的乳铁蛋白ｃＤＮＡ。核苷酸序列测定证明,该ｃＤＮＡ 所编码的氨基酸与前人论证的人乳铁蛋白的氨基酸序列完全一致。将该ｃＤＮＡ 构建成植物表达载体ｐ３５ＳｈＬＦｃ,经根癌农杆菌ＬＢＡ４４０４ 感染烟草叶盘,获得具有卡那霉素抗性的烟草植株,ＰＣＲ检测和Ｓｏｕｔｈｅｒｎ杂交表明,人乳铁蛋白基因已整合到烟草基因组中。Ｎｏｒｔｈｅｒｎ 杂交与Ｗｅｓｔｅｒｎ 免疫印迹分析,检测到人乳铁蛋白基因在转化烟草中表达。体外实验证明,人乳铁蛋白基因在烟草中的表达产物对植物致病菌烟草火叶病菌、水稻白叶枯病菌有一定抑制作用     

Isolation and Some Physical and Chemical Properties of Elastase and Cathepsin G from Dog Neutrophils

A new method for isolation of leukocyte serine proteinases has been developed. Elastase (EC 3.4.21.37) and cathepsin G (EC 3.4.21.20) have been isolated from dog neutrophils and purified to homogeneous state. The results of inhibitor analysis indicate that the enzymes belong to the group of serine proteinases. Some physical and chemical characteristics of the purified enzymes have been determined. The molecular weights of the enzymes are 24.5-26 kD for the elastase and 23.5-25.5 kD for the cathepsin G. The cathepsin G is a glycoprotein, while the elastase molecule lacks carbohydrate components. The cathepsin G exhibits a broad pH optimum of catalytic activity in the range of 7.0-9.0; the pH optimum for the elastase is 8.0-8.5. The Michaelis constant of the elastase for N-t-Boc-L-alanine p-nitrophenyl ester is 0.10 mM; the Michaelis constant of the cathepsin G for N-benzoyl-L-tyrosine ethyl ester is 0.42 mM.     

Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota

The objective of the present study was to characterize lactobacilli isolates from the feces of male Wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity, antibiotic susceptibility and in vitro aggregation. Based on their morphological and biochemical characteristics, four potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened. The isolates showed good tolerance to stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities against the pathogens tested as assessed in neutral pH culture supernatants. All lactobacilli isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the isolate CS4 and CS7 were found to possess stronger cell surface traits such as hydrophobicity, auto‐aggregation and co‐aggregation capacity. In addition, CS4 and CS7 had greater β‐galactosidase activities than the others. Biochemical tests and 16S rRNA gene sequencing confirmed that CS2, CS3, CS4 and CS7 are Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4 and L. plantarum PJ7 are ideal in vitro probiotic candidates and require further in vivo evaluation.     

黄粉虫抗菌肽的提取及其生化特性的研究

【目的】测定黄粉虫Tenebrio molitor Linnaeus幼虫抗菌肽提取液的浓度、抑菌活性及其部分生化特性和凝血效应。【方法】本实验用浓度为1×108 CFU/m L大肠杆菌Escherichia coli诱导5龄黄粉虫幼虫,分别在诱导12、24、36、48、60和72 h后提取其中的抗菌肽,并用考马斯亮兰法测定抗菌肽粗提液蛋白的浓度,并用滤纸片法测定其抑菌活性,同时对其热稳定性、反复冻融稳定性、蛋白酶稳定性及不同p H对其活性的影响等生化特性及凝血效应进行了探究。【结果】经大肠杆菌诱导的黄粉虫抗菌肽粗提液的蛋白浓度均显著高于未诱导的黄粉虫组(P<0.01),且在诱导48 h时产生的抗菌肽提取液蛋白的浓度最高,产生的抑菌圈直径也显著高于未诱导的黄粉虫组(P<0.05),生化特性的测定结果显示,黄粉虫抗菌肽有较好的热稳定性、酶稳定性及酸碱稳定性,反复冻溶后对其抑菌活性影响不大,并且无凝血效应。【结论】大肠杆菌可以刺激黄粉虫的免疫系统,增加抗菌肽的表达量,使其产生浓度高、活性强的抗菌肽,且生化特性较稳定。本研究对黄粉虫抗菌肽作为绿色抗生素用于畜牧养殖业的进一步开发与利用提供了科学的理论依据。     

Neutrophil dysregulation during sepsis: an overview and update

Sepsis remains a leading cause of death worldwide, despite advances in critical care, and understanding of the pathophysiology and treatment strategies. No specific therapy or drugs are available for sepsis. Neutrophils play a critical role in controlling infection under normal conditions, and it is suggested that their migration and antimicrobial activity are impaired during sepsis which contribute to the dysregulation of immune responses. Recent studies further demonstrated that interruption or reversal of the impaired migration and antimicrobial function of neutrophils improves the outcome of sepsis in animal models. In this review, we provide an overview of the associated mediators and signal pathways involved which govern the survival, migration and antimicrobial function of neutrophils in sepsis, and discuss the potential of neutrophils as a target to specifically diagnose and/or predict the outcome of sepsis.     

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections

Aims: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, bla_OXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla_OXA‐31 in a canine Ps. aeruginosa isolate.     

Activity optimization of an undecapeptide analogue derived from a frog-skin antimicrobial peptide

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH₂) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH₂) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.     

Mini-review: Lactoferrin: a bioinspired,anti-biofilm therapeutic

Medically relevant biofilms have gained a significant level of interest, in part because of the epidemic rise in obesity and an aging population in the developed world. The associated comorbidities of chronic wounds such as pressure ulcers, venous leg ulcers, and diabetic foot wounds remain recalcitrant to the therapies available currently. Development of chronicity in the wound is due primarily to an inability to complete the wound healing process owing to the presence of a bioburden, specifically bacterial biofilms. New therapies are clearly needed which specifically target biofilms. Lactoferrin is a multifaceted molecule of the innate immune system found primarily in milk. While further investigation is warranted to elucidate mechanisms of action, in vitro analyses of lactoferrin and its derivatives have demonstrated that these complex molecules are structurally and functionally well suited to address the heterogeneity of bacterial biofilms. In addition, use of lactoferrin and its derivatives has proven promising in the clinic.