人工合成抗菌肽对常见口腔感染菌及口腔肿瘤细胞系杀伤作用的研究

人工合成具有抗菌活性的七肽,并分离常见口腔感染菌株,体外培养口腔肿瘤细胞系KB.将抗菌肽制备成不同浓度的药敏纸片,通过药敏纸片定量法,观察抑菌环直径.将不同浓度的抗菌肽溶液加至细胞培养体系中,培养24～48 h后,收获细胞分别进行细胞凋亡检测及细胞增殖检测.发现当抗菌肽药敏纸片在100、300 μg/片时,对常见口腔感染菌的抑菌环直径与阴性对照相比均有统计学差异;人工合成两段的抗菌肽对人口腔肿瘤细胞系KB均有明显的促凋亡作用.认为人工合成抗菌肽体外可有效抑制常见口腔感染菌株的感染,并对口腔肿瘤细胞有明显的促进凋亡作用.     

贯众中活性组分对金黄色葡萄球菌抑制作用的研究

目的 分离贯众中活性组分,并研究其对老年肺部感染金黄色葡萄球菌的抑制作用.方法 采用pH3的95%乙醇进行回流提取,制成粗提液,并以石油醚、乙酸乙酯萃取,纸片法筛选活性部分,低压液相色谱做精分离,根据分离图谱的不同吸收峰位置来收集不同组分.采用三氯化铁+铁氰化钾多酚定性实验和Folin-酚法进行组分的总多酚定量,以液体试管稀释法测定不同组分对老年肺部感染患者痰中分离的金黄色葡萄球菌的抑制作用.结果 贯众95%乙醇粗提液和乙酸乙酯萃取部分都具有较强的抑制作用,将后者经低压液相色谱分离后共获得13个组分,组分2、3、4、8、9、11、12和13均具有较强的抑菌及杀菌作用,其组分13的抑菌作用最强,其MIC为4.29 μg/ml,MBC为8.58 μg/ml.结论 贯众经乙醇粗提并且乙酸乙酯萃取部分中含有多种活性组分,具有较强的抑制及杀灭金黄色葡球菌作用.     

库伦驴血液白细胞抗菌肽提取及其对牛乳腺炎主要致病菌的抗菌活性研究

目的验证库伦驴血液白细胞抗菌肽对牛乳腺炎无乳链球菌、金黄色葡萄球菌及大肠埃希菌的体外抗菌活性。方法利用乙酸萃取法提取库伦驴血液白细胞抗菌肽,制备药敏试纸片,用平皿药敏纸片法检测其抗菌活性。结果库伦驴血液白细胞抗菌肽对无乳链球菌、金黄色葡萄球菌及大肠埃希菌具有抑菌效果,43.60mg/ml抗菌肽作用24h抑菌环直径分别为14、15和17mm。结论库伦驴白细胞抗菌肽对无乳链球菌、金黄色葡萄球菌、大肠埃希菌有抑菌活性,可用于上述致病菌感染所致奶牛乳腺炎的治疗。     

抗菌肽的研究与应用

抗菌肽是具有广谱抗细菌、病毒和真菌的相对分子质量较小的物质.他们由生物体基因组DNA编码,在机体天然免疫中扮演着重要角色.其在临床应用的特性包括广谱抗微生物活性、迅速的杀菌活性、产生抗药性的可能性低.同时,抗菌肽也存在着不利的方面,包括成本高、稳定性差、药物动力学及毒理学不明.随着传统抗菌素的耐药菌的增加,迫切需要开发出新型的抗微生物药应用于临床治疗.目前,人们关注最多的就是抗菌肽.该文就抗菌肽的研究及应用作一综述.     

家蝇消化腺抗菌肽生物活性的研究

目的研究家蝇消化腺抗菌肽抗菌活性和细胞毒性。方法针刺细菌感染法诱导家蝇幼虫表达抗菌肽,采用显微解剖的方法解剖并收集成蝇中肠,用超声破壁低温离心等技术提取、纯化抗菌肽,用提取的抗菌肽进行抑菌试验及细胞毒性试验。结果金黄葡萄球菌诱导的家蝇中肠抗菌肽抑菌环直径为1.3 cm,正常成蝇中肠抗菌肽抑菌环直径为1.1 cm,青霉素及蛆血淋巴抗菌肽抑菌环直径分别为1.0和<0.5 cm。细胞毒性试验中,各浓度抗菌肽组细胞生长正常。讨论中肠抗菌肽抑菌活性优于蝇蛆血淋巴抗菌肽的抑菌活性,且细胞毒性低。     

家蝇幼虫分泌物抗菌肽的分离及其部分特性

目的 分离家蝇（Musca domestica）幼虫分泌物抗菌肽并研究其部分理化特性。 方法 用超滤、固相萃取、反相高效液相色谱（RP-HPLC）分离纯化家蝇幼虫分泌物抗菌肽；测定抗菌肽对多株革兰氏阴性菌及革兰氏阳性菌的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）；比较其在不同pH值（pH 6.0～9.0）、不同阳离子浓度（Mg²⁺ ：（0.5×10-3～10.0×10-3）mol/L，Na⁺、K⁺：（10×10^-3～100×10^-3）mol/L）及不同小鼠血清浓度（12.5％～75％）环境中抗菌活性的变化。 结果 超滤分离获得的活性蛋白相对分子质量为Mr 3 000～30 000，该蛋白经20％、30％、70％及80％乙腈洗脱，其组分均有活性，其中以70％乙腈洗脱组分活性较强且稳定。进一步用RP?鄄HPLC从70％洗脱组分中纯化出两个抗菌活性肽。抗菌肽对大肠埃希菌、绿脓杆菌、金黄色葡萄球菌及枯草芽孢杆菌的MIC值分别为32.738、16.369、65.475 及32.738 μg/ml 。在培养基pH值为6～9、不同阳离子浓度及不同小鼠血清浓度的环境中，各实验组的A ₅₇₀增加量均小于0.05，而对照组的A570增加量多为0.3以上，各实验组与对照组比较，其差异均具有统计学意义（P值均<0.01）。 结论 用固相萃取等方法从家蝇幼虫分泌物中可快速分离出具有较强活性的抗菌肽。该抗菌肽在不同条件下均具有较好的抗菌效果。     

以肌无力为首发症状的金黄色葡萄球菌肺炎、败血症一例

患者 ,男性 ,18岁 ,学生。以肢体无力、疼痛 1周就诊。患者既往体健 ,入院前 10天受凉后出现头痛和流涕 ,自服感冒通治疗后上述症状缓解。发病前无皮肤、软组织感染史 ,无应用大剂量激素、巴比妥类药物、链霉素及其他氨基糖甙类抗生素史。患者入院前 1周无明显诱因出现右侧腹股沟区疼痛并放射至大腿 ,进行性加重伴四肢无力 ,以双下肢及右上肢明显 ,行走困难 ,经止痛治疗效果不明显。入院前 3天出现心悸、气促、进行性呼吸困难 ,诊断为心肌炎 ,静脉点滴先锋霉素、氟美松及能量合剂 ,上述症状持续。入院前 2天开始周身皮肤出现多发脓疱疹。入…     

金黄色葡萄球菌耐药率与抗菌药物使用量的相关性分析

正clinical practice. Methods A retrospective analysis of the frequency of use of eight types of antibacterial agents and the drug-resistant rate of Staphylococcus aureus was performed in our hospital from 2014 to 2018,and the correlation between the antibacterial drug cumulative defined daily doses(DDDs) and the drug-resistant rate of Staphylococcus aureus was analyzed using Pearson correlation analysis. Results The penicillin resistance rate of Staphylococcus aureus was positively correlated with DDDs of the third-generation cephalosporins (r=0. 912) and quinolones (r=0. 940) antibacterials(P 0. 05). The rate of oxacillin resistance was positively correlated with DDDs of the compound preparations containing enzyme inhibitors(r=0. 902,P 0. 05). The resistance rate of Staphylococcus aureus to levofloxacin was highly positively correlated with DDDs of the first-generation cephalosporins(r=0. 949) and macrolides(r=0. 861) antibacterials(P 0. 05),and was highly negatively correlated with DDDs of the compound preparations containing enzyme inhibitors(r=-0. 852,P 0. 05). The clindamycin resistance rate were highly positively correlated with DDDs of the first-generation cephalosporins(r=0. 921,P 0. 05). Conclusion There is a certain correla-     

筛选家蝇抗菌肽相关基因的oligo探针设计

目的设计筛选家蝇抗菌肽相关基因的寡核苷酸(oligonucleotide)探针。方法用生物学软件ArrayDesig-ner2.0,结合NCBI开发的免费生物信息学软件,对GenBank数据库中部分昆虫抗菌肽基因编码区保守序列设计特异性高、Tm值接近、长度均一的oligo探针。结果共设计出372条oligo探针,长度为50bp,GC含量为40%～60%,Tm为70～75℃。结论用GenBank数据库资源,结合生物学软件Array Designer2.0及NCBI中相关生物信息学软件能快速而有效地设计出筛选家蝇抗菌肽基因的探针。     

重组超抗原金黄色葡萄球菌肠毒素B基因的克隆和序列分析

目的　采用基因工程技术制备金黄色葡萄球菌B型肠毒素 (SEB)。方法　根据SEB已知序列 ,设计一对引物 ,用PCR方法从金黄色葡萄球菌染色体DNA上扩增出基因片段 ,克隆到原核表达质粒 pET32a上 ,转化大肠埃希菌JM10 9感受态细胞 ,经酶切和PCR鉴定 ,然后进行测序。结果　PCR扩增产物大小为 74 0bp ,重组质粒经双酶切PCR鉴定表明已正确重组 ,测序结果与已知序列基本吻合。结论　成功地克隆了金黄色葡萄球菌B型肠毒素 ,为下一步研究发病机制奠定基础。     

Evaluation of antimicrobial peptide LL-37 for treatment of Staphylococcus aureus biofilm on titanium plate

The antimicrobial peptide LL-37 belongs to the cathelicidin family and is one of the few human bactericidal peptides with potent antistaphylococcal activity. Staphylococcus aureus is one of the main infection bacteria in orthopedic implant therapy. Biofilm formation after bacterial infection brings more and more severe test for clinical antiinfection treatment.However, there are few studies on LL-37 in S. aureus infection of prosthesis. In this work, addition to research the antibacterial activity and the inhibitory effect on bacterial adhesion of LL-37, an in vitro model of S. aureus biofilm formation on titanium alloy surface was established to observe the inhibitory effect of LL-37.The results showed that LL-37 has a strong antibacterial effect on S. aureus in vitro, and the minimum inhibitory concentration (MIC) is about 0.62 μΜ. Moreover, LL-37 has significant impact on the adhesion of S. aureus when the concentration ≥0.16 μM and significant anti-staphylococcal biofilm effects on static biofilm models at the concentration of 0.31 to 10 μM. Additionally, LL-37 at 5 μM had a significant destructive effect on S. aureus biofilm (P < .05) that formed on the titanium alloy surface.This study further confirmed the role of LL-37 in the process of S. aureus infection, including antimicrobial activities, inhibition of bacterial adhesion, and inhibition of mature biofilm. LL-37 can significantly destroy the stable biofilm structure on the titanium alloy surface in vitro, which may provide a new way for refractory infection caused by S. aureus in titanium alloy prosthesis infection.     

家蝇抗菌肽MAF-1A的串联表达与抗白念珠菌体外活性检测

目的 构建家蝇抗菌肽MAF-1A基因原核串联表达体系,在大肠杆菌中表达具有抗菌活性的MAF-1A。方法 根据大肠杆菌密码子的偏嗜性进行密码子优化,设计并合成含有5个拷贝的MAF-1A串联基因序列;将合成的串联基因序列克隆到表达载体pET28a并转化大肠杆菌Roseeta(DE3),应用IPTG诱导表达5×MAF-1A串联重组蛋白;通过SDS-PAGE电泳、Western Blot对表达产物进行分析;用肠激酶专一性切割经Ni-NTA纯化后的5×MAF-1A重组串联蛋白,得到MAF-1A单体;采用微量稀释法检测MAF-1A单体对白念珠菌的体外抗菌活性。结果 5×MAF-1A蛋白在大肠杆菌中呈可溶性表达,28 ℃、0.8 mmol/L IPTG诱导12 h可达最大表达量;酶切后的抗菌肽MAF-1A单体对白念珠菌的MIC和MBC分别为0.5 mg/mL、1.0 mg/mL。结论 成功构建MAF-1A原核串联表达系统,重组表达的MAF-1A对白念珠菌具有较高的抑杀活性。     

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae. Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Multidrug-resistant (MDR) bacterial infections have become a major threat to human health (1–3). Mortality rates from infections caused by gram-negative bacteria, specifically Klebsiella pneumoniae, are on the rise owing to the lack of effective antibiotics to treat the emergent MDR strains (4–7). The capsule of K. pneumoniae is composed of extracellular polysaccharides that promote infection by masking the bacteria from immune recognition and provide an especially potent barrier against peptide-based antimicrobials, including innate host defense peptides and last-resort polymyxin antibiotics (8–14).Antimicrobial peptides are commonly amphipathic, with both a charged and a hydrophobic character (15). The anionic nature of the bacterial capsule promotes an electrostatic attraction to cationic antimicrobial peptides, and peptide hydrophobicity has been proposed to enhance capsule binding through nonionic interactions (9, 12, 16). Interaction with the bacterial capsule is thought to induce structural changes that cause sequestration of antimicrobial peptides to prevent them from reaching their bacterial membrane target (16, 17). While the bacterial capsule inhibits host defense peptides and polymyxins, a few amphipathic antimicrobial peptides have been identified that can retain activity against capsulated K. pneumoniae (18–21). However, it is not known what enables some peptides to avoid sequestration by the capsule of K. pneumoniae while the capsule effectively neutralizes our innate host defense peptides with similar physicochemical properties. This lack of knowledge prevents us from understanding how to bypass the capsule barrier that K. pneumoniae uses to avoid our innate immune response and last-resort treatment options.Here we characterize the synthetic evolution of a peptide inhibited by capsule to a peptide with potent activity against capsulated K. pneumoniae. Remarkably, our results indicate that rather than reduced interactions, our active peptide retains binding to capsule and undergoes conformational changes associated with capsule aggregation. We present a model in which peptide-driven sequestration of capsule disrupts this barrier and reduces its ability to protect K. pneumoniae against antimicrobial attack. These findings provide insight into improving antimicrobial peptide activity against K. pneumoniae and may help strengthen our understanding of the inability of innate host defense peptides to act on capsulated bacteria.     

From the Cover: A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrus australiasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr. Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

Citrus Huanglongbing (HLB), also known as citrus greening, is caused by the vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas). It is the most destructive disease threatening citrus industries worldwide (1, 2), and thus far, no cure has been discovered. Current management strategies include insecticide application to control the transmission vector Asian citrus psyllids (ACP) and antibiotics treatment to inhibit CLas (3), but neither of these could control HLB effectively. Since the first report of HLB in Florida in 2005, citrus acreage and production in Florida decreased by 38% and 74%, respectively (2, 4). The disease has spread to most citrus-producing states, including Texas and California. Along with drastic losses in fruit production, increasing chemical applications to control the vector and the bacteria have raised costs significantly, making citrus production for growers unsustainable. In severely affected areas, such as Florida, effective therapy is demanded because disease eradication is impractical. In recently impacted areas, such as California, the focus remains on preventing new infections. Hence, innovative therapeutic and preventive strategies to combat this lethal citrus disease are urgently needed to ensure the survival of the citrus industry.One of the most effective and ecofriendly strategies to combat pathogen infection is to utilize existing plant innate immunity–related genes from disease resistant or tolerant varieties for plant protection. Upon pathogen infection, plant defense response genes undergo expression reprogramming to trigger plant innate immunity. Plant endogenous small RNAs play a pivotal role in this regulatory process (5, 6). In addition, primary pathogen infection or application of some phytohormone analogs and chemicals, such as salicylic acid (SA) analogs, could induce systemic acquired resistance or defense priming in plants, which can promote faster and stronger host immune responses upon subsequent pathogen challenges (7, 8).Although all commercially important citrus varieties are susceptible to HLB (9, 10), HLB tolerance has been observed in some hybrids [e.g., US-942 and Sydney hybrid 72 (11, 12)] and close citrus relatives (e.g., Microcitrus australiasica, Eremocitrus glauca, and Poncirus trifoliata) (13). By comparative expression analysis of small RNAs and messenger RNAs (mRNAs) between HLB-sensitive cultivars and HLB-tolerant citrus hybrids and relatives (11, 12), we identified a list of candidate natural defense genes potentially responsible for HLB tolerance (14). One of the candidate regulators is a novel antimicrobial peptide (AMP), which we named “stable antimicrobial peptide” (SAMP). Here, we demonstrate that SAMP not only has the antimicrobial activity but also has the priming activity and can induce citrus systemic defense responses. This dual-functional SAMP can reduce CLas titer and suppress disease symptoms in HLB-positive trees and activate plant systemic defense responses against new infection.     

Inoculum effect of antimicrobial peptides

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 10⁵ cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide–cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.

The minimum inhibitory concentration (MIC) is one of the most common measures for the efficacy of antimicrobial compounds (1, 2). According to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the MIC is the lowest drug concentration that abolishes in vitro bacterial growth during a short period (typically 20 h) when using a standard initial cell density (inoculum) of ∼5 × 10⁵ colony-forming units (CFU)/mL in assays performed in broth (with an acceptable range of 2 × 10⁵ to 8 × 10⁵ CFU/mL for CLSI and 3 × 10⁵ to 7 × 10⁵ CFU/mL for EUCAST) (3–5). The choice of a specific value for the inoculum to be applied is dictated by a need for standardizing the assay in clinical practice (2), albeit bacterial cell densities in clinically relevant infections in vivo range from 1 CFU/mL to 10⁹ CFU/mL (in soft tissue or peritoneal infections) (6–8).Soon after the introduction of penicillin for civilian use in the 1940s, it was realized that the active concentration of antibiotics might need to be increased significantly when higher bacterial cell densities are inoculated in the assay medium (9, 10), a phenomenon termed the “inoculum effect” (IE) (11). The IE can be caused by different mechanisms (12, 13), including enzymatic degradation of the drug (11, 13), a simple consequence of the number of available drug molecules per cell (14, 15), or inhibition of antibiotics by intracellular material released by dead cells, causing enhanced survival of the remaining bacterial population (16). Traditionally, an IE has been defined as a change in MIC greater than or equal to eightfold when an inoculum 100-fold greater than the CLSI recommendation is used, but recent studies have shown that even subtle differences in inoculum may have a dramatic effect on MIC values (17). The IE is commonly examined by determining the MIC; in this assay, the cell density varies by several orders of magnitude with respect to the initial inoculum, during the many hours in which the bacteria are allowed to grow. However, an IE has been demonstrated also under conditions of constant cell density (12).High-density bacterial infections, including septic bloodstream and urinary tract infections, endocarditis, and abscesses, are quite prevalent and lack efficacious therapies (18). Although some reports have contested the therapeutic relevance of the IE (19, 20), several studies have demonstrated its clinical significance, showing that the MICs determined in the standardized assay were ineffective in the clinical treatment of high-density infections (12, 21–28). In some cases, a concentration 1,000-fold higher than the MIC is required to cure the infection (22, 23).While the IE is well characterized for traditional antibiotics, little is known about this phenomenon for other antimicrobial compounds. Antimicrobial peptides (AMPs), sometimes referred to as “host defense peptides,” are produced by all living organisms as a first line of defense against pathogens (29–31). These peptides can have many functions (32), but most of them exert direct bactericidal effects that typically involve perturbation of the membrane integrity of microbial cells (31, 33). The majority of known AMPs are short, amphipathic, and cationic peptides capable of binding selectively to the anionic membranes of bacterial cells (34–37). Most AMPs accumulate on the outer leaflet of cell membranes, thereby perturbing their surface tension (36, 38). When a threshold of membrane-bound molecules is reached, the stress is released by the formation of pores or other membrane defects. This mechanism of action has been termed the “carpet” model (39, 40), and it makes the development of bacterial resistance particularly difficult (30, 41, 42). Therefore, AMPs represent promising lead compounds in the fight against multidrug-resistant bacteria (30, 42, 43), which constitute a dramatically increasing worldwide threat (44), and several peptides are undergoing clinical trials (43).Considering their characteristic mechanism of action as compared to that of commonly used antibiotics, the existence of a pronounced IE is not obvious in the case of AMPs. Surprisingly, the IE within this class of molecules has been investigated only in a handful of studies (45–50), which are summarized in Table 1. In some of these reports (46, 49), the minimum bactericidal concentrations (MBCs; i.e., the minimum drug concentration killing more than 99.9% of the original bacterial cells) (51), rather than the MICs, were measured. While Jones (46) determined the MBC under normal growth conditions, in our previous report (49) we used a minimal medium that ensured a constant cell density. Different media were used also for the MIC assays: salt-free Luria broth (47), Müller–Hinton broth (MHB) (48), or 3–morpholinopropane–1–sulfonic acid (MOPS)–based rich defined media (RDM) (50). In another study concerning the activity of MSI–94 against Pseudomonas aeruginosa (52), quantitative MIC or MBC determinations were not performed, and hence, it is not included in Table 1. However, the time–kill curves obtained at different cell densities were indicative of a significant IE.Table 1.Literature studies of IE for AMPs

家蝇抗菌肽MAF-1A体外抗甲型流感病毒活性及机制研究

目的 探讨家蝇抗菌肽MAF-1A体外抗甲型流感病毒(IAV)活性及其潜在机制。方法 通过观察CPE、MTT法及qRT-PCR评价MAF-1A体外抗IAV活性,采用MTT法测定MAF-1A对MDCK细胞的毒性;利用透射电镜(TEM)技术、血凝抑制试验和神经氨酸酶抑制试验进一步分析MAF-1A抗IAV的作用机制。结果 MAF-1A对IAV 的半数有效浓度(EC₅₀)为(89.8±2.97)μg/mL,而对 MDCK 细胞的毒性较小;MAF-1A可直接破坏IAV形态结构的完整性;浓度为1.56 μg/mL的MAF-1A即可抑制IAV引起的红细胞凝集;对神经氨酸酶具有抑制作用,IC₅₀为(134.7±10.31)μg/mL。结论 抗菌肽MAF-1A具有体外抗IAV活性,除能直接破坏IAV的结构外,还可能通过与血凝素 HA1 亚基结合、抑制神经氨酸酶的活性而阻止IAV的感染,提示MAF-1A具有多靶点抗IAV的作用。     

Precision-guided antimicrobial peptide as a targeted modulator of human microbial ecology

One major challenge to studying human microbiome and its associated diseases is the lack of effective tools to achieve targeted modulation of individual species and study its ecological function within multispecies communities. Here, we show that C16G2, a specifically targeted antimicrobial peptide, was able to selectively kill cariogenic pathogen Streptococcus mutans with high efficacy within a human saliva-derived in vitro oral multispecies community. Importantly, a significant shift in the overall microbial structure of the C16G2-treated community was revealed after a 24-h recovery period: several bacterial species with metabolic dependency or physical interactions with S. mutans suffered drastic reduction in their abundance, whereas S. mutans’ natural competitors, including health-associated Streptococci, became dominant. This study demonstrates the use of targeted antimicrobials to modulate the microbiome structure allowing insights into the key community role of specific bacterial species and also indicates the therapeutic potential of C16G2 to achieve a healthy oral microbiome.Human microbiome research revealed that every human body contains a variety of microbial communities on various mucosal surfaces that consist of thousands of different microbial species (1–4). Disturbance from host and environmental factors may alter the composition and abundance of these microbial species, leading to various polymicrobial diseases (5–8). With the complexity of these multispecies microbial communities, it is very difficult to determine the functions of individual species contributing to observed physiological and pathological changes, making it one of the most challenging issues in human microbiome research. This study aims to develop and validate a new tool to address this important issue.The indigenous microbial flora of the human oral cavity consists of over 700 different species of bacteria, with over 100 present in any individual (4, 9, 10). Most bacteria help promote a healthy oral environment by stimulating the immune system and preventing the invasion of pathogenic species (11–13). One pathogenic bacterium, Streptococcus mutans, is predominantly responsible for tooth decay worldwide (14). The carious lesions that S. mutans can cause are generally not considered life-threatening, but they result in an economic burden that leaves many cases in underdeveloped areas untreated, resulting in tooth extraction as the only remedy (15). Currently, there is no effective treatment for S. mutans. Broad-spectrum antibiotics administered to the oral cavity result in destruction of the entire oral bacterial flora, making it prone to reinfection by S. mutans, which reestablishes at pretreatment levels.To combat S. mutans infection, our group developed C16G2, a synthetic peptide that belongs to a new class of antimicrobials called specifically targeted antimicrobial peptides that can achieve targeted killing of selected pathogens (16). A typical targeted antimicrobial peptide molecule consists of two functionally independent moieties conjoined in a linear peptide sequence: a nonspecific antimicrobial peptide serves as the killing moiety, whereas a species-specific binding peptide comprises the targeting moiety that provides specific binding to a selected pathogen and facilitates the targeted delivery of the attached antimicrobial peptide. Previous studies showed that C16G2 was potent against S. mutans grown in liquid or biofilm states. It displays targeted killing of S. mutans within a three-species biofilm without affecting closely related noncariogenic oral streptococci, including Streptococcus sanguinis and Streptococcus gordonii (16). Additional study showed that C16G2 has a mechanism of action similar to that of traditional antimicrobial peptides and kills S. mutans through disruption of the cell membrane followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans but not against other noncariogenic oral streptococci (17).In this study, we further investigated the antimicrobial specificity of C16G2 by expanding the panel of testing bacterial species to include more oral streptococci species closely related to S. mutans, nonstreptococcal oral species, and nonoral representatives. To explore its potential clinical application, a saliva-derived in vitro model containing over 100 species approaching the diversity and overall metabolic functionality of the human oral microbiome (18) was used for examining the selective antimicrobial activity of C16G2 against S. mutans within a multispecies community. The main goal of this study is to investigate the impact of targeted removal of S. mutans on the overall structure of a multispecies oral microbial community and provide proof of concept data for using targeted antimicrobials to modulate the microbiome structure and study the ecological role of specific bacterial species.     

家蝇抗菌物质诱导表达后的电泳分析

目的探讨家蝇抗菌肽的诱导及电泳分离方法。方法家蝇三龄幼虫针刺体壁诱导后,第48h提取其血淋巴,置于100℃、80℃水浴10s、30s、1min、3min、5min等不同时间后,观察不同温度处理不同时间后的去杂蛋白效果和对溶壁微球菌的抑菌效果,并对各样品进行电泳分离。结果血淋巴100℃水浴10s、30s、1min仍有抑菌活性,处理3min和5min无抑菌活性;80℃水浴30s-5min均有抑菌活性,电泳显示100℃处理3min和5min的样品与其他相比缺少2条蛋白区带。结论针刺诱导的家蝇抗菌肽分离时血淋巴预处理采用80℃水浴1min去杂蛋白效果较好,诱导的血淋巴100℃处理3min和5min即丧失抗菌活性,电泳显示缺少2条区带,此2条差异区带可能为抗菌肽组份。     

Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes

Current influenza virus vaccines protect mostly against homologous virus strains; thus, regular immunization with updated vaccine formulations is necessary to guard against the virus' hallmark remodeling of regions that mediate neutralization. Development of a broadly protective influenza vaccine would mark a significant advance in human infectious diseases research. Antibodies with broad neutralizing activity (nAbs) against multiple influenza virus strains or subtypes have been reported to bind the stalk of the viral hemagglutinin, suggesting that a vaccine based on this region could elicit a broadly protective immune response. Here we describe a hemagglutinin subunit 2 protein (HA2)-based synthetic peptide vaccine that provides protection in mice against influenza viruses of the structurally divergent subtypes H3N2, H1N1, and H5N1. The immunogen is based on the binding site of the recently described nAb 12D1, which neutralizes H3 subtype viruses, demonstrates protective activity in vivo, and, in contrast to a majority of described nAbs, appears to bind to residues within a single α-helical portion of the HA2 protein. Our data further demonstrate that the specific design of our immunogen is integral in the induction of broadly active anti-hemagglutinin antibodies. These results provide proof of concept for an HA2-based influenza vaccine that could diminish the threat of pandemic influenza disease and generally reduce the significance of influenza viruses as human pathogens.