2. 龙口海关, 烟台 265700;
3. 江苏省动物重要疫病与人畜共患病防控协同创新中心, 扬州 225009
2. Long Kou Customhouse, Yantai 265700, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
奶牛乳腺炎是限制全球乳制品行业发展最重要的疾病之一，常引起牛奶产量、质量下降。长期以来，抗生素疗法引起的细菌耐药性和食品安全问题也给人们带来了巨大困扰[1]。金黄色葡萄球菌(Staphylococcus aureus，SA)是引起奶牛慢性、亚临床型乳腺炎的重要病原体，在牛群中具有传染性[2]。奶牛乳腺是一种外分泌腺，也是机体公共黏膜免疫系统的重要组成部分。乳房天然免疫系统和适应性免疫系统在乳房炎过程中起抗感染作用[3-4]。SA感染奶牛乳腺分3个阶段：1)黏附乳头皮肤和皮下基质；2)侵入、破坏乳腺组织和细胞；3)逃避宿主免疫系统并大量增殖。据统计，在SA致奶牛乳腺炎过程中至少有25种毒素、15种黏附蛋白、20种免疫逃避分子发挥重要作用[5]。
随着对SA与机体免疫系统相互作用研究的不断深入，多种免疫逃避作用被发现参与SA致病过程。近年研究的SA免疫逃避机制主要有：1)适应宿主；2)损伤宿主免疫功能；3)内化作用；4)降低菌体免疫原性。
1 适应宿主通过进化基因组学研究发现，SA通过可移动遗传元件的获得和核心基因组的突变，获得宿主特异性适应和高度遗传多样性[6]。而宿主细胞对出现适应性的SA株的免疫反应也表现出谱系特异性[7-9]。有研究指出，SA可在宿主内进化为sigB缺陷型(sigB因子可与多种RNA聚合酶结合以调控基因表达[10])，该变异株毒性降低、蛋白水解活性和生物膜形成能力增强，可在宿主内持续存在[11]。宿主适应还包括菌株代谢与毒力的适应、形成小菌落突变株等。
1.1 SA代谢与毒力适应近期研究发现，SA中的一些代谢相关分子在菌体暴露于宿主免疫系统或其他应激时具有调节自身代谢与毒力的功能。Goncheva等[12]发现，嘌呤生物合成调节因子R (purine biosynthesis regulator R, PurR)缺失突变SA株在小鼠全身感染模型中具有强致病力，这种强致病性与菌体高表达毒力因子纤维连接蛋白结合蛋白(fibrinogen binding proteins, FnBP)有关，且该突变可在菌体暴露于机体免疫系统时出现，以利于菌体存活。Sause等[13]发现，PurR可与多种毒力因子(如FnBP、杀白细胞素、α-毒素等)基因启动子直接结合，并调节其表达，PurR缺失突变时毒力因子在转录、蛋白质水平均明显升高。彭琦[14]也证明，SA代谢和毒力变化与“持留菌(bacterial persisters)”的形成有关。
此外，SA分泌的主要脂肪酶(glycerol ester hydrolase，Geh)不仅能水解环境中的长链脂质以供菌体营养，还能水解菌体表面脂蛋白，以逃避机体免疫识别[15]。Tan[16-17]等还发现，SA脂肪酸合成基因lipA参与脂肪酸、脂酰丙酮酸脱氢酶(lipoyl-pyruvate dehydrogenase, lipoyl-PDH)的产生，而纳摩尔量的lipoyl-PDH可与脂蛋白竞争性结合TLR2，阻断TLR2依赖的巨噬细胞激活，从而实现菌体的免疫逃逸。
毒素-抗毒素(toxin-antitoxin, TA)系统是细菌的遗传组件，由2个共表达的基因组成，分别编码稳定的毒素蛋白和易降解的抗毒素，过表达的毒素蛋白使细菌生长停滞或者死亡，而抗毒素则可中和其毒性。二者相互作用、精密调节细菌毒力、生存状态，并且对细菌耐药性的产生有重要意义[18]。其中，Ⅰ型TA系统中编码毒素蛋白的基因通常与编码抗毒素sRNA的基因位置相邻、方向相反，有基因重叠现象[19]。由SA致病岛(pathogenicity islands, PIs)表达的7种sRNA (regulatory RNAs)，分别被命名为SprA-G (small pathogenicity island RNA, Spr)，其中，SprA1/SprA1AS和SprG1/SprF1属于Ⅰ型TA系统。Germain-Amiot等[20]在SA中鉴定出与SprA1/SprA1AS核苷酸相似的SprA2/SprA2ASⅠ型TA系统。SprA2指导合成毒素肽A2 (peptide A2, PepA2)，过量PepA2在菌体内可引起细菌生长停止或死亡，在细胞间质内则对宿主细胞(如中性粒细胞、红细胞等)有严重毒性；反式编码的SprA2AS抗毒素能阻止核糖体装载到顺式SprA2上，阻止PepA2过度表达。在渗透应激反应过程中，SprA2AS表达下降，同时PepA2过度表达，造成细菌本身死亡。而释放到细胞间质的PepA2破坏宿主细胞以利于群体定植、转移、感染[20]。SprA2/SprA2AS与SprA1/SprA1AS间不存在交叉调节。Riffaud等[21]发现，SprG1/SprF1的3个核心基因组拷贝(SprG2/SprF2、SprG3/SprF3、SprG4/SprF4)在SA受到高渗应激或吞噬作用诱导的氧化应激时也发生上述作用，但各拷贝间可能存在交叉调节。总之，TA系统对于SA在宿主体内环境中持续存在有重要意义。
1.2 小菌落突变株(small colony variants, SCV)SCV是SA在机体免疫系统或抗生素作用下的一种持续存活能力提高的存在形式，主要表现为新陈代谢、生长繁殖变慢，毒力降低，生物膜形成能力增强[22-23]。从慢性SA乳腺炎病例中有时可分离出SCV，其特点是菌落小、无色、非溶血性、缓慢凝固酶阳性、不发酵甘露糖，可恢复至野生株表型[24]。Wong Fok Lung等[25]发现，SCV能有效刺激宿主糖酵解和线粒体活性氧的产生，并引发一种与caspase无关的细胞坏死性凋亡，造成宿主损伤。同时，SCV无法有效激活宿主免疫反应，可在非吞噬细胞[如奶牛乳腺上皮细胞(bovine mammary epithelial cells, BMEC)]中持续存在，从而造成牛群的慢性感染[24]。关于SCV的形成机制，许多研究者认为与抗生素等不利条件下SA呼吸链组分功能的损伤或丧失有关，呼吸链瘫痪，ATP合成受阻，SA表型随之向SCV发生转变。此过程的遗传学基础可能是catA、menB、menD、hemA、hemB、hemH、thyA、fusA等基因的突变[26-27]。在SA奶牛乳腺炎过程中，SCV形成及其表型回复的具体机制仍待进一步研究。
2 损伤宿主免疫功能SA除可通过调节自身毒力、代谢、持续生存相关基因的激活和关闭来适应宿主以获得自身或菌群的持续存在外，也可通过分泌外泌酶主动破坏机体免疫功能。
2.1 杀白细胞素(leukocidins, Luk)杀白细胞素是主要靶向中性粒细胞(neutrophil, NE)的双组分成孔毒素。奶牛乳腺炎SA分离株主要携带LukED’、LukMF’基因[28]。LukMF’是分泌最多、活性最高的牛NE毒素，其通过识别牛NE表面的趋化因子受体(chemokine receptor1, CCR1)发挥杀伤作用，乳腺炎严重程度与乳汁中LukMF’含量呈正相关[29]。LukED’可通过结合红细胞上的Duffy抗原趋化因子受体(duffy antigen receptor for chemokines, DARC)，破坏红细胞以获取铁源。DARC也是LukED’对内皮细胞的关键致死性靶点，而DARC缺陷的人内皮细胞对该毒素产生抗性[30]。另外，杀白细胞素对NK细胞、树突细胞、T细胞等免疫细胞也具有杀伤作用[31]。杀白细胞素在机体内对免疫机能的影响仍待探索。
2.2 SA超抗原SA超抗原包括肠毒素家族(staphylococcal enterotoxins, SEs)、肠毒素样家族(staphylococcal enterotoxin like toxin, SELs)以及中毒性休克综合征毒素1 (toxic shock syndrome toxin-1, TSST-1)[32]。SA超抗原可与T细胞受体Vβ结构域以及主要相容性复合体Ⅱ型分子(major histocompatibility complex classⅡ molecules, MHC Ⅱ)直接结合，促进T细胞有丝分裂，以及促炎因子(如IL-1、IL-2、IL-6、TNF-α、IFN-γ等)的大量释放，造成炎性损伤，也破坏机体有效适应性免疫反应的发生。研究者检测超抗原激活的Vβ-特异性T细胞谱发现，一些菌株已进化出一系列超抗原来激活整个牛T细胞库，以实现SA免疫逃避[33]。
SA超抗原中肠毒素种类较多、危害大。Fang等[34]发现，肠毒素C以剂量依赖性方式诱导乳腺炎性细胞浸润以及IL-1β、IL-6等细胞因子表达，注射抗肠毒素C抗体对小鼠SA乳腺炎模型的乳腺炎症和组织损伤有特异性抑制作用。奶牛腺房炎病例中分离出的肠毒素能非特异性刺激T细胞增殖并高水平表达TNF-α，最终使反应性T细胞失能、凋亡、缺失，造成机体免疫损伤[35-36]。另外，肠毒素H可在体外诱导BMEC凋亡，增强淋巴细胞的增殖活性[35]；肠毒素B可以促进T细胞、B细胞、成纤维细胞、肥大细胞IL-4、IL-5表达，抑制TGF-β、IL-10表达，同时调节类花生酸代谢，诱导机体糖皮质激素的缺乏；Hayes等[37]还观察到，肠毒素B在人鼻息肉外植体模型中诱导肥大细胞向上皮聚集，且促进SA被肥大细胞胞外DNA陷阱捕获并吞噬内化，从而实现菌体胞内增殖。
2.3 α毒素20%~50%奶牛乳腺炎SA菌株可产生α毒素。α毒素是一种重要的成孔毒素，能够与细胞膜结合形成穿膜孔，使细胞因分子泄漏而死亡[38]。宿主细胞可通过内吞跨膜孔复合体以逃避成孔毒素的杀伤作用[39]。Shah等[40]发现，解整合素金属蛋白酶(a disintegrin and metalloproteinase domain-containing protein 10, ADAM10)、普列克底物蛋白同源结构蛋白7 (pleckstrin homology domain-containing family A member 7, PLEKHA7)、四跨膜蛋白(tetraspanin Tsp33)、丝状肌动蛋白结合蛋白(afadin)等宿主蛋白是影响α毒素宿主毒性的重要因素。α毒素可通过与上述蛋白作用而锁定于胞间连接，使跨膜孔得以稳定形成，并且阻止细胞对跨膜孔的内吞，造成细胞死亡[40-41]。Lee等[42]提出, 在SA皮肤感染中出现的适应性免疫缺陷可能与α毒素引起DC细胞丢失有关，而用α-毒素免疫小鼠后，SA再感染小鼠皮肤时感染局部DC细胞累积，抗原特异性T细胞水平增加，小鼠免疫力提高。
2.4 细胞毒性脱氧腺苷(deoxyadenosine, dAdo)通常情况下，活化的NE可释放含有核酸、线粒体DNA、抗菌肽等物质的胞外陷阱(neutrophil extracellular trap, NETs)来捕杀细菌[43]。但SA可通过释放蛋白酶和核酸酶降解NETs来逃避捕杀[43]，伴随着NETs降解而产生的脱氧腺苷酸(deoxyadenosine monophosphate, dAMP)可被SA腺苷合成酶A (adenosine synthase A, adsA)转化为对吞噬细胞有较强毒性的dAdo，进而损伤机体免疫功能，使SA持续感染[44]。Winstel等[45]用CRISPER-cas9筛选出的缺乏平衡核苷转运体1 (equilibrative transporter family member1, ENT1)、腺苷激酶(adenosine kinase, ADK)、脱氧胞苷激酶(deoxycytidine kinase, DCK)的人巨噬细胞对dAdo处理耐受(细胞凋亡减少)，且突变体细胞质中未检测到dAdo；Winstel等[45]推测，dAdo在ENT1介导下摄入巨噬细胞，然后在嘌呤补救途径激酶ADK和DCK作用下转化为dADP、dATP并累积，最后使巨噬细胞在caspase-3介导下发生凋亡。
3 内化作用(cellular internalization)经内化作用进入吞噬或非吞噬细胞是SA逃避机体免疫因子和抗菌药物的重要手段。SA最重要的侵入毒力因子FnBP A和B可通过与宿主细胞膜α5β1整合素结合，促进SA内化[46]。Ashraf等[46]指出, SA也可通过聚集因子A (clumping factor A, CLFA)与BMEC的膜联蛋白A2 (annexin A2)相互作用，介导细菌内化。Cai等[47]还发现, SA内化入巨噬细胞后可引起微管相关蛋白1轻链3-Ⅱ (microtubule associated protein 1 light chain 3 - Ⅱ, LC3)、死骨片蛋白1 (sequestosome 1, p62)表达增加，自噬体形成；但同时，自噬体与溶酶体的融合被阻断，自噬体不断累积，使SA得以在巨噬细胞内持续存活和增殖。Wang等[48]发现, BMEC中也存在相似现象。
4 降低免疫原性 4.1 磷壁酸SA磷壁酸分为脂磷壁酸(lipoteichoic acid, LTA)和壁磷壁酸(wall teichoic acid, WTA)。LTA能在乳腺炎过程中造成血乳屏障损伤[49]，也参与生物膜形成，促进细菌的局部黏附和定殖[50]。近年来，WTA在SA免疫逃逸过程中的作用被广泛研究。WTA由聚磷酸核糖醇(poly-ribitol-phosphate, RboP)重复单元组成，D-丙氨酸(D-alanine, D-Ala)和N-乙酰葡萄糖胺(N-acetylglucosamine, GlcNAc)修饰[51]。Gerlach等[52]在WTA合成基因中鉴定出一种由噬菌体插入后编码的β-糖基转移酶Tarp，其可在RboP的C3位加入GlcNAc，催化C3GlcNAc WTA的形成；C3GlcNAc WTA诱导的小鼠免疫球蛋白G水平明显低于标准糖基转移酶Tars修饰的C4GlcNAc WTA；在同时表达Tars和Tarp的SA中，Tarp修饰的C3GlcNAc WTA占主导，此时WTA与抗体结合降低，抗体介导的调理、吞噬过程被抑制，菌体得以逃避免疫；Tarp还可以恢复WTA糖基化缺陷株的糖基化以及β-内酰胺耐药性；另外，有研究指出，部分家畜相关的CC5、CC398耐甲氧西林SA株感染携带了Tarp基因的前噬菌体，同时在鉴别出的一个前噬菌体中发现了免疫逃避簇基因[52-55]。上述结果都表明, Tarp降低了WTA的免疫原性，且在SA免疫逃避和抗生素耐药过程中有重要作用。
4.2 胞外囊泡(extracellular vesicles, EV)SA生长过程中释放的EV，被认为是向宿主传递毒力因子的纳米载体，与生物膜、耐药性形成和菌种间交流有关。EV免疫原性较低，在小鼠乳腺中引起的炎症反应与热灭活SA和LTA相当[56]。但体外试验发现，EV可被人巨噬细胞内化，并由其携带的毒素引起细胞炎症反应和损伤[57]。
4.3 酚溶性调节蛋白(phenol soluble modulins, PSM)PSM被证明能裂解宿主细胞、引起宿主炎症反应、促进生物被膜稳定性，对菌体毒力和免疫逃避有重要作用[58]。Deplanche等[59]认为, PSM是SA感染BMEC时炎性因子表达程度较低的关键因素，与PSM表达株相比，PSM缺失突变株处理BMEC后细胞分泌的IL-6、IL-8、IL-32(参与DC细胞成熟)显著升高。PSM也被发现与SA胞外囊泡形成有关[60]。
PSM家族部分成员能形成功能性淀粉样蛋白(functional amyloids)，但其构象与活性的关系仍有争议。22残基肽PSMα3 (the 22-residue peptide PSMα3)是PSM家族毒性最强的成员，为两亲性α螺旋(amphipathic helices)结构。Tayeb-Fligelman E等[61]指出，此螺旋需进一步形成长的、无分支的交叉α淀粉样纤维(cross-α amyloid-like fibril)才能实现其生物学功能；有研究发现，两株PSMα3纤维化缺失的突变体对T细胞表现出较低的细胞毒性，而野生株则较强[61]。同样的，纤维化依赖性细胞毒性作用也被发现于人胚胎肾细胞，表明PSMα3是非细胞特异性杀伤毒素，其毒性可能源于螺旋自组装(self-assemble)，并在细胞膜上形成巨大的两亲性薄片(amphipathic sheets)，造成膜变形[61]。Yao等[62]虽然也认同PSMα3的毒性不依赖于与靶蛋白或手性大分子的立体特异性结合，而是基于膜破裂的猜想，但认为其毒性源于可溶性而非纤维素样形式。淀粉样蛋白可以参与人类神经系统疾病(如帕金森病)的病理过程，但细菌中的PSMα3是否以纤维素样形式发挥作用仍未可知。
5 展望目前，关于SA毒力因子的研究主要集中于单一毒素的体外试验，这限制了对SA毒素在生物体内不同致病阶段的分泌情况、协同作用以及与宿主相互作用等重要致病机理的认识，因此，基于活体组织和体内试验的研究、毒素间的联合作用研究需要得到重视。另外，应在修饰改造已知抗原和筛选适合佐剂的基础上，继续探索对多谱系SA具有共同保护作用的抗原。
SA毒力因子与免疫逃避作用的深入研究，将进一步指出毒力因子与机体免疫的复杂相互作用，促进免疫细胞或分子过继转移[63]、抗体靶向的纳米抗菌剂[64]、免疫模拟设计细胞[65-66]等精准抗菌治疗研究的发展。SA在生物体中的免疫逃避机制也可能涉及局部微生物群间交流、宿主神经免疫通路[67]等，对于开发益生菌[68-69]、细菌素、抗菌肽等生物抗菌制剂以及用药策略方面有一定指导意义。
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