The authors would like to thank members of the Krachler group for critical reading of the manuscript. DHS, DV and AMK were funded by the Biotechnology and Biological Sciences Research Council (BB/L007916/1), FA was funded by a Republic of Iraq Ministry of Higher Education and Scientific Research Scholarship and NPS was funded by a CONICYT Scholarship.

蛋白质1.化学偶联聚合物微球<ol><li>巯基胺定向耦合 注意：此协议是适用于包含蛋白质胺官能化聚合物珠粒，使用磺基琥珀酰亚胺4-（对-maleimidophenyl）丁酸酯（磺基SMPB），为交联剂（图1）的半胱氨酸偶联。 <img alt="图1" src="/files/ftp_upload/53400/53400fig1.jpg" /> 图1.交联用于蛋白质的定向硫醇-胺偶联到聚合物珠的策略。胺改性的聚苯乙烯珠粒激活与磺基SMPB。马来酰亚胺反应了免费的半胱氨酸以定向耦合蛋白珠。 <a href="https://www.jove.com/files/ftp_upload/53400/53400fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>制备试剂的： <ol><li>制备的PBS（100mM磷酸钠，150mM的氯化钠，pH值7.0）和高压釜中。 </l我&gt; <li>在即将使用前制备100倍的股票的TCEP（0.5M或287毫克/毫升在PBS中）（三（2-羧乙基）膦）。 </li><li>准备5倍的股票磺基SMPB（10毫米或4.58毫克/毫升卫生署2 O）在使用前。 </li><li>准备了10倍的库存（500毫米或88毫克/毫升在PBS，pH 7.0）中的半胱氨酸即将使用前。 </li></ol></li><li>珠激活： <ol><li>轻轻倒转混合珠悬浮液和所需珠悬浮液（例如，12ml）中的量转移到无菌1.5毫升管含1ml无菌PBS，pH为7.0。 </li><li>轻轻吸液管上下离心在离心（16,000 XG 2分钟）洗珠和沉淀。 </li><li>小心取出用吸管，弃去上清液。重悬沉淀珠在1ml新鲜无菌PBS，并重复洗涤步骤。重悬珠颗粒0.8毫升的PBS。 </li><li>加200毫升新制备的10mM磺基SMPB的，得到最终concent日粮2毫米。 </li><li>孵育珠悬浮液1小时，在25℃在旋转轮。 </li></ol></li><li>蛋白降低： <ol><li>在活化步骤中的潜伏期，制备该蛋白质为以下偶合步骤，以便可以立即加入到活化的珠子。 注意：虽然此还原步骤并不总是需要的GST（谷胱甘肽S-转移酶）融合蛋白，建议以确保高耦合效率。 <ol><li>检查蛋白质浓度，并调整到所需的偶联反应中的最终浓度。注意：6毫蛋白在PBS中，并以1毫升的体积，通常使用。 </li><li>添加TCEP库存，得到5mM的终浓度。孵育在室温30分钟的溶液中。 注意：反应混合物可直接用于下面的偶联反应。 </li><li>保留蛋白质溶液少量（几毫升），用于确定亲<html>蛋白浓度和耦合效率计算（见第2节）。 </li></ol></li></ol></li><li>蛋白质偶合步骤： <ol><li>沉淀活化珠通过离心（2分钟，16000×g离心在微量），并在1毫升新鲜无菌PBS洗涤沉淀一次。 </li><li>重悬沉淀制备的蛋白质溶液（如1毫升）中，以得到所需蛋白质浓度。 注意：在偶联步骤中的蛋白质浓度将取决于平均耦合效率（参见测定偶联效率的第2部分）和所需的耦合密度（如计算1.1.4.3）。效率约为85％，和所需的最终浓度5mM的在10倍的珠悬浮液，所以在偶联步骤蛋白质浓度应为约6毫米。 </li><li>使用下列公式计算的耦合密度： JPG“/&gt;其中 ρC偶联密度[蛋白质分子数/纳米2]， 蛋白质浓蛋白质浓度[毫克/毫升]， 蛋白质男女蛋白分子量[大] 珠浓珠浓度[珠数/ L] ð珠径[纳米2] 阿伏伽德罗数</li><li>使用耦合密度计算平均配体间距： <img alt="公式（2）" src="/files/ftp_upload/53400/53400eq2.jpg" /></li><li>孵育该蛋白质 - 珠悬浮液2小时，在25℃在旋转轮。 注意：有些在RT蛋白可能不稳定。在这些情况下，反应可在4℃CO / N。 </li><li>停用剩余的活化的基团上的小珠通过将半胱氨酸股票的50mM的终浓度和孵育悬浮液30分钟，在25℃在旋转轮。通过离心（2分钟16,000 XG在微量）沉淀珠。 </li><li>保留上清用于确定蛋白质浓度和耦合效率计算（见第2节）。 </li><li>在1ml与1ml的PBS重悬洗两次珠粒料新鲜的PBS，以得到最终产物。 注：上述协议通常给予1 ml的偶联蛋白质，在5mM的蛋白质终浓度，这可被用作一个10倍的库存为随后的实验（见第3节）。 </li><li>要继续协议第3条，工作与珠的股票为100毫升/毫升，或500纳米的蛋白质终浓度。注：一个很好的起点为这个再修改则e会是2×10 12珠/毫升，产生3×10 -4的蛋白质/ nm 2的平均密度耦合或57纳米的上的2毫米直径的珠的平均间距。 </li></ol></li></ol></li><li>巯基羧基定向耦合 注意：此协议是适于耦合含有蛋白质羧基官能化聚苯乙烯珠半胱氨酸。羧基部分第一次被激活使用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）/ N-羟基琥珀酰亚胺（NHS），胺改性，然后交联使用磺基SMPB（ 图2）。 <img alt="图2" src="/files/ftp_upload/53400/53400fig2.jpg" /> 图2.交联用于蛋白到聚合物珠的定向硫醇羧基偶联策略。羧基聚苯乙烯珠粒首先激活用EDC和改性NHS以形成半稳定NHS-酯。乙基二的后续胺耦合胺的结果在自由胺基，它可与磺基SMPB。马来酰亚胺激活，则珠可以免费半胱氨酸反应，定向耦合蛋白珠。 <a href="https://www.jove.com/files/ftp_upload/53400/53400fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>制备试剂的： <ol><li>制备的PBS（100mM磷酸钠，150mM的氯化钠，pH值7.0）和高压釜中。 </li><li>制备100倍的股票即将使用之前的TCEP（0.5M或287毫克/毫升在PBS中，）。 </li><li>准备了10倍的库存中（20mM，或4mg / ml，在PBS中）的EDC（1-乙基-3-（3-二甲基氨基丙基）碳二亚胺）在即将使用前。 </li><li>准备了10倍的库存（50毫或6毫克/毫升在PBS中）的NHS（N-羟基琥珀酰亚胺）在使用前立即。 </li><li>准备5倍的股票磺基SMPB（10毫米或4.58毫克/毫升卫生署2 O）在使用前。 </li><li>立即b制备半胱氨酸的10倍的库存（500毫米或88毫克/毫升在PBS，pH 7.0）中安伏使用。 </li></ol></li><li>珠激活： <ol><li>混合珠悬浮液轻轻倒转和所需珠悬浮液（例如，12ml）中的量转移到无菌1.5毫升管含1ml无菌PBS，pH为7.0。 </li><li>轻轻吸液管上下离心在离心（16,000 XG 2分钟）洗珠和沉淀。 </li><li>小心取出用吸管，弃去上清液。 </li><li>重悬沉淀珠在1ml新鲜无菌PBS，并重复洗涤步骤。 </li><li>重悬珠颗粒0.8毫升的PBS。 </li><li>加入100毫升10倍EDC股票（2毫米终浓度），并立即百毫升10倍NHS原液（5毫米终浓度）至珠悬浮液。 </li><li>孵育珠悬浮液30分钟，在25℃下在旋转的轮子。 </li><li>于0.8ml新鲜无菌PBS于1ml PBS中重悬的一次洗珠。 </li><li>加200 ml的乙二胺和增量乌瓦特珠悬浮液1小时，在25℃在旋转轮。 </li><li>用1ml的PBS洗涤一次珠子和重悬珠子在0.8毫升新鲜的PBS。 </li><li>从第1.1.2.4（珠活化硫代SMPB）根据第1.1.2所述继续并按第1.1（巯基胺定向耦合）描述的协议的其余部分。在相同的那些部分1.1中描述的方式执行的蛋白制剂与蛋白质偶联步骤。 注意：使用此协议的典型耦合效率略低相比第1.1节，因此调整初始蛋白质浓度相应地达到同样的耦合密度（约75％）。 </li></ol></li></ol></li></ol> 2.确定耦合效率注意：要确定蛋白质浓度，使用Bradford试剂10和比色分析如下： <ol><li>轻轻倒转Bradford试剂到e<html>试剂的nsure均匀性。 </li><li>使用10毫克/毫升BSA原液，制备蛋白质标准覆盖的浓度为0.1至1.5毫克/毫升BSA的缓冲液中。 </li><li>加入250 ml的布拉德福德试剂向96孔板的孔中。准备足够的井所有的样品，蛋白质的标准和阴性对照（只缓冲）。 </li><li>加入5 ml的蛋白质样品（蛋白质标准或缓冲器，用于阴性对照）在试剂中的96孔板中。 </li><li>孵育板在定轨摇床上在室温下进行10分钟。 </li><li>用酶标仪测定吸光度在595nm处。 </li><li>生成BSA浓度与A595纳米的标准曲线，并用它来确定初始和上清液样品中蛋白质的浓度。 </li><li>计算耦合蛋白的浓度如下： <img alt="公式3" src="/files/ftp_upload/53400/53400eq3.jpg" /></li><li>计算偶联效率为： 00eq4.jpg“/&gt; </li></ol>在竞争性试验3.用珠子耦合粘附的<ol><li>制备： <ol><li>种子每Hela细胞的孔1ml在浓度为150,000个细胞/毫升到24孔板的竞争性测定的前一天，以使细胞在实验开始前，达到约80％汇合。 </li><li>设置每种实验条件下，一式三份。包括井阴性对照（无竞争过程中加入检测细菌），阳性对照和裂解控件（毒性实验）（耦合到融合标签只能在竞争测定加控制珠）。 </li><li>接种5ml的海洋LB（MLB）文化与V的新的殖民地副溶血性弧菌和成长O / N在30℃下，抖。 </li><li>准备足够的珠子耦合MAM，如第1（耦合）来描述​​。允许100毫升10倍珠子库存每孔。 </li></ol></li><li>比赛分析： <ol><li>在competit的天离子实验，测量细菌培养物的OD 600。 </li><li>由不含添加剂稀释细菌培养成无色的DMEM制备感染媒体，预先加热到37℃，含10％v / v的珠悬浮液（或者粘附偶联珠或对照珠），以得到10.以MOI制备1 ml的/孔和每个样品10-20％过量体积。注意：对于上面提到的条件（24孔板， 副溶血性弧菌 ，MOI 10），O / N培养物的必要量，以每孔（毫升/毫升）计算为3 / OD 600的培养物加入。例如，1毫升感染培养基中通常含有100个毫升珠粒悬浮液中，几毫升细菌培养物，并补足到1毫升无色，未补充的DMEM。 </li><li>从井除去旧培养基，并通过添加1毫升无菌PBS预温至37℃至各孔洗涤培养的Hela细胞。 </li><li>删除PBS和每孔加入1毫升传染媒介。同时设立对照，加入解续癌宁含有0.1％的Triton X-100（裂解控制，仅需要细胞毒性测量）控制珠子和细菌（阳性对照）或粘附素珠和无细菌（负对照），或DMEM。 </li><li>孵育板在组织培养培养箱中于37℃所需的时间量（例如，4小时的细胞毒性测量或1小时为粘附测量）。 注意：两个细菌粘附和细胞毒性上的宿主细胞可以被用作读数抑制的效力。如果细胞毒性和粘附测量时间点重合，这两个测定法可以使用​​的样品来自相同井进行，因为细胞毒性，使用培养物上清和附着测定使用从剩余的细胞层来源的样品来确定。 </li></ol></li><li>细胞毒性测试： <ol><li>在指定的时间点（例如，4小时后的感染），从每个24孔并转移到除去三次200毫升一个96孔板中。 </li><li>旋转96孔板在1500×g离心5分钟，并转移100从每毫升早已进入一个新的96孔板中。加入100 ml期间感染实验的96孔板中一式三份的孔中新鲜使用的媒体的（这些将被用作空白）。 </li><li>进行使用LDH细胞毒性检测试剂盒，并按照制造商的说明乳酸脱氢酶（LDH）释放测定。 <ol><li>简要地说，计算所需的25增量试剂的量（例如，如果62样品被测量，使足够的试剂混合物75等）。 </li><li>例如，对于100个样本，混合11.25毫升试剂A的250微升试剂B.反转，不要旋涡，以避免起泡。 </li><li>把该混合物在一个容器，以便能够移液用多通道移液器。加入100微升的试剂混合物的每个样品。 </li><li>孵育板在RT和读取吸光度上酶标仪490nm处为10，20，30分钟。 </l我&gt; <li>分析的量，裂解对照样品的吸收是高，但仍是板读数器（典型地，2-3个吸收单位）的线性范围内的数据集。 </li><li>表述结果％的细胞毒性，使用下列公式进行转换： <img alt="公式5" src="/files/ftp_upload/53400/53400eq5.jpg" /></li></ol></li></ol></li><li>测量细菌粘附的： <ol><li>在指定的时间点（例如，1小时后感染），从细胞层中取出介质。 </li><li>彻底无菌的，预温热的PBS（至少3-4次洗涤的1ml的PBS各）洗涤细胞层，以除去任何未附着的细胞。 </li><li>裂解宿主细胞加入1ml无菌1％体积/体积的Triton X-100溶液的PBS每孔。孵育板在37℃下5分钟。 </li><li>吸取各样品上下数次转印每个内容以及以分离1.5 ml的管之前。制备样品的10倍系列稀释成无菌PBS（例如，使用100个ml样品和900毫升的PBS）。 </li><li>板100 ml的上MLB琼脂每一样品和散布使用细胞吊具。优化该稀释液根据细菌菌株和时间点到板。注意：对于所描述的实验设置，10 5或10 6倍稀释液得到的CFU的一个适当的数目。 </li><li>孵育板在37°CO / N和菌落计数列举细菌。 </li></ol></li></ol>

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	<li>Lim, J., Stones, D. H., Hawley, C. A., Watson, C. A., Krachler, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivalent+Adhesion+Molecule+7+clusters+act+as+signaling+platform+for+host+cellular+GTPase+activation+and+facilitate+epithelial+barrier+dysfunction.">Multivalent Adhesion Molecule 7 clusters act as signaling platform for host cellular GTPase activation and facilitate epithelial barrier dysfunction.</a> PLoS Pathog. 10 (9), e1004421 (2014).</li><li>Krachler, A. M., Ham, H., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+adhesion+factor+multivalent+adhesion+molecule+7+initiates+host+cell+binding+during+infection+by+gram-negative+pathogens.">Outer membrane adhesion factor multivalent adhesion molecule 7 initiates host cell binding during infection by gram-negative pathogens.</a> Proc Natl Acad Sci USA. 108 (28), 11614-11619 (2011).</li><li>Krachler, A. M., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+the+interaction+between+bacterial+adhesin+multivalent+adhesion+molecule+7+(MAM7)+protein+and+its+host+cell+ligands.">Functional characterization of the interaction between bacterial adhesin multivalent adhesion molecule 7 (MAM7) protein and its host cell ligands.</a> J Biol Chem. 286 (45), 38939-38947 (2011).</li><li>Joh, D., Speziale, P., Gurusiddappa, S., Manor, J., Hook, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+specificities+of+the+staphylococcal+and+streptococcal+fibronectin-binding+microbial+surface+components+recognizing+adhesive+matrix+molecules.">Multiple specificities of the staphylococcal and streptococcal fibronectin-binding microbial surface components recognizing adhesive matrix molecules.</a> Eur J Biochem. 258 (2), 897-905 (1998).</li><li>Bingham, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+fibronectin-binding+sites+from+Staphylococcus+aureus+FnBPA+in+complex+with+fibronectin+domains.">Crystal structures of fibronectin-binding sites from Staphylococcus aureus FnBPA in complex with fibronectin domains.</a> Proc Natl Acad Sci U.S.A. 105 (34), 12254-12258 (2008).</li><li>Ensenberger, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interactions+between+F1+adhesin+of+Streptococcus+pyogenes+and+N-terminal+modules+of+fibronectin.">Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin.</a> J Biol Chem. 276 (38), 35606-35613 (2001).</li><li>Hawley, C. A., Watson, C. A., Orth, K., Krachler, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+MAM7+peptide-based+inhibitor+of+Staphylococcus+aureus+adhesion+does+not+interfere+with+in+vitro+host+cell+function.">A MAM7 peptide-based inhibitor of Staphylococcus aureus adhesion does not interfere with in vitro host cell function.</a> PLoS One. 8 (11), e81216 (2013).</li><li>Krachler, A. M., Ham, H., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnabout+is+fair+play:+use+of+the+bacterial+Multivalent+Adhesion+Molecule+7+as+an+antimicrobial+agent.">Turnabout is fair play: use of the bacterial Multivalent Adhesion Molecule 7 as an antimicrobial agent.</a> Virulence. 3 (1), 68-71 (2012).</li><li>Krachler, A. M., Mende, K., Murray, C., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+multivalent+adhesion+molecule+7-based+inhibition+of+multidrug-resistant+bacteria+isolated+from+wounded+military+personnel.">In vitro characterization of multivalent adhesion molecule 7-based inhibition of multidrug-resistant bacteria isolated from wounded military personnel.</a> Virulence. 3 (4), 389-399 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

The authors have nothing to disclose.

此，我们描述两个协议，可分别用于偶联含硫醇的蛋白质含胺或羧化物修饰的聚苯乙烯珠子。由于容易的程序，硫醇 - 胺偶联是优选的，但根据所期望的珠规范（直径，荧光性），使用胺官能化珠可能不是可能的，并且因此我们包括了一个协议，该协议将羧酸转换 - 到一个胺部分给研究者在选择支架的最大的灵活性。虽然这两个硫醇-胺和硫醇羧基耦合工作含有蛋白质，定向耦合任何半胱氨酸（即每其N-末端的蛋白质，模仿表面显示的固定化）需要无半胱氨酸残基的蛋白质，即制造作为GST融合蛋白质，或包含由现场引入单个末端半胱氨酸残定向诱变。如果有多个无功半胱氨酸载蛋白质内，这将导致随机的固定化，可能妨碍蛋白质的功能。许多细菌粘附不含有半胱氨酸自然。对于其他人，这也可以通过位点定向诱变除去，虽然这将需要大量的测定，以确保本机的结构和功能被保留在突变体。对于GST融合蛋白，纯化的GST-标签耦合到珠可作为一个合适的阴性对照。使用未偶联的珠子作为对照应该避免，因为这些通常具有更高的倾向，聚集在一起或粘附细胞非特异性。的简化版本的协议1.2，只用EDC，可以用来偶联蛋白质羧基官能化的聚合物珠，但在这种情况下，耦合发生经由在蛋白质的伯胺，因此，并不能保证定向耦合。 TCEP作为还原剂必须不替换为其它市售常用的还原剂，如dithiothreitol（DTT）或2-巯基乙醇（BME），作为其中包含的硫醇将在硫醇 - 马来酰亚胺偶联步骤与蛋白质偶联竞争。 PBS可以与其它缓冲液替换，但考虑下面的因素：缓冲器可能不包含伯胺（所谓的含Tris缓冲液不适合）。使用含非常低（&lt;10mM）的盐浓度的缓冲区导致珠聚集也应避免。蛋白质纯度也是在此过程中要考虑，而且，以实现高品质的数据的一个重要因素，纯蛋白应该被使用。我们经常纯化蛋白在多个步骤，其中包括至少一个亲和纯化和凝胶过滤步骤，但在某些情况下，离子交换层析是作为第三步骤。其结果是用于偶联蛋白质的纯度通常为90％或更高是，作为判断通过SDS-PAGE。 建议确定的蛋白浓度在初始反应混合物以及第反应结束后，É上清液。这将有助于确定珠偶联蛋白的表观浓度，并因此耦合密度。这两个值的判定也将允许耦合效率的计算。这可以在制备在随后的反应中的起始蛋白溶液，以达到所需的最终浓度和偶联密度加以考虑。 Bradford试剂特别适合用于测定蛋白质浓度的前和耦合反应之后，为none物质在反应干扰染料复合物形成在所使用的浓度。如果较低的蛋白质浓度将被使用，这种方法可能必须通过更灵敏的检测方法来代替，但是注意有要支付给它必须是与包含在偶合反应中的物质相容的事实。此外，还建议使用新鲜配制的试剂和处理粉状试剂小心（例如，存储在密封容器中，并使用二氧化硅珠，以避免图的水分的试剂），因为试剂将影响耦合效率的质量。如果偶联效率低于预期，可能的补救措施包括增加初始卷边和蛋白浓度。如果正在使用较高浓度，的偶合试剂的浓度必须按比例增加，以保证足够的摩尔过量。修改珠/蛋白浓度是朝着优化通常是一个更好的一步，而不是增加反应时间。由于胎圈连接的协议是漫长的，我们通常准备大批量的材料。该悬浮液的等分试样可被管理单元在液氮中冷冻并保存在-20℃下数月。解冻等分试样不应该再冷冻，并应保持在4℃和在1-2天内使用。然而，这将与用作最初应测试的小批量的蛋白质的性质和稳定性而有所不同。 副溶血性弧菌的能力，使用任一在细菌附着的降低的宿主细胞或降低的细胞毒性作为一读出。在这两种情况下，准备和竞争测定法遵循相同的协议。根据读出，不同V的菌株副溶血正在使用：细胞毒性菌株POR1用于细胞毒性测定，而对非细胞毒性菌株POR2用于测量细菌粘附，由于细胞死亡和细胞分离危及程序用于定量附着细菌。 最初，补偿装置天信实验设置为逐步的协议，其中，宿主细胞是第一预孵育，然后加入细菌的珠。对于V.溶血性弧菌和所使用的珠的规格（2微米珠耦合到MAM7），这两个小珠和细菌可以在同一时间加入，而不改变所得到的细胞毒性。 也就是说，在这个实验装置，珠outcompete细菌为宿主细胞结合。取决于所使用的细菌种类和胎圈几何形状，可能有很好的理由用于保持珠粘附和细菌感染作为两个独立的步骤。例如，感染细胞与非活动细菌，将板除了感染介质后通常离心。然而，应该避免含珠混悬液板离心，因为这导致了小珠的细胞层上的高度分布不均匀。如果使用更小的颗粒尺寸，珠将需要更长的时间来解决在细胞表面上，在这种情况下，苏fficient时间应该允许前感染微珠附着。当细菌粘附用作读出，样品应在时间点采取其中宿主细胞不显著由感染菌株损坏，如细胞脱离和溶解可损害附着细菌的定量。 代替通过稀释电镀列举细菌粘附的，样品可替代地处理用于成像（ 图5）。在这种情况下，组织培养细胞应接种到玻璃盖玻片，而不是直接进入孔中。此外，荧光珠和细菌表达的荧光蛋白可以被使用，随着感染特定的宿主细胞的标记。例如，竞争实验通常使用荧光红色罗丹明 - 鬼笔染色宿主细胞的肌动蛋白细胞骨架和荧光蓝色的珠子和荧光评估由感染引起，形态变化以及成像绿色（GFP表达或SYTO18染色）的细菌。 的珠偶联粘附素， 包括金黄色葡萄球菌FnBPA，酿脓链球菌的F1 FUD和副溶血性弧菌 MAM甲范围，已被用作仿生材料，研究粘附，粘附抑制和附着于贡献病原体介导的细胞毒性1，7，8。一种使用这种方法的优点是易于附着事件的可视化的，因为是在很宽的范围的颜色（例如，蓝色，荧光红，蓝，绿，橙）可用作支架的聚合物珠。因此，直接蛋白质标记，其可以与功能干扰，能够避免。此外，表面耦合模仿在细菌表面粘附的多价展示，从而反映了多种生理上相关的构象相比可溶性蛋白。 相比于使用完整的细菌或bacteri研究人突变，珠办法规避与细菌生长相关的问题。例如，长期（例如，O / N）细菌粘附到主机使用完整的细菌细胞的研究是经常会影响由伴随细菌生长现象-生长培养基和营养物耗尽的酸化的宿主细胞产生负面影响，和细菌复制最终危及成像质量。 最近，利用珠耦合黏附已扩大到包括其作为对参与信号细菌附着的下游过程宿主细胞因子亲和纯化工具的使用。V.副溶血 MAM7，通过结合在宿主细胞膜磷脂酸，触发器RhoA的活性和肌动蛋白的重排1，3。MAM偶联珠被用于纯化和鉴定参与组装为MAM宿主细胞的结果的信令平台蛋白结合。由于珠可很容易地从上清液分离通过短的离心步骤，和感兴趣的蛋白质共价偶联，这是一个很好的方法，实现了从污染蛋白质分离和富集相关蛋白复合物，它可用于下游应用如蛋白质组学或西印迹。

Dissecting the interactions between bacterial adhesins and host surface receptors at the host-pathogen interface is an essential step towards our understanding of the underlying mechanisms driving bacterial adhesion. Ultimately, this will help us to identify strategies to interfere with these processes during infections. In conducting such studies, we often face a dilemma: Biochemical and biophysical analysis of the molecular mechanisms of adhesin binding requires their separation from other cell wall components, which may interfere with adhesion events. On the other hand, the use of recombinant soluble proteins over-simplifies the adhesion event, by disregarding protein anchoring in the bacterial cell wall and multivalency of binding achieved through bacterial surface display. Equally, from the host cell&#8217;s perspective, encountering and binding to single adhesin molecules does not always have the same impact in terms of plasma membrane organization, membrane fluidity and receptor clustering1 and this, ultimately, makes it difficult to evaluate the impact of adhesion on host cellular signaling and the outcome of bacteria-host interactions. 
Recently a method was devised, whereby recombinant purified adhesins or adhesin fragments are directionally and covalently coupled to polymer particles similar in size to bacteria, thus mimicking bacterial surface display. This approach has been used on a range of different adhesins, from Gram-negative Multivalent Adhesion Molecules (MAMs)2, 3, to Gram-positive adhesins including Staphylococcus aureus fibronectin binding protein (FnBPA)4, 5 and Streptococcus pyogenes F1 6, 7. This method has allowed the dissection of adhesin fragments important for host cell binding, identification of host surface receptors and determination of their behavior on the host cell surface, while taking both binding affinity and avidity into consideration 1, 8. Additionally, this approach has been used to investigate the efficacy of immobilized adhesins and derivatives as inhibitors of host-pathogen interactions 8, 9. It has been demonstrated that surface coupled derivatives of the Vibrio parahaemolyticus Multivalent Adhesion Molecule (MAM) 7 can be used to attenuate a range of bacterial infections in vitro, including those caused by multidrug-resistant pathogens such as Acinetobacter baumannii and methicillin-resistant S. aureus (MRSA) 7, 9. 
Herein, we describe different chemistries which can be used to immobilize adhesins to commercially available functionalized polystyrene particles. Due to the wide array of available surface functionalities, labels and particle sizes, these are a useful scaffold for the production of bacteriomimetic materials to investigate adhesin-host interactions. We further describe methods for the initial characterization of the coupling reaction, and for the calculation of important properties of the resulting materials. Finally, the use of bead-coupled adhesins as competitive inhibitors of in vitro bacterial infections is discussed as an example of their application, as well as the future scope and limitations of this approach.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td>Sigma</td>
			<td>646547</td>
			<td>Danger; corrosive 
			can be bought as solution or freshly prepared</td>
		</tr>
		<tr>
			<td>Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB)</td>
			<td>Fisher</td>
			<td>PN22317</td>
			<td>Danger; toxic</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>30089</td>
			<td>Danger; toxic</td>
		</tr>
		<tr>
			<td>Latex beads, amine-modified polystyrene, fluorescent blue - aqueous suspension, 2.0&#160;&#956;m mean particle size</td>
			<td>Sigma</td>
			<td>L0280</td>
			<td>harmful; 
			a range of alternative particle sizes and colors is available</td>
		</tr>
		<tr>
			<td>1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)</td>
			<td>Thermo scientific</td>
			<td>22980</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Thermo scientific</td>
			<td>24500</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylate-modified polystyrene beads</td>
			<td>Sigma</td>
			<td>CLB9</td>
			<td>harmful; 
			a range of alternative particle sizes and colors is available</td>
		</tr>
		<tr>
			<td>Ethylenediamine</td>
			<td>Sigma</td>
			<td>E26266</td>
			<td>Danger; flammable, corrosive, toxic, sensitizing</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Promega</td>
			<td>W3841</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Sigma</td>
			<td>B6916</td>
			<td>Danger; corrosive and toxic</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose - With 4500 mg/L glucose and sodium bicarbonate, without&#160;L-glutamine, sodium pyruvate, and phenol red, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>D1145</td>
			<td>for infection experiments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td>for maintenance of cultured host cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, heat inactivated</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td>supplement for tissue culture medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin - 
			Solution stabilized, with 10,000 units penicillin and 10&#160;mg streptomycin/mL, sterile-filtered, BioReagent, suitable for cell culture</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td>supplement for tissue culture medium</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH Cytotoxicity detection kit</td>
			<td>Takara</td>
			<td>MK401</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>reagent reservoirs (25ml)</td>
			<td>SLS</td>
			<td>F267660</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Greiner</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greiner</td>
			<td>655182</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>haemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plate reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tissue culture facilities</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>multichannel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

biomimetic materials to characterize bacteria-host interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

该五副溶血性弧菌黏附MAM7包含七个串联哺乳动物细胞进入（MCE）参与识别主机表面受体的结构域。我们使用耦合到重组聚苯乙烯珠，纯化片段包含或者所有七个串联MCE域（MAM7）或只有第一MCE域（MAM1）中测试这些材料与五竞争的能力副溶血弧菌为宿主细胞结合，将所得这些材料作为粘附抑制剂的功效。 Hela细胞感染了五 4小时（图3）之后副溶血性弧菌菌株POR1，和细胞毒性从体外感染引起进行评价。 Hela细胞用0.1％的Triton X-100（阳性裂解对照）治疗导致完整细胞裂解，显示未感染细胞的细胞毒性的非常低的水平。 在体外感染未处理的细胞与POR1的导致非常高水平的细胞裂解，而这被抑制MAM7共upled珠但不MAM1耦合或GST-耦合控制珠（ 图3）。 <img alt="图3" src="/files/ftp_upload/53400/53400fig3.jpg" /> 在竞争分析副溶血性弧菌图3.鉴定诱导的细胞毒性的上皮细胞，细胞用V.治疗副溶血性弧菌 （VP），标有（+），或无细菌（ - ）在不同的竞争实体（化合物）的存在所指示。这些措施包括或者没有补偿。 （ - ），MAM7珠（MAM7），MAM1珠（MAM1）或GST对照珠（GST）。作为对照，将细胞治疗要么用Triton X-100（TRITON，裂解对照），或不感染（uninf）。如在第3节中所述4小时后LDH释放测定和结果标准化为对照的Triton（100％）和空白（0％），如上所述。结果是平均值±SEM从一式三份实验。 <a href="https://www.jove.com/files/ftp_upload/53400/53400fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 五，枚举副溶血性弧菌附着于未处理HeLa细胞，或孵育MAM7-，MAM1-，或GST控制珠，细胞透露，MAM7珠而不是MAM1-或GST-控制珠outcompete V.副溶血用于附着到宿主细胞表面受体（图4）。 <img alt="图4" src="/files/ftp_upload/53400/53400fig4.jpg" /> 细菌附着的过程中竞争实验图4表征。Hela细胞感染了五副溶血 POR2（VP +），在不存在（ - ）或存在竞争实体如下：MAM7珠（MAM7），MAM1珠（MAM1）或GST对照珠（GST）（化合物）。细菌粘附测定1小时后，如<html>在第3所述的结果平均值±SEM从一式三份实验。手段（FLTR在CFU /毫升）为2.3010 6，1.5310 5，2.0510 6 2.1210 6。 <a href="https://www.jove.com/files/ftp_upload/53400/53400fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 粘附耦合至荧光团标记珠粒导致模仿细菌粘附于宿主细胞并用于细胞成像（ 图5）的有力工具材料的产生。使用MAM7偶联荧光蓝色珠，我们特征MAM7介导的附着的上皮细胞的过程。 MAM7的附着宿主细胞导致肌动蛋白和重排形成应力纤维，其中共可视使用罗丹明-鬼笔环肽染色的F-肌动蛋白（图5B）的。 _upload / 53400 / 53400fig5.jpg“/&gt; 的MAM7耦合珠Hela细胞成像的目的标记图5.编制和使用荧光的仿生珠。（A）暂停荧光蓝色仿生珠（右管，10倍的股票）和缓冲控制（左管）。（B）附件细胞导致肌动蛋白重排和应力纤维的形成。荧光蓝色的珠子附件和产生的应力纤维（红色，沾罗丹明鬼笔）由显微镜成像。酒吧，10微米。在B组图片进行了调整，从林等人 1并根据知识共享署名许可转载。 <a href="https://www.jove.com/files/ftp_upload/53400/53400fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Biomimetic Materials to Characterize Bacteria-host Interactions at JoVE.com

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

biomimetic-materials-to-characterize-bacteria-host-interactions

Research

JoVE Journal

Immunology and Infection

9.2K 次观看.  University of Birmingham. Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors. 

视频: Biomimetic Materials to Characterize Bacteria-host Interactions

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

使用γ-干扰素酶酶联免疫检测的特点人类乳头状瘤病毒的新型T-细胞表位

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

科研

免疫与感染

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学：生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...