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><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&lt;br/&gt; 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&amp;nbsp;&amp;mu;m mean particle size</td><td>Sigma</td><td>L0280</td><td>harmful;&lt;br/&gt; 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;&lt;br/&gt; 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&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - high glucose - With 4,500 mg/L glucose and sodium bicarbonate, without&amp;nbsp;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 -&lt;br/&gt; Solution stabilized, with 10,000 units penicillin and 10&amp;nbsp;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

9.3K 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

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

体外肠道宿主-微生物界面合成细菌联合的可行性评价

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

科研

生物化学

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

免疫与感染

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

自动、高通量检测细菌对宿主细胞的粘附

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

生物学

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

用于研究体外宿主-病原体相互作用的器官型组织模型系统

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

<meta charset="utf8"></head><body>测量各种细菌的迁移和生物膜形成

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

测量各种细菌的迁移和生物膜形成

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

仿生材料表征细菌宿主相互作用

摘要

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体外肠道宿主-微生物界面合成细菌联合的可行性评价

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单细胞物种间细菌相互作用的动力学可视化

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

自动、高通量检测细菌对宿主细胞的粘附

采用共培养，以检测化学介导的种间相互作用

用于研究体外宿主-病原体相互作用的器官型组织模型系统

测量各种细菌的迁移和生物膜形成

仿生材料表征细菌宿主相互作用

摘要

探索更多视频

体外肠道宿主-微生物界面合成细菌联合的可行性评价

牙龈卟啉单作为模式生物的评价厌氧细菌的相互作用与宿主细胞

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

微生物相互作用研究的高通量共培养测定

单细胞物种间细菌相互作用的动力学可视化

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

自动、高通量检测细菌对宿主细胞的粘附

采用共培养，以检测化学介导的种间相互作用

用于研究体外宿主-病原体相互作用的器官型组织模型系统

测量各种细菌的迁移和生物膜形成

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用