A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.
A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.
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DOI：
10.1371/journal.ppat.1001102
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发表时间：
2010-09-16
期刊：
影响因子：
6.7
通讯作者：
Schoolnik GK
中科院分区：
文献类型：
--
作者：
Nielsen AT;Dolganov NA;Rasmussen T;Otto G;Miller MC;Felt SA;Torreilles S;Schoolnik GK
在宿主-病原体相互作用中一个基本但未回答的问题是感染期间毒力基因表达的时间、定位和群体分布。在这里，微阵列和在原位单细胞表达方法被用来研究霍乱弧菌的生长和毒力基因的表达在感染的兔结扎回肠袢模型的霍乱。编码毒素协同调节菌毛（TCP）和霍乱毒素（CT）的基因在邻近上皮表面的细菌感染过程的早期就强烈表达。发现增加的生长与毒力基因表达共定位。TCP的重复亚基tcpA的表达的显着异质性，观察感染过程的后期。tcpA的表达，研究在单细胞中的均匀介质中，证明单峰诱导tcpA后，加入碳酸氢盐，毒力基因表达的化学诱导剂。在进入稳定期的过程中发生了引人注目的人口分叉：一个亚群继续表达tcpA，而在其他亚群的表达下降。ctxA，编码CT的A亚基，和toxT，编码毒力基因表达的近端主调节因子也表现出分叉表型。发现分叉表型是可逆的，表观遗传的，并且在去除碳酸氢盐后持续存在，这些特征与双稳态开关一致。该开关需要正反馈回路控制ToxT表达和CRP-cAMP复合物在进入稳定期期间的形成。在体内也证明了这种tcpA开关的关键特征，在感染后期的腔液中观察到tcpA表达的显著异质性。当这种液体被稀释到人工海水中时，细菌聚集体持续表达tcpA很长一段时间。这种对毒力基因表达的控制指向了一种机制，这种机制可以产生一个霍乱弧菌亚群，该亚群继续在霍乱患者的米水粪便中产生TCP和CT。大多数病原微生物以逐步的方式感染：宿主表面的定殖之后是宿主组织的入侵和损伤，并且在感染过程的后期，发生向其他宿主的传播。在宿主体内停留期间，病原体产生重要的毒力决定因子，并经常迅速复制，导致其生物量的巨大扩张。虽然这种情况也是建立在霍乱弧菌，一个潜在的致命的肠道疾病的原因，它以前没有能够准确地确定何时或何地在肠道中产生的毒力决定因素。我们通过研究单个霍乱弧菌在小肠感染过程中毒力基因的表达来解决这个问题。发现毒力基因在感染过程的早期被靠近上皮表面的细菌强烈表达。增加的复制率也局限于上皮表面。在感染的后期阶段，霍乱弧菌群体分为两个部分：一个亚群继续表达毒力基因，而这些基因在另一个亚群中被沉默。控制毒力基因持续产生的遗传程序可能介导霍乱患者粪便中高感染性细菌亚群的持续存在。
A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients. Most pathogenic microorganisms infect in a stepwise manner: colonization of host surfaces is followed by invasion and injury of host tissues and, late in the infectious process, dissemination to other hosts occurs. During its residence in the host, the pathogen produces essential virulence determinants and often replicates rapidly, leading to a vast expansion of its biomass. Although this scenario is well established also for Vibrio cholerae, the cause of a potentially fatal diarrheal illness, it has not previously been possible to identify precisely when or where virulence determinants are produced in the intestine. We addressed this question by investigating the expression of virulence genes by individual V. cholerae during infection of the small intestine. Virulence genes were found to be powerfully expressed early in the infectious process by bacteria in close proximity to epithelial surfaces. Increased replication rates were also localized to epithelial surfaces. During later stages of the infection, the population of V. cholerae bifurcates into two fractions: one subpopulation continues to express virulence genes, whereas these genes are silenced in the other subpopulation. The genetic program controlling the continued production of virulence genes may mediate the persistence of a hyper-infectious subpopulation of bacteria in the stools of cholera patients.
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