Requirements For Bacterial Colonization Of Animal Tissue

描述（由申请人提供）：生物膜，基质相关细菌的组织聚集体，增强细菌在包括宿主组织在内的表面定植的能力。生物膜中的细菌是大多数医院获得性感染的原因，包括那些源于导管等医疗设备的感染，并且对抗微生物药物表现出显著增加的耐药性，从而降低了抗生素治疗的有效性。虽然目前正在研究许多细菌形成和分散生物膜的能力，但我们能够在实验室培养和自然感染动物模型中检查由费氏弧菌形成生物膜的作用。我们已经表明，生物膜的形成代表了V. fischeri在其宿主Euprymna scoles的共生定殖过程中开始的关键早期步骤。共生器官表面的生物膜样聚集和随后的定植都依赖于我们最近发现的18个基因簇（syp）及其调控因子。syp簇包括多糖生物合成基因和一些新的调控因子。syp的诱导促进了共生生物膜的形成和定植，而syp的缺失则破坏了这两者。引人注目的是，这些原位定植表型与实验室培养中容易观察到的生物膜表型密切相关。因此，该模型为我们提供了一个例外的机会，通过对该基因座及其调控因子的遗传和生化分析，来发展和测试关于生物膜在真核宿主细菌定植中的作用的假设。迄今为止，我们已经发现了费氏弧菌用于控制生物膜形成的复杂调控回路，包括激活剂和抑制剂，从而使其成为了解如何控制生物膜形成的丰富模型。控制生物膜的形成，以及细菌对其宿主的额外反应，是理解使费氏弧菌成功穿越自然屏障的发育变化的关键组成部分。因此，我们建议进一步探索费氏弧菌对其宿主的反应，特别是探究对生物膜形成至关重要的syp位点的诱导是如何被调节的（Aim 1）。此外，我们将询问新的反应调节因子SypE如何控制生物膜的形成，SypE (a)在生物膜形成中发挥积极和消极的作用，(b)预计表达丝氨酸激酶和磷酸酶活性，(c)可能通过信号转导级联控制（Aim 2）。最后，我们的证据表明其他因素有助于生物膜的形成，因此我们建议识别这些因素并确定它们在共生生物膜形成和定植中的作用（Aim 3）。在每个目标中，我们建议检查体内观察到的表型与原位发生的表型之间的相关性。这个强大的工具，比较体内和原位生物膜表型的能力，结合对生物膜形成能力受复杂调控的模式生物的研究，有可能揭示尚未被更传统的生物膜形成模型所识别的环境特异性控制。公共卫生相关性：细菌细胞可以与自身和微生物群落中的其他细菌结合，称为生物膜，这些细菌对抗生素等抗微生物疗法表现出更强的耐药性。虽然生物膜正在实验室中进行深入研究，但很少有模型可以将实验室中生物膜的形成与动物宿主中自然发生的生物膜形成进行比较。其中之一是我们的模型，即费氏弧菌与其鱿鱼宿主之间的共生关系，该模型揭示了实验室生物膜与细菌-宿主相互作用过程中形成的生物膜之间的明确相关性，以及我们建议在此进一步研究的复杂调控控制。

DESCRIPTION (provided by applicant): Biofilms, organized aggregates of matrix-associated bacteria, enhance the ability of bacteria to colonize surfaces, including host tissues. Bacteria in biofilms are responsible for the majority of hospital-acquired infections, including those stemming from medical devices such as catheters, and exhibit substantially increased resistance to anti-microbials, thus diminishing the effectiveness of antibiotic treatment. While numerous bacteria are currently being studied for their ability to form and disperse from biofilms, we are able to examine the role of biofilm formation by Vibrio fischeri both in laboratory culture and in a natural animal model of infection. We have shown that biofilm formation represents a critical early step during initiation by V. fischeri of symbiotic colonization of its host, the squid Euprymna scolopes. Both biofilm-like aggregation on the surface of the symbiotic organ and subsequent colonization depend upon an 18 gene cluster (syp) that we have recently discovered as well as its regulators. The syp cluster includes polysaccharide biosynthesis genes and several novel regulators. Induction of syp enhances symbiotic biofilm formation and colonization, while loss of syp disrupts both. Strikingly, these in situ colonization phenotypes are tightly correlated with biofilm phenotypes readily observable in laboratory culture. This model thus affords us an exception opportunity to develop and test hypotheses about the role of biofilms in bacterial colonization of a eukaryotic host through genetic and biochemical analysis of this locus and its regulators. To date, we have uncovered a complex regulatory circuitry used by V. fischeri to control biofilm formation, including both activators and inhibitors, thus making it a rich model for understanding how biofilm formation can be controlled. Control of biofilm formation, as well as additional responses of the bacterium to its host, are key components in understanding the developmental changes that enable V. fischeri to successfully navigate natural barriers to colonization. We therefore propose to further explore how V. fischeri responds to its host, and particularly ask how induction of the syp locus, essential for biofilm formation, is regulated (Aim 1). In addition, we will ask how biofilm formation is controlled by the novel response regulator SypE, which (a) plays both positive and negative roles in biofilm formation, (b) is predicted to express serine kinase and phosphatase activities, and (c) is likely controlled through a signal transduction cascade (Aim 2). Finally, our evidence suggests that other factors contribute to biofilm formation, and thus we propose to identify these factors and determine their roles in symbiotic biofilm formation and colonization (Aim 3). In each of the aims, we propose to examine the correlation between phenotypes observed in vivo with those that occur in situ. This powerful tool, the ability to compare in vivo and in situ biofilm phenotypes, combined with the study of a model organism whose biofilm formation capability is under complex regulatory control, has the potential to reveal insights into environment-specific control not yet identified by the more traditional models of biofilm formation. PUBLIC HEALTH RELEVANCE: Bacterial cells can associate with themselves and other bacteria in microbial communities called biofilms, which exhibit increased resistance to anti-microbial therapies such as antibiotics. While biofilms are being intensively studied in the laboratory, few models exist in which biofilm formation in the lab can be compared to those that occur naturally in an animal host. One of these is our model, the symbiosis between Vibrio fischeri and its squid host, which has revealed a clear correlation between biofilms in lab and those formed during bacteria-host interactions, as well as complex regulatory control that we propose to investigate further here.