Requirements For Bacterial Colonization Of Animal Tissue

描述(申请人提供)：生物膜，基质相关细菌的有组织的聚集体，增强细菌在表面定居的能力，包括宿主组织。生物被膜中的细菌是大多数医院获得性感染的罪魁祸首，包括来自导管等医疗设备的感染，并对抗菌素表现出极大的耐药性，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生物膜和从生物膜中扩散的能力，但我们能够在实验室培养和自然动物感染模型中研究费氏弧菌生物膜形成的作用。我们已经证明，生物膜的形成是费氏弧菌启动其宿主乌贼Euprymna scolope共生定植的关键早期步骤。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一个18基因簇(SYP)及其调控因子。SYP簇包括多糖生物合成基因和几个新的调节因子。SYP的诱导增强了共生生物膜的形成和定植，而SYP的丧失则破坏了这两者。值得注意的是，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，这个模型为我们提供了一个例外的机会，通过对生物膜基因及其调节因子的遗传和生化分析，来开发和测试关于生物膜在真核宿主的细菌定植中所起作用的假设。到目前为止，我们已经发现了费斯切里杆菌用来控制生物膜形成的复杂调控电路，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，以及细菌对宿主的额外反应，是理解使费氏弧菌能够成功穿越自然屏障进行定居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宿主的反应，特别是询问对生物膜形成至关重要的SYP基因的诱导是如何调控的(目标1)。此外，我们还将询问新的反应调节因子SypE是如何控制生物膜的形成的，它(A)在生物膜的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，以及(C)可能通过信号转导级联反应控制(目标2)。最后，我们的证据表明，其他因素有助于生物膜的形成，因此我们建议确定这些因素，并确定它们在共生生物膜形成和定植中的作用(目标3)。在每个目标中，我们建议检查在体内观察到的表型与发生在原位的表型之间的相关性。这一强大的工具能够比较体内和原位的生物被膜表型，结合对其生物被膜形成能力受到复杂调控的模式生物的研究，有可能揭示出更传统的生物被膜形成模型尚未确定的环境特定控制的见解。与公共卫生相关：细菌细胞可以与自己和被称为生物膜的微生物群落中的其他细菌联系在一起，这些微生物群落对抗生素等抗微生物疗法表现出更强的耐药性。虽然实验室正在对生物膜进行深入的研究，但很少有模型可以将实验室中的生物膜形成与动物宿主中自然形成的生物膜进行比较。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中的生物膜与细菌与宿主相互作用过程中形成的生物膜之间的明显相关性，以及我们建议在这里进一步研究的复杂监管控制。

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.