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Our long-term goal is to understand the molecular mechanisms involved in biofilm formation in order to develop new strategies to prevent bacterial biofilms. The objective of this proposal is to determine the contribution of T2SS to biofilm formation in Gram-negative human and plant pathogens. <P>Our central-hypothesis is that c-di-GMP regulation of the T2SS operon leads to pseudopili production and increased biofilm formation. This hypothesis is based upon our preliminary results in V. cholerae showing that overproduction of T2SS pseudopili, containing the pseudopilin protein EpsG, increases biofilm formation, loss of T2SS abolishes biofilm formation, extracellular pseudopili are present in the biofilm matrix, and c-di-GMP induces expression of the genes encoding the T2SS. <P>To test our central-hypothesis, we propose the following three specific aims: <br/>Aim 1. Visualize T2SS pseudopili in V. cholerae biofilms. We have observed extracellular EpsG in WT biofilms of V. cholerae. To determine if this EpsG exists as extracellular pseudopili, we will specifically label and examine extracellular EpsG from biofilm grown bacteria using confocal and transmission electron microscopy. <br/>Aim 2. Characterize the regulatory mechanisms that lead to increase in EpsG production. We have determined that high levels of c-di-GMP promote pseudopili formation. Moreover, we have identified two promoters in the T2SS eps operon, one of which is induced by c-di-GMP. We will use RT-PCR and reporter gene expression to determine the regulatory elements that control expression of the eps genes and determine how this regulation impacts pseudopili production and biofilm formation. <br/>Aim 3. Examine if the role of the T2SS in biofilm formation is widely conserved. To explore if the contribution of T2SS pseudopili to biofilm formation is widespread and if c-di-GMP similarly regulates T2SS in other bacterial pathogens, we will examine the regulation of T2SS by c-di-GMP and the role of the T2SS in biofilm formation of Pseudomonas aeruginosa Pseudomonas syringae and Klebsiella pneumoniae. Although the role of T2SS in bacterial infection has long been appreciated, these systems are only thought to contribute to acute disease through secretion of extracellular virulence factors. Here, we propose experiments that will be the first to explore in depth the regulation of T2SS by c-di-GMP and its role in bacterial biofilm formation. Our collaborative team is well situated to address these questions as we have expertise in T2SS, c-di-GMP signaling, and biofilm formation. These innovative experiments will examine if T2SS pseudopili provide a structural component to bacterial biofilms. The initial studies will be carried out with V. cholerae as we have expertise, strains, and genetic tools available for this organism, but our ultimate goal is to determine if the contribution of T2SS to biofilm formation is widely conserved. We expect our results will uncover a novel mechanism important in bacterial biofilm formation, which could lead to new strategies to inhibit biofilm formation and bacterial disease.
Non-Technical Summary:<br/>
Biofilm formation by bacteria is a significant problem for human and animal health, for plant health and agriculture and even for industry. Infectious biofilms are responsible for chronic disease and contamination of artificial medical devices. Biofilms are difficult to treat because they are highly tolerant to antibiotic therapy and resist clearance by the host's cell immune system. They contribute to transmission of infections as evidenced by epidemics caused by enterohemorragic E. coli transmitted not only on meat products, but also on vegetables. They contribute to problems of many industries clogging pipes and other liquid transmission equipment. It is of critical importance, therefore, to understand the mechanisms and regulation of biofilm formation to be able to develop new strategies to disrupt this process. Several genetic and biochemical factors have been implicated in the process of biofilm formation. Our preliminary data have indicated that Type 2 Secretion System (T2SS) present in most Gram-negative bacteria is essential for this process. We have established that in our model organism, Vibrio cholerae, a component of T2SS, protein EpsG, forms extracellular appendages called pseudopili that are essential for formation of biofilms by these bacteria. We hypothesize that these pseudopili provide structure to the matrix, enabling biofilm formation. We hypothesize further that in many other Gram-negative bacteria T2SS contributes to biofilm formation by similar mechanisms. Here, we propose research to test this hypothesis and define the role of T2SS pseudopili in bacterial biofilm formation. We expect this research will uncover novel mechanisms by which bacteria form biofilms that can lead to the development of new strategies to disrupt this process.
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Approach:<br/>
Aim 1. Visualize T2SS pseudopili in V. cholerae biofilms. We will grow biofilms of WT, delta-epsG, epsG overexpression, delta-hapR, and qrgB overexpression in a flow cell system, label extracellular EpsG as described above, and image stained biofilms using an Laser Scanning Confocal Microscope. These experiments will determine if pseudopili structures are natural components of the V. cholerae biofilm matrix. Next these strains will be harvested from a biofilm and visualized by Transmission Electron Microscopy (TEM) through immune-gold labelling.
<br/>Aim 2. Characterize the regulatory mechanisms that lead to increase EpsG production. We will shear the external pseudopili from the intact cells by passage through a syringe needle, separate the pseudopili by differential centrifugation and determine the EpsG content by quantitative Western Blot. Comparison of cells wit a delta-epsG mutation, a delta-hapR mutation (repressor of c-di-GMP production) and overexpression of qrgB gene (production of c-di-GMP) will determine if these regulatory signals are involved in production of external pseudopili. We will compare production of mRNA between WT, the delta-hapR mutant, and the delta-flrA mutant. Both WT and the delta-flrA mutants will be examined at low and high c-di-GMP levels using qrgB overexpression. These data will be used to compare expression of each gene between the different strains as well as relative expression of the different genes within one strain. If these expression studies support our hypothesis, we will further test this hypothesis by generating an expression construct that encodes the genes epsG-N under the control of the Ptac promoter. We will express these genes at different levels in a delta-epsG-N deletion mutant and measure secretion, biofilm formation, and hyper-pseudopili production by TEM. We expect that different ratios of the genes at the start of the operon (epsC-F) versus the genes in the latter half of the operon (epsG-N) will promote external pseudopili production and increased biofilm formation.
<br/>Aim 3. Examine if the role of the T2SS in biofilm formation is widely conserved. We will determine if P. aeruginosa, P.syringae and K. pneumoniae produce pilus structures in the biofilm matrix as described above for V. cholerae. We will generate lux transcriptional fusions of the DNA sequences upstream of the epsG homolog and epsC homologs ofthese bacteria and examine if they exhibit transcription activity in their parent organisms. We will determine if these promoters are induced by c-di-GMP. This will be checked by quantification of intracellular c-di-GMP levels ba a technique routine in our laboratories. We have generated expression vectors for all 40 V. cholerae GGDEFs and know their relative activity in V. cholerae. This experiment will determine if c-di-GMP exerts transcription control of the T2SS in other bacteria and if these bacteria also encode the internal promoter located upstream of epsG. In addition, in-frame deletions of the orthologs of any regulatory proteins that we isolate in Section C3.2 from V. cholerae will be similarly checked for a role in the regulation of these promoters.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: Type 2 Secretion System (T2SS) of Vibrio cholerae is composed of 13 proteins 12 of which are encoded by contiguous genes on the chromosome I in the eps operon. Our results indicate that T2SS forms extracellular appendages, composed of the protein EpsG. These appendages, called pseudopili, are formed even if the epsG gene is expressed at wild type levels. These structures seem to be important for the formation of biofilm matrix components. We have also observed that production of extracellular pseudopili is a regulated process. Our results show that quorum sensing (QS), the process of cell-cell communication mediated by the secretion and detection of small chemical signals known as autoinducers, controls pseudopili formation. Similar, the second messenger signal cyclic di-GMP (c-di-GMP) induces pseudopili production. C-di-GMP is a newly appreciated second messenger signal that promotes a sessile, biofilm forming lifestyle and inhibits a planktonic, motile existence. C-di-GMP is widespread in bacteria as enzymes predicted to synthesize and degrade c-di-GMP are predicted in the genomes of 85% of all bacteria. In every organism that has been examined, c-di-GMP is essential for biofilm formation. Moreover, it is clear that c-di-GMP impacts a wide array of fundamental bacterial behaviors in addition to biofilm formation including cell cycle propagation, development, fimbriae synthesis, Type Three Secretion, RNA modulation, stress response, bacterial predation, and virulence. Overexpression of epsG led to increased biofilm formation whereas mutation of epsG dramatically reducd the ability of WT to form biofilms. This result showed that the T2SS is required for V. cholerae biofilm formation. Immunodetection showed the presence of EpsG in both extracellular matrix of biofilms and in extracellular substance sheared of cells. Moreover, we have determined that the production of extracellular EpsG is under c-di-GMP regulation. Our results added T2SSs to the growing list of bacterial behaviors modulated by c-di-GMP, and lead us to the hypothesis that c-di-GMP promotes T2SS pseudopili formation through regulation of the T2SS operon.
<br/>PARTICIPANTS: Group of Dr. M. Bagdasarian: 1. Michael Bagdasarian - Principal Investigator 2. Mira Bagdasarian - Associate Researcher 3. Andrew Smedley - Undergraduate Researcher Group of Dr. C.M. Waters: 1. C.M. Waters - Principal Investigator 2. R. Sloup - Graduate student 2. A. Konal - Undergraduate Researcher
<br/>TARGET AUDIENCES: Researchers involved in development of antibacterial strategies to combat human, animal or plant infections and antibacterial strategies to combat biofilm fouling of surfaces.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: Biofilm formation by bacteria is a significant problem for human and animal health, for plant health and agriculture and even for industry. Infectious biofilms are responsible for chronic disease and contamination of artificial medical devices. Biofilms are difficult to treat because they are highly tolerant to antibiotic therapy and resist clearance by the host's cell immune system. They contribute to transmission of infections as evidenced by epidemics caused by enterohemorragic E. coli transmitted not only on meat products, but also on vegetables. They contribute to problems of many industries clogging pipes and other liquid transmission equipment. It is of critical importance, therefore, to understand the mechanisms and regulation of biofilm formation to be able to develop new strategies to disrupt this process. Several genetic and biochemical factors have been implicated in the process of biofilm formation. Our results have indicated that Type 2 Secretion System (T2SS) present in most Gram-negative bacteria is essential for this process. We have established that in our model organism, Vibrio cholerae, a component of T2SS, protein EpsG, forms extracellular appendages called pseudopili that are essential for formation of biofilms by these bacteria. We hypothesize that these pseudopili provide structure to the matrix, enabling biofilm formation. We hypothesize further that in many other Gram-negative bacteria T2SS contributes to biofilm formation by similar mechanisms. These findings will help to design new therapeutic and preventive strategies to fight biofilm formation in medicine, agriculture and technology.