Progress 04/01/12 to 09/30/15
Outputs
Target Audience:Researchers, medical or veterinary doctors or agricultural workers involved in development of antibacterial strategies to combat human, animal or plant infections resulting or being agravated by biofilm formation. Biologists or engineers developing antibacterial strategies to combat biofilm fouling of surfaces in diverse engineering applications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Publications, two seminars at Michigan State University, two posters: one at Michigan Symposium of ASM in Ann Arbor, MI and one at the General ASM Meeting in 2013. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
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. It was confirmed by ultracentrifugation and by electron microscopy that pseudopili are protruding on the surface of the Vibrio cells. It was also confirmed that eps operon is regulated by c-di-GMP. the product of epsG gene was also detected in the extracellular matrix of biofilms by imunologocal tests with anti-EpsG antibodies. We postulate, therefore, that pseudopili are normal components of biofilms and they presumably help the cells to adher to the surfaces. We have shown that EpsG overexpression increases biofilm formation and mutation of epsG reduces biofilm formation. These observations lead us to hypothesize that EpsG pseudopili form a component of the biofilm matrix. An alternative model is that overexpression of EpsG and mutation of epsG alters secretion of an extracellular factor involved in biofilm formation. However, using a well characterized protease assay, we have determined that overproduction of EpsG, accomplished with the aid of epsG inserted in an overexpression vector, has no impact on secretion, even though this plasmid is able to complement mutations in epsG. Thus, our results suggest T2SS increases biofilm formation through the production of pseudopili and not alteration of secretion.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: 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 indicated 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. Changes/Problems: The establishment of the role of EpsG in biofilm formation was complicated by the discovery that other cellular proteins, that constitute the matrix of biofilm are cecreted by the T2SS. What opportunities for training and professional development has the project provided? Understanding of how T2SS helps form biofilms has an imprtant significance in developing methods to prevent biofilm formation. These can be anti-Eps antibodies or inhibitors that will serve as drugs against biofilm formation in medicine or industrial applications. How have the results been disseminated to communities of interest? Publications. (manuscript in preparation). What do you plan to do during the next reporting period to accomplish the goals? Understand whether EpsE functiones to initiate or to help promote the biofilm formation.
Impacts
What was accomplished under these goals?
It was confirmed by ultracentrifugation and by electron microscopy that pseudopili are protruding on the surface of the Vibrio cells. It was also confirmed that eps operon is regulated by c-di-GMP. the product of epsG gene was also detected in the extracellular matrix of biofilms by imunologocal tests with anti-EpsG antibodies. We postulate, therefore, that pseudopili are normal components of biofilms and they presumably help the cells to adher to the surfaces.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Researchers, medical or veterinary doctors or agricultural workers involved in development of antibacterial strategies to combat human, animal or plant infections resulting or being agravated by biofilm formation. Biologists or engineers developing antibacterial strategies to combat biofilm fouling of surfaces in diverse engineering applications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? 1. One graduate student is working on this project 2. Three undergraduate students were trained through the research of this project in the report periode. How have the results been disseminated to communities of interest? The results were reported in: 1. Two seminars at Michigan State University 2. Two posters. One at Michigan Symposium of ASM in Ann Arbor and one at the General ASM Meeting in 2013. 3. A manuscript is being prepared for publication in J. Bacteriol. What do you plan to do during the next reporting period to accomplish the goals? We intend to visualize the extracellular portions of the EpsG pseudopilus by cryo-electron-microscopy and by immuno-detection in a fraction sheared off the surface of the cells under different regulatory conditions.
Impacts
What was accomplished under these goals?
We have shown that EpsG overexpression increases biofilm formation and mutation of epsG reduces biofilm formation. These observations lead us to hypothesize that EpsG pseudopili form a component of the biofilm matrix. An alternative model is that overexpression of EpsG and mutation of epsG alters secretion of an extracellular factor involved in biofilm formation. However, using a well characterized protease assay, we have determined that overproduction of EpsG, accomplished with the aid of epsG inserted in an overexpression vector, has no impact on secretion, even though this plasmid is able to complement mutations in epsG. Thus, our results suggest T2SS increases biofilm formation through the production of pseudopili and not alteration of secretion
Publications
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Progress 01/01/12 to 12/31/12
Outputs
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. 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 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
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.
Publications
- Strozen, T. G., Stanley, H., Gu, Y., Boyd, J., Bagdasarian, M., Sandkvist, M. and . Howard, S. P. 2011. Role of the GspAB complex in secretin assembly and function of the Type II secretion system of Aeromonas and Vibrio species. J. Bacteriol 193:2322-2331.
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