Citation Spotlight: BugBuster® Protein Extraction Reagent for Efficient Protein Extraction from Bacterial Pathogens
BugBuster® Protein Extraction Reagents are widely used for gentle extraction of Escherichia coli proteins in preparation for recombinant protein purification. However, they also have applications for bacterial species other than E. coli. In the following examples, researchers used BugBuster® Protein Extraction Reagent with microbes ranging from the opportunistic human pathogen Psuedomonas aeruginosa to Yersinia pestis, the pathogen responsible for bubonic plague. Downstream applications were diverse, including direct enzymatic assays, analysis of large multimeric complexes, and protein identification by mass spectrometry. These studies illustrate the versatility of BugBuster Protein Extraction Reagents for microbiological research.
Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa
Olga Zaborina1, Francois Lepine2, Gaoping Xiao3, Vesta Valuckaite4, Yimei Chen5, Terry Li6, Mae Ciancio4, Alex Zaborin1, Elaine O. Petrof4, Jerrold R. Turner7, Laurence G. Rahme3, Eugene Chang4, and John C. Alverdy1
2 Institut National de la Recherche Scientifique (INRS)–Institut Armand-Frappier, Laval, Quebec, Canada; 3 Department of Surgery, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA; and 1 Department of Surgery, 4 Department of Medicine, 5 Department of Biochemistry and Molecular Biology, 6 Department of Immunohistochemistry, and 7 Department of Pathology, Pritzker School of Medicine, University of Chicago, Chicago, IL, USA
The Gram-negative bacterium Psuedomonas aeruginosa is an opportunistic human pathogen, taking advantage of weakened host immunity to mount severe and often fatal infections. Like many bacterial pathogens, the hostdefensive armory of P. aeruginosa can be triggered by host- or pathogen-generated compounds that activate quorum sensing (QS) pathways. Since severe physiological stress in humans can cause the release of morphine-like compounds such as dynorphin, the authors hypothesized that endogenous opioids could influence QS in P. aeruginosa. Initial experiments showed that the κ-opioid receptor agonist U-50,488 stimulated production of the QS molecule pyocyanin (PCN) in P. aeruginosa PAO1. Precursors of PCN are synthesized by the pqsABCDE operon under control of the transcriptional activator MvfR. The authors used a series of pqsABCDE- and mvfR-lacZ reporter strains for rapid assessment. BugBuster® Master Mix was used to prepare protein extracts from cells treated or untreated with U-50,488, and extracts were assayed directly for β-galactosidase activity. Finding that U-50,488 did result in expression of pqsABCDE, the authors followed up with in vivo tests using mouse models. Ultimately, the researchers demonstrated that dynorphin produced in the host’s intestine leads to enhanced virulence of P. aeruginosa.
The full article in PLoS Pathogens, March 2007, 3(3): e35. https://doi.org/10.1371/journal.ppat.0030035
Identification and type III-dependent secretion of the Yersinia pestis insecticidal-like proteins
Inessa Gendlina1,2, Kiara G. Held1, Sara Schesser Bartra2, Byron M. Gallis3, Catalin E. Doneanu3, David R. Goodlett3, Gregory V. Plano2, and Carleen M. Collins1
1 Department of Microbiology and 3 Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA; 2 Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, USA.
Yersinia pestis, the causative agent of bubonic plague, is infamous amongst historians and clinical microbiologists alike. Transmission of Y. pestis from mammal to mammal occurs via fleas. When fleas are infected with Y. pestis, bacteria accumulate at the proventriculus between the esophagus and midgut, blocking off their digestive tract. Literally starving to death, the fleas attempt to regurgitate the blockage in an effort to feed - thereby spreading the pathogen. The authors noted the presence of Y. pestis genes (yitA, yitB, yitC, yipA, yipB, and the regulatory gene yitR) homologous to an insecticidal Toxin complex (Tc) occurring in another insect pathogen, Photorhabdus luminescens. In P. luminescens, Tc proteins form large multimeric complexes that damage the insect midgut epithelia. The authors used BugBuster® Protein Extraction Reagent to prepare protein extract from Y. pestis wild-type, yit, and yip deletion strains, and analyzed the extracts by native PAGE. Gentle extraction achieved with BugBuster® Protein Extraction Reagent facilitated the integrity of multimeric complexes. A large molecular-weight complex was absent in several deletion strains missing key members of the gene cluster. Subsequent Western blot analysis of complexes eluted from the gel samples of the parental Y. pestis strain indicated presence of YitA, YitB, and YitC in the complex. The authors made larger amounts of extract for preparative native PAGE, and tryptic digestions of proteins excised from the gel were analyzed by LC/ESI-MS/MS. Gendlina et al. confirmed presence of YitA, YitB, YitC, YipA, and YipB in the complex, and hypothesized that these proteins regulate pathogen transmission by affecting the flea infection process.
The full article in Molecular Microbiology, June 2007, 64(5): 1214-1227. https://doi.org/10.1111/j.1365-2958.2007.05729.x
A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenunated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen
Wen-Tssann Liu1, Hui-Ling Hsu1, Chung-Chih Liang1, Chuan-Chang Chuang1, Huang-Chi Lin1, and Yu-Tien Liu2
1 Institute of Preventative Medicine and 2 Institute of Microbiology and Immunology, National Defence Medical Center, Taipei, Taiwan
Liu et al. utilized Salmonella enterica for heterologous expression of Yersinia pestis proteins. In addition to causing bubonic plague, Y. pestis also causes pneumonic plague. The pneumonic form of the disease is spread by human-to-human contact, and the pathogen is of concern for its potential as a biological weapon. The currently available Y. pestis vaccine (killed whole-cell) does not provide adequate coverage for pneumonic plague. Pathogenic Salmonella serovar Typhimurium is a major cause of food poisoning; however, live attenunated strains have been used as carriers of heterologous antigens from viral, bacterial, or fungal pathogens for the purpose of immunization. In pilot studies, the authors used an attenuated strain of Salmonella enterica serovar Typhimurium expressing Y. pestis F1 or V antigen to immunize mice. After creating strains of S. enteria serovar Typhimurium harboring plasmids encoding secreted forms of Y. pestis F1 (strain X85MF1) or V (strain X85V) antigen, the authors used BugBuster® Protein Extraction Reagent to prepare cell lysates. Concentrated media fractions and cell pellet lysate samples were analyzed by Western blot using anti-F1 or anti-FV monoclonal antibodies. The authors showed that both strains expressed the antigens, and that they were secreted, with signal present in both the media samples and the cell pellet samples. This confirmation allowed them to proceed with mouse immunization studies. Immunization conferred 20–80% protection against lethal challenge with Y. pestis, depending on strain and immunization procedure.
The full article in FEMS Immunol. Med. Microbiol., Oct. 2007, 51(1):58-69. https://doi.org/10.1111/j.1574-695x.2007.00280.x