Researchers in China have used proteomic technologies to identify six proteins that may play key roles in making Escherichia coli resistant to streptomycin, a discovery that may shed additional light on antibiotic resistance and offer hints on how to develop new drugs that can avoid these defenses.
In recent years, drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, or MRSA, have grabbed headlines with reports of small but fatal community outbreaks.
In a 2005 analysis, the Centers for Disease Control and Prevention found that more than 94,000 people in the US came down with a “serious” MRSA infection in 2005, and nearly 19,000 died from it that year, according to a report published last year in the Journal of the American Medical Association.
While drug companies are working to develop new treatments, the relative little return on investment in developing new antibiotics, even those that thwart resistance, means that there are no solutions in sight.
But using 2D gel electrophoresis followed by Western blotting, researchers at Sun Yat-Sen University in Guangzhou, China, have identified six proteins that were differentially expressed in streptomycin-resistant E. coli. Then by characterizing them, they were able to determine the proteins’ roles in streptomycin resistance. The research, which the researchers claim is the first of its kind, also identified an interaction network for the proteins.
In their work, the researchers focused on intrinsic antibiotic resistance and the role of outer membrane proteins. “The intrinsic resistance of gram-negative bacteria is largely dependent on the constitutive or inducible changes of active efflux system and porins, especially the changes at the level of protein expression of outer membrane proteins,” they write in an article describing their work, published Aug. 5 in the online edition of the Journal of Proteome Research.
They added that despite studies showing that the bacterial outer membrane subproteome rather than a single outer membrane proteome protein plays a role in intrinsic antibiotic resistance, “little is known about the key proteins and the interaction network of the altered [outer membrane] proteins.
“The questions we wanted to answer in the present study were, one, which [outer membrane] protein[s] may play a key role in [streptomycin resistance], and, two, how altered outer membrane proteins interacted with each other for the regulation of [streptomycin] resistance,” they said.
In an e-mail to ProteoMonitor, Xuan-Xian Peng, corresponding author of the study, said that in continuing research he and his colleagues are doing further 2D gel electrophoresis analysis of the network and investigating whether there are additional proteins that may be up- or down-regulated.
“The questions we wanted to answer in the present study were one, which [outer membrane] protein[s] may play a key role in [streptomycin] resistance, and two, how altered outer membrane proteins interacted with each other for the regulation of [streptomycin] resistance.”
“We are sure that down- and up-regulation of additional outer membrane proteins could be identified,” he said, but “the roles of these additional outer membrane proteins in the interaction network are not clear.”
He added that while his study looked at streptomycin and E. coli, the same strategy they used could be employed to other antibiotics and other pathogens.
For their work, they used E. coli K12 BW25113 and strains that had been modified by genetic deletion. They developed a method to study the effect of altered proteins on the antibiotic-resistant ability using the original E. coli that had been genetically deleted that were propagated in the medium with streptomycin resistance.
“This method was based on the hypothesis that absence of an antibiotic-resistant gene may result in change in antimicrobial susceptibility of the mutant strain,” the researchers wrote.
They used 2-DE to identify altered outer membrane proteins in the sarcosine-insoluble fraction of streptomycin-resistant E. coli, selected from the original E. coli strain. Of about 60 protein spots visualized, 14 were found to have “significant changes” in protein expression. The 14 spots corresponded to seven proteins including five that are outer membrane proteins, TolC, FadL, OmpT, OmpW, and LamB. One protein, Dps is a protein whose location is unknown, and one, AceA is a cytoplasm protein.
TolC, OmpT, and LamB were found to be up-regulated while FadL, OmpW, and Dps were down-regulated compared to the original E. coli strain. The results were then confirmed by Western blotting.
They then functionally characterized the six proteins and their interaction networks using gene-deleted E. coli strains with streptomycin resistance and their original strains.
Based on their analysis of the original strains, the researchers said that TolC, OmpT and Dps “might be functionally and directly linked to [streptomycin] resistance, and TolC and OmpT might positively and Dps negatively contribute to the regulation of [streptomycin] resistance.”
In the gene-deleted samples, they hypothesized based on their findings that FadL, TolC, and OmpT may play the most important roles in streptomycin resistance, but overall their results “suggest that the deletion of the six [outer membrane proteins] may result in [streptomycin] resistance,” and each may play important roles in how the bacterium regulates streptomycin resistance.
While previous studies have reported that TolC control efflux-mediated multi-drug resistance including streptomycin in Salmonella enterica serovar and Typhimurium DT104, this study is the first performing a functional characterization of the other five proteins they identified in the outer membrane as being differentially expressed in the streptomycin-resistant E. coli strain, according to the authors.
Other studies have indicated they are involved in other antibiotic resistance in E. coli, and their study indicates that TolC, OmpT, Dps, FadL are functionally linked to streptomycin resistance.
The authors also add that Dps was found to be a negative regulator in the network. “This novel mechanism of negative regulation may open new [avenues] to the control of antibiotic-resistant bacteria,” they said.
For the last part of their study, the researchers investigated whether an interaction network of the altered outer membrane proteins exists by isolating the membrane proteins and testing the effects of the absence of each altered outer membrane protein on the other five proteins. Their results, they said, suggest such a network does, in fact, exist.
For example, the up-regulation or deletion of TolC led to the elevation of Dps and OmpW. They note, however, that TolC was unaffected by the absence of the other five proteins, suggesting it may be an independent and upstream protein in the network. They also found that Dps was the only protein that interacted with all of the other proteins, suggesting that TolC and Dps may play central roles in the network.
“That TolC did not upregulate when other proteins were absent indicates that TolC pumped antibiotics out of these cells more independently,” Peng said in his e-mail. “Contrary to this, Dps is affected by most of the other proteins. Thus, it may play a transfer (conversion, switching) role in this response.”
He and his colleagues’ data suggests that OmpT and LamB may also play important roles in the regulation of other proteins in the network as they were related to four and five other proteins, respectively and both “might locate at the center of the network.
“The network identified clearly specifies and strengthens the roles and importance of the six altered [outer membrane] proteins in the regulation of [streptomycin] resistance,” the authors said, and their overall findings “may provide novel insights into the mechanism of [streptomycin] resistance in E. coli.”