Discovery at the VICB
A Dual Signaling Pathway to Antibiotic Resistance
By: Carol A. Rouzer, VICB Communications
Published: January 18, 2017
Cooperation between two-component systems leads to polymixin B resistance in uropathogenic Escherichia coli
An alarming trend seen in recent years in the healthcare setting is the rise in multi-drug resistant Enterobacteriaceae. For example in the past year, there have been reports of uropathogenic Escherichia coli (UPEC) and Klebsiella pneumoniae that are resistant to almost all available antibiotics, including the cationic polypeptides polymixin B (PMB) and colistin. PMB and colistin bind to the bacterial membrane and act as detergents, disrupting membrane structure. Bacterial lipopolysaccharide (LPS) is an important structural component of the outer membrane of Gram-negative bacteria. In addition to its structural role, LPS helps bacteria resist the toxic effects of some antibiotics, such as PMB. In the majority of cases, bacteria gain resistance to these cationic polypeptides by acquiring genes that code for enzymes that modify the bacterial LPS and block the polypeptides from accessing the cell membrane. Now, Vanderbilt Institute of Chemical Biology member Maria Hadjifrangiskou and her laboratory reveal new evidence supporting the presence of an intrinsic mechanism in strains of UPEC, that provides temporary tolerance to PMB, via the interaction of two signaling pathways [K. R. Guckes, E. J. Breland, et al., (2017) Sci. Signal., 10, eaag1775].
An important mechanism by which bacteria sense and respond to their environment is through two-component system- (TCS)-mediated signaling. As their name implies, TCSs comprise a minimum of two key proteins, a membrane-bound histidine kinase receptor and a response regulator. Upon detecting a specific signal, the histidine kinase dimer autophosphorylates a conserved histidine residue in its cytoplasmic, C-terminal region. It then serves as a phospho-donor, transferring the phosphate group to an aspartate residue of the response regulator protein. In most cases, phosphorylation of the response regulator leads to dimerization and unmasking of DNA binding ability, enabling the response regulator to act as a transcription factor. The histidine kinases of bacterial TCSs are notably different from mammalian kinases, in that they can also act as phosphatases, providing a mechanism for down-regulation of the TCS-mediated signaling cascade. Most research on TCSs has focused on signaling that is restricted to interactions between the histidine kinase and its cognate response regulator. However, increasing evidence suggests that cross-talk can occur between components of two separate TCSs.
QseBC is a well-known TCS that responds to the mammalian hormones epinephrine and norepinephrine as well as autoinducer-3, a signaling molecule that bacteria use to communicate with each other. The key components of QseBC are the histidine kinase QseC and the response regulator QseB (Figure 1). Prior work in UPEC had shown that deletion of the gene encoding QseC (qseC) led to aberrant phosphorylation of QseB by the histidine kinase PmrB of the PmrAB TCS. The result was dysregulation of gene expression in the bacteria, leading to a loss of virulence. If the gene encoding PmrB (pmrB) was also deleted in these bacteria, the aberrant gene expression returned to normal. Researchers hypothesized that the phosphatase activity of QseC may be required to prevent abnormal levels of QseB phosphorylation. Whether or not this is true, however, these findings clearly indicated that cross-talk can occur between the PmrAB and QseBC TCSs. Further evidence of this interaction came from experiments showing that the PmrAB response regulator, PmrA, can bind to the promoter of the qseBC operon.
FIGURE 1. Simplified outline of the QseBC two-component system. A stimulus [in this case autoinducer 3, norepinephrine, or epinephrine (gray circles)] interacts with QseC, leading to autophosphorylation of a conserved histidine residue. The phosphate is then transferred to an aspartate residue on QseB, which dimerizes and acts as a transcription factor to modulate the expression of target genes. Figure reprinted by permission from Macmillan Publishers Ltd from N. E. Kimes, et al. (2012) The ISME Journal, 6, 835. Copyright 2012.
These initial findings led the Hadjifrangiskou laboratory to more thoroughly explore the possible physiological consequences of cross-talk between the two TCSs. Knowing that the PmrB histidine kinase can respond to ferric ion, they started their studies by examining the effects of ferric ion exposure on the expression of the qseBC operon, which is self-regulated by QseB. They found that, indeed, ferric ion caused a marked and rapid induction of qseBC expression in the wild-type UPEC clinical isolate UTI89. They further showed that this response was essentially eliminated by deletion of the genes for any single component of either the QseBC or PmrAB TCS. These results supported the hypothesis that ferric ion acts to regulate qseBC gene expression by a mechanism that requires an interaction between the two TCSs.
The failure of epinephrine to alter the response of qseBC expression to ferric ion suggested that the histidine kinase most likely responsible for initiating the response was PmrB. To test this hypothesis, the investigators examined the ability of PmrB to phosphorylate QseB and PmrA in response to ferric ion in vitro. They found that both response regulators were subject to PmrB-mediated phosphorylation and that ferric ion markedly stimulated the reaction in each case.
The expression of the YibD glycosyltransferase, which mediates LPS modification in response to cationic polypeptide stress, is known to be regulated by exposure to ferric ion in some bacteria. This led the investigators to test the hypothesis that QseBC and PrmAB might cooperate to regulate ferric ion-stimulated yibD gene expression in UPEC. They confirmed that exposure to the ion caused a surge in yibD expression in UTI89 and that the surge was abolished by deletion of pmrA and reduced by 80% by deletion of qseB. Mobility shift assays confirmed that the phosphorylated forms of both PmrA and QseB bind to the yibD promoter region, supporting a role for both response regulators in modulation of yibD transcription.
Past research revealed that PMB-triggered LPS alterations were modulated, at least in part, by the PmrAB TCS. This led the investigators to hypothesize that QseBC might also play a role. They found little difference in the baseline sensitivity of wild-type UTI89 versus UTI89 lacking both components of either PmrAB or QseBC. However, deletion of both qseC and pmrA resulted in increased resistance to PMB. The researchers explained this finding by noting that this combination of deletions promotes crosstalk between PmrB and QseB. Supporting their hypothesis was the finding that deletion of qseB eliminated the PMB resistance observed in the presence of deletions of both qseC and pmrA. Of even greater interest was their finding that exposure of wild-type UTI89 to ferric ion increased its resistance to PMB. However, this response was markedly reduced upon deletion of either pmrA or qseB, findings that strongly support the hypothesis that PmrAB and QseBC cooperate to regulate LPS alterations upon exposure to PMB. Further observations demonstrated, however, that various UPEC strains differed widely in terms of their baseline sensitivity to PMB and their ability to respond to ferric ion with increased PMB resistance. Further work will be required to delineate the mechanism of this variability among bacterial strains.
The investigators concurred that PmrAB and QseBC interact to regulate gene expression in a number of target systems. This is one of the first demonstrations of physiologically relevant collaboration between two TCSs. Figure 2 outlines a possible mechanism by which PmrAB and QseBC work together to regulate the expression of yibD. The investigators note that they have strong evidence that the PmrB histidine kinase phosphorylates both PmrA and QseB; however, even though their data suggest that QseC is required for cooperation between the TCSs, the role of this histidine kinase remains a mystery. This will be a focus of exciting future research.
FIGURE 2. Proposed mechanism for the cross-talk between the QseBC and PmrAB two component systems in response to ferric ion. (A) In the basal state, the bacterial cell expresses both the histidine kinases (QseC and PmrB) and the response regulators (QseB and PrmA) of both systems, but phosphorylation is minimal, and expression of the target gene that encodes for YibD is low. (B) Addition of ferric ion leads to dimerization and activation of PmrB, resulting in autophosphorylation of histidine residues. The phosphates are then transferred to aspartate residues on PmrB and QseB, both of which dimerize and interact with the promoter of the gene for YibD, resulting in increased transcription.