PmrD Is Required for Modifications to Escherichia coli Endotoxin That Promote Antimicrobial Resistance

Abstract

In Salmonella enterica, PmrD is a connector protein that links the two-component systems PhoP-PhoQ and PmrA-PmrB. While Escherichia coli encodes a PmrD homolog, it is thought to be incapable of connecting PhoPQ and PmrAB in this organism due to functional divergence from the S. enterica protein. However, our laboratory previously observed that low concentrations of Mg 2؉ , a PhoPQ-activating signal, leads to the induction of PmrAB-dependent lipid A modifications in wild-type E. coli ( These modifications include phosphoethanolamine (pEtN) and 4-amino-4-deoxy-L-arabinose (L-Ara4N), which promote bacterial resistance to cationic antimicrobial peptides (CAMPs) when affixed to lipid A. Here, we demonstrate that pmrD is required for modification of the lipid A domain of E. coli lipopolysaccharide (LPS) under low-Mg 2؉ growth conditions. Further, RNA sequencing shows that E. coli pmrD influences the expression of pmrA and its downstream targets, including genes coding for the modification enzymes that transfer pEtN and L-Ara4N to the lipid A molecule. In line with these findings, a pmrD mutant is dramatically impaired in survival compared with the wild-type strain when exposed to the CAMP polymyxin B. Notably, we also reveal the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system. These results illuminate a more complex network of protein interactions surrounding activation of PhoPQ and PmrAB in E. coli than previously understood. Bacteria often encounter adverse conditions that threaten survival in an unpredictable environment. The first line of defense for most Gram-negative bacteria is the outer membrane, which contains lipopolysaccharide (LPS) in the outer leaflet that interfaces with the surroundings (1). LPS is a multicomponent macromolecule anchored to the bacterial membrane via its lipid A domain, a potent activator of the host innate immune response (2, 3). In the presence of environmental stressors, numerous Gram negatives have evolved machinery to modify the lipid A moiety with chemical groups that promote bacterial survival by creating a fortified, more resistant outer membrane (4).Lipid A modifications often are regulated by complex twocomponent system (TCS) protein networks that coordinate detection of various signals with targeted transcriptional regulation. A typical TCS consists of a sensor histidine kinase that detects specific environmental signals and a cognate response regulator, which carries out changes in expression of a subset of genes known as its regulon. Upon recognition of a given signal, the sensor first autophosphorylates and then phosphorylates the response regulator, causing it to activate or repress gene expression within the regulon. When the signal is no longer present or detectable, the sensor deactivates the response regulator by dephosphorylation, thereby terminating transcriptional control of the affected genes (2, 5, 6). The research here involves two such systems, PhoP-PhoQ (PhoPQ) and PmrA-PmrB ...

Mechanisms of Increased Resistance to Chlorhexidine and Cross-Resistance to Colistin following Exposure of Klebsiella pneumoniae Clinical Isolates to Chlorhexidine

“…While expression levels of pmrAB may be regulated by PhoPQ (through PmrD), pmrAB is also transcribed, independently of PhoPQ, from a constitutive promoter within the upstream pagB gene (26). Expression of pmrD may be subject to regulation by a second, unknown system based on the observation that in E. coli expression of pmrD was not abolished following inactivation of phoPQ (35). In K. pneumoniae, upregulation of pmrD following adaptation to colistin was not always linked to upregulation of phoQ (36).…”

Colistin in Pig Production: Chemistry, Mechanism of Antibacterial Action, Microbial Resistance Emergence, and One Health Perspectives

“…PmrB and PhoQ are sensor cytoplasmic membranes activated respectively by high concentrations of Fe 3+ and low pH and by low concentrations of Mg 2+ and Ca 2+ or certain antimicrobial peptides (McPhee et al, 2003; Rubin et al, 2015). In colistin resistant Salmonella , apart from an environmental stimuli such as low Mg 2+ concentration, a mutation in the PmrA/PmrB and/or PhoP/PhoQ TCS is the major mechanism involved in LPS modification (Olaitan et al, 2014).…”

“…Moreover, it has been demonstrated that the sRNA MgrR of E. coli was also involved in the regulation of lipid A modification (Moon and Gottesman, 2009). Most recently, Rubin et al (2015) have shown that in E. coli , another unknown bacterial system activates PmrD under low Mg 2+ conditions to promote lipid A modification, even in the absence of PhoPQ.…”

Defensive remodeling: How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides

“…In Gram-positive bacteria GraRS (ApsSR) is another TCS that is known to be activated by AMPs and is involved in AMP resistance. [57,107,160,177,186,187,200] …”

“…These systems have homologous systems in several Gram-negative bacteria, but the interaction between them in different species can be different. For example in E. coli another unknown bacterial system is involved in PmrAB activation in a PmrD dependent manner [160]. Both PhoPQ and PmrAB were found to be essential for the virulence of many bacteria species [143,161,162].…”