The interplay of antibiotic resistance and bacterial stress responses in phosphate starvation condition

Abstract

During the past decades, antibiotic resistance has led to increasing failure in the treatment of bacterial infectious diseases and the mortality rate associated with invasive surgeries. The molecular mechanisms of antibiotic resistance are relatively well studied. However, the causative environmental, clinical, and physiological factors are complex and the underlying mechanisms are less understood. Recent studies have suggested that bacterial responses to various stresses can lead to the development of antibiotic resistance, but the molecular mechanisms and physiological implications of this biological phenomenon are poorly understood. Phosphate (Pi) starvation is a stress condition prevalent in many environmental and host niches of bacteria. Previous research has revealed alterations of the expression of genes involved in Pi acquisition and metabolism under this condition. However, a functional proteomics of bacterial responses to Pi starvation is lacking. In this study, I present a high-throughput proteomic study on the global protein expression changes in response to Pi starvation in Escherichia coli K-12 MG1655. Several classes of proteins which function outside of Pi metabolism has been identified, such as lipid A modification enzymes and drug efflux pump, which suggested a connection of Pi starvation and antibiotic resistance. My study showed that several genes involved in L-Ara4N modification of lipid A (arnABC and ugd) are up-regulated in E. coli during Pi starvation. The expression change of these genes is found to be regulated by the BasSR two-component system, which might be activated by elevated cellular iron content during Pi starvation. Consistent with the protein expression results, MS analysis revealed that lipid A molecules in E. coli are modified by L-Ara4N during Pi starvation, and deletion of basSR abolished this modification. Further studies showed that E. coli cells display high-level resistance to cationic antibiotics (polymyxin B/E) in this condition. Similar enhanced resistance to cationic antibiotics during Pi starvation is also observed in pathogenic E. coli strains (O157:H7 and four urinary-tract-infection associated clinic strains), Salmonella enterica serovar Typhimurium (ATCC14028) and Pseudomonas aeruginosa (PAO1), suggesting that this phenotype is conserved in these gram-negative bacterial species. Next, my study showed that Pi starvation also lead to up-regulation of the multidrug resistance efflux pump MdtEF. I found that the transcription of mdtEF during Pi starvation is initiated from the P1 and P3 regions of the promoter of gadE-mdtEF operon, and the transcription of mdtEF from P1 region is regulated by the acid-response regulator GadE and sigma factor RpoS, while transcription of mdtEF from P3 region requires the function of Pi-response regulator PhoB and RpoS. Physiological studies showed that the system is necessary to protect the bacterium from bile salts and SDS damage in E. coli cells lacking the housekeeping pump acrAB during Pi starvation. Together my studies revealed novel adaptation strategies E. coli utilizes during its response to Pi starvation and advanced our understanding of the development of antibiotic resistance during the response to this stress condition. Given the prevalence of Pi depletion in many natural, physiological, and clinical settings, these studies will contribute to the management of drug resistance in these contexts.