Investigating the Contribution of Iron Acquisition to Serratia marcescens Pathogenesis during Bloodstream Infection

Abstract

Although largely considered an environmental pathogen given its ability to infect insects and survive in nature, Serratia marcescens is an important cause of multi-drug resistant bloodstream infections with increasing prevalence. With its current status as an emerging pathogen, little is known about factors contributing to S. marcescens infection specifically regarding those that aid in iron acquisition. Iron uptake is well established in the field as being crucial for bacterial colonization and pathogenesis since it is an essential protein co-factor in many cellular functions. However, mammalian hosts tightly regulate iron, making it difficult for pathogens to access. Therefore, bacteria employ specialized systems to gather iron, including the use of siderophores, heme uptake, ferrous iron receptors, and others. The overarching goal of this dissertation was to comprehensively characterize the iron-uptake systems in a bloodstream isolate of S. marcescens, UMH9. Iron-depleted RNA-sequencing was used to identify all the genes regulated by iron concentration in UMH9. Through these experiments, seven independent iron acquisition systems were identified, including two siderophore loci and two previously described heme uptake systems. Seven additional orphan transporters were also identified. Further studies in this dissertation focused on characterizing the siderophore and heme uptake loci. Two siderophore systems, cbs and sch, identified by RNA-seq in UMH9, named after previously described siderophore loci to which they have similarity, were characterized. Deletions were made in the non-ribosomal peptide synthetase (NRPS) machinery in each siderophore locus and together in a double mutant. In vitro growth in iron-poor conditions revealed that the double NRPS mutant in both siderophore loci had defective growth, but still produced siderophore by chrome azurol S (CAS) and mass spectrometry. The single NRPS mutants behaved similarly to the wild type strain, suggesting that the deleted NRPS genes potentially have redundant roles in siderophore transport. These findings challenge the traditional exclusive role of NRPSs in siderophore biosynthesis. The siderophores were then examined from a pathogenesis perspective. Mutations were made deleting the entire siderophore loci individually and in combination. Mutants lacking the full sch locus lost their ability to chelate iron as quantified by the CAS assay, while the cbs single mutant retained wild-type activity. Using mass spectrometry, the chelating siderophore was found to be serratiochelin (sch) while the other was chrysobactin (cbs). Sch-mutants had defective growth in iron-limited conditions and deletion of the sch gene cluster resulted in attenuation of UMH9 in the mouse bacteria model. Therefore, serratiochelin is necessary for full virulence during bloodstream infection. The S. marcescens heme uptake systems, hem and has, were also studied. Deletion mutants were made in each system individually and together in a double mutant. Growth conditions supplemented with heme revealed that mutants with intact hem system experienced an abnormally long lag phase before reaching exponential growth and saturation. This suggests that the hem system may contribute to heme toxicity. Whole genome sequencing revealed new insights into potential heme detoxification mechanisms. Interestingly, the double heme uptake mutant did not display any in vivo fitness defects or attenuation, suggesting a nonsignificant contribution of heme uptake to UMH9 pathogenesis during bacteremia. Altogether, these studies provide an in-depth characterization of two major classes of iron acquisition, siderophore and heme uptake, and their contribution to pathogenesis in Serratia marcescens. This work challenges canonical understandings of Gram-negative iron acquisition and provides new insights into the virulence of emerging pathogens.