In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Acquisition of iron underpins the ability of pathogens to cause disease and Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection. In this study, we sought to address the knowledge gap that exists regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis utilizes diverse genome encoded iron acquisition mechanisms to satisfy its need for this nutrient. Indeed, S. lugdunensis can usurp hydroxamate siderophores, and staphyloferrin A and B from S. aureus, using the fhuC ATPase-encoding gene. Acquisition of catechol siderophores and catecholamine stress hormones necessitates the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitates the feoAB locus. Heme iron is acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice we demonstrate that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci, we further demonstrate that only a strain mutated for all of isd, fhuC, sst-1, and feo, versus combination mutants carrying wild type copies of any one of those loci, was attenuated in its ability to proliferate to high numbers in kidneys. Taken together our data reveal that S. lugdunensis requires a repertoire of both heme and non-heme iron acquisition mechanisms to proliferate during systemic infection of mammals

Site-specific adaptation mechanisms of an oral pathobiont in the oral and gut mucosae

Periodontal inflammation leads to oral dysbiosis with the expansion of oral pathobionts. Besides the pathogenic role of oral pathobionts during periodontal inflammation, studies have revealed that oral pathobionts contribute to diseases in distant organs beyond the oral mucosa. For example, the oral pathobiont Klebsiella aerogenes, which accumulates in the oral mucosa during periodontitis in mice, can exacerbate colitis when it ectopically colonizes the gastrointestinal tract. However, the precise mechanisms by which oral pathobionts establish their colonization in extra-oral mucosal sites remains incompletely understood. We performed high-throughput in vivo genetic screening to identify fitness genes required for the adaptation of the oral pathobiont K. aerogenes to different mucosal sites – the oral and gut mucosae – in the steady state and during inflammation. In addition, the global transcriptome of K. aerogenes in different environments was analyzed. We determined that K. aerogenes employs genes related to iron acquisition and chaperone usher pili, which are encoded on a newly identified genomic locus named “locus of colonization in the inflamed gut” (LIG), for adaptation in the gut mucosa, particularly during inflammation. In contrast, the LIG virulence factors are not required for K. aerogenes to adapt to the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche as compared to their original niche.

Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.

Disentangling the Evolutionary History of Feo, the Major Ferrous Iron Transport System in Bacteria

Feo, a ferrous iron transport system composed of three proteins (FeoA, -B, and -C), is the most prevalent bacterial iron transporter. It plays an important role in iron acquisition in low-oxygen environments and some host-pathogen interactions.

Lysosomal iron recycling in mouse macrophages is dependent upon both reductases LcytB and Steap3

Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase Six-transmembrane epithelial antigen of the prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CrispR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and Tfrc1 suggesting cells were iron limited. We show transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89 supporting a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89, led to increased intracellular UTI89 proliferation suggesting that the endolysosomal system is retaining Fe3+ that can be used for intravesicular pathogen proliferation. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

Biosynthesis Pathways, Transport Mechanisms and Biotechnological Applications of Fungal Siderophores

Iron (Fe) is the fourth most abundant element on earth and represents an essential nutrient for life. As a fundamental mineral element for cell growth and development, iron is available for uptake as ferric ions, which are usually oxidized into complex oxyhydroxide polymers, insoluble under aerobic conditions. In these conditions, the bioavailability of iron is dramatically reduced. As a result, microorganisms face problems of iron acquisition, especially under low concentrations of this element. However, some microbes have evolved mechanisms for obtaining ferric irons from the extracellular medium or environment by forming small molecules often regarded as siderophores. Siderophores are high affinity iron-binding molecules produced by a repertoire of proteins found in the cytoplasm of cyanobacteria, bacteria, fungi, and plants. Common groups of siderophores include hydroxamates, catecholates, carboxylates, and hydroximates. The hydroxamate siderophores are commonly synthesized by fungi. L-ornithine is a biosynthetic precursor of siderophores, which is synthesized from multimodular large enzyme complexes through non-ribosomal peptide synthetases (NRPSs), while siderophore-Fe chelators cell wall mannoproteins (FIT1, FIT2, and FIT3) help the retention of siderophores. S. cerevisiae, for example, can express these proteins in two genetically separate systems (reductive and nonreductive) in the plasma membrane. These proteins can convert Fe (III) into Fe (II) by a ferrous-specific metalloreductase enzyme complex and flavin reductases (FREs). However, regulation of the siderophore through Fur Box protein on the DNA promoter region and its activation or repression depend primarily on the Fe availability in the external medium. Siderophores are essential due to their wide range of applications in biotechnology, medicine, bioremediation of heavy metal polluted environments, biocontrol of plant pathogens, and plant growth enhancement.

Siderophore-Mediated Iron Acquisition Plays a Critical Role in Biofilm Formation and Survival of Staphylococcus epidermidis Within the Host

Iron acquisition through siderophores, a class of small, potent iron-chelating organic molecules, is a widely spread strategy among pathogens to survive in the iron-restricted environment found in the host. Although these molecules have been implicated in the pathogenesis of several species, there is currently no comprehensive study addressing siderophore production in Staphylococcus epidermidis. Staphylococcus epidermidis is an innocuous skin commensal bacterium. The species, though, has emerged as a leading cause of implant-associated infections, significantly supported by an inherent ability to form biofilms. The process of adaptation from skin niche environments to the hostile conditions during invasion is yet not fully understood. Herein, we addressed the possible role of siderophore production in S. epidermidis virulence. We first identified and deleted a siderophore homolog locus, sfaABCD, and provided evidence for its involvement in iron acquisition. Our findings further suggested the involvement of siderophores in the protection against oxidative stress-induced damage and demonstrated the in vivo relevance of a siderophore-mediated iron acquisition during S. epidermidis infections. Conclusively, this study addressed, for the first time in this species, the underlying mechanisms of siderophore production, highlighting the importance of a siderophore-mediated iron acquisition under host relevant conditions and, most importantly, its contribution to survival within the host.

Genome placement of alpha-haemolysin cluster is associated with alpha-haemolysin sequence variation, adhesin and iron acquisition factor profile of Escherichia coli

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli . Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

Commensal Pseudomonas protect Arabidopsis thaliana from a coexisting pathogen via multiple lineage-dependent mechanisms

Plants are protected from pathogens not only by their own immunity but often also by colonizing commensal microbes. In Arabidopsis thaliana, a group of cryptically pathogenic Pseudomonas strains often dominates local populations. This group coexists in nature with commensal Pseudomonas strains that can blunt the deleterious effects of the pathogens in the laboratory. We have investigated the interaction between one of the Pseudomonas pathogens and 99 naturally co-occurring commensals, finding plant protection to be common among non-pathogenic Pseudomonas. While protective ability is enriched in one specific lineage, there is also a substantial variation for this trait among isolates of this lineage. These functional differences do not align with core-genome phylogenies, suggesting repeated gene inactivation or loss as causal. Using genome-wide association, we discovered that different bacterial genes are linked to plant protection in each lineage. We validated a protective role of several lineage-specific genes by gene inactivation, highlighting iron acquisition and biofilm formation as prominent mechanisms of plant protection in this Pseudomonas lineage. Collectively, our work illustrates the importance of functional redundancy in plant protective traits across an important group of commensal bacteria.