yersiniabactin and Disease-Models--Animal

There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64-labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.

Topics: Animals; Bacteria; Bacterial Infections; Copper; Disease Models, Animal; Echocardiography; Female; Mice; Mice, Inbred BALB C; Molecular Imaging; Phenols; Positron-Emission Tomography; Siderophores; Thiazoles

The genus Escherichia is composed of several species and cryptic clades, including E. coli, which behaves as a vertebrate gut commensal, but also as an opportunistic pathogen involved in both diarrheic and extra-intestinal diseases. To characterize the genetic determinants of extra-intestinal virulence within the genus, we carried out an unbiased genome-wide association study (GWAS) on 370 commensal, pathogenic and environmental strains representative of the Escherichia genus phylogenetic diversity and including E. albertii (n = 7), E. fergusonii (n = 5), Escherichia clades (n = 32) and E. coli (n = 326), tested in a mouse model of sepsis. We found that the presence of the high-pathogenicity island (HPI), a ~35 kbp gene island encoding the yersiniabactin siderophore, is highly associated with death in mice, surpassing other associated genetic factors also related to iron uptake, such as the aerobactin and the sitABCD operons. We confirmed the association in vivo by deleting key genes of the HPI in E. coli strains in two phylogenetic backgrounds. We then searched for correlations between virulence, iron capture systems and in vitro growth in a subset of E. coli strains (N = 186) previously phenotyped across growth conditions, including antibiotics and other chemical and physical stressors. We found that virulence and iron capture systems are positively correlated with growth in the presence of numerous antibiotics, probably due to co-selection of virulence and resistance. We also found negative correlations between virulence, iron uptake systems and growth in the presence of specific antibiotics (i.e. cefsulodin and tobramycin), which hints at potential "collateral sensitivities" associated with intrinsic virulence. This study points to the major role of iron capture systems in the extra-intestinal virulence of the genus Escherichia.

Topics: Animals; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Genetic Variation; Genome-Wide Association Study; Genomic Islands; Humans; Iron; Mice; Phenols; Phylogeny; Sepsis; Siderophores; Thiazoles; Virulence

Escherichia coli sequence type 69 (ST69; "clonal group A") is an important extraintestinal pathogen. To clarify the yersiniabactin siderophore system's role in ST69's extraintestinal virulence we compared a wild-type ST69 cystitis isolate, isogenic irp2 (yersiniabactin) mutants, and irp2-complemented mutants in murine models of sepsis and urinary tract infection (UTI). irp2 mutants were attenuated mildly in the UTI model and profoundly in the sepsis model. In both models, complementation with a functional copy of irp2 restored full parental virulence. These findings suggest that in ST69 the yersiniabactin system has a minor role in urovirulence and a major role in sepsis causation.

Topics: Animals; Cystitis; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Female; Gene Deletion; Genetic Complementation Test; Iron Regulatory Protein 2; Mice; Mutation; Phenols; Sepsis; Thiazoles; Urinary Tract Infections; Virulence; Virulence Factors

The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 10(6)-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs.

Topics: Animals; ATP-Binding Cassette Transporters; Bacterial Proteins; Cystitis; Disease Models, Animal; Escherichia coli; Female; Gene Deletion; Genetic Complementation Test; Mice, Inbred C3H; Phenols; Siderophores; Thiazoles; Virulence Factors

The genotoxin colibactin, synthesized by Escherichia coli, is a secondary metabolite belonging to the chemical family of hybrid polyketide/nonribosomal peptide compounds. It is produced by a complex biosynthetic assembly line encoded by the pks pathogenicity island. The presence of this large cluster of genes in the E. coli genome is invariably associated with the high-pathogenicity island, encoding the siderophore yersiniabactin, which belongs to the same chemical family as colibactin. The E. coli heat shock protein HtpG (Hsp90Ec) is the bacterial homolog of the eukaryotic molecular chaperone Hsp90, which is involved in the protection of cellular proteins against a variety of environmental stresses. In contrast to eukaryotic Hsp90, the functions and client proteins of Hsp90Ec are poorly known. Here, we demonstrated that production of colibactin and yersiniabactin is abolished in the absence of Hsp90Ec We further characterized an interplay between the Hsp90Ec molecular chaperone and the ClpQ protease involved in colibactin and yersiniabactin synthesis. Finally, we demonstrated that Hsp90Ec is required for the full in vivo virulence of extraintestinal pathogenic E. coli This is the first report highlighting the role of heat shock protein Hps90Ec in the production of two secondary metabolites involved in E. coli virulence.

Topics: Animals; Disease Models, Animal; Endopeptidase Clp; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Female; Gene Deletion; HSP90 Heat-Shock Proteins; Mice, Inbred C57BL; Mutagens; Peptides; Phenols; Polyketides; Protein Interaction Mapping; Rats, Wistar; Siderophores; Thiazoles; Virulence

Bacterial pathogens must overcome host sequestration of zinc (Zn(2+) ), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn(2+) by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn(2+) -deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn(2+) acquisition. Studies with the Zn(2+) -dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn(2+) . However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe(3+) acquisition by Ybt, are not needed for Ybt-dependent Zn(2+) uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn(2+) uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model.

Topics: Animals; ATP-Binding Cassette Transporters; Disease Models, Animal; Mice; Phenols; Plague; Sepsis; Thiazoles; Virulence; Virulence Factors; Yersinia pestis; Zinc

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and β-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Topics: Acute-Phase Proteins; Animals; Blood; Disease Models, Animal; DNA, Bacterial; Feces; Humans; Immunologic Factors; Klebsiella Infections; Klebsiella pneumoniae; Lipocalin-2; Lipocalins; Mass Spectrometry; Mice; Mice, Inbred C57BL; Mice, Knockout; Oncogene Proteins; Phenols; Polymerase Chain Reaction; Proto-Oncogene Proteins; Respiratory System; Respiratory Tract Infections; Thiazoles; Urine; Virulence; Virulence Factors

Hypoxia inducible factor-1 (HIF-1) is the key transcriptional regulator during adaptation to hypoxia. Recent studies provide evidence for HIF-1 activation during bacterial infections. However, molecular details of how bacteria activate HIF-1 remain unclear. Here, we pursued the role of bacterial siderophores in HIF-1 activation during infection with Enterobacteriaceae.. In vivo, HIF-1 activation and HIF-1-dependent gene induction in Peyer's patches were analyzed after orogastric infection with Yersinia enterocolitica. The course of an orogastric Y enterocolitica infection was determined using mice with a deletion of HIF-1alpha in the intestine. In vitro, the mechanism of HIF-1 activation was analyzed in infections with Y enterocolitica, Salmonella enterica subsp enterica, and Enterobacter aerogenes.. Infection of mice with Y enterocolitica led to functional activation of HIF-1 in Peyer's patches. Because mice with deletion of HIF-1alpha in the intestinal epithelium showed a significantly higher susceptibility to orogastric Y enterocolitica infections, bacterial HIF-1 activation appears to represent a host defense mechanism. Additional studies with Y enterocolitica, S enterica subsp enterica, or E aerogenes, and, moreover, application of their siderophores (yersiniabactin, salmochelin, aerobactin) caused a robust, dose-dependent HIF-1 response in human epithelia and endothelia, independent of cellular hypoxia. HIF-1 activation occurs most likely because of inhibition of prolylhydroxylase activity and is abolished upon infection with siderophore uptake deficient bacteria.. Taken together, this study reveals what we believe to be a previously unrecognized role of bacterial siderophores for hypoxia-independent activation of HIF-1 during infection with human pathogenic bacteria.

Topics: Animals; Caco-2 Cells; Cell Hypoxia; Disease Models, Animal; Endothelial Cells; Enterobacter aerogenes; Enterobacteriaceae; Epithelial Cells; Female; Gene Expression Regulation; HeLa Cells; Humans; Hydroxamic Acids; Hydroxylation; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Oxygen; Peyer's Patches; Phenols; Procollagen-Proline Dioxygenase; Salmonella enterica; Siderophores; Thiazoles; Time Factors; Transcriptional Activation; Up-Regulation; Yersinia enterocolitica; Yersinia Infections