Yersiniabactin is a quorum-sensing autoinducer and siderophore in uropathogenic Escherichia coli.

Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

Yersiniabactin is a quorum sensing autoinducer and siderophore in uropathogenic Escherichia coli.

Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all E. coli encode the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally encode the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared growth of different isogenic siderophore biosynthesis mutants in the presence of transferrin, a human iron-binding protein. We observed that the Ybt system does not compensate for loss of the Ent system during siderophore-dependent, low density growth. Using transcriptional and product analysis, we found that this non-redundancy is attributable to a density-dependent transcriptional stimulation cycle in which Ybt assume an additional autoinducer function. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. The efficiency of this arrangement may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales. IMPORTANCE: Urinary tract infections (UTIs) are one of the most common human bacterial infections encountered by physicians. Adaptations that increase the pathogenic potential of commensal microbes such as E.coli are of great interest. One potential adaptation observed in clinical isolates is accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth conditions, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

The iron hand of uropathogenic Escherichia coli: the role of transition metal control in virulence.

The role of iron as a critical nutrient in pathogenic bacteria is widely regarded as having driven selection for iron acquisition systems among uropathogenic Escherichia coli (UPEC) isolates. Carriage of multiple transition metal acquisition systems in UPEC suggests that the human urinary tract manipulates metal-ion availability in many ways to resist infection. For siderophore systems in particular, recent studies have identified new roles for siderophore copper binding as well as production of siderophore-like inhibitors of iron uptake by other, competing bacterial species. Among these is a process of nutritional passivation of metal ions, in which uropathogens access these vital nutrients while simultaneously protecting themselves from their toxic potential. Here, we review these new findings within the current understanding of UPEC transition metal acquisition.

Virus entry and replication in the brain precedes blood-brain barrier disruption during intranasal alphavirus infection.

Viral infections of the central nervous system (CNS) are often associated with blood-brain barrier (BBB) disruption, yet the impact of virus replication and immune cell recruitment on BBB integrity are incompletely understood. Using two-photon microscopy, we demonstrate that Venezuelan equine encephalitis virus (VEEV) strain TC83-GFP, a GFP expressing, attenuated strain with a G3A mutation within the 5' UTR that is associated with increased sensitivity to type I interferons (IFNs), does not directly impact BBB permeability. Following intranasal infection of both wild-type and IFN-induced protein with tetratricopeptide repeats 1 (IFIT1)-deficient mice, which fail to block TC83-specific RNA translation, virus spreads to the olfactory bulb and cortex via migration along axonal tracts of neurons originating from the olfactory neuroepithelium. Global dissemination of virus in the CNS by 2days post-infection (dpi) was associated with increased BBB permeability in the olfactory bulb, but not in the cortex or hindbrain, where permeability only increased after the recruitment of CX3CR1+ and CCR2+ mononuclear cells on 6 dpi, which corresponded with tight junction loss and claudin 5 redistribution. Importantly, despite higher levels of viral replication, similar results were obtained in IFIT1-deficient mice. These findings indicate that TC83 gains CNS access via anterograde axonal migration without directly altering BBB function and that mononuclear and endothelial cell interactions may underlie BBB disruption during alphavirus encephalitis.

Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.

Cutting edge: identification of autoreactive CD4+ and CD8+ T cell subsets resistant to PD-1 pathway blockade.

Programmed death-1 (PD-1) promotes T cell tolerance. Despite therapeutically targeting this pathway for chronic infections and tumors, little is known about how different T cell subsets are affected during blockade. We examined PD-1/PD ligand 1 (PD-L1) regulation of self-antigen-specific CD4 and CD8 T cells in autoimmune-susceptible models. PD-L1 blockade increased insulin-specific effector CD4 T cells in type 1 diabetes. However, anergic islet-specific CD4 T cells were resistant to PD-L1 blockade. Additionally, PD-L1 was critical for induction, but not maintenance, of CD8 T cell intestinal tolerance. PD-L1 blockade enhanced functionality of effector T cells, whereas established tolerant or anergic T cells were not dependent on PD-1/PD-L1 signaling to remain unresponsive. This highlights the existence of Ag-experienced T cell subsets that do not rely on PD-1/PD-L1 regulation. These findings illustrate how positive treatment outcomes and autoimmunity development during PD-1/PD-L1 inhibition are linked to the differentiation state of a T cell.