Hfe Permease and Haemophilus influenzae Manganese Homeostasis.

Haemophilus influenzae is a commensal of the human upper respiratory tract that can infect diverse host niches due, at least in part, to its ability to withstand both endogenous and host-mediated oxidative stresses. Here, we show that hfeA, a gene previously linked to iron import, is essential for H. influenzae manganese recruitment via the HfeBCD transporter. Structural analyses show that metal binding in HfeA uses a unique mechanism that involves substantial rotation of the C-terminal lobe of the protein. Disruption of hfeA reduced H. influenzae manganese acquisition and was associated with decreased growth under aerobic conditions, impaired manganese-superoxide dismutase activity, reduced survival in macrophages, and changes in biofilm production in the presence of superoxide. Collectively, this work shows that HfeA contributes to H. influenzae manganese acquisition and virulence attributes. High conservation of the hfeABCD permease in Haemophilus species suggests that it may serve similar roles in other pathogenic Pasteurellaceae.

The Impact of Chromate on Pseudomonas aeruginosa Molybdenum Homeostasis.

Acquisition of the trace-element molybdenum via the high-affinity ATP-binding cassette permease ModABC is essential for Pseudomonas aeruginosa respiration in anaerobic and microaerophilic environments. This study determined the X-ray crystal structures of the molybdenum-recruiting solute-binding protein ModA from P. aeruginosa PAO1 in the metal-free state and bound to the group 6 metal oxyanions molybdate, tungstate, and chromate. Pseudomonas aeruginosa PAO1 ModA has a non-contiguous dual-hinged bilobal structure with a single metal-binding site positioned between the two domains. Metal binding results in a 22° relative rotation of the two lobes with the oxyanions coordinated by four residues, that contribute six hydrogen bonds, distinct from ModA orthologues that feature an additional oxyanion-binding residue. Analysis of 485 Pseudomonas ModA sequences revealed conservation of the metal-binding residues and ß-sheet structural elements, highlighting their contribution to protein structure and function. Despite the capacity of ModA to bind chromate, deletion of modA did not affect P. aeruginosa PAO1 sensitivity to chromate toxicity nor impact cellular accumulation of chromate. Exposure to sub-inhibitory concentrations of chromate broadly perturbed P. aeruginosa metal homeostasis and, unexpectedly, was associated with an increase in ModA-mediated molybdenum uptake. Elemental analyses of the proteome from anaerobically grown P. aeruginosa revealed that, despite the increase in cellular molybdenum upon chromate exposure, distribution of the metal within the proteome was substantially perturbed. This suggested that molybdoprotein cofactor acquisition may be disrupted, consistent with the potent toxicity of chromate under anaerobic conditions. Collectively, these data reveal a complex relationship between chromate toxicity, molybdenum homeostasis and anaerobic respiration.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.