Conformation of the Solute-Binding Protein AdcAII Influences Zinc Uptake in Streptococcus pneumoniae.

Streptococcus pneumoniae scavenges essential zinc ions from the host during colonization and infection. This is achieved by the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins (SBPs), AdcA and AdcAII. It has been established that AdcAII serves a greater role during initial infection, but the molecular details of how the protein selectively acquires Zn(II) remain poorly understood. This can be attributed to the refractory nature of metal-free AdcAII to high-resolution structural determination techniques. Here, we overcome this issue by separately mutating the Zn(II)-coordinating residues and performing a combination of structural and biochemical analyses on the variant proteins. Structural analyses of Zn(II)-bound AdcAII variants revealed that specific regions within the protein underwent conformational changes via direct coupling to each of the metal-binding residues. Quantitative in vitro metal-binding assays combined with affinity determination and phenotypic growth assays revealed that each of the four Zn(II)-coordinating residues contributes to metal binding by AdcAII. Intriguingly, the phenotypic growth impact of the mutant adcAII alleles was, in general, independent of affinity, suggesting that the Zn(II)-bound conformation of the SBP is crucial for efficacious metal uptake. Collectively, these data highlight the intimate coupling of ligand affinity with protein conformational change in ligand-receptor proteins and provide a putative mechanism for AdcAII. These findings provide further mechanistic insight into the structural and functional diversity of SBPs that is broadly applicable to other prokaryotes

Riverscape Patterns among Years of Juvenile Coho Salmon in Midcoastal Oregon: Implications for Conservation

Patterns of salmon distribution throughout a riverscape may be expected to change over time in response to environmental conditions and population sizes. Changing patterns of use, including identification of consistently occupied locations, are informative for conservation and recovery planning. We explored interannual patterns of distribution by juvenile Coho Salmon Oncorhynchus kisutch in 11 subbasins on the midcoast of Oregon. We found that juvenile Coho Salmon distribution expanded and contracted around stream sections that were continuously occupied (core areas). Timing of expansion or contraction was synchronous among subbasins and appeared to be related to the size of the parental spawning run. Juvenile distribution expanded from core areas when adults were abundant and contracted into core areas at lower adult abundances. The “intrinsic potential” of stream sections to support high quality habitat for Coho Salmon also appeared to inform distribution patterns. In most subbasins, when populations expanded, they moved into areas of high intrinsic potential. We identified areas that were consistently used by juvenile Coho Salmon which may be important target locations for conservation and restoration. Our study contributes to the body of work that seeks to explain the processes behind spatial and temporal patterns of freshwater habitat use by salmonids in the Pacific Northwest, thereby enhancing understanding of the complexity of biological and environmental interactions over broad scales

The role of ZntA in Klebsiella pneumoniae zinc homeostasis

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Essential to the colonization and infection by K. pneumoniae is the acquisition of nutrients, such as the transition metal ion zinc. Zinc has crucial structural and catalytic roles in the proteome of all organisms. Nevertheless, in excess, it has the potential to mediate significant toxicity by dysregulating the homeostasis of other transition elements, disrupting enzymatic processes, and perturbing metalloprotein cofactor acquisition. Here, we sought to elucidate the zinc detoxification mechanisms of K. pneumoniae, which remain poorly defined. Using the representative K. pneumoniae AJ218 strain, we showed that the P-type ATPase, ZntA, which is upregulated in response to cellular zinc stress, was the primary zinc efflux pathway. Deletion of zntA rendered K. pneumoniae AJ218 highly susceptible to exogenous zinc stress and manifested as an impaired growth phenotype and increased cellular accumulation of the metal. Loss of zntA also increased sensitivity to cadmium stress, indicating a role for this efflux pathway in cadmium resistance. Disruption of zinc homeostasis in the K. pneumoniae AJ218 ΔzntA strain also impacted manganese and iron homeostasis and was associated with increased production of biofilm. Collectively, this work showed the critical role of ZntA in K. pneumoniae zinc tolerance and provided a foundation for further studies on zinc homeostasis and the future development of novel antimicrobials to target this pathway. IMPORTANCE: Klebsiella pneumoniae is a leading cause of healthcare-associated infections, including pneumonia, urinary tract infections, and sepsis. Treatment of K. pneumoniae infections is becoming increasingly challenging due to high levels of antibiotic resistance and the rising prevalence of carbapenem-resistant, extended-spectrum β-lactamases producing strains. Zinc is essential to the colonization and infection by many bacterial pathogens but toxic in excess. This work described the first dissection of the pathways associated with resisting extracellular zinc stress in K. pneumoniae. This study revealed that the P-type ATPase ZntA was highly upregulated in response to exogenous zinc stress and played a major role in maintaining bacterial metal homeostasis. Knowledge of how this major bacterial pathogen resists zinc stress provided a foundation for antimicrobial development studies to target and abrogate their essential function.Eve A. Maunders, Katherine Ganio, Andrew J. Hayes, Stephanie L. Neville, Mark R. Davies, Richard A. Strugnell, Christopher A. McDevitt, Aimee Ta

A Trap-Door Mechanism for Zinc Acquisition by Streptococcus pneumoniae AdcA.

Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes. IMPORTANCE Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined. We report the first structure of a two-domain lipoprotein, AdcA from S. pneumoniae, and use computational, spectroscopic, and microbiological approaches to provide new insights into the functional basis of zinc recruitment. Our findings reveal that AdcA employs a novel mechanism for zinc binding that we have termed the “trap-door” mechanism, and we show how the static metal-binding site of the protein, which confers its selectivity for zinc ions, is combined with a dynamic surface element to facilitate zinc recruitment and import into the bacterium. Together, these findings expand our understanding of how bacteria acquire zinc from the environment and provide a foundation for inhibiting this process, through antimicrobial targeting of the dynamic structural elements to block bacterial zinc scavenging

Multiomics modeling of the immunome, transcriptome, microbiome, proteome and metabolome adaptations during human pregnancy.

MotivationMultiple biological clocks govern a healthy pregnancy. These biological mechanisms produce immunologic, metabolomic, proteomic, genomic and microbiomic adaptations during the course of pregnancy. Modeling the chronology of these adaptations during full-term pregnancy provides the frameworks for future studies examining deviations implicated in pregnancy-related pathologies including preterm birth and preeclampsia.ResultsWe performed a multiomics analysis of 51 samples from 17 pregnant women, delivering at term. The datasets included measurements from the immunome, transcriptome, microbiome, proteome and metabolome of samples obtained simultaneously from the same patients. Multivariate predictive modeling using the Elastic Net (EN) algorithm was used to measure the ability of each dataset to predict gestational age. Using stacked generalization, these datasets were combined into a single model. This model not only significantly increased predictive power by combining all datasets, but also revealed novel interactions between different biological modalities. Future work includes expansion of the cohort to preterm-enriched populations and in vivo analysis of immune-modulating interventions based on the mechanisms identified.Availability and implementationDatasets and scripts for reproduction of results are available through: https://nalab.stanford.edu/multiomics-pregnancy/.Supplementary informationSupplementary data are available at Bioinformatics online

Systemic Immunologic Consequences of Chronic Periodontitis

Chronic periodontitis (ChP) is a prevalent inflammatory disease affecting 46% of the US population. ChP produces a profound local inflammatory response to dysbiotic oral microbiota that leads to destruction of alveolar bone and tooth loss. ChP is also associated with systemic illnesses, including cardiovascular diseases, malignancies, and adverse pregnancy outcomes. However, the mechanisms underlying these adverse health outcomes are poorly understood. In this prospective cohort study, we used a highly multiplex mass cytometry immunoassay to perform an in-depth analysis of the systemic consequences of ChP in patients before (n = 28) and after (n = 16) periodontal treatment. A high-dimensional analysis of intracellular signaling networks revealed immune system–wide dysfunctions differentiating patients with ChP from healthy controls. Notably, we observed exaggerated proinflammatory responses to Porphyromonas gingivalis–derived lipopolysaccharide in circulating neutrophils and monocytes from patients with ChP. Simultaneously, natural killer cell responses to inflammatory cytokines were attenuated. Importantly, the immune alterations associated with ChP were no longer detectable 3 wk after periodontal treatment. Our findings demarcate systemic and cell-specific immune dysfunctions in patients with ChP, which can be temporarily reversed by the local treatment of ChP. Future studies in larger cohorts are needed to test the boundaries of generalizability of our results

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 β-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.Eve A. Maunders, Dalton H. Y. Ngu, Katherine Ganio, Sheikh I. Hossain, Bryan Y. J. Lim, Michael G. Leeming, Zhenyao Luo, Aimee Tan, Evelyne Deplazes, Boštjan Kobe, and Christopher A. McDevit

Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions

An immune clock of human pregnancy

The maintenance of pregnancy relies on finely tuned immune adaptations. We demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using mass cytometry, the abundance and functional responses of all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. Cell signaling-based Elastic Net, a regularized regression method adapted from the elastic net algorithm, was developed to infer and prospectively validate a predictive model of interrelated immune events that accurately captures the chronology of pregnancy. Model components highlighted existing knowledge and revealed previously unreported biology, including a critical role for the interleukin-2-dependent STAT5ab signaling pathway in modulating T cell function during pregnancy. These findings unravel the precise timing of immunological events occurring during a term pregnancy and provide the analytical framework to identify immunological deviations implicated in pregnancy-related pathologies

Single-cell Analysis of the Neonatal Immune System Across the Gestational Age Continuum

Although most causes of death and morbidity in premature infants are related to immune maladaptation, the premature immune system remains poorly understood. We provide a comprehensive single-cell depiction of the neonatal immune system at birth across the spectrum of viable gestational age (GA), ranging from 25 weeks to term. A mass cytometry immunoassay interrogated all major immune cell subsets, including signaling activity and responsiveness to stimulation. An elastic net model described the relationship between GA and immunome (R=0.85, p=8.75e-14), and unsupervised clustering highlighted previously unrecognized GA-dependent immune dynamics, including decreasing basal MAP-kinase/NFkB signaling in antigen presenting cells; increasing responsiveness of cytotoxic lymphocytes to interferon-a; and decreasing frequency of regulatory and invariant T cells, including NKT cells and MAIT cells. Knowledge gained from the analysis of the neonatal immune landscape across GA provides a mechanistic framework to understand the unique susceptibility of preterm infants to both hyper-inflammatory diseases and infections