Culture conditions and sample preparation methods affect spectrum quality and reproducibility during profiling of Staphylococcus aureus with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) has emerged as a promising tool to rapidly characterize Staphylococcus aureus. Different protocols have been employed, but effects of experimental factors, such as culture condition and sample preparation, on spectrum quality and reproducibility have not been rigorously examined. We applied MALDI‐TOF MS to characterize a model system consisting of five methicillin‐sensitive (MSSA) and five methicillin‐resistant S. aureus isolates (MRSA) under two culture conditions (agar and broth) and using two sample preparation methods [intact cell method and protein extraction method (PEM)]. The effects of these treatments on spectrum quality and reproducibility were quantified. PEM facilitated increases in the number of peaks and mass range width. Broth cultures further improved spectrum quality in terms of increasing the number of peaks. In addition, PEM increased reproducibility in samples prepared using identical culture conditions. MALDI imaging data suggested that the improvement in reproducibility may result from a more homogeneous distribution of sample associated with the broth/PEM treatment. Broth/PEM treatment also yielded the highest rate (96%) of correct classification for MRSA. Taken together, these results suggest that broth/PEM maximizes the performance of MALDI‐TOF MS to characterize S. aureus.

Significance and Impact of the Study

Two culture conditions (agar or broth) and two sample preparation methods (intact cell or protein extraction) were evaluated for their effects on profiling of Staphylococcus aureus using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Results indicated that MALDI‐enabled profiling of S. aureus is most effective when cultures are grown in broth and processed using a protein extraction‐based approach. These findings should enhance future efforts to maximize the performance of this approach to characterize strains of S. aureus.     

NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor

Rapid internalization of the cell surface low density lipoprotein (LDL) receptor requires the first 22 amino acids of the cytoplasmic domain (residues 790-811), which must include an aromatic residue at position 807. In the human LDL receptor, this position is part of a tetrameric sequence, NPVY. A similar tetramer, NPXY (where X stands for any amino acid), is conserved in LDL receptors from six species (including Xenopus laevis) and in two members of the LDL receptor gene family, human LDL receptor-related protein and rat GP330. To determine whether the NPXY sequence is necessary for coated pit-mediated internalization, we used oligonucleotide-directed mutagenesis to substitute alanines for individual amino acids in the cytoplasmic tail of the human LDL receptor. Substitution of alanine for Asn804, Pro805, or Tyr807 (but not Val806) markedly reduced internalization. Only one other amino acid in the cytoplasmic 22-mer (Phe802) was important for internalization. A review of published data revealed NPXY sequences in cytoplasmic domains of at least 10 other cell surface proteins, including tyrosine kinase-linked receptors of the epidermal growth factor and insulin receptor family, the beta-subunits of three integrin receptors, and the amyloid A4 precursor protein. This occurrence is much more frequent than would be expected by chance alone. The possibility of a conditional role for the NPXY sequence in ligand-independent internalization of these proteins is discussed.     

Intraspecific variation in immune gene expression and heritable symbiont density

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These ‘biotypes’ have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system’s complex dual role in interacting with both beneficial and harmful microbes.     

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.     

Stem and leaf hydraulics of congeneric tree species from adjacent tropical savanna and forest ecosystems

Leaf and stem functional traits related to plant water relations were studied for six congeneric species pairs, each composed of one tree species typical of savanna habitats and another typical of adjacent forest habitats, to determine whether there were intrinsic differences in plant hydraulics between these two functional types. Only individuals growing in savanna habitats were studied. Most stem traits, including wood density, the xylem water potential at 50% loss of hydraulic conductivity, sapwood area specific conductivity, and leaf area specific conductivity did not differ significantly between savanna and forest species. However, maximum leaf hydraulic conductance (K _leaf) and leaf capacitance tended to be higher in savanna species. Predawn leaf water potential and leaf mass per area were also higher in savanna species in all congeneric pairs. Hydraulic vulnerability curves of stems and leaves indicated that leaves were more vulnerable to drought-induced cavitation than terminal branches regardless of genus. The midday K _leaf values estimated from leaf vulnerability curves were very low implying that daily embolism repair may occur in leaves. An electric circuit analog model predicted that, compared to forest species, savanna species took longer for their leaf water potentials to drop from predawn values to values corresponding to 50% loss of K _leaf or to the turgor loss points, suggesting that savanna species were more buffered from changes in leaf water potential. The results of this study suggest that the relative success of savanna over forest species in savanna is related in part to their ability to cope with drought, which is determined more by leaf than by stem hydraulic traits. Variation among genera accounted for a large proportion of the total variance in most traits, which indicates that, despite different selective pressures in savanna and forest habitats, phylogeny has a stronger effect than habitat in determining most hydraulic traits.     

The salt‐sensitive structure and zinc inhibition of Borrelia burgdorferi protease BbHtrA

HtrA serine proteases are highly conserved and essential ATP‐independent proteases with chaperone activity. Bacteria express a variable number of HtrA homologues that contribute to the virulence and pathogenicity of bacterial pathogens. Lyme disease spirochetes possess a single HtrA protease homologue, Borrelia burgdorferi HtrA (BbHtrA). Previous studies established that, like the human orthologue HtrA1, BbHtrA is proteolytically active against numerous extracellular proteins in vitro. In this study, we utilized size exclusion chromatography and blue native polyacrylamide gel electrophoresis (BN‐PAGE) to demonstrate BbHtrA oligomeric structures that were substrate independent and salt sensitive. Examination of the influence of transition metals on the activity of BbHtrA revealed that this protease is inhibited by Zn²⁺ > Cu²⁺ > Mn²⁺. Extending this analysis to two other HtrA proteases, E. coli DegP and HtrA1, revealed that all three HtrA proteases were reversibly inhibited by ZnCl₂ at all micro molar concentrations examined. Commercial inhibitors for HtrA proteases are not available and physiologic HtrA inhibitors are unknown. Our observation of conserved zinc inhibition of HtrA proteases will facilitate structural and functional studies of additional members of this important class of proteases.     

Environmental and physiological regulation of transpiration in tropical forest gap species: the influence of boundary layer and hydraulic properties

Environmental and physiological regulation of transpiration were examined in several gap-colonizing shrub and tree species during two consecutive dry seasons in a moist, lowland tropical forest on Barro Colorado Island, Panama. Whole plant transpiration, stomatal and total vapor phase (stomatal + boundary layer) conductance, plant water potential and environmental variables were measured concurrently. This allowed control of transpiration (E) to be partitioned quantitatively between stomatal (g _s) and boundary layer (g _b) conductance and permitted the impact of invividual environmental and physiological variables on stomatal behavior and E to be assessed. Wind speed in treefall gap sites was often below the 0.25 m s^–1 stalling speed of the anemometer used and was rarely above 0.5 m s^–1, resulting in uniformly low g _b (c. 200–300 mmol m^–2 s^–1) among all species studied regardless of leaf size. Stomatal conductance was typically equal to or somewhat greater than g _b. This strongly decoupled E from control by stomata, so that in Miconia argentea a 10% change in g _s when g _s was near its mean value was predicted to yield only a 2.5% change in E. Porometric estimates of E, obtained as the product of g _s and the leaf-bulk air vapor pressure difference (VPD) without taking g _b into account, were up to 300% higher than actual E determined from sap flow measurements. Porometry was thus inadequate as a means of assessing the physiological consequences of stomatal behavior in different gap colonizing species. Stomatal responses to humidity strongly limited the increase in E with increasing evaporative demand. Stomata of all species studied appeared to respond to increasing evaporative demand in the same manner when the leaf surface was selected as the reference point for determination of external vapor pressure and when simultaneous variation of light and leaf-air VPD was taken into account. This result suggests that contrasting stomatal responses to similar leaf-bulk air VPD may be governed as much by the external boundary layer as by intrinsic physiological differences among species. Both E and g _s initially increased sharply with increasing leaf area-specific total hydraulic conductance of the soil/root/leaf pathway (G _t), becoming asymptotic at higher values of G _t. For both E and g _s a unique relationship appeared to describe the response of all species to variations in G _t. The relatively weak correlation observed between g _s and midday leaf water potential suggested that stomatal adjustment to variations in water availability coordinated E with water transport efficiency rather than bulk leaf water status.     

Ruminococcal cellulosome systems from rumen to human

A cellulolytic fiber‐degrading bacterium, Ruminococcus champanellensis, was isolated from human faecal samples, and its genome was recently sequenced. Bioinformatic analysis of the R. champanellensis genome revealed numerous cohesin and dockerin modules, the basic elements of the cellulosome, and manual sequencing of partially sequenced genomic segments revealed two large tandem scaffoldin‐coding genes that form part of a gene cluster. Representative R. champanellensis dockerins were tested against putative cohesins, and the results revealed three different cohesin–dockerin binding profiles which implied two major types of cellulosome architectures: (i) an intricate cell‐bound system and (ii) a simplistic cell‐free system composed of a single cohesin‐containing scaffoldin. The cell‐bound system can adopt various enzymatic architectures, ranging from a single enzyme to a large enzymatic complex comprising up to 11 enzymes. The variety of cellulosomal components together with adaptor proteins may infer a very tight regulation of its components. The cellulosome system of the human gut bacterium R. champanellensis closely resembles that of the bovine rumen bacterium Ruminococcus flavefaciens. The two species contain orthologous gene clusters comprising fundamental components of cellulosome architecture. Since R. champanellensis is the only human colonic bacterium known to degrade crystalline cellulose, it may thus represent a keystone species in the human gut.     

Shifts in the composition and distribution of Pacific Arctic larval fish assemblages in response to rapid ecosystem change

The Pacific Arctic marine ecosystem has undergone rapid changes in recent years due to ocean warming, sea ice loss, and increased northward transport of Pacific-origin waters into the Arctic. These climate-mediated changes have been linked to range shifts of juvenile and adult subarctic (boreal) and Arctic fish populations, though it is unclear whether distributional changes are also occurring during the early life stages. We analyzed larval fish abundance and distribution data sampled in late summer from 2010 to 2019 in two interconnected Pacific Arctic ecosystems: the northern Bering Sea and Chukchi Sea, to determine whether recent warming and loss of sea ice has restricted habitat for Arctic species and altered larval fish assemblage composition from Arctic- to boreal-associated taxa. Multivariate analyses revealed the presence of three distinct multi-species assemblages across all years: (1) a boreal assemblage dominated by yellowfin sole (Limanda aspera), capelin (Mallotus catervarius), and walleye pollock (Gadus chalcogrammus); (2) an Arctic assemblage composed of Arctic cod (Boreogadus saida) and other common Arctic species; and (3) a mixed assemblage composed of the dominant species from the other two assemblages. We found that the wind- and current-driven northward advection of warmer, subarctic waters and the unprecedented low-ice conditions observed in the northern Bering and Chukchi seas beginning in 2017 and persisting into 2018 and 2019 have precipitated community-wide shifts, with the boreal larval fish assemblage expanding northward and offshore and the Arctic assemblage retreating poleward. We conclude that Arctic warming is most significantly driving changes in abundance at the leading and trailing edges of the Chukchi Sea larval fish community as boreal species increase in abundance and Arctic species decline. Our analyses document how quickly larval fish assemblages respond to environmental change and reveal that the impacts of Arctic borealization on fish community composition spans multiple life stages over large spatial scales.     

Age-associated adipose tissue inflammation promotes monocyte chemotaxis and enhances atherosclerosis

Although aging enhances atherosclerosis, we do not know if this occurs via alterations in circulating immune cells, lipid metabolism, vasculature, or adipose tissue. Here, we examined whether aging exerts a direct pro-atherogenic effect on adipose tissue in mice. After demonstrating that aging augmented the inflammatory profile of visceral but not subcutaneous adipose tissue, we transplanted visceral fat from young or aged mice onto the right carotid artery of Ldlr^−/− recipients. Aged fat transplants not only increased atherosclerotic plaque size with increased macrophage numbers in the adjacent carotid artery, but also in distal vascular territories, indicating that aging of the adipose tissue enhances atherosclerosis via secreted factors. By depleting macrophages from the visceral fat, we identified that adipose tissue macrophages are major contributors of the secreted factors. To identify these inflammatory factors, we found that aged fat transplants secreted increased levels of the inflammatory mediators TNFα, CXCL2, and CCL2, which synergized to promote monocyte chemotaxis. Importantly, the combined blockade of these inflammatory mediators impeded the ability of aged fat transplants to enhance atherosclerosis. In conclusion, our study reveals that aging enhances atherosclerosis via increased inflammation of visceral fat. Our study suggests that future therapies targeting the visceral fat may reduce atherosclerosis disease burden in the expanding older population.