Benjamin Katz’s research while affiliated with Kansas State University and other places

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal β-hairpin into the MPO active site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N-terminus adopts a β-hairpin confirmation upon binding to MPO, solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, while the N-terminal β-hairpin of SPIN accounts for ~55% of the buried surface area in the SPIN/MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N-terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins.

The colony of eusocial bee Apis mellifera has a reproductive queen and sterile workers performing tasks such as brood care and foraging. Chemical communication plays a crucial role in the maintenance of sociability in bees with many compounds released by the exocrine glands. The Dufour’s gland is a non-paired gland associated with the sting apparatus with important functions in the communication between members of the colony, releasing volatile chemicals that influence workers roles and tasks. However, the protein content in this gland is not well studied. This study identified differentially expressed proteins in the Dufour’s glands of nurse and forager workers of A. mellifera through 2D-gel electrophoresis and mass spectrometry. A total of 131 spots showed different expression between nurse and forager bees, and 28 proteins were identified. The identified proteins were categorized into different functions groups including protein, carbohydrate, energy and lipid metabolisms, cytoskeleton-associated proteins, detoxification, homeostasis, cell communication, constitutive and allergen. This study provides new insights of the protein content in the Dufour’s gland contributing to a more complete understanding of the biological functions of this gland in honeybees.

List of identified proteins spots in Dufour’s gland of Apis mellifera workers with Image Master. Up-regulated and down-regulated refer to proteins with higher and lower expression, respectively, in nurse worker in comparison with forager ones. (PDF)

The extracellular adherence protein (Eap) plays a crucial role in pathogenesis and survival of Staphylococcus aureus by inhibiting the classical and lectin pathways of complement. We have previously shown that Eap binds with nanomolar affinity to complement C4b and disrupts the initial interaction between C4b and C2, thereby inhibiting formation of the classical and lectin pathway C3 pro-convertase. Although an underlying mechanism has been identified, the structural basis for Eap binding to C4b is poorly understood. Here, we show that Eap domains 3 and 4 each contain a low-affinity, but saturable binding site for C4b. Taking advantage of the high lysine content of Eap, we used a zero-length crosslinking approach to map the Eap binding site to both the α'- and γ-chains of C4b. We also probed the C4b/Eap interface through a chemical footprinting approach involving lysine modification, proteolytic digestion, and mass spectrometry. This identified seven lysines in Eap that undergo changes in solvent exposure upon C4b binding. We found that simultaneous mutation of these lysines to either alanine or glutamate diminished C4b binding and complement inhibition by Eap. Together, our results provide insight into Eap recognition of C4b, and suggest that the repeating domains that comprise Eap are capable of multiple ligand-binding modes. This article is protected by copyright. All rights reserved.

Siderophore nutrition tests with Caulobacter crescentus strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. C. crescentus did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [⁵⁹Fe]apoferrichrome and ⁵⁹Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd], 1 nM; Michaelis-Menten constant [KM], 0.1 nM; Vmax, 19 pMol/10⁹ cells/min) were similar to those of Escherichia coli FhuA. Transport properties for ⁵⁹Fe-citrate were similar to those of E. coli FecA (KM, 5.3 nM; Vmax, 29 pMol/10⁹ cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028, 00138, 02277, and 03023 as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to Escherichia coli FepA and BtuB. A Δ02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for heme/hemoglobin utilization). IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus, identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by C. crescentus, its heme/hemoglobin transporter (HutA).

The ubiquitous soilborne and plant-pathogenic basidiomycete Rhizoctonia solani, although classified as a single species, is a complex of anastomosis groups (AGs) that cause disease in a broad range of higher plants. Here, we investigated the persistent co-isolation of bacteria with R. solani from brown patch-infected, cool-season turfgrasses, and report the presence of endo-hyphal bacteria, related to members in the genus Enterobacter, in an isolate of R. solani AG 2-2IIIB. The intracellular localization of the bacteria was corroborated by fluorescence, confocal and electron microscopy, and DNA analysis. Furthermore, the Enterobacter sp., which is rod-shaped in the free-living form, exists as an L-form (spheroid) within the fungus, a phenomenon not previously reported in endosymbionts. Our findings also indicate that the bacterium is required for full virulence of R. solani on creeping bentgrass and production of wild type levels of the toxin phenylacetic acid in fungal cultures. The possible presence of ...

Importance: Extracellular virus particles transmit infection between organisms, but within infected hosts, intercellular infection can be spread by additional mechanisms. In this study, we described an alternative pathway for intercellular transmission of PRRSV, in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases and structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response, and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito.

Sorghum is a staple crop consumed in some regions of Africa and Asia, where vitamin A deficiency is prevalent. The objective of this study was to identify and quantify the carotenoids by LC‐MS in the diversity of phenotypic sorghum accessions. Totally, five carotenoids (α‐carotene, β‐carotene, lutein, zeaxanthin, and β‐crypoxanthin) were identified. Top three carotenoids (lutein, zeaxanthin and β‐carotene) accounted for more than half of the total carotenoids were quantitated. A high content of total carotenoids was detected in the sorghum accessions with yellow endosperm (PI533943, PI655996, PI656096, PI656010), but high β‐carotene content was found in the accessions with brown or yellow pericarp (PI656010, PI655996, PI656096). PI656010 possessed the highest content of both carotenoids and β‐carotene up to 43.7 and 18.6 ng/g dry weight, respectively. No carotenoids were detectable in the accessions with white/red pericarp or white endosperm (PI656112, PI297104). Developing an association of phenotypic diversity of sorghum varieties with carotenoid profile may contribute to a healthy benefit in vitamin A deficiency prevention. Support or Funding Information Supported by USDA Cooperative KS511‐1001903

We previously selected an anthocyanin‐enriched purple‐fleshed sweet potato P40 that prevented colorectal cancer in a murine model. The objectives of this study was to characterize anthocyanins in P40 shoots used as common edible greens. Eight anthocyanins have been identified and quantified in leaves of P40 by HPLC‐MS/MS, whereas fourteen anthocyanins have been found in the control white‐fleshed Bonita and orange‐fleshed Beauregard. The three most abundant anthocyanins were cyanidin 3‐caffeoyl‐p‐hydroxybenzoyl sophoroside‐5‐glucoside, peonidin 3‐caffeoyl sophoroside‐5‐glucoside, and cyanidin 3‐(6″‐caffeoyl‐6″‐feruloylsophoroside)‐5‐glucoside, which comprised up to 58% of total anthocyanins in leaves of P40. The content of anthocyanins in leaves of P40 (17 mg/kg dry matter) was much lower than that in the flesh (13,000 mg/kg dry matter). No anthocyanin was detectable in the stem. The content of anthocyanins in P40 greens was also much lower than that in the Bonita (563 mg/kg dry matter) and Beauregard (334 mg/kg dry matter). The total phenolic content in leaves of P40, as measured by Folin‐Ciocalteu, was 36.8±4.8 mg GAE/g dry weight, compared with Bonita at 46.7±2.1mg GAE/g dry weight and Beauregard at 41.2±5.0 mg GAE/g dry weight Support or Funding Information supported by USDA Cooperative KS511‐1001903