The IL-8 protease SpyCEP/ScpC of group A Streptococcus promotes resistance to neutrophil killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.     

Effect of inoculum density,nitrogen source and saprophytic fungi on Fusarium wilt of Mexican lime

Summary Mexican lime seedlings were inoculated with 0, 500, 1000, 2000, 4000 and 8000 microconidia ofFusarium oxysporum f. sp.citri per gram of potting media. The percent infection and mean disease severity rating increased with increasing inoculum density of the pathogen. In potting mix infested withAspergillus ochraceus, Penicillium funiculosum andTrichoderma harzianum at 5000 conidia per gram 2 weeks prior to infestation withF. oxysporum f. sp.citri at 0, 1000, 4000, and 8000 microconidia per gram,A. ochraceus reduced,P. funiculosum increased andT. harzianum had no effect on disease severity or pathogen population. OnlyP. funiculosum showed antagonistic activityin vitro against the pathogen. Disease severity and pathogen propagule densitites were greater and pH was lower in potting media fertilized with NH₄–N than in media fertilized with NO₃–N.Portion of M. S. thesis submitted by the senior author to the Graduate School, University of Florida, Gainesville. Florida Agricultural Experiment Stations Journal Series No. 4221.     

Protein abundance of AKT and ERK pathway components governs cell type‐specific regulation of proliferation

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.     

Inducible expression of erythrocyte band 3protein

A permanent cell line with inducible expression of the humananion exchanger protein 1 (hAE1) was constructed in a derivative ofhuman embryonic kidney cells (HEK-293). In the absence of the inducer,muristerone A, the new cell line had no detectable hAE1 protein byWestern analysis or additional³⁶Cl flux. Increasing dose andincubation time with muristerone A increased the amount of protein(both unglycosylated and glycosylated). The4,4'-dinitrostilbene-2,2'-disulfonate(DNDS)-inhibitable rapid Cl exchange flux was increased up to40-fold in induced cells compared with noninduced cells. There was noDNDS-inhibitable rapid flux component in noninduced cells. This resultdemonstrates inducible expression of a new rapid Cl transport pathwaythat is DNDS sensitive. The additional transport of³⁶Cl and³⁵SO₄had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 µM DNDS,2) activation of³⁶Cl efflux by external Cl with aconcentration producing half-maximal effect of 4.8 mM,3) activation of³⁶Cl efflux by external anionsthat was selective in the orderNO₃ = Cl > Br > I, and4) activation of³⁵SO₄influx by external protons. Under the assumption that the turnovernumbers of hAE1 were the same as in erythrocytes, there was good agreement (±3-fold) between the number of copies ofglycosylated hAE1 and the induced tracer fluxes. This is the firstexpression of hAE1 in a mammalian system to track the kineticcharacteristics of the native protein.

     

Caspase substrates

The relatively common occurrence of sequences within proteins that match the consensus substrate specificity of caspases in intracellular proteins suggests a multitude of substrates in vivo - somewhere in the order of several hundred in humans alone. Indeed, the list of proteins that are reported to be cleaved by caspases in vitro proliferates rapidly. However, only a few of these proteins have been rigorously established as biologically or pathologically relevant, bona fide substrates in vivo. Many of them probably simply represent 'innocent bystanders' or erroneous assignments. In this review we discuss concepts of caspase substrate recognition and specificity, give resources for the discovery and annotation of caspase substrates, and highlight some specific human or mouse proteins where there is strong evidence for biologic or pathologic relevance.     

Probing glycomics

The study of protein-carbohydrate interactions is one central theme of glycomics research. The challenges encountered when investigating these interactions have resulted in an approach that studies saccharides through the enzymes that process them. Proteins and their function are often probed by manipulating the genes that encode them. Efforts in proteoglycomics exploring protein-binding properties and the enzymatic modification of carbohydrates have intensified, and synthetic tools, including activity- and affinity-based probes, have enhanced our understanding of the roles of carbohydrates in biology.