Andrea Villarino

Inhibition of Mycobacterium tuberculosis tyrosine phosphatase PtpA by synthetic chalcones: Kinetics, molecular modeling, toxicity and effect on growth

Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world, and it is estimated that one-third of the worlds population is infected with Mycobacterium tuberculosis. Among a series of tested compounds, we have recently identified five synthetic chalcones which inhibit the activity of M. tuberculosis protein tyrosine phosphatase A (PtpA), an enzyme associated with M. tuberculosis infectivity. Kinetic studies demonstrated that these compounds are reversible competitive inhibitors. In this work we also carried out the analysis of the molecular recognition of these inhibitors on their macromolecular target, PtpA, through molecular modeling. We observed that the predominant determinants responsible for the inhibitory activity of the chalcones are the positions of the two methoxyl groups at the A-ring, that establish hydrogen bonds with the amino acid residues Arg17, His49, and Thr12 in the active site of PtpA, and the substitution of the phenyl ring for a 2-naphthyl group as B-ring, that undergoes pi stacking hydrophobic interaction with the Trp48 residue from PtpA. Interestingly, reduction of mycobacterial survival in human macrophages upon inhibitor treatment suggests their potential use as novel therapeutics. The biological activity, synthetic versatility, and low cost are clear advantages of this new class of potential tuberculostatic agents.

The FHA-containing protein GarA acts as a phosphorylation-dependent molecular switch in mycobacterial signaling.

MINT‐6804218: GarA (uniprotkb:P64897) and GarA (uniprotkb:P64897) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

Cellular activities in ultra-violet killed Escherichia coli

In this work we analyze the physiological state of cells after lethal-UV dose disinfection using independent metabolic markers. Through the detection of some metabolic activities we proved that cell lysis does not immediately follow death in UV-irradiated Escherichia coli K12 cells.

New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state

The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

Russian sturgeon cultured in a subtropical climate shows weaken innate defences and a chronic stress response

Abstract Russian sturgeon (Acipenser gueldenstaedtii) has been successfully farmed in Uruguay for the past ten years. However, during the Uruguayan summer fish endure high water temperatures and increased bacterial infections that threaten aquaculture. Our understanding of sturgeons immune system and its interplay with environmental factors like temperature is almost unknown. This study analysed the way in which seasonal variations affect enzymatic blood components of Russian sturgeons innate defences, including the serum alternative complement pathway (ACP), ceruloplasmin (Cp) and lysozyme activities. Results showed that summertime conditions in the farm altered these defences in different ways, inducing a significant decrease in ACP and Cp, and an increase in lysozyme. In addition, serum levels of total protein and cortisol decreased in summer, suggesting a chronic stress response was induced in parallel. Subsequently, we analysed whether the increase in water river temperature during summer could account for the observed results. To that end, we acclimated juvenile sturgeons to mild (18 °C) or warm (24 °C) temperatures for 37 days. Like in summer, sturgeons exposed to 24 °C showed lower levels of serum ACP, Cp and total proteins, together with a progressive decrease in body weight and increased fish mortality. Administration of an immunostimulant containing Se and Zn slightly reverted the temperature‐induced effects on sturgeons defences. Altogether, our study provides novel data on various physiological parameters of the Russian sturgeon and highlights the impact warm temperature has on stress and innate immunity in this chondrostean fish. HighlightsWe describe novel data on Russian sturgeons innate defences and stress response.Uruguayan summer conditions weaken sturgeons innate soluble components.Weakened defences correlated with a chronic stress response.Long‐term exposure to 24 °C reproduces summer‐induced alterations.

OH1 from Orf Virus: A New Tyrosine Phosphatase that Displays Distinct Structural Features and Triple Substrate Specificity

Viral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homolog in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual-specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual-specificity phosphatase and reveals its ability to dephosphorylate phosphatidylinositol 3,5-bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation.

AQUACULTURE OF RUSSIAN STURGEON IN URUGUAY: DECREASE OF THE INNATE DEFENSES IN SUMMER AND ITS ASSOCIATION WITH HIGH TEMPERATURES

Sturgeons (family Acipenseridae) are fish species of high ecological and economic value. Unfortunately, sturgeon species are highly endangered; overfishing and pollution have led to a drastic decline of natural reserves. Aquaculture is of decisive importance for the conservation and restoration of sturgeon populations, representing also an activity with valuable socio-economic impact. Acipenser gueldensteadtii (Russian sturgeon) is one of the most cultured sturgeon specie worldwide. In Uruguay, sturgeon farming has been successful, but a high susceptibility to bacterial infection has been observed during summer. We studied the health status of Russian sturgeons, cultured during winter and summer seasons in an Uruguayan farm, by analyzing the activity of some innate immune components, including serum ceruloplasmin (Cp), the alternative complement pathway (ACP) and lysozyme. Adult females exhibited a low serum level of Cp and ACP, and a slight increase of lysozyme level during summer. Since Cp and ACP activities depend strongly on liver synthesis, we explored whether their alterations were linked to a chronic stress response. Serum protein levels were significantly lower in adult sturgeons during summer, but no significant differences were observed in cortisol. Therefore, a chronic stress response might have altered liver metabolism, interfering with the synthesis of constitutive proteins, including Cp and ACP components. Examination of the contribution of temperature changes to the observed effects was carried out by analyzing the same indicators in young sturgeons cultured at 180C or 240C during 37 days. In addition, the effect of oral administration of a nutritional supplement containing Se, Zn and a yeast-derived immunostimulator (Biopack, Alltech) was analyzed. Exposure to 240C caused a high mortality and a significant decrease in fish growth and serum protein levels, but not changes in cortisol. Cp and ACP activities were significantly diminished at 240C. Results resemble those obtained for sturgeon reared in summer. Biopack administration enhanced fish growth, Cp, and ACP activities in fishes cultured at 180C, although failed to reverse the detrimental effects observed at 240C. In contrast, Biopack seems to revert the modest increase in lysozyme activity found at 240C. Overall, the higher susceptibility of sturgeons to bacterial infections may be caused by the usual temperatures reported during the Uruguayan summer, which would lead to decrease innate defenses as Cp and ACP. Biopack supplementation may benefit sturgeon health status, but further studies are needed to determine if it could prevent defense deficiencies provoked by high temperatures.