Bcl-xL targeting eliminates ageing tumor-promoting neutrophils and inhibits lung tumor growth.

Elevated peripheral blood and tumor-infiltrating neutrophils are often associated with a poor patient prognosis. However, therapeutic strategies to target these cells are difficult to implement due to the life-threatening risk of neutropenia. In a genetically engineered mouse model of lung adenocarcinoma, tumor-associated neutrophils (TAN) demonstrate tumor-supportive capacities and have a prolonged lifespan compared to circulating neutrophils. Here, we show that tumor cell-derived GM-CSF triggers the expression of the anti-apoptotic Bcl-xL protein and enhances neutrophil survival through JAK/STAT signaling. Targeting Bcl-xL activity with a specific BH3 mimetic, A-1331852, blocked the induced neutrophil survival without impacting their normal lifespan. Specifically, oral administration with A-1331852 decreased TAN survival and abundance, and reduced tumor growth without causing neutropenia. We also show that G-CSF, a drug used to combat neutropenia in patients receiving chemotherapy, increased the proportion of young TANs and augmented the anti-tumor effect resulting from Bcl-xL blockade. Finally, our human tumor data indicate the same role for Bcl-xL on pro-tumoral neutrophil survival. These results altogether provide preclinical evidence for safe neutrophil targeting based on their aberrant intra-tumor longevity.

SifA SUMOylation governs Salmonella Typhimurium intracellular survival via modulation of lysosomal function.

One of the mechanisms shaping the pathophysiology during the infection of enteric pathogen Salmonella Typhimurium is host PTM machinery utilization by the pathogen encoded effectors. Salmonella Typhimurium (S. Tm) during infection in host cells thrives in a vacuolated compartment, Salmonella containing vacuole (SCV), which sequentially acquires host endosomal and lysosomal markers. Long tubular structures, called as Salmonella induced filaments (SIFs), are further generated by S. Tm, which are known to be required for SCV's nutrient acquisition, membrane maintenance and stability. A tightly coordinated interaction involving prominent effector SifA and various host adapters PLEKHM1, PLEKHM2 and Rab GTPases govern SCV integrity and SIF formation. Here, we report for the first time that the functional regulation of SifA is modulated by PTM SUMOylation at its 11th lysine. S. Tm expressing SUMOylation deficient lysine 11 mutants of SifA (SifAK11R) is defective in intracellular proliferation due to compromised SIF formation and enhanced lysosomal acidification. Furthermore, murine competitive index experiments reveal defective in vivo proliferation and weakened virulence of SifAK11R mutant. Concisely, our data reveal that SifAK11R mutant nearly behaves like a SifA knockout strain which impacts Rab9-MPR mediated lysosomal acidification pathway, the outcome of which culminates in reduced bacterial load in in vitro and in vivo infection model systems. Our results bring forth a novel pathogen-host crosstalk mechanism where the SUMOylation of effector SifA regulated S. Tm intracellular survival.

Activation of epigenetic regulator KDM6B by Salmonella Typhimurium enables chronic infections.

Non-typhoidal Salmonella (NTS) infections result in self limiting gastroenteritis except in rare cases wherein manifestations of chronic infections can occur. Strategies employed by Salmonella to thrive in hostile environments of host during chronic infections are complex and multifaceted. In chronic state, a coordinated action of bacterial effectors allows reprogramming of macrophages to M2 subtype and thereby creating a permissible replicative niche. The mechanistic details of these processes are not fully known. In the current study we identified, histone H3-lysine 27 trimethylation (H3K27me3)-specific demethylase, KDM6B to be upregulated in both cell culture and in murine model of Salmonella infection. KDM6B recruitment upon infection exhibited an associated loss of overall H3K27me3 in host cells and was Salmonella SPI1 effectors coordinated. ChIP-qRT-PCR array analysis revealed several new gene promoter targets of KDM6B demethylase activity including PPARδ, a crucial regulator of fatty acid oxidation pathway and Salmonella-persistent infections. Furthermore, pharmacological inhibition of KDM6B demethylase activity with GSKJ4 in chronic Salmonella infection mice model led to a significant reduction in pathogen load and M2 macrophage polarization in peripheral lymphoid organs. The following work thus reveals Salmonella effector-mediated epigenetic reprogramming of macrophages responsible for its long-term survival and chronic carriage.

Molecular determinants of peaceful coexistence versus invasiveness of non-Typhoidal Salmonella: Implications in long-term side-effects.

The genus Salmonella represents a wide range of strains including Typhoidal and Non-Typhoidal Salmonella (NTS) isolates that exhibit illnesses of varied pathophysiologies. The more frequent NTS ensues a self-limiting enterocolitis with rare occasions of bacteremia or systemic infections. These self-limiting Salmonella strains are capable of subverting and dampening the host immune system to achieve a more prolonged survival inside the host system thus leading to chronic manifestations. Notably, emergence of new invasive NTS isolates known as invasive Non-Typhoidal Salmonella (iNTS) have worsened the disease burden significantly in some parts of the world. NTS strains adapt to attain persister phenotype intracellularly and cause relapsing infections. These chronic infections, in susceptible hosts, are also capable of causing diseases like IBS, IBD, reactive arthritis, gallbladder cancer and colorectal cancer. The present understanding of molecular mechanism of how these chronic infections are manifested is quite limited. The current work is an effort to review the prevailing knowledge emanating from a large volume of research focusing on various forms of NTS infections including those that cause localized, systemic and persistent disease. The review will further dwell into the understanding of how this pathogen contributes to the associated long term sequelae.

A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

SUMOylation pathway alteration coupled with downregulation of SUMO E2 enzyme at mucosal epithelium modulates inflammation in inflammatory bowel disease.

Post-translational modification pathways such as SUMOylation are integral to all cellular processes and tissue homeostasis. We investigated the possible involvement of SUMOylation in the epithelial signalling in Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD). Initially in a murine model of IBD, induced by dextran-sulfate-sodium (DSS mice), we observed inflammation accompanied by a lowering of global SUMOylation of colonic epithelium. The observed SUMOylation alteration was due to a decrease in the sole SUMO E2 enzyme (Ubc9). Mass-spectrometric analysis revealed the existence of a distinct SUMOylome (SUMO-conjugated proteome) in DSS mice with alteration of key cellular regulators, including master kinase Akt1. Knocking-down of Ubc9 in epithelial cells resulted in dramatic activation of inflammatory gene expression, a phenomenon that acted via reduction in Akt1 and its SUMOylated form. Importantly, a strong decrease in Ubc9 and Akt1 was also seen in endoscopic biopsy samples (N = 66) of human CD and UC patients. Furthermore, patients with maximum disease indices were always accompanied by severely lowered Ubc9 or SUMOylated-Akt1. Mucosal tissues with severely compromised Ubc9 function displayed higher levels of pro-inflammatory cytokines and compromised wound-healing markers. Thus, our results reveal an important and previously undescribed role for the SUMOylation pathway involving Ubc9 and Akt1 in modulation of epithelial inflammatory signalling in IBD.

Glycoside hydrolase production by Aspergillus terreus CM20 using mixture design approach for enhanced enzymatic saccharification of alkali pretreated paddy straw.

A successful lignocellulosic ethanol production process needs to address the technological impediments such as cost-competitiveness and sustainability of the process. Effective biomass utilization requires a repertoire of enzymes including various accessory enzymes. Developing an enzyme preparation with defined hydrolytic activities can circumvent the need for supplementing cellulases with accessory enzymes for enhanced hydrolysis. With this objective, mixture design approach was used in the present study to enhance glycoside hydrolase production of a fungal isolate, Aspergillus terreus CM20, by determining the proportion of different lignocellulosic components as enzyme inducers in the culture medium. A mixture of paddy straw and wheat straw (1.42:1.58) resulted in improved cellulolytic activities. The precipitated crude enzyme showed higher CMCase (365.03 18 IU g-1), FPase (161.48 IU g-1), avicelase (15.46 IU g-1), ß-glucosidase (920.92 IU g-1) and xylanase (9627.79 IU g-1) activities. The potential of the crude enzyme for saccharification of alkali pretreated paddy straw was also tested. Under optimum conditions, saccharification released 25.0 g L-1 of fermentable sugars. This indicates the superiority of the crude enzyme produced with respect to its hydrolytic enzyme components.

Salmonella Engages Host MicroRNAs To Modulate SUMOylation: a New Arsenal for Intracellular Survival.

Posttranslational modifications (PTMs) can alter many fundamental properties of a protein. One or combinations of them have been known to regulate the dynamics of many cellular pathways and consequently regulate all vital processes. Understandably, pathogens have evolved sophisticated strategies to subvert these mechanisms to achieve instantaneous control over host functions. Here, we present the first report of modulation by intestinal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) of host SUMOylation, a PTM pathway central to all fundamental cellular processes. Both in cell culture and in a mouse model, we observed that S. Typhimurium infection led to a dynamic SUMO-conjugated proteome alteration. The intracellular survival of S. Typhimurium was dependent on SUMO status as revealed by reduced infection and Salmonella-induced filaments (SIFs) in SUMO-upregulated cells. S. Typhimurium-dependent SUMO modulation was seen as a result of depletion of crucial SUMO pathway enzymes Ubc-9 and PIAS1, at both the protein and the transcript levels. Mechanistically, depletion of Ubc-9 relied on upregulation of small noncoding RNAs miR30c and miR30e during S. Typhimurium infection. This was necessary and sufficient for both down-modulation of Ubc-9 and a successful infection. Thus, we demonstrate a novel strategy of pathogen-mediated perturbation of host SUMOylation, an integral mechanism underlying S. Typhimurium infection and intracellular survival.

Optimization of Enzymatic Saccharification of Alkali Pretreated Parthenium sp. Using Response Surface Methodology.

Parthenium sp. is a noxious weed which threatens the environment and biodiversity due to its rapid invasion. This lignocellulosic weed was investigated for its potential in biofuel production by subjecting it to mild alkali pretreatment followed by enzymatic saccharification which resulted in significant amount of fermentable sugar yield (76.6%). Optimization of enzymatic hydrolysis variables such as temperature, pH, enzyme, and substrate loading was carried out using central composite design (CCD) in response to surface methodology (RSM) to achieve the maximum saccharification yield. Data obtained from RSM was validated using ANOVA. After the optimization process, a model was proposed with predicted value of 80.08% saccharification yield under optimum conditions which was confirmed by the experimental value of 85.80%. This illustrated a good agreement between predicted and experimental response (saccharification yield). The saccharification yield was enhanced by enzyme loading and reduced by temperature and substrate loading. This study reveals that under optimized condition, sugar yield was significantly increased which was higher than earlier reports and promises the use of Parthenium sp. biomass as a feedstock for bioethanol production.

Comparative efficiency of different pretreatment methods on enzymatic digestibility of Parthenium sp.

The potential of Parthenium sp. as a feedstock for enzymatic saccharification was investigated by using chemical and biological pretreatment methods. Mainly chemical pretreatments (acid and alkali) were compared with biological pretreatment with lignolytic fungi Marasmiellus palmivorus PK-27. Structural and chemical changes as well as crystallinity of cellulose were examined through scanning electron microscopy, fourier transform infra red and X-ray diffraction analysis, respectively after pretreatment. Total reducing sugar released during enzymatic saccharification of pretreated substrates was also evaluated. Among the pretreatment methods, alkali (1% NaOH) treated substrate showed high recovery of acid perceptible polymerised lignin (7.53 ± 0.5 mg/g) and significantly higher amount of reducing sugar (513.1 ± 41.0 mg/gds) compared to uninoculated Parthenium (163.4 ± 21.2) after 48 h of hydrolysis. This is the first report of lignolytic enzyme production from M. palmivorus, prevalent in oil palm plantations in Malaysia and its application in biological delignification of Parthenium sp. Alkali (1% NaOH) treatment proves to be the suitable method of pretreatment for lignin recovery and enhanced yield of reducing sugar which may be used for bioethanol production from Parthenium sp.

Harnessing the hydrolytic potential of phytopathogenic fungus Phoma exigua ITCC 2049 for saccharification of lignocellulosic biomass.

Phytopathogenic fungi develop unique systems for fast invasion by producing hydrolases, which may be explored as a source of hydrolytic enzymes for biofuel research. The present work deals with evaluation of a potato pathogen Phoma exigua ITCC 2049 for its potential to produce cellulase and xylanase enzyme. Taguchi methodology was applied to reveal the influence and contribution of five important factors (carbon source, organic and inorganic nitrogen source, surfactant, and pH) on hydrolytic enzyme production by Phoma. Cultivation of fungus under optimized condition produced endoglucanase (37.00 IU/ml), FPase (1.13 IU/ml), ß-glucosidase (2.67 IU/ml) and xylanase (24.92 IU/ml) within 8 days of submerged fermentation. Saccharification of biopretreated Parthenium and paddy straw with cocktail of Phoma secretome supplemented with commercial ß-glucosidase resulted in the significantly higher reducing sugar yield (651.04-698.11 mg/gds). This study proves the potential of Phoma as an alternative source of enzymes for biomass saccharification.

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.