Repression of Inflammasome by Francisella tularensis during Early Stages of Infection

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Development of a Multivalent Subunit Vaccine against Tularemia Using Tobacco Mosaic Virus (TMV) Based Delivery System

Francisella tularensis is a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD₁₀₀) doses of F. tularensis LVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensis antigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensis proteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens.     

Plasmid-Mediated Sulfamethoxazole Resistance Encoded by the sul2 Gene in the Multidrug-Resistant Shigella flexneri 2a Isolated from Patients with Acute Diarrhea in Dhaka,Bangladesh

In this study, mechanisms of plasmid-mediated sulfamethoxazole resistances in the clinical strains of multi-drug resistant (MDR) Shigella flexneri 2a were elucidated for the first time in Bangladesh. From 2006 to 2011, a total of 200 S. flexneri 2a strains were randomly selected from the stock of the Enteric and Food Microbiology Laboratory of icddr,b. Antimicrobial susceptibility of the strains showed 73%, 98%, 93%, 58%, 98%, 64% and 4% resistance to trimethoprim-sulfamethoxazole, nalidixic acid, ampicillin, erythromycin, tetracycline, ciprofloxacin and ceftriaxone respectively. Plasmid profiling revealed heterogeneous patterns and interestingly, all the trimethoprim-sulfamethoxazole resistant (SXT^R) strains yielded a distinct 4.3 MDa plasmid compared to that of the trimethoprim-sulfamethoxazole susceptible (SXT^S) strains. Curing of this 4.3 MDa plasmid resulted in the susceptibility to sulfamethoxazole alone suggesting the involvement of this plasmid in the resistance of sulfamethoxazole. Moreover, PCR analysis showed the presence of sul2 gene in SXT^R strains which is absent in SXT^S strains as well as in the 4.3 MDa plasmid-cured derivatives, confirming the involvement of sul2 in the resistance of sulfamethoxazole. Furthermore, pulsed-field gel electrophoresis (PFGE) analysis revealed that both the SXT^R and SXT^S strains were clonal. This study will significantly contributes to the knowledge on acquired drug resistance of the mostly prevalent S. flexneri 2a and further warrants continuous monitoring of the prevalence and correlation of this resistance determinants amongst the clinical isolates of Shigella and other enteric pathogens around the world to provide effective clinical management of the disease.     

Evidence of an alternative route of cellobiase secretion in the presence of brefeldin A in the filamentous fungus Termitomyces clypeatus

Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 microgram/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50°C, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.     

Oxidative stress,DNA damage,and the telomeric complex as therapeutic targets in acute neurodegeneration

Oxidative stress has been identified as an important contributor to neurodegeneration associated with acute CNS injuries and diseases such as spinal cord injury (SCI), traumatic brain injury (TBI), and ischemic stroke. In this review, we briefly detail the damaging effects of oxidative stress (lipid peroxidation, protein oxidation, etc.) with a particular emphasis on DNA damage. Evidence for DNA damage in acute CNS injuries is presented along with its downstream effects on neuronal viability. In particular, unchecked oxidative DNA damage initiates a series of signaling events (e.g. activation of p53 and PARP-1, cell cycle re-activation) which have been shown to promote neuronal loss following CNS injury. These findings suggest that preventing DNA damage might be an effective way to promote neuronal survival and enhance neurological recovery in these conditions. Finally, we identify the telomere and telomere-associated proteins (e.g. telomerase) as novel therapeutic targets in the treatment of neurodegeneration due to their ability to modulate the neuronal response to both oxidative stress and DNA damage.     

SU5416 and EGCG Work Synergistically and Inhibit Angiogenic and Survival Factors and Induce Cell Cycle Arrest to Promote Apoptosis in Human Malignant Neuroblastoma SH-SY5Y and SK-N-BE2 Cells

Malignant neuroblastomas are solid tumors in children. Available therapeutic agents are not highly effective for treatment of malignant neuroblastomas. Therefore, new treatment strategies are urgently needed. We tested the efficacy of combination of SU5416 (SU), an inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2), and (−)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, for controlling growth of human malignant neuroblastoma SH-SY5Y and SK-N-BE2 cells. Combination of 20 μM SU and 50 μM EGCG synergistically inhibited cell survival, suppressed expression of VEGFR-2, inhibited cell migration, caused cell cycle arrest, and induced apoptosis. Combination of SU and EGCG effectively blocked angiogenic and survival pathways and modulated expression of cell cycle regulators. Apoptosis was induced by down regulation of Bcl-2, activation of caspase-3, and cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP). Taken together, this combination of drugs can be a promising therapeutic strategy for controlling the growth of human malignant neuroblastoma cells.     

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as "undescribed" or "novel" taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.