CpxA/R-Controlled Nitroreductase Expression as Target for Combinatorial Therapy against Uropathogens by Promoting Reactive Oxygen Species Generation.

The antibiotic resistances emerged in uropathogens lead to accumulative treatment failure and recurrent episodes of urinary tract infection (UTI), necessitating more innovative therapeutics to curb UTI before systematic infection. In the current study, the combination of amikacin and nitrofurantoin is found to synergistically eradicate Gram-negative uropathogens in vitro and in vivo. The mechanistic analysis demonstrates that the amikacin, as an aminoglycoside, induced bacterial envelope stress by introducing mistranslated proteins, thereby constitutively activating the cpxA/R two-component system (Cpx signaling). The activation of Cpx signaling stimulates the expression of bacterial major nitroreductases (nfsA/nfsB) through soxS/marA regulons. As a result, the CpxA/R-dependent nitroreductases overexpression generates considerable quantity of lethal reactive intermediates via nitroreduction and promotes the prodrug activation of nitrofurantoin. As such, these actions together disrupt the bacterial cellular redox balance with excessively-produced reactive oxygen species (ROS) as "Domino effect", accelerating the clearance of uropathogens. Although aminoglycosides are used as proof-of-principle to elucidate the mechanism, the synergy between nitrofurantoin is generally applicable to other Cpx stimuli. To summarize, this study highlights the potential of aminoglycoside-nitrofurantoin combination to replenish the arsenal against recurrent Gram-negative uropathogens and shed light on the Cpx signaling-controlled nitroreductase as a potential target to manipulate the antibiotic susceptibility.

Antibiotic resistance and virulence characteristics of four carbapenem-resistant Klebsiella pneumoniae strains coharbouring blaKPC and blaNDM based on whole genome sequences from a tertiary general teaching hospital in central China between 2019 and 2021.

OBJECTIVE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a worldwide health issue that poses a serious threat to public health. This study summarizes the clinical features of four patients with CRKP coproducing NDM and KPC infections and further analyses the molecular typing, resistance and virulence factors of the four CRKP strains. METHODS: Of the twenty-two CRKP isolates, four strains coharbouring blaKPC and blaNDM isolated from four patients were screened by Sanger sequencing between October 2019 and April 2021. Demographics, clinical and pathological data of the four patients were collected through electronic medical records. Antimicrobial susceptibility testing, biofilm formation assays and serum bactericidal assays were performed on the four isolates. The antibiotic resistance and virulence genes were investigated by whole-genome sequencing. Sequence types (STs) were determined by multilocus sequence typing, and serotypes were identified by wzi gene sequencing. RESULTS: Three patients recovered, and one patient stopped treatment. Four strains were multiple carbapenemase producers: KPC-2, NDM-4, SME-5 and IMI-4 coproducer; KPC-2, NDM-1 and SME-3 coproducer; KPC-2, NDM-1 and IMI-3 coproducer; KPC-2 and NDM-5 coproducer. They also harboured ESBL genes and mutations in the efflux pump regulator genes. They were multidrug resistant but sensitive to tigecycline and colistin. Four isolates had moderate biofilm-forming abilities and carried various virulence genes, including siderophores, type 1 fimbriae and E. coli common pilus. Only the NO. 3 strain was resistant to the serum. The STs and serotypes of the four strains were ST11 and KL64, ST337 and none, ST307 and KL102KL149KL155, and ST29 and K54, respectively. CONCLUSION: Four CRKP strains coharbouring blaKPC and blaNDM also carried other carbapenemase genes. Notably, the NO. 1 isolate carrying four carbapenemase genes has not been reported globally until now. Four strains exhibited a high level of resistance to multiple antibiotics. Additionally, three of the four patients were exposed to invasive medical devices that provided an environment for biofilm formation. Meanwhile, three strains with adhesion genes as moderate biofilm formers might form biofilms resulting in long hospital stays, increasing therapeutic difficulty, and even treatment failure. This study reminds clinicians that CRKP strains with multiple carbapenemase genes emerged in our hospital, and stronger measures should be taken to the control of nosocomial infections.

A comprehensive review on microbial l-asparaginase: Bioprocessing, characterization, and industrial applications.

l-Asparaginase (E.C.3.5.1.1.) is a vital enzyme that hydrolyzes l-asparagine to l-aspartic acid and ammonia. This property of l-asparaginase inhibits the protein synthesis in cancer cells, making l-asparaginase a mainstay of pediatric chemotherapy practices to treat acute lymphoblastic leukemia (ALL) patients. l-Asparaginase is also recognized as one of the important food processing agent. The removal of asparagine by l-asparaginase leads to the reduction of acrylamide formation in fried food items. l-Asparaginase is produced by various organisms including animals, plants, and microorganisms, however, only microorganisms that produce a substantial amount of this enzyme are of commercial significance. The commercial l-asparaginase for healthcare applications is chiefly derived from Escherichia coli and Erwinia chrysanthemi. A high rate of hypersensitivity and adverse reactions limits the long-term clinical use of l-asparaginase. Present review provides thorough information on microbial l-asparaginase bioprocess optimization including submerged fermentation and solid-state fermentation for l-asparaginase production, downstream purification, its characterization, and issues related to the clinical application including toxicity and hypersensitivity. Here, we have highlighted the bioprocess techniques that can produce improved and economically viable yields of l-asparaginase from promising microbial sources in the current scenario where there is an urgent need for alternate l-asparaginase with less adverse effects.

Eye Drop Instillation of the Rac1 Modulator CNF1 Attenuates Retinal Gliosis and Ameliorates Visual Performance in a Rat Model of Hypertensive Retinopathy.

In hypertensive retinopathy, the retinal damage due to high blood pressure is accompanied by increased expression of Glial Fibrillary Acidic Protein (GFAP), which indicates a role of neuroinflammatory processes in such a retinopathy. Proteins belonging to the Rho GTPase family, particularly Rac1, are involved in the activation of Müller glia and in the progression of photoreceptor degeneration, and may thus represent a novel candidate for therapeutic intervention following central nervous system inflammation. In this paper, we have observed that topical administration as eye drops of Cytotoxic Necrotizing Factor 1 (CNF1), a Rho GTPase modulator, surprisingly improves electrophysiological and behavioral visual performances in aged spontaneously hypertensive rats. Furthermore, such functional improvement is accompanied by a reduction of Rac1 activity and retinal GFAP expression. Our results suggest that Rac1 inhibition through CNF1 topical administration may represent a new strategy to target retinal gliosis.

Bacterial Protein Mimetic of Peptide Hormone as a New Class of Protein- based Drugs.

Specific peptide molecules classified as hormones, neuropeptides and cytokines are involved in intercellular signaling regulating various physiological processes in all organs and tissues. This justifies the peptidergic signaling as an attractive pharmacological target. Recently, a protein mimetic of a peptide hormone has been identified in Escherichia coli suggesting the potential use of specific bacterial proteins as a new type of peptide-like drugs. We review the scientific rational and technological approaches leading to the identification of the E. coli caseinolytic protease B (ClpB) homologue protein as a conformational mimetic of α-melanocyte-stimulating hormone (α-MSH), a melanocortin peptide critically involved in the regulation of energy homeostasis in humans and animals. Theoretical and experimental backgrounds for the validation of bacterial ClpB as a potential drug are discussed based on the known E. coli ClpB amino acid sequence homology with α-MSH. Using in silico analysis, we show that other protein sources containing similar to E. coli ClpB α-MSH-like epitopes with potential biological activity may exist in Enterobacteriaceae and in some Brassicaceae. Thus, the original approach leading to the identification of E. coli ClpB as an α-MSH mimetic protein can be applied for the identification of mimetic proteins of other peptide hormones and development of a new type of peptide-like protein-based drugs.

Bacterial Toxins and Targeted Brain Therapy: New Insights from Cytotoxic Necrotizing Factor 1 (CNF1).

Pathogenic bacteria produce toxins to promote host invasion and, therefore, their survival. The extreme potency and specificity of these toxins confer to this category of proteins an exceptionally strong potential for therapeutic exploitation. In this review, we deal with cytotoxic necrotizing factor (CNF1), a cytotoxin produced by Escherichia coli affecting fundamental cellular processes, including cytoskeletal dynamics, cell cycle progression, transcriptional regulation, cell survival and migration. First, we provide an overview of the mechanisms of action of CNF1 in target cells. Next, we focus on the potential use of CNF1 as a pharmacological treatment in central nervous system's diseases. CNF1 appears to impact neuronal morphology, physiology, and plasticity and displays an antineoplastic activity on brain tumors. The ability to preserve neural functionality and, at the same time, to trigger senescence and death of proliferating glioma cells, makes CNF1 an encouraging new strategy for the treatment of brain tumors.

New therapeutics from Nature: The odd case of the bacterial cytotoxic necrotizing factor 1.

Natural products may represent a rich source of new drugs. The enthusiasm toward this topic has recently been fueled by the 2015 Nobel Prize in Physiology or Medicine, awarded for the discovery of avermectin and artemisinin, natural products from Bacteria and Plantae, respectively, which have targeted one of the major global health issues, the parasitic diseases. Specifically, bacteria either living in the environment or colonizing our body may produce compounds of unexpected biomedical value with the potentiality to be employed as therapeutic drugs. In this review, the fascinating history of CNF1, a protein toxin produced by pathogenic strains of Escherichia coli, is divulged. Even if produced by bacteria responsible for a variety of diseases, CNF1 can behave as a promising benefactor to mankind. By modulating the Rho GTPases, this bacterial product plays a key role in organizing the actin cytoskeleton, enhancing synaptic plasticity and brain energy level, rescuing cognitive deficits, reducing glioma growth in experimental animals. These abilities strongly suggest the need to proceed with the studies on this odd drug in order to pave the way toward clinical trials.

Immunization with recombinant Salmonella expressing SspH2-EscI protects mice against wild type Salmonella infection.

BACKGROUND: Enhancing caspase-1 activation in macrophages is helpful for the clearance of intracellular bacteria in mice. Our previous studies have shown that EscI, an inner rod protein of type III system in E. coli can enhance caspase-1 activation. The purpose of this study was to further analyze the prospect of EscI in the vaccine design. RESULTS: A recombinant Salmonella expressing SspH2-EscI fusion protein using the promotor of Salmonella effector SspH2, X4550(pYA3334-P-SspH2-EscI), was constructed. A control recombinant Salmonella expressing SspH2 only X4550(pYA3334-P-SspH2) was also constructed. In the early stage of in vitro infection of mouse peritoneal macrophages, X4550(pYA3334-P-SspH2-EscI) could significantly (P < 0.05) enhance intracellular caspase-1 activation and pyroptotic cell death of macrophages, when compared with X4550(pYA3334-P-SspH2). Except for the intracellular pH value, the levels of reactive oxygen species, intracellular concentration of calcium ions, nitric oxide and mitochondrial membrane potential in macrophages were not significantly different between the cells infected with X4550(pYA3334-P-SspH2-EscI) and those infected with X4550(pYA3334-P-SspH2). Besides, only lower inflammatory cytokines secretion was induced by X4550(pYA3334-P-SspH2-EscI) than X4550(pYA3334-P-SspH2). After intravenous immunization of mice (1 × 106 cfu/mouse), the colonization of X4550(pYA3334-P-SspH2-EscI) in mice was significantly limited at one week post immunization (wpi), when compared with X4550(pYA3334-P-SspH2) (P < 0.05). The population of activated CD8+T lymphocytes in mouse spleens induced by X4550(pYA3334-P-SspH2-EscI) was lower than that induced by X4550(pYA3334-P-SspH2) at 2-3 wpi, and the ratio of CD4+T cells to CD8+T cells decreased. The blood coagulation assay indicated that no significant difference was found between X4550(pYA3334-P-SspH2-EscI) and uninfected control, while X4550(pYA3334-P-SspH2) could induce the quick coagulation. Notably, immunization of X4550(pYA3334-P-SspH2-EscI) could limit the colonization of challenged Salmonella strains in the early stage of infection and provide more effective protection. CONCLUSION: The activation of caspase-1 in macrophages by EscI can be used in the design of live attenuated Salmonella vaccine candidate.

Expression of Salmonella typhimurium and Escherichia coli flagellin protein and its functional characterization as an adjuvant.

BACKGROUND: Flagellin is the major structural protein monomer of bacterial flagella. Flagellin through binding to its receptor and activation of antigen presenting cells stimulates the innate and adaptive immune responses. Flagellin is used as an effective systemic or mucosal adjuvant to stimulate the immune system. Recently, the therapeutic and protective role of flagellin in some infectious diseases and cancers has been investigated. In this study, we cloned the fliC genes from Salmonella typhimurium and Escherichia coli into pET-28a vector and investigated their expression in the prokaryotic system. METHODS: The fliC genes of S. typhimurium and E. coli were amplified by PCR with a specific oligonucleotide primer set. thse were cloned into the pET-28a vector and the recombinant pET-28a-fliC plasmids were successfully transformed into the E. coli strain BL-21(DE3). The expression of flagellin proteins in the prokaryotic cells were evaluated. Finally, Transcription of TNF-α mRNA was confirmed using Real-time PCR. RESULTS: The expression of proteins in the prokaryotic cells were approved by SDS-PAGE and western blotting method. Further, the functional characterization of flagellin proteins were evaluated using their ability to induce increased m-RNA expression of pro-inflammatory cytokine. CONCLUSIONS: The flagellin proteins were expressed in the prokaryotic system. These proteins can be used to link target antigens as an effective adjuvant for future DNA vaccine studies. Purified recombinant proteins in this study can also be used for therapeutic and prophylactic purposes.

Prolonged versus standard native E. coli asparaginase therapy in childhood acute lymphoblastic leukemia and non-Hodgkin lymphoma: final results of the EORTC-CLG randomized phase III trial 58951.

Asparaginase is an essential component of combination chemotherapy for childhood acute lymphoblastic leukemia and non-Hodgkin lymphoma. The value of asparaginase was further addressed in a group of non-very high-risk patients by comparing prolonged (long-asparaginase) versus standard (short-asparaginase) native E. coli asparaginase treatment in a randomized part of the phase III 58951 trial of the European Organization for Research and Treatment of Cancer Children's Leukemia Group. The main endpoint was disease-free survival. Overall, 1,552 patients were randomly assigned to long-asparaginase (775 patients) or short-asparaginase (777 patients). Patients with grade ≥2 allergy to native E. coli asparaginase were switched to equivalent doses of Erwinia or pegylated E. coli asparaginase. The 8-year disease-free survival rate (±standard error) was 87.0±1.3% in the long-asparaginase group and 84.4±1.4% in the short-asparaginase group (hazard ratio: 0.87; P=0.33) and the 8-year overall survival rate was 92.6±1.0% and 91.3±1.2% respectively (hazard ratio: 0.89; P=0.53). An exploratory analysis suggested that the impact of long-asparaginase was beneficial in the National Cancer Institute standard-risk group with regards to disease-free survival (hazard ratio: 0.70; P=0.057), but far less so with regards to overall survival (hazard ratio: 0.89). The incidences of grade 3-4 infection during consolidation (25.2% versus 14.4%) and late intensification (22.6% versus 15.9%) and the incidence of grade 2-4 allergy were higher in the long-asparaginase arm (30% versus 21%). Prolonged native E. coli asparaginase therapy in consolidation and late intensification for our non-very high-risk patients did not improve overall outcome but led to an increase in infections and allergy. This trial was registered at www.clinicaltrials.gov as #NCT00003728.

Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme.

BACKGROUND: Chemotherapy is an effective option to treat recurrent or metastatic cancer but its debilitating side-effects limit the dose and time of exposure. Prodrugs that can be activated locally by an activating enzyme can minimize collateral damage from chemotherapy. We previously demonstrated the efficacy of a poly-L-lysine-based theranostic nanoplex containing bacterial cytosine deaminase (bCD) that locally converted 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil in MDA-MB-231 primary tumor xenografts. MATERIALS AND METHODS: Here we used a more effective variant of bCD to target metastatic red fluorescence protein expressing MDA-MB-435 cells in the lungs. We used an intravenous injection of tumor cells and monitored tumor growth in the lungs for 5 weeks by which time metastatic nodules were detected with optical imaging. The animals were then treated with the bCD-nanoplex and 5-FC. RESULTS: We observed a significant decrease in metastatic burden with a single dose of the enzyme-nanoplex and two consecutive prodrug injections. CONCLUSION: These results are a first step towards the longitudinal evaluation of such a strategy with multiple doses. Additionally, the enzyme can be directly coupled to imaging reporters to time prodrug administration for the detection and treatment of aggressive metastatic cancer.

Therapeutic effects of the Rho GTPase modulator CNF1 in a model of Parkinson's disease.

Recent evidence suggests an early involvement of dopaminergic (DA) processes and terminals in Parkinson's disease (PD). The arborization of neurons depends on the actin cytoskeleton, which in turn is regulated by small GTPases of the Rho family, encompassing Rho, Rac and Cdc42 subfamilies. Indeed, some reports point to a role for Rac and Cdc42 signaling in the pathophysiology of inherited parkinsonisms. We thus investigated the potential therapeutic effect of the modulation of cerebral Rho GTPases in PD. Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa protein toxin produced by Escherichia coli, permanently activates RhoA, Rac1 and Cdc42 in intact cells. We report that the modulation of Rho GTPases by CNF1 results in hypertrophy of DA cell processes of cultured substantia nigra neurons, including increase in length, branching and varicosity. In vivo, the treatment corrects long-standing motor and biochemical asymmetries and restores degenerated nigrostriatal DA tissue after 6-hydroxydopamine lesion. We conclude that the pharmacological modulation of Rho GTPases shows neurorestorative potential and represents a promising avenue in the treatment PD. The study also suggests that naturally occurring molecules acting on Rho GTPase signaling, such as some bacterial protein toxins, might play a role in the development of PD.

Cell-to-cell propagation of the bacterial toxin CNF1 via extracellular vesicles: potential impact on the therapeutic use of the toxin.

Eukaryotic cells secrete extracellular vesicles (EVs), either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1). Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.

Redesigned Escherichia coli cytosine deaminase: a new facet of suicide gene therapy.

BACKGROUND: The Escherichia coli cytosine deaminase (CD)/5-fluorocytosine (5-FC) approach emerges as a potential aid for suicide gene therapy in the field of modern cancer treatment. However, the poor binding affinity of CD towards 5-FC compared to the natural substrate cytosine limits its application for successful suicide gene therapy. Redesigning a bacterial mutant CD with site-directed mutagenesis showed higher potency compare to wild-type CD (wtCD) in vitro. In the present study, we conducted a comparative analysis of F186W mutant and wtCD in a human lung cancer cell line (A549). METHODS AND RESULTS: A comparative investigation was initiated with cell viability analyses by MTT and trypan blue dye exclusion assays on A549 cells transfected with wtCD and F186W genes. The mode of cell death was confirmed by acridine Orange/ethidium Bromide dual staining. Furthermore, flow cytometric assessments were performed by cell cycle analysis and caspase 3 assay. The experimental results showed a drug dependent decrease in cell viability; interestingly, mutant (F186W) reached IC50 at a much lower concentration of prodrug (5-FC) than wtCD. Cell cycle analysis showed that G1 arrest of a larger population of 5-FC treated F186W transfected cells, in contrast to that of wtCD under similar conditions. The caspase 3 assay revealed progression and execution of apoptosis. CONCLUSIONS: We report a novel bacterial CD mutant that provided a superior alternate to the wtCD suicide gene. The F186W mutant required a much lower dose of 5-FC to reach its IC50 , thus minimizing the systemic side effects of large doses of 5-FC as required for wtCD.

Modulation of Rho GTPases rescues brain mitochondrial dysfunction, cognitive deficits and aberrant synaptic plasticity in female mice modeling Rett syndrome.

Rho GTPases are molecules critically involved in neuronal plasticity and cognition. We have previously reported that modulation of brain Rho GTPases by the bacterial toxin CNF1 rescues the neurobehavioral phenotype in MeCP2-308 male mice, a model of Rett syndrome (RTT). RTT is a rare X-linked neurodevelopmental disorder and a genetic cause of intellectual disability, for which no effective therapy is available. Mitochondrial dysfunction has been proposed to be involved in the mechanism of the disease pathogenesis. Here we demonstrate that modulation of Rho GTPases by CNF1 rescues the reduced mitochondrial ATP production via oxidative phosphorylation in the brain of MeCP2-308 heterozygous female mice, the condition which more closely recapitulates that of RTT patients. In RTT mouse brain, CNF1 also restores the alterations in the activity of the mitochondrial respiratory chain (MRC) complexes and of ATP synthase, the molecular machinery responsible for the majority of cell energy production. Such effects were achieved through the upregulation of the protein content of those MRC complexes subunits, which were defective in RTT mouse brain. Restored mitochondrial functionality was accompanied by the rescue of deficits in cognitive function (spatial reference memory in the Barnes maze), synaptic plasticity (long-term potentiation) and Tyr1472 phosphorylation of GluN2B, which was abnormally enhanced in the hippocampus of RTT mice. Present findings bring into light previously unknown functional mitochondrial alterations in the brain of female mice modeling RTT and provide the first evidence that RTT brain mitochondrial dysfunction can be rescued by modulation of Rho GTPases.

The B subunit of Escherichia coli enterotoxin helps control the in vivo growth of solid tumors expressing the Epstein-Barr virus latent membrane protein 2A.

Latent membrane protein 2A (LMP2A) is expressed on almost all Epstein-Barr virus (EBV)-associated tumors and is a potential target for immunotherapeutic intervention and vaccination. However, LMP2A is not efficiently processed and presented on major histocompatibility antigens class I molecules to generate potent cytotoxic T-lymphocytes (CTL) responses capable of killing these tumors. The B subunit of Escherichia coli enterotoxin (EtxB), causes rapid internalization and processing of membrane-bound LMP2A on EBV-infected B cells, and facilitates loading of processed-LMP2A peptides onto MHC class I. This re-directed trafficking/delivery of LMP2A to the MHC class I machinery enhances recognition and killing by LMP2A-specific CTL in vitro. To test the potential of EtxB to enhance immune targeting of LMP2A expressed in solid tumors, we generated a murine tumor model (Renca-LMP2A), in which LMP2A is expressed as a transgenic neoantigen on a renal carcinoma (Renca) cell line and forms solid tumors when injected subcutaneously into BALB/c mice. The data show that in BALB/c mice which have only low levels of peripheral K(d)-LMP2A-specific CD8(+) T cells, merely a transient inhibition of tumor growth is achieved compared with naïve mice; suggesting that there is suboptimal LMP2A-specifc CTL recognition and poorly targeted tumor killing. However, importantly, treatment of these mice with EtxB led to a significant delay in the onset of tumor growth and significantly lower tumor volumes compared with similar mice that did not receive EtxB. Moreover, this remarkable effect of EtxB was achieved despite progressive reduction in tumor expression of LMP2A and MHC class I molecules. These data clearly demonstrate the potential efficacy of EtxB as a novel therapeutic agent that could render EBV-associated tumors susceptible to immune control.

Protection from hemolytic uremic syndrome by eyedrop vaccination with modified enterohemorrhagic E. coli outer membrane vesicles.

We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs) is effective for protecting against hemolytic uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB). Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB) subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.

A prospective study on drug monitoring of PEGasparaginase and Erwinia asparaginase and asparaginase antibodies in pediatric acute lymphoblastic leukemia.

This study prospectively analyzed the efficacy of very prolonged courses of pegylated Escherichia coli asparaginase (PEGasparaginase) and Erwinia asparaginase in pediatric acute lymphoblastic leukemia (ALL) patients. Patients received 15 PEGasparaginase infusions (2500 IU/m(2) every 2 weeks) in intensification after receiving native E coli asparaginase in induction. In case of allergy to or silent inactivation of PEGasparaginase, Erwinia asparaginase (20 000 IU/m(2) 2-3 times weekly) was given. Eighty-nine patients were enrolled in the PEGasparaginase study. Twenty (22%) of the PEGasparaginase-treated patients developed an allergy; 7 (8%) showed silent inactivation. The PEGasparaginase level was 0 in all allergic patients (grade 1-4). Patients without hypersensitivity to PEGasparaginase had serum mean trough levels of 899 U/L. Fifty-nine patients were included in the Erwinia asparaginase study; 2 (3%) developed an allergy and none silent inactivation. Ninety-six percent had at least 1 trough level ≥100 U/L. The serum asparagine level was not always completely depleted with Erwinia asparaginase in contrast to PEGasparaginase. The presence of asparaginase antibodies was related to allergies and silent inactivation, but with low specificity (64%). Use of native E coli asparaginase in induction leads to high hypersensitivity rates to PEGasparaginase in intensification. Therefore, PEGasparaginase should be used upfront in induction, and we suggest that the dose could be lowered. Switching to Erwinia asparaginase leads to effective asparaginase levels in most patients. Therapeutic drug monitoring has been added to our ALL-11 protocol to individualize asparaginase therapy.

Inhibition of conidiophore development in Aspergillus fumigatus by an Escherichia coli DH5α strain, a promising antifungal candidate against aspergillosis.

The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigated secretory and cytosolic proteins of non-pathogenic bacterial species (mostly belonging to human microbiome) for antifungal potential against A. fumigatus, A. flavus and A. niger. Our preliminary results revealed that cytosolic proteins of E. coli DH5α were most active and the less toxic against various pathogenic isolates of A. fumigatus (the major pathogenic species), depicting a minimum inhibitory concentration (MIC) of 62.50 µg/mL, 62.50 µg/mL and 12.50 µg/disc using microbroth dilution assay (MDA), percentage spore germination inhibition assay (PSGI) and disc diffusion assay (DDA), respectively. E. coli protein was non-toxic against human erythrocytes at doses up to 1000 µg/mL as compared to standard drug, amphotericin B which lysed 100% of erythrocytes at a concentration of 37.50 µg/mL. Time kill analysis proved it to be fungicidal in a concentration and time-dependent manner. Scanning electron microscopic studies (SEM) were carried out to prevail what kind of damage it causes to A. fumigatus. SEM results reported that conidiophore (structures forming conidia) development was halted as a major consequence, reducing the number of conidiophores to insignificant values as well as alteration in their morphological attributes. This feature may contribute to the development of new prevention strategies against Aspergillus infections. Hyphal atrophy was also observed, evidenced by shrinking and flattening of hyphal walls and reduced, abrupt hyphal branching. Such actions may effectively reduce the invasive ability of Aspergillus as well as it can sterilize the fungal burden by obstructing the conidiation pathway of A. fumigatus. Hence, E. coli DH5α, being a commensal species, can lead to the development of antifungal molecule with novel targets in fungal metabolism, which will help in combating the antifungal resistance and toxicity associated with current therapy.