Anti-biofilm activity of caffeine against uropathogenic E. coli is mediated by curli biogenesis.

Biofilms are assemblages of sessile microorganisms that form an extracellular matrix around themselves and mediate attachment to surfaces. The major component of the extracellular matrix of Uropathogenic E. coli and other Enterobacteriaceae are curli fibers, making biofilms robust and resistant to antimicrobials. It is therefore imperative to screen antibiofilm compounds that can impair biofilm formation. In the present study, we investigated the curli-dependent antibiofilm activity of caffeine against UPEC strain CFT073 and commensal strain E. coli K-12MG1655.Caffeine significantly reduced the biofilm formation of both UPEC and E. coli K-12 by 86.58% and 91.80% respectively at 48 mM caffeine as determined by Crystal Violet assay. These results were further confirmed by fluorescence microscopy and Scanning Electron Microscope (SEM). Caffeine significantly reduced the cytotoxicity and survivability of UPEC. Molecular docking analysis revealed a strong interaction between caffeine and curli regulator protein (Csg D) of E. coli. The qRT-PCR data also showed significant downregulation in the expression of CsgBA and the CsgDEFG operon at both 24 mM and 48 mM caffeine. The findings revealed that caffeine could inhibit E. coli biofilm formation by regulating curli assembly and thus may be used as an alternative therapeutic strategy for the treatment of chronic E. coli biofilm-related infections.

Local cytokine/chemokine profiles in BALB/c and C57BL/6 mice in response to T. vaginalis infection.

Trichomonas vaginalis is the causative agent of Trichomoniasis (a sexually transmitted infection). Recent reports have shown that stimulation of cellular immunity can reduce trichomoniasis infection. Animal studies are essential to understanding the pathogenesis of infection and developing new potential drugs and vaccines to treat the infection. Therefore, we have tried to understand the pathogenesis of T. vaginalis infection by investigating the differences in the expression of chemokine/cytokine levels in vaginal and cervical tissues of BALB/c and C57BL/6 mice. Different pathological symptoms, like desquamation, neutrophil infiltration, and hemorrhage, were recorded in BALB/c and C57BL/6 in response to T. vaginalis infection. Vaginal and cervical tissues of BALB/c showed these symptoms on 2nd dpi, which became severe on 7th dpi and turned to mild or normal till 14th dpi compared to C57BL/6 strain. Immunohistochemistry in the vagina and cervical tissues of BALB/c and C57BL/6 mice was done to assess cytokines at different time intervals post-infection. Significant expression of Interleukin-1ß (IL-1ß) (a pro-inflammatory cytokine) was found in BALB/c compared to the C57BL/6 mice, on 7th dpi and 2nd dpi in vaginal and cervical tissues, respectively. Higher expression of MIP-2 (neutrophil chemoattractant) was observed in the vaginal tissues of BALB/c mice on 7th dpi compared to the C57BL/6 group. In addition, higher expression of TGF-ß (immune-suppressor) was observed on 7th dpi in the vaginal tissue of BALB/c mice. The present study demonstrates that more pathological signs of T. vaginalis infection developed in BALB/c mice than C57BL/6 mice. Also, significant levels of IL-1ß and MIP-2 were measured in BALB/c mice in response to T. vaginalis compared to C57BL/6.

Latent Upregulation of Nlrp3, Nlrc4 and Aim2 Differentiates between Asymptomatic and Symptomatic Trichomonas vaginalis Infection.

Trichomonas vaginalis is a parasitic protozoan that causes trichomoniasis. The involvement of NLRP3 inflammasome in trichomoniasis has been discussed in recent studies. The present study aimed to find out the involvement of Nlrp3, Nlrc4, and Aim2 in the BALB/c mouse model infected with symptomatic and asymptomatic isolates of T. vaginalis by quantitative real-time PCR and immunohistochemistry. Our results showed a significantly increased expression of Nlrp3 in the vaginal tissue of the symptomatic group on the 2nd dpi and 14th dpi in the asymptomatic group, respectively. The cervical tissue of asymptomatic groups expressed higher Nlrp3 on 14th dpi than the symptomatic group. The Nlrc4 was expressed on 14th dpi in the vaginal and cervical tissues of mice infected with asymptomatic group as compared to the symptomatic group. Aim2 expression in vaginal tissue was highest at early time points in both the infected groups as compared to controls. However, in cervical tissues, a significant increase of Aim2 expression was observed on 14th dpi in asymptomatic as compared to the symptomatic group. The significantly higher expression of caspase-1 and caspase-4 was observed in cervical tissues of the asymptomatic group on 14th dpi as compared to the symptomatic group, respectively. All NLRs together resulted in higher IL-1ß expression in the vaginal tissues of the symptomatic and asymptomatic groups. We conclude from this study that early expression of Nlrp3, Nlrc4, and Aim2 was seen in the symptomatic group as compared to the late-onset asymptomatic in the vaginal and cervical tissues.

Targeting of Uropathogenic Escherichia coli papG gene using CRISPR-dot nanocomplex reduced virulence of UPEC.

Urinary tract infections (UTI) are the most common infectious diseases in the world. It is becoming increasingly tough to treat because of emergence of antibiotic resistance. So, there is an exigency to develop novel anti-virulence therapeutics to combat multi-drug resistance pathogenic strains. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) discovery has revolutionized the gene editing technology for targeted approach. The greatest obstacle for CRISPR/Cas9 is cargo delivery systems and both viral and plasmid methods have disadvantages. Here, we report a highly efficient novel CRISPR based gene editing strategy, CRISPR-dots for targeting virulence factor Fimbrial Adhesion (papG gene), the bacterial adhesion molecule. Carbon quantum dots (CQD) were used as a delivery vehicle for Cas9 and gRNA into CFT073, a UPEC strain. CQDs were covalently conjugated to cas9 and papG-targeted guide RNA (gRNA) forming a nanocomplex CRISPR-dots (Cri-dots) as confirmed by DLS and transmission electron microscopy. Cri-dots-papG significantly targeted papG as demonstrated by decrease in the expression of papG.Further papG deficient UPEC had significantly reduced adherence ability and biofilm forming ability as demonstrated by fluorescence microscopy and scanning electron microscopy. Also, papG deficient UPEC had reduced virulence as shown by significantly increased survival of Caenorhabditis elegans (C. elegans) worms compared to UPEC. Our findings suggest that targeting of papG gene using Cri-dots nanocomplexes significantly reduced the pathogenicity of UPEC. Thus, Cri-dots nanocomplex offer a novel anti-bacterial strategy against multi-drug resistant UPEC.

α-Hemolysin of uropathogenic E. coli regulates NLRP3 inflammasome activation and mitochondrial dysfunction in THP-1 macrophages.

Hemolysin expressing UPEC strains have been associated with severe advanced kidney pathologies, such as cystitis and pyelonephritis, which are associated with an inflammatory response. Macrophages play an important role in regulating an inflammatory response during a urinary tract infection. We have studied the role of purified recombinant α-hemolysin in inducing inflammatory responses and cell death in macrophages. Acylation at lysine residues through HlyC is known to activate proHlyA into a fully functional pore-forming toxin, HlyA. It was observed that active α-hemolysin (HlyA) induced cleavage of caspase-1 leading to the maturation of IL-1ß, while inactive α-hemolysin (proHlyA) failed to do so in THP-1 derived macrophages. HlyA also promotes deubiquitination, oligomerization, and activation of the NLRP3 inflammasome, which was found to be dependent on potassium efflux. We have also observed the co-localization of NLRP3 within mitochondria during HlyA stimulations. Moreover, blocking of potassium efflux improved the mitochondrial health in addition to a decreased inflammatory response. Our study demonstrates that HlyA stimulation caused perturbance in potassium homeostasis, which led to the mitochondrial dysfunction followed by an acute inflammatory response, resulting in cell death. However, the repletion of intracellular potassium stores could avoid HlyA induced macrophage cell death. The findings of this study will help to understand the mechanism of α-hemolysin induced inflammatory response and cell death.

Data showing differential expression of Monocyte chemoattractant protein-1 in response to symptomatic and asymptomatic T. vaginalis infection.

Trichomoniasis is caused by Trichomonas vaginalis (a protozoan parasite). About 80% of the infected cases remain asymptomatic [1]. The differential response of showing symptoms or no symptoms is not yet explored. However, some studies gave us some insights on the pathogenesis of trichomonas and also about host defense mechanism. Host secretes pro-inflammatory cytokines and chemokines to evade infection. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a strong chemoattractant of monocytes, NK-cells and T-lymphocytes. Many reports have shown high MCP-1 levels during trichomonas infection [2], [3], [4], [5] in human prostate stromal myofibroblast cells (WPMY-1), HeLa cells, vaginal epithelial cells (VECs) but levels in response to symptomatic and asymptomatic isolates is not yet reported. In this article, we have reported MCP-1 levels in the vaginal washes and serum samples of BALB/c mouse infected with symptomatic and asymptomatic T. vaginalis isolates for different time points. We found higher levels of MCP-1 in vaginal washes of symptomatic group on 2nd day post infection (dpi) than control uninfected group. While on 4th dpi and 14th dpi, higher levels of MCP-1 in vaginal washes was observed in asymptomatic group as compared to control group. However, significant level of MCP-1 was observed in asymptomatic group on 14th dpi as compared to symptomatic group in vaginal washes. We have also observed significantly higher levels of MCP-1 in the serum samples of symptomatic group on 2nd, 4th and 14th dpi as compared to control group. A higher level of MCP-1 was found at all the time points in serum samples of asymptomatic group as compared to control group. Interestingly, a significant higher level of MCP-1 was found in the serum samples of BALB/c mice in asymptomatic as compared to symptomatic group. The MCP-1 levels in both vaginal washes and serum were significantly higher in asymptomatic group at later time points.

Involvement of NLRP3 and NLRC4 Inflammasome in Uropathogenic E. coli Mediated Urinary Tract Infections.

BACKGROUND: Inflammatory response during urinary tract infection (UTI) is mediated by innate immune defense. Nod like receptors (NLRs) have been proposed to work simultaneously beside TLR pathways to mediate pro-inflammatory response and maintain tissue homeostasis. Some in vitro reports have showed the involvement of NLRP3 inflammasome during uropathogenic Escherichia coli (UPEC) mediated UTI. So we have sought to determine the status of various inflammasomes and their components in UPEC mediated UTI. METHODS: A total of 186 females experiencing the first episode of UTI were recruited for the study and forty were found to be positive for UPEC (≥105 CFU/ml) in their urine (N = 40). Further, we analyzed the expression of NLRP3, NLRC4, NAIP, AIM2, ASC, CASPASE-4, and CASPASE-1 gene at mRNA and protein level in the blood of UPEC confirmed study subjects through real time qPCR and immunoblotting. Healthy females (N = 40) visiting the OPD for health checkups, family planning advice and subjects undergoing routine medical examinations, were recruited as healthy control subjects. Pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α and MCP-1) were measured in the plasma of patients and controls through ELISA. For investigation of the involvement of NLRC4 and NLRP3 inflammasome, in vitro studies were performed using co-immunoprecipitation and confocal microscopy. RESULTS: Most of the inflammatory regulators studied (i.e., NLRP3, NAIP, NLRC4, ASC, and CASPASE-1) were found to be up-regulated at both mRNA and protein levels in the UPEC infected UTI patients. Also, pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, TNF-α, and MCP-1) were found to be up-regulated in the patients group. However, no significant difference was observed in the expression of AIM2 and CASPASE-4 genes at both mRNA and protein levels. Further, in vitro studies have shown the involvement of NLRC4 inflammasome in UPEC infected THP1 derived macrophages. CONCLUSION: Involvement of NLRP3 and NLRC4 inflammasomes in UPEC infected UTI is evident from our findings. This is the first report showing levels of inflammasome and its components in UTI patients suggesting a possible role during UPEC mediated UTI. We have also reported the involvement of NLRC4 inflammasome for the first time during UTI infection.

Alarming levels of antimicrobial resistance among sepsis patients admitted to ICU in a tertiary care hospital in India - a case control retrospective study.

Background: Hospital acquired infections (HAI) are principal threats to the patients of intensive care units. An increase in the antimicrobial resistance (AMR) observed in gram negative bacteria is a great challenge to deal with. HAI and AMR lead to prolonged hospitalization and additional doses of anti-microbial treatment affecting patient's fitness and finances. Present study was undertaken to determine the pathotypes, genetic diversity and the antimicrobial resistance of E.coli in isolates from the patients admitted to intensive care unit at a tertiary care hospital in Delhi, India. Methods: E.coli isolates (N = 77) obtained from the blood culture of patients diagnosed with sepsis and the isolates (N = 71) from the stool culture of patients admitted in intensive care unit (ICU) but not diagnosed with sepsis were investigated for their pathotypes, adherence patterns and genetic diversity by Enterobacterial Repeated Intergenic Consensus-polymerase chain reaction (ERIC-PCR). A Kirby-Bauer Disc diffusion test and antimicrobial susceptibility assays were performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Extended-spectrum ß-lactamase (ESBL) genes and sequence type 131 (ST131) clone were characterised genotypically by gene-specific PCRs. Results: Pathotypes analysis revealed 46 and 16% of the blood E.coli isolates were ETEC and EAEC respectively, in contrast to the fecal isolates wherein 22% of the isolates were ETEC and 28.5% were EAEC. EPEC, STEC and EIEC pathotypes were not detected in blood or fecal isolates. Of all the isolates studied, more than 90% of the blood and 70% of the fecal isolates were found to be resistant to cephalosporins. On the other hand, 68% of blood and 44% of the fecal isolates were found to be ESBL producers. Interestingly 83% of the blood isolates contained CTX-M15, whereas only 21% of them contained CTX-M9 genes. On the other hand CTX-M15 genes were found in 90% and CTX-M9 genes were found in 63% of the fecal isolates. Conclusion: The antimicrobial resistant profile found in this study is alarming and poses a great threat to public health. Apparently an increased antimicrobial resistance to the extensively used cephalosporins is affecting an optimal drug therapy for patients. In addition, the presence of catheters, prolonged duration of stay in the hospital and poor hygienic conditions due to infrequent urination of the patient can lead to an additional vulnerability. Therefore continuous surveillance and rational use of antibiotics along with effective hygienic measures are urgently recommended in such settings.

Data showing levels of interleukin-1ß and nitric oxide in the plasma of uropathogenic E. coli infected UTI patients.

Urinary tract infections (UTI) are a major cause of morbidity, affecting at least four million women worldwide, 65-75% of these infections are caused by Uropathogenic Escherichia coli (UPEC) (Foxman, 2010) [1]. Repertoire of virulence factors carried by UPEC provides the ability to precede urinary tract and additionally they provoke pro-inflammatory responses (Cirl et al., 2008; Verma et al., 2016) [2], [3]. In context to UPEC infected UTI patients, the levels of pro-inflammatory cytokine IL-1ß and enzymatic antioxidant nitric oxide (NO) have not been reported worldwide till date, including India. In this data article, we report for the first time the levels of IL-1ß and nitric oxide in the plasma of UPEC infected UTI patients. Data includes a profile of pro-inflammatory cytokine IL-1ß and NO in the plasma of the confirmed UPEC infected UTI patients (N = 30) versus healthy controls (N = 40) from the present pilot study. The levels of IL-1ß in plasma were significantly higher (p < 0.0001) in patients (252.3 ± 6.49 pg/ml) as compared to healthy controls (127.6 ± 3.98 pg/ml), whereas plasma levels of NO were significantly lower (p < 0.0001) in UPEC infected UTI patients (60.29 ± 1.1 µM) as compared to healthy controls (106.3 ± 8.75 µM).

Whole-Genome Shotgun Sequence of Escherichia coli Strain MN067 from India, a Commensal Bacterium with Potent Pathogenic Ability.

Escherichia coli is one of the most frequently prevalent pathogens, causing infections in health care settings throughout the world. Here, we report the whole-genome sequence of MN067, a commensal bacterium with a pathogenic potential.

Inflammasomes and Their Role in Innate Immunity of Sexually Transmitted Infections.

Inflammasomes are multiprotein complexes present in the cytosol as pattern recognition receptors or as sensors of damage-associated molecular patterns. After recognition of microbe-associated molecular patterns or host-derived danger signals, nucleotide oligomerization domain-like receptors oligomerize to form inflammasomes. The activation of inflammasomes results in an alarm, which is raised to alert adjacent cells through the processing and release of a number of other substrates present in the cytosol. A wide array of inflammasomes and their adapter molecules have been identified in the host's innate immune system in response to various pathogens. Components of specific pathogens activate different inflammasomes, which once activated in response to pathogen-induced infection, induce the activation of caspases, and the release of mature forms of interleukin-1ß (IL-1ß) and IL-18. Identifying the mechanisms underlying pathogen-induced inflammasome activation is important if we are to develop novel therapeutic strategies to target sexually transmitted infections (STIs) related pathogens. This information is currently lacking in literature. In this review, we have discussed the role of various inflammasomes in sensing different STIs, as well as the beneficial or detrimental effects of inflammasome signaling in host resistance. Additionally, we have discussed both canonical and non-canonical processing of IL-1ß induced with respect to particular infections. Overall, these findings transform our understanding of both the basic biology and clinical relevance of inflammasomes.

Kinetics of cytokine profile in response to Mycobacterium bovis BCG and Streptococcus pyogenes activated cells.

The infection of epithelial cells is a necessary step for Mycobacterium bovis BCG dissemination, but the mechanism of mycobacterial epithelial interactions is not completely understood. Similarly, Streptococcus pyogenes is a strictly human pathogen that favorably colonizes the skin and the pharynx. Effective cytokine secretion is essential in order to fabricate a suitable inflammatory response against an infection. In this data article, the cytokine profile in BCG and S. pyogenes activated THP-1 cell line in media after the acute phase of infection by ELISA is described. The interleukin-8 level was increased in response to both BCG and S. pyogenes, but was quite prominent after 24 h and further increased upto 72 h post infection. On the other hand, an increase in IL-6 response to S. pyogenes was observed while there was no response to BCG even after 48 h of infection. A low level of TNF-α was detected upon BCG and S. pyogenes infection.

Data showing phenotypic profile of uropathogenic Escherichia coli isolates from sepsis patients.

Bacterial virulence factors (VFs) influence the site and severity of urinary tract infections (UTI) and further leading to sepsis infection. Phenotypic characterisation of VFs specific to sepsis Escherichia coli strains has not been characterized in Indian population till date. In this data article, we have described important VFs of uropathogenic E. coli (UPEC) that is P fim, Type-1 fim, cell surface hydrophobicity, mannose resistant haemagglutination/mannose sensitive haemagglutination (MRHA/MSHA) expression and α-haemolysin production. The data includes a profile of the five VFs investigated in E. coli isolates from sepsis patients (N=78) and control group (N=50) from non-sepsis subjects. We found that P fim phenotype was expressed in 25.3% of E. coli isolates from sepsis patients, whereas Type-1 fimbriae was detected in 30.5%. Cell surface hydrophobicity phenotype was present in 30.5%, α-haemolysin in 26.3% and MRHA/MSHA in 22.1% of sepsis E. coli isolates. None of the control E. coli isolates showed presence of these phenotypes. The combined phenotypic profile of all the five VFs was significantly higher in sepsis patients as compared to the control group.

Inhibition of grade dependent autophagy in urothelial carcinoma increases cell death under nutritional limiting condition and potentiates the cytotoxicity of chemotherapeutic agent.

PURPOSE: We evaluated the status of autophagy in different grades of urothelial carcinoma and explored autophagy modulators as a potential adjunctive therapeutic agent for urothelial carcinoma. MATERIALS AND METHODS: The study was performed in tumor tissue from patients with low and high grade urothelial carcinoma, in normal urothelial tissue and in the T24 cell line. Autophagic vesicles and the expression of various autophagic proteins were studied in tissue samples by transmission electron microscopy and Western blot, respectively. The effect of autophagy induction and inhibition was evaluated by measuring AMPK and mTOR expression, cell viability and mitochondrial membrane potential. The therapeutic implication of autophagy was studied using cisplatin alone or combined with an autophagy inhibitor. RESULTS: High grade urothelial carcinoma showed a higher number of autophagic vesicles and significantly higher expression of autophagic proteins. Upon starvation cells cultured from high and low grade urothelial carcinoma demonstrated significant autophagy induction associated with AMPK activation and mTOR inhibition. AMPK inhibition decreased the autophagic response and increased cell death. Autophagy inhibition by wortmannin, 3-methyladenine and chloroquine increased mitochondrial hypopolarization as well as caspase-9 and 3 dependent cell death. Combined treatment with cisplatin and an autophagy inhibitor resulted in greater cell death than cisplatin treatment alone. CONCLUSIONS: Autophagy is related to urothelial carcinoma grade and regulated via the AMPK pathway for tumor cell survival. Autophagy inhibition leads to cancer cell death through an intrinsic apoptotic pathway. The potential application of autophagy inhibitors as an adjunct to chemotherapy for urothelial carcinoma must be explored.

The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells.

BACKGROUND: MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis. RESULTS: 5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16/MTGR1 promoters by the leukemia associated AML1-ETO fusion gene may have a role in hematopoietic dysfunction of leukemia. CONCLUSIONS: An evolutionary conserved GATA binding site is critical in transcriptional regulation of the MTG16 promoter. In contrast, the MTGR1 gene depends on a GC-box-rich sequence for transcriptional regulation and possible ubiquitous expression. Our results demonstrate that the ETO homologue promoters are regulated differently consistent with hematopoietic cell-type- specific expression and function.

Persistent malignant stem cells in del(5q) myelodysplasia in remission.

BACKGROUND: The in vivo clinical significance of malignant stem cells remains unclear. METHODS: Patients who have the 5q deletion (del[5q]) myelodysplastic syndrome (interstitial deletions involving the long arm of chromosome 5) have complete clinical and cytogenetic remissions in response to lenalidomide treatment, but they often have relapse. To determine whether the persistence of rare but distinct malignant stem cells accounts for such relapses, we examined bone marrow specimens obtained from seven patients with the del(5q) myelodysplastic syndrome who became transfusion-independent while receiving lenalidomide treatment and entered cytogenetic remission. RESULTS: Virtually all CD34+, CD38+ progenitor cells and stem cells that were positive for CD34 and CD90, with undetectable or low CD38 (CD38−/low), had the 5q deletion before treatment. Although lenalidomide efficiently reduced these progenitors in patients in complete remission, a larger fraction of the minor, quiescent, CD34+,CD38-/low, CD90+ del(5q) stem cells as well as functionally defined del(5q) stem cells remained distinctly resistant to lenalidomide. Over time, lenalidomide resistance developed in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. CONCLUSIONS: In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)

The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells.

BACKGROUND: The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function. RESULTS: A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. CONCLUSIONS: We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression.

Transcriptional repression by leukaemia-associated ETO family members can be independent of oligomerization and coexpressed hSIN3B and N-CoR.

The leukaemia-associated eight-twenty-one (ETO) family members ETO, MTG16 (Myeloid Translocation Gene on chromosome 16) and MTGR1 (Myeloid Transforming Gene-Related protein1) are putative transcriptional repressor proteins, which form complexes with coregulatory nuclear corepressors such as SIN3 (SWI-Independent) and N-CoR (Nuclear receptor Co Repressor). In acute myeloid leukaemia (AML), fusion proteins involving the transcription factor AML1 and corepressors ETO or MTG16 are recurrently found. We investigated transcriptional repression by the ETO family members ETO and MTG16 with attention to the conserved Nervy Homology Regions (NHRs) and the interacting corepressors human SIN3B (hSIN3B) and N-CoR. Transcriptional repression was examined in a cell line by a GAL4-thymidine kinase luciferase reporter to which the corepressors were tethered through a binding domain. ETO- and MTG16-mediated repression was found to be independent of deletion of the oligomerization NHR2, but deletion of NHR4 and in particular combined deletion of NHR2 and NHR4 lowered the capacity for repression. An interaction was observed between the corepressors hSIN3B and N-CoR and these two proteins cooperated for transcriptional repression independent of co-transfected ETO and MTG16. Transcriptional repression mediated by ETO and MTG16 was only slightly strengthened by coexpression of hSIN3B or N-CoR and was dependent on HDAC activity. Our data indicate that ETO family member-mediated oligomerization and repression can be distinct events and that interaction between ETO family members and hSIN3B or N-CoR may not necessarily strengthen transcriptional repression.

The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues.

BACKGROUND: SIN3 (SWI-Independent) is part of a transcriptional deacetylase complex, which generally mediates the formation of repressive chromatin. The purpose of this work was to study possible interactions between corepressors human SIN3B (hSIN3B) and the ETO homologues - ETO (eight twenty-one), MTG16 (myeloid-transforming gene 16) and MTGR1 (MTG-related protein 1). In addition, the subnuclear localization of the hSIN3B and the ETO homologues was also examined. RESULTS: A ubiquitous expression of hSIN3B was observed in adult and fetal tissues. Results with both ectopically expressed proteins in COS-7 cells and endogeneous proteins in the K562 human erytholeukemia cell line demonstrated interactions between hSIN3B and ETO or MTG16 but not MTGR1. Furthermore, nuclear extract of primary placental cells showed complexes between hSIN3B and ETO. The interaction between hSIN3B and ETO required an intact amino-terminus of ETO and the NHR2 domain. A nucleolar localization of hSIN3B and all the ETO homologues was demonstrated upon overexpression in COS-7 cells, and confirmed for the endogeneously expressed proteins in K562 cells. However, hSIN3B did not colocalize or interact with the leukemia-associated AML1 -ETO. CONCLUSION: Our data from protein-protein interactions and immunolocalization experiments support that hSIN3B is a potential member of a corepressor complex involving selective ETO homologues.