Cytosolic Proliferation Research Articles

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in various water and land environments and organisms, including insects and mammals. Some P. alcalifaciens strains encode gene homologs of virulence factors found in pathogenic Enterobacterales members, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are pathogenic determinants in P. alcalifaciens is not known. In this study, we investigated P. alcalifaciens-host interactions at the cellular level, focusing on the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. A large plasmid that is present in a subset of P. alcalifaciens strains, primarily isolated from diarrheal patients, encodes for T3SS1a. We show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, lyses its internalization vacuole, and proliferates in the cytosol. This triggers caspase-4-dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis, and cytosolic proliferation is host cell type-specific, playing a more prominent role in intestinal epithelial cells than in macrophages or insect cells. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa and induces mild epithelial damage with negligible fluid accumulation in a T3SS1a- and T3SS1b-independent manner. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS 1b is widespread in Providencia spp. and encoded on the chromosome. T3SS 1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS 1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS 1a or T3SS 1b was seen in the calf infection model. However, T3SS 1b was required for the rapid killing of Drosophila melanogaster . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Controlled Activity of the Salmonella Invasion-Associated Injectisome Reveals Its Intracellular Role in the Cytosolic Population.

ABSTRACTThe Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella-containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular Salmonella.

Abstract 2561: Contextual combination of PARP inhibitors with p110beta inhibitors: Functional logistics to tame PTEN null tumors

Abstract Background: Hyper-activation of the PI3K pathway due to a functional loss of PTEN is a characteristic feature of many tumors including TNBC and GBM. Although p110b rarely undergoes activating mutations in these cancers, loss of PTEN function makes these tumor cells dependent specifically on p110b. In a mechanism-based study we have recently established an integral role of the PI3K pathway in DNA damage repair (DDR)-mediated anti-tumor efficacy of PARP inhibitor wherein we have demonstrated that PTEN-nullness confers in vivo sensitivity to the combination as compared to tumors carrying PTEN WT (De et al., Neoplasia, 2014). Thus here we hypothesize that PTEN-null tumors will be more susceptible to a combination of p110b inhibitor (p110b i) plus PARP inhibitor (PARP i). Method: To test our hypothesis we used PTEN-null TNBC and GBM models. Isogenic cell lines as well as SiRNA-directed PTEN-knockdown cells were tested for the (a) IC50 (MTT) (b) real-time confluence assay (Incucyte) (c) cell cycle (flow cytometry) (d) clonogenecity (ON-TOP assay) (e) cytosolic proliferation and apoptotic signals and (f) nuclear HRD signals for DDR using 3 p110b i (TGX221, AZD6482, GSK2636771) and 5 PARPi (BMN673, Niraparib, Olaparib, Rucaparib, Veliparib) (procured from Selleckchem, USA). Results: Individually (1) AZD6482 was the most effective among p110b i in controlling the cell cycle when tested in PTEN null U87MG GBM cells and (2) AZD6482 had the lowest dose (1-10 uM) for achieving the minimum effective dose (MED) in the confluence studies in TNBC and GBM models as compared to other p110b i. The MED of 5 PARPi for TNBC and GBM cells were determined by clonogenic assay (7-10 days). The most potent among 5 PARPi was BMN673 with a dose range of 100 nM-500 nM. Based on the effectiveness of individual drugs, we used a combination dose range of 500 nM-10 uM of 5 PARPi with 10 uM fixed dose of p110b i, AZD6482, and tested the effect of this combination using (1) real time apoptosis assay (Incucyte) and (2) 3D clonogenic assay. The combination of each PARPi with AZD6482 was more effective in perturbing clonogenic growth of PTEN-null TNBC and GBM cells as compared to the individual agent(s) which can be corroborated with the increased apoptosis in the cells treated with the combination and their effect on cell cycle. Significance: Better understanding of the mechanistic involvement of the combination of p110b i and PARPi in BRCA-competent PTEN null tumors may provide a novel target for future therapeutic intervention and provide insight to PTEN-mediated carcinogenesis in these tumors which eventually will extend the realm of PARP i use in cancer therapy. Citation Format: Jennifer H. Carlson, Yuliang Sun, Xiaoqian Lin, Pradip De, Brian Leyland-Jones, Nandini Dey. Contextual combination of PARP inhibitors with p110beta inhibitors: Functional logistics to tame PTEN null tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2561. doi:10.1158/1538-7445.AM2015-2561