Transcription Factors in Prostate Cancer: Insights for Disease Development and Diagnostic and Therapeutic Approaches.

Transcription factors (TFs) are proteins essential for the regulation of gene expression, and they regulate the genes involved in different cellular processes, such as proliferation, differentiation, survival, and apoptosis. Although their expression is essential in normal physiological conditions, abnormal regulation of TFs plays critical role in several diseases, including cancer. In prostate cancer, the most common malignancy in men, TFs are known to play crucial roles in the initiation, progression, and resistance to therapy of the disease. Understanding the interplay between these TFs and their downstream targets provides insights into the molecular basis of prostate cancer pathogenesis. In this review, we discuss the involvement of key TFs, including the E26 Transformation-Specific (ETS) Family (ERG and SPDEF), NF-κB, Activating Protein-1 (AP-1), MYC, and androgen receptor (AR), in prostate cancer while focusing on the molecular mechanisms involved in prostate cancer development. We also discuss emerging diagnostic strategies, early detection, and risk stratification using TFs. Furthermore, we explore the development of therapeutic interventions targeting TF pathways, including the use of small molecule inhibitors, gene therapies, and immunotherapies, aimed at disrupting oncogenic TF signaling and improving patient outcomes. Understanding the complex regulation of TFs in prostate cancer provides valuable insights into disease biology, which ultimately may lead to advancing precision approaches for patients.

DCUN1D1 Is an Essential Regulator of Prostate Cancer Proliferation and Tumour Growth That Acts through Neddylation of Cullin 1, 3, 4A and 5 and Deregulation of Wnt/Catenin Pathway.

Defective in cullin neddylation 1 domain containing 1 (DCUN1D1) is an E3 ligase for the neddylation, a post-translational process similar to and occurring in parallel to ubiquitin proteasome pathway. Although established as an oncogene in a variety of squamous cell carcinomas, the precise role of DCUN1D1 in prostate cancer (PCa) has not been previously explored thoroughly. Here, we investigated the role of DCUN1D1 in PCa and demonstrated that DCUN1D1 is upregulated in cell lines as well as human tissue samples. Inhibition of DCUN1D1 significantly reduced PCa cell proliferation and migration and remarkably inhibited xenograft formation in mice. Applying both genomics and proteomics approaches, we provide novel information about the DCUN1D1 mechanism of action. We identified CUL3, CUL4B, RBX1, CAND1 and RPS19 proteins as DCUN1D1 binding partners. Our analysis also revealed the dysregulation of genes associated with cellular growth and proliferation, developmental, cell death and cancer pathways and the WNT/ß-catenin pathway as potential mechanisms. Inhibition of DCUN1D1 leads to the inactivation of ß-catenin through its phosphorylation and degradation which inhibits the downstream action of ß-catenin, reducing its interaction with Lef1 in the Lef1/TCF complex that regulates Wnt target gene expression. Together our data point to an essential role of the DCUN1D1 protein in PCa which can be explored for potential targeted therapy.

The Role of the Receptor Tyrosine Kinase Axl in Carcinogenesis and Development of Therapeutic Resistance: An Overview of Molecular Mechanisms and Future Applications.

Resistance to chemotherapeutic agents by cancer cells has remained a major obstacle in the successful treatment of various cancers. Numerous factors such as DNA damage repair, cell death inhibition, epithelial-mesenchymal transition, and evasion of apoptosis have all been implicated in the promotion of chemoresistance. The receptor tyrosine kinase Axl, a member of the TAM family (which includes TYRO3 and MER), plays an important role in the regulation of cellular processes such as proliferation, motility, survival, and immunologic response. The overexpression of Axl is reported in several solid and hematological malignancies, including non-small cell lung, prostate, breast, liver and gastric cancers, and acute myeloid leukaemia. The overexpression of Axl is associated with poor prognosis and the development of resistance to therapy. Reports show that Axl overexpression confers drug resistance in lung cancer and advances the emergence of tolerant cells. Axl is, therefore, an important candidate as a prognostic biomarker and target for anticancer therapies. In this review, we discuss the consequence of Axl upregulation in cancers, provide evidence for its role in cancer progression and the development of drug resistance. We will also discuss the therapeutic potential of Axl in the treatment of cancer.

In silico characterization of microRNAs-like sequences in the genome of Paracoccidioides brasiliensis.

Eukaryotic cells have different mechanisms of post-transcriptional regulation. Among these mechanisms, microRNAs promote regulation of targets by cleavage or degradation of the mRNA. Fungi of the Paracoccidioides complex are the etiological agents of the main systemic mycosis of Latin America. These fungi present a plasticity to adapt and survive in different conditions, and the presence of microRNAs-like molecules could be part of the mechanisms that provide such plasticity. MicroRNAs produced by the host influence the progression of this mycosis in the lungs besides regulating targets involved in apoptosis in macrophage, activation of T and B cells and the production of cytokines. Therefore, this work analyzed the presence of regions in the genome of this fungus with a potential to encode microRNAs-like molecules. Here we show by analysis of sequence similarity the presence of 18 regions, putatively coding for microRNAs-like molecules in the Paracoccidioides brasiliensis genome. We also described the conservation of dicer and argonaut proteins and the cognate transcripts induced in the yeast parasitic phase. This work represents a starting point for the analysis of the presence of those molecules in the morphological stages of the fungus and their role in fungal development.

Dihydroartemisinin inhibits prostate cancer via JARID2/miR-7/miR-34a-dependent downregulation of Axl.

Axl expression is deregulated in several cancer types, predicts poor overall patient survival and is linked to resistance to drug therapy. Here, we evaluated a library of natural compounds for inhibitors of Axl and identified dihydroartemisinin, the active principle of the anti-malarial drug artemisinin, as an Axl-inhibitor in prostate cancer. Dihydroartemisinin blocks Axl expression leading to apoptosis, decrease in cell proliferation, migration, and tumor development of prostate cancer cells. Dihydroartemisinin treatment synergizes with docetaxel, a standard of care in metastatic prostate cancer increasing overall survival of mice with human xenografts. Dihydroartemisinin control of miR-34a and miR-7 expression leads to inhibition of Axl expression in a process at least partially dependent on regulation of chromatin via methylation of histone H3 lysine 27 residues by Jumonji, AT-rich interaction domain containing 2 (JARID2), and the enhancer of zeste homolog 2. Our discovery of a previously unidentified miR-34a/miR-7/JARID2 pathway controlling dihydroartemisinin effects on Axl expression and inhibition of cancer cell proliferation, migration, invasion, and tumor formation provides new molecular mechanistic insights into dihydroartemisinin anticancer effect on prostate cancer with potential therapeutic implications.

The Dual Role of TAM Receptors in Autoimmune Diseases and Cancer: An Overview.

Receptor tyrosine kinases (RTKs) regulate cellular processes by converting signals from the extracellular environment to the cytoplasm and nucleus. Tyro3, Axl, and Mer (TAM) receptors form an RTK family that plays an intricate role in tissue maintenance, phagocytosis, and inflammation as well as cell proliferation, survival, migration, and development. Defects in TAM signaling are associated with numerous autoimmune diseases and different types of cancers. Here, we review the structure of TAM receptors, their ligands, and their biological functions. We discuss the role of TAM receptors and soluble circulating TAM receptors in the autoimmune diseases systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Lastly, we discuss the effect of TAM receptor deregulation in cancer and explore the therapeutic potential of TAM receptors in the treatment of diseases.

Cell Wall Synthesis, Development of Hyphae and Metabolic Pathways Are Processes Potentially Regulated by MicroRNAs Produced Between the Morphological Stages of Paracoccidioides brasiliensis.

MicroRNAs are molecules involved in post-transcriptional gene regulation. In pathogenic fungi, microRNAs have been described at different morphological stages by regulating targets involved in processes such as morphogenesis and energy production. Members of the Paracoccidioides complex are the main etiological agents of a systemic mycosis in Latin America. Fungi of the Paracoccidioides complex present a wide range of plasticity to colonize different niches. In response to environmental changes these fungi undergo a morphological switch, remodel their cellular metabolism and modulate structural cell wall components. However, the underlying mechanisms regulating the gene expression is not well understood. By using high performance sequencing and bioinformatics analyses, this work characterizes microRNAs produced by Paracoccidioides brasiliensis. Here, we demonstrated that the transcript encoding proteins involved in microRNA biogenesis were differentially expressed in each morphological stage. In addition, 49 microRNAs were identified in cDNA libraries with 44 differentially regulated among the libraries. Sixteen microRNAs were differentially regulated in comparison to the mycelium in the mycelium-to-yeast transition phase. The yeast parasitic phase revealed a complete remodeling of the expression of these small RNAs. Analyses of targets of the induced microRNAs, from the different libraries, revealed that these molecules may potentially regulate in the cell wall, by repressing genes involved in the synthesis and degradation of glucans and chitin. Furthermore, mRNAs involved in cellular metabolism and development were predicted to be regulated by microRNAs. Therefore, this work describes a putative post transcriptional regulation, mediated by microRNAs in P. brasiliensis and its influence on the adaptive processes of thermal dimorphic fungus.

Potential role of P2X7R in esophageal squamous cell carcinoma proliferation.

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5'-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.

GADD45α and γ interaction with CDK11p58 regulates SPDEF protein stability and SPDEF-mediated effects on cancer cell migration.

The epithelium-specific Ets transcription factor, SPDEF, plays a critical role in metastasis of prostate and breast cancer cells. While enhanced SPDEF expression blocks migration and invasion, knockdown of SPDEF expression enhances migration, invasion, and metastasis of cancer cells. SPDEF expression and activation is tightly regulated in cancer cells; however, the precise mechanism of SPDEF regulation has not been explored in detail. In this study we provide evidence that the cell cycle kinase CDK11p58, a protein involved in G2/M transition and degradation of several transcription factors, directly interacts with and phosphorylates SPDEF on serine residues, leading to subsequent ubiquitination and degradation of SPDEF through the proteasome pathway. As a consequence of CDK11p58 mediated degradation of SPDEF, this loss of SPDEF protein results in increased prostate cancer cell migration and invasion. In contrast, knockdown of CDK11p58 protein expression by interfering RNA or SPDEF overexpression inhibit migration and invasion of cancer cells. We demonstrate that CDK11p58 mediated degradation of SPDEF is attenuated by Growth Arrest and DNA damage-inducible 45 (GADD45) α and , two proteins inducing G2/M cell cycle arrest. We show that GADD45 α and γ, directly interact with CDK11p58 and thereby inhibit CDK11p58 activity, and consequentially SPDEF phosphorylation and degradation, ultimately reducing prostate cancer cell migration and invasion. Our findings provide new mechanistic insights into the complex regulation of SPDEF activity linked to cancer metastasis and characterize a previously unidentified SPDEF/CDK11p58/GADD45α/γ pathway that controls SPDEF protein stability and SPDEF-mediated effects on cancer cell migration and invasion.

Discovery of novel candidate urinary protein biomarkers for prostate cancer in a multiethnic cohort of South African patients via label-free mass spectrometry.

PURPOSE: Improvement in diagnostic accuracy of prostate cancer (PCa) progression using MS-based methods to analyze biomarkers in our African, Caucasian, and Mixed Ancestry patients can advance early detection and treatment monitoring. EXPERIMENTAL DESIGN: MS-based proteomic analysis of pooled (N = 36) and individual samples (N = 45) of PCa, benign prostatic hyperplasia, normal healthy controls, and patients with other uropathies was used to identify differences in proteomics profile. Samples were analyzed for potential biomarkers and proteome coverage in African, Caucasian, and Mixed Ancestry PCa patients. RESULTS: A total of 1102 and 5595 protein groups and nonredundant peptides, respectively, were identified in the pooling experiments (FDR = 0.01). Twenty potential biomarkers in PCa were identified and fold differences ± 2SD were observed in 17 proteins using intensity-based absolute quantification. Analysis of 45 individual samples yielded 1545 and 9991 protein groups and nonredundant peptides, respectively. Seventy-three (73) proteins groups, including existing putative PCa biomarkers, were found to be potential biomarkers of PCa by label-free quantification and demonstrated ethnic trends within our PCa cohort. CONCLUSION AND CLINICAL RELEVANCE: Urinary proteomics is a promising route to PCa biomarker discovery and may serve as source of ethnic-related biomarkers of PCa.

Inactivation of GSK3ß and activation of NF-κB pathway via Axl represents an important mediator of tumorigenesis in esophageal squamous cell carcinoma.

The receptor tyrosine kinase Axl has been described as an oncogene, and its deregulation has been implicated in the progression of several human cancers. While the role of Axl in esophageal adenocarcinoma has been addressed, there is no information about its role in esophageal squamous cell carcinoma (OSCC). In the current report, we identified, for the first time, deregulation of Axl expression in OSCC. Axl is consistently overexpressed in OSCC cell lines and human tumor samples, mainly in advanced stages of the disease. Blockage of Axl gene expression by small interfering RNA inhibits cell survival, proliferation, migration, and invasion in vitro and esophageal tumor growth in vivo. Additionally, repression of Axl expression results in Akt-dependent inhibition of pivotal genes involved in the nuclear factor-kappaB (NF-κB) pathway and in the induction of glycogen synthase kinase 3ß (GSK3ß) activity, resulting in loss of mesenchymal markers and induction of epithelial markers. Furthermore, treatment of esophageal cancer cells with the Akt inhibitor wortmannin inhibits NF-κB signaling, induces GSK3ß activity, and blocks OSCC cell proliferation in an Axl-dependent manner. Taken together, our results establish a clear role for Axl in OSCC tumorigenesis with potential therapeutic implications.

Aberrant methylation of the MSH3 promoter and distal enhancer in esophageal cancer patients exposed to first-hand tobacco smoke.

PURPOSE: Polymorphisms in MSH3 gene confer risk of esophageal cancer when in combination with tobacco smoke exposure. The purpose of this study was to investigate the methylation status of MSH3 gene in esophageal cancer patients in order to further elucidate possible role of MSH3 in esophageal tumorigenesis. METHODS: We applied nested methylation-specific polymerase chain reaction to investigate the methylation status of the MSH3 promoter in tumors and matching adjacent normal-looking tissues of 84 esophageal cancer patients from a high-risk South African population. The Cancer Genome Atlas data were used to examine DNA methylation profiles at 17 CpG sites located in the MSH3 locus. RESULTS: Overall, promoter methylation was detected in 91.9 % of tumors, which was significantly higher compared to 76.0 % in adjacent normal-looking esophageal tissues (P = 0.008). When samples were grouped according to different demographics (including age, gender and ethnicity) and smoking status of patients, methylation frequencies were found to be significantly higher in tumor tissues of Black subjects (P = 0.024), patients of 55-65 years of age (P = 0.032), males (P = 0.037) and tobacco smokers (P = 0.015). Furthermore, methylation of the MSH3 promoter was significantly more frequent in tumor samples from smokers compared to tumor samples from non-smokers [odds ratio (OR) = 31.9, P = 0.031]. The TCGA data confirmed significantly higher DNA methylation level at the MSH3 promoter region in tumors (P = 0.0024). In addition, we found evidence of an aberrantly methylated putative MSH3-associated distal enhancer element. CONCLUSION: Our results suggest that methylation of MSH3 together with exposure to tobacco smoke is involved in esophageal carcinogenesis. Due to the active role of the MSH3 protein in modulating chemosensitivity of cells, methylation of MSH3 should further be examined in association with the outcome of esophageal cancer treatment using anticancer drugs.

Computational repositioning and preclinical validation of pentamidine for renal cell cancer.

Although early stages of clear cell renal cell carcinoma (ccRCC) are curable, survival outcome for metastatic ccRCC remains poor. We previously established a highly accurate signature of differentially expressed genes that distinguish ccRCC from normal kidney. The purpose of this study was to apply a new individualized bioinformatics analysis (IBA) strategy to these transcriptome data in conjunction with Gene Set Enrichment Analysis of the Connectivity Map (C-MAP) database to identify and reposition FDA-approved drugs for anticancer therapy. Here, we demonstrate that one of the drugs predicted to revert the RCC gene signature toward normal kidney, pentamidine, is effective against RCC cells in culture and in a RCC xenograft model. ccRCC-specific gene expression signatures of individual patients were used to query the C-MAP software. Eight drugs with negative correlation and P-value <0.05 were analyzed for efficacy against RCC in vitro and in vivo. Our data demonstrate consistency across most patients with ccRCC for the set of high-scoring drugs. Most of the selected high-scoring drugs potently induce apoptosis in RCC cells. Several drugs also demonstrate selectivity for Von Hippel-Lindau negative RCC cells. Most importantly, at least one of these drugs, pentamidine, slows tumor growth in the 786-O human ccRCC xenograft mouse model. Our findings suggest that pentamidine might be a new therapeutic agent to be combined with current standard-of-care regimens for patients with metastatic ccRCC and support our notion that IBA combined with C-MAP analysis enables repurposing of FDA-approved drugs for potential anti-RCC therapy.

Gut adhesive Bacillus subtilis spores as a platform for mucosal delivery of antigens.

Bacillus subtilis spores have been used as safe and heat-resistant antigen delivery vectors. Nonetheless, the oral administration of spores typically induces weak immune responses to the passenger antigens, which may be attributed to the fast transit through the gastrointestinal tract. To overcome this limitation, we have developed B. subtilis spores capable of binding to the gut epithelium by means of expressing bacterial adhesins on the spore surface. The resulting spores bound to in vitro intestinal cells, showed a longer transit through the mouse intestinal tract, and interacted with Peyer's patch cells. The adhesive spores increased the systemic and secreted antibody responses to the Streptococcus mutans P1 protein, used as a model antigen, following oral, intranasal, and sublingual administration. Additionally, P1-specific antibodies efficiently inhibited the adhesion of the oral pathogen Streptococcus mutans to abiotic surfaces. These results support the use of gut-colonizing B. subtilis spores as a new platform for the mucosal delivery of vaccine antigens.

The receptor tyrosine kinase Axl in cancer: biological functions and therapeutic implications.

The receptor tyrosine kinase Axl has been implicated in the malignancy of different types of cancer. Emerging evidence of Axl upregulation in numerous cancers, as well as reports demonstrating that its inhibition blocks tumor formation in animal models, highlight the importance of Axl as a new potential therapeutic target. Furthermore, recent data demonstrate that Axl plays a pivotal role in resistance to chemotherapeutic regimens. In this review we discuss the functions of Axl and its regulation and role in cancer development, resistance to therapy, and its importance as a potential drug target, focusing on acute myeloid leukemia, breast, prostate and non-small cell lung cancers.

JunD-mediated repression of GADD45α and γ regulates escape from cell death in prostate cancer.

The AP-1 transcription factor complex has been implicated in a variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. We previously established that activation of the AP-1 family member JunD contributes to deregulated expression of the anti-apoptotic IL-6 gene in prostate cancer cells. We now show that inhibition of JunD in prostate cancer cells results in GADD45α- and γ-dependent induction of cell death and inhibition of tumor growth that is mediated at least partially via c-Jun N-terminal kinase (JNK) and p38 kinase activation. Apoptosis induction by dominant negative JunD and JNK and p38 kinase activation are impeded upon knock down of GADD45α and γ expression by small interfering RNA, most vividly demonstrating the central role of GADD45α and γ in JunD-mediated escape of prostate cancer cells from programmed cell death.

Induction of neutralizing antibodies in mice immunized with an amino-terminal polypeptide of Streptococcus mutans P1 protein produced by a recombinant Bacillus subtilis strain.

The oral pathogen Streptococcus mutans expresses a surface protein, P1, which interacts with the salivary pellicle on the tooth surface or with fluid-phase saliva, resulting in bacterial adhesion or aggregation, respectively. P1 is a target of protective immunity. Its N-terminal region has been associated with adhesion and aggregation functions and contains epitopes recognized by efficacious antibodies. In this study, we used Bacillus subtilis, a gram-positive expression host, to produce a recombinant N-terminal polypeptide of P1 (P1(39-512)) derived from the S. mutans strain UA159. Purified P1(39-512) reacted with an anti-full-length P1 antiserum as well as one raised against intact S. mutans cells, indicating preserved antigenicity. Immunization of mice with soluble and heat-denatured P1(39-512) induced antibodies that reacted specifically with native P1 on the surface of S. mutans cells. The anti-P1(39-512) antiserum was as effective at blocking saliva-mediated aggregation of S. mutans cells and better at blocking bacterial adhesion to saliva-coated plastic surfaces compared with the anti-full-length P1 antiserum. In addition, adsorption of the anti-P1 antiserum with P1(39-512) eliminated its ability to block the adhesion of S. mutans cells to abiotic surfaces. The present results indicate that P1(39-512), expressed and purified from a recombinant B. subtilis strain, maintains important immunological features of the native protein and represents an additional tool for the development of anticaries vaccines.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit.

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle.

Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles.

Recombinant Bacillus subtilis strains, either in the form of spores or vegetative cells, may be employed as safe and low-cost vaccine vehicles. In this study, we studied the role of promoter sequences and antigen-sorting signals on the immunogenicity based on previously constructed B. subtilis episomal expression systems. Mice orally immunized with spores or cells encoding the B subunit of the heat labile toxin (LTB), originally expressed by some enterotoxigenic Escherichia coli (ETEC) strains, under control of the stress-inducible gsiB promoter developed higher anti-LTB serum IgG and fecal IgA responses with regard to vaccine strains transformed with plasmids encoding the antigen under control of IPTG-inducible (Pspac) or constitutive (PlepA) promoters. Moreover, surface expression of the vaccine antigen under the control of the PgsiB promoter enhanced the immunogenicity of vegetative cells, while intracellular accumulation of LTB led to higher antibody responses in mice orally immunized with recombinant B. subtilis spores. Specific anti-LTB antibodies raised in vaccinated mice recognized and neutralized in vitro the native toxin produced by ETEC strains. Nonetheless, only mice orally immunized with recombinant B. subtilis strains, either as vegetative cells or spores, expressing intracellular LTB under the control of the gsiB promoter conferred partial protection to lethal challenges with purified LT. The present report further demonstrates that B. subtilis plasmid-based heterologous protein expression systems are adequate for antigen delivery via the oral route.