Negative Regulation of Autophagy during Macrophage Infection by Mycobacterium bovis BCG via Protein Kinase C Activation.

Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.

Murine RAW Macrophages Are a Suitable Model to Study the CD3 Signaling in Myeloid Cells.

In recent years, a growing body of evidence has shown the presence of a subpopulation of macrophages that express CD3, especially in the context of mycobacterial infections. Despite these findings, the function of these cells has been poorly understood. Furthermore, the low frequency of CD3+ macrophages in humans limits the study of this subpopulation. This work aimed to evaluate the expression of CD3 in a murine macrophage cell line and its potential for the study of CD3 signaling. The murine macrophage cell line RAW was used to evaluate CD3 expression at the transcriptional and protein levels and the effect of in vitro infection with the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) on these. Our data showed that RAW macrophages express CD3, both the ε and ζ chains, and it is further increased at the transcriptional level after BCG infection. Furthermore, our data suggest that CD3 can be found on the cell surface and intracellularly. However, this molecule is internalized constantly, mainly after activation with anti-CD3 stimulus, but interestingly, it is stably maintained at the transcriptional level. Finally, signaling proteins such as NFAT1, c-Jun, and IKK-α are highly expressed in RAW macrophages. They may play a role in the CD3-controlled signaling pathway to deliver inflammatory cytokines such as TNF and IL-6. Our study provides evidence to support that RAW cells are a suitable model to study the function and signaling of the CD3 complex in myeloid cells.

A Novel Zinc Exporter CtpG Enhances Resistance to Zinc Toxicity and Survival in Mycobacterium bovis.

Zinc is a microelement essential for the growth of almost all organisms, but it is toxic at high concentrations and represents an antimicrobial strategy for macrophages. Mycobacterium tuberculosis and Mycobacterium bovis are two well-known intracellular pathogens with strong environmental adaptability, including zinc toxicity. However, the signaling pathway and molecular mechanisms on sensing and resistance to zinc toxicity remains unclear in mycobacteria. Here, we first report that P1B-type ATPase CtpG acts as a zinc efflux transporter and characterize a novel CmtR-CtpG-Zn2+ regulatory pathway that enhances mycobacterial resistance to zinc toxicity. We found that zinc upregulates ctpG expression via transcription factor CmtR and stimulates the ATPase activity of CtpG. The APC residues in TM6 is essential for CtpG to export zinc and enhance M. bovis BCG resistance to zinc toxicity. During infection, CtpG inhibits zinc accumulation in the mycobacteria, and aids bacterial survival in THP-1 macrophage and mice with elevated inflammatory responses. Our findings revealed the existence of a novel regulatory pathway on mycobacteria responding to and adapting to host-mediated zinc toxicity. IMPORTANCE Tuberculosis is caused by the bacillus Mycobacterium tuberculosis and is one of the major sources of mortality. M. tuberculosis has developed unique mechanisms to adapt to host environments, including zinc deficiency and toxicity, during infection. However, the molecular mechanism by which mycobacteria promote detoxification of zinc, and the associated signaling pathways remains largely unclear. In this study, we first report that P1B-type ATPase CtpG acts as a zinc efflux transporter and characterize a novel CmtR-CtpG-Zn2+ regulatory pathway that enhances mycobacterial resistance to zinc toxicity in M. bovis. Our findings reveal the existence of a novel excess zinc-triggered signaling circuit, provide new insights into mycobacterial adaptation to the host environment during infection, and might be useful targets for the treatment of tuberculosis.

T Cell Interactions in Mycobacterial Granulomas: Non-Specific T Cells Regulate Mycobacteria-Specific T Cells in Granulomatous Lesions.

Infections with pathogenic mycobacteria are controlled by the formation of a unique structure known as a granuloma. The granuloma represents a host-pathogen interface where bacteria are killed and confined by the host response, but also where bacteria persist. Previous work has demonstrated that the T cell repertoire is heterogenous even at the single granuloma level. However, further work using pigeon cytochrome C (PCC) epitope-tagged BCG (PCC-BCG) and PCC-specific 5CC7 RAG-/- TCR transgenic (Tg) mice has demonstrated that a monoclonal T cell population is able to control infection. At the chronic stage of infection, granuloma-infiltrating T cells remain highly activated in wild-type mice, while T cells in the monoclonal T cell mice are anergic. We hypothesized that addition of an acutely activated non-specific T cell to the monoclonal T cell system could recapitulate the wild-type phenotype. Here we report that activated non-specific T cells have access to the granuloma and deliver a set of cytokines and chemokines to the lesions. Strikingly, non-specific T cells rescue BCG-specific T cells from anergy and enhance the function of BCG-specific T cells in the granuloma in the chronic phase of infection when bacterial antigen load is low. In addition, we find that these same non-specific T cells have an inhibitory effect on systemic BCG-specific T cells. Taken together, these data suggest that T cells non-specific for granuloma-inducing agents can alter the function of granuloma-specific T cells and have important roles in mycobacterial immunity and other granulomatous disorders.

Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity.

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Impact of knockdown LincRNA-Cox2 on apoptosis of macrophage infected with Bacillus Calmette-Guérin.

Mycobacterium tuberculosis (Mtb)-induced apoptosis of alveolar macrophages plays an important role in the pathogenesis of tuberculosis. Previous studies indicated that massive LncRNAs could deteriorate MTB invasion or latent infection by regulating macrophage's apoptosis. However, whether LincRNA-Cox2 is involved in apoptosis of macrophage infected with Mtb is unclear. In this study, we found Bacillus Calmette-Guerin(BCG)infection induced cell apoptosis with a increasing LincRNA-Cox2 expression in RAW264.7 cells. Furthermore, the activation of TLR signal pathway elevated the expression of lincRNA-Cox2. In this regard, we used small interfering RNA to explore the role of LincRNA-Cox2 on regulating apoptosis of RAW264.7 cells infected with BCG. The results showed that si-LincRNA-Cox2 was capable of increased the expression of apoptosis-associated proteins and accumulation of ROS in BCG-infected RAW264.7 cells. Mechanically, si-LincRNA-Cox2 facilitated BCG-induced macrophage apoptosis by activating the intrinsic apoptotic pathway as well as increased the genes expression of PERK/eIF2α/CHOP. These results provide novel insights into host-pathogen interactions and highlight the potential role of LincRNA-Cox2 in regulating apoptosis induced by BCG-infection.

Acid Sphingomyelinase Contributes to the Control of Mycobacterial Infection via a Signaling Cascade Leading from Reactive Oxygen Species to Cathepsin D.

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the most severe diseases worldwide. The initial pulmonary localization of the pathogen often develops into systemic infection with high lethality. The present work investigated the role of sphingolipids, specifically the function of acid sphingomyelinase (Asm) and ceramide, in infection of murine macrophages in vitro and mice in vivo with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In vitro, we investigated macrophages from wild-type (wt) and Asm deficient (Asm-/-) mice to define signaling events induced by BCG infection and mediated by Asm. We demonstrate that infection of wt macrophages results in activation of Asm, which increases reactive oxygen species (ROS) via stimulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. ROS promote BCG degradation by cathepsin D. Asm deficiency in macrophages abrogates these effects. In vivo studies reveal that wt mice rapidly control BCG infection, while Asm-/- mice fail to control the infection and kill the bacteria. Transplantation of wt macrophages into Asm-/- mice reversed their susceptibility to BCG, demonstrating the importance of Asm in macrophages for defense against BCG. These findings indicate that Asm is important for the control of BCG infection.

Involvement of NF-κB in the reversal of CYP3A down-regulation induced by sea buckthorn in BCG-induced rats.

Previous studies reported that sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae, HRP) exhibits hepatoprotective effects via its anti-inflammatory and antioxidant properties as well as its inhibitory effects on collagen synthesis. However, it is unclear whether this hepatoprotective effect is also achieved by regulating liver drug metabolism enzyme pathways. Herein, we examined the regulatory effect of HRP on cytochrome P450 3A (CYP3A) in rats with immune liver injury, and explored the molecular mechanism of its hepatoprotective effect. Rat models of immunological liver injury were induced by intravenous injections of Bacillus Calmette-Guerin (BCG; 125 mg kg-1; 2 wks). Specific protein levels were detected by ELISA or western blot, and CYP3A mRNA expression was detected by RT-PCR. High-performance liquid chromatography (HPLC) detected relative changes in CYP3A metabolic activity based on the rates of 1-hydroxylation of the probe drug midazolam (MDZ). BCG pretreatment (125 mg kg-1) significantly down-regulated liver CYP3A protein expression compared with the control, metabolic activity, and transcription levels while up-regulating liver NF-κB, IL-1ß, TNF-α and iNOS. HRP intervention (ED50: 78 mg kg-1) moderately reversed NF-κB, inflammatory cytokines, and iNOS activation in a dose-dependent manner (P < 0.05), and suppressed CYP3A down-regulation (P < 0.05); thereby partially alleviating liver injury. During immune liver injury, HRP may reverse CYP3A down-regulation by inhibiting NF-κB signal transduction, and protect liver function, which involves regulation of enzymes transcriptionally, translationally and post-translationally. The discovery that NF-κB is a molecular target of HRP may initiate the development and optimization of a clinical therapeutic approach to mitigate hepatitis B and other immunity-related liver diseases.

Does Mycobacterium bovis persist in cattle in a non-replicative latent state as Mycobacterium tuberculosis in human beings?

Members of the Mycobacterium tuberculosis complex (MTBC) are responsible for tuberculosis in several mammals. In this complex, Mycobacterium tuberculosis and Mycobacterium bovis, which are closely related, show host preference for humans and cattle, respectively. Although human and bovine tuberculosis are clinically similar, M. tuberculosis mostly causes latent infection in humans, whereas M. bovis frequently leads to an acute infection in cattle. This review attempts to connect the pathology in experimental animal models as well as the cellular responses to M. bovis and M. tuberculosis regarding the differences in protein expression and regulatory mechanisms of both pathogens that could explain their apparent divergent latency behaviour. The occurrence of latent bovine tuberculosis (bTB) would represent a serious complication for the eradication of the disease in cattle, with the risk of onward transmission to humans. Thus, understanding the physiological events that may lead to the state of latency in bTB could assist in the development of appropriate prevention and control tools.

Poor stimulation of bovine dendritic cells by Mycobacterium bovis culture supernatant and surface extract is associated with decreased activation of ERK and NF-κB and higher expression of SOCS1 and 3.

The cell envelope of pathogenic mycobacteria interfaces with the host. As such, the interaction of bacterial products localized at or released from the cell surface with the host's immune system can determine the fate of the bacterium in its host. In this study, the effects of three different types of Mycobacterium bovis cell envelope fractions-purified protein derivative, total cell wall lipids and culture supernatant and surface extract-on bovine dendritic cells were assessed. We found that the culture supernatant and surface extract fraction induced little to no production of the pro-inflammatory cytokines TNF-α and IL-12 in bovine dendritic cells. Moreover, this muted response was associated with poor activation of ERK and NF-κB, both of which are critical for the pro-inflammatory response. Furthermore, culture supernatant and surface extract treatment increased the expression of suppressor of cytokine signaling 1 and 3, both of which are negative regulators of pro-inflammatory signaling, in bovine dendritic cells. These observations taken together suggest the M. bovis culture supernatant and surface extract fraction contain immunomodulatory molecules that may aid in M. bovis pathogenesis.

Cutting Edge: TNF Is Essential for Mycobacteria-Induced MINCLE Expression, Macrophage Activation, and Th17 Adjuvanticity.

TNF blockade is a successful treatment for human autoimmune disorders like rheumatoid arthritis and inflammatory bowel disease yet increases susceptibility to tuberculosis and other infections. The C-type lectin receptors (CLR) MINCLE, MCL, and DECTIN-2 are expressed on myeloid cells and sense mycobacterial cell wall glycolipids. In this study, we show that TNF is sufficient to upregulate MINCLE, MCL, and DECTIN-2 in macrophages. TNF signaling through TNFR1 p55 was required for upregulation of these CLR and for cytokine secretion in macrophages stimulated with the MINCLE ligand trehalose-6,6-dibehenate or infected with Mycobacterium bovis bacillus Calmette-Guérin. The Th17 response to immunization with the MINCLE-dependent adjuvant trehalose-6,6-dibehenate was specifically abrogated in TNF-deficient mice and strongly attenuated by TNF blockade with etanercept. Together, interference with production or signaling of TNF antagonized the expression of DECTIN-2 family CLR, thwarting vaccine responses and possibly increasing infection risk.

ESAT-6 regulates autophagous response through SOD-2 and as a result induces intracellular survival of Mycobacterium bovis BCG.

Mycobacterium is known for subverting the host defense machinery, and one such mechanism is the inhibition of autophagy. Here, we have demonstrated that Mycobacterium tuberculosis (MTB) secretes a virulence factor; an early secretory antigenic target protein (ESAT-6) into the phagosome, which induces the expression and activity of mitochondrial superoxide dismutase (SOD-2) of macrophages. Using a series of experiments, and Mycobacterium bovis BCG as a model strain (where ESAT-6 protein is not expressed), we have delineated that the protein regulates SOD-2 of macrophages. The expression and augmentation of SOD-2 activity were confirmed by either incubating the macrophages with ESAT-6 protein, transfection of macrophage by esat6 gene using a eukaryotic promoter vector, or by infection with different mycobacterial strains. The induction of acidification of phagosomal compartment containing bacteria was observed in cells that express low levels of SOD-2. This was further confirmed by observing a significant decrease in the M. bovis BCG intracellular load in the sod-2 knocked-down macrophages.

Busting biofilms: free-living amoebae disrupt preformed methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis biofilms.

Biofilm-associated infections are difficult to eradicate because of their ability to tolerate antibiotics and evade host immune responses. Amoebae and/or their secreted products may provide alternative strategies to inhibit and disperse biofilms on biotic and abiotic surfaces. We evaluated the potential of five predatory amoebae - Acanthamoeba castellanii, Acanthamoeba lenticulata, Acanthamoeba polyphaga, Vermamoeba vermiformis and Dictyostelium discoideum - and their cell-free secretions to disrupt biofilms formed by methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium bovis. The biofilm biomass produced by MRSA and M. bovis was significantly reduced when co-incubated with A. castellanii, A. lenticulata and A. polyphaga, and their corresponding cell-free supernatants (CFS). Acanthamoeba spp. generally produced CFS that mediated biofilm dispersal rather than directly killing the bacteria; however, A. polyphaga CFS demonstrated active killing of MRSA planktonic cells when the bacteria were present at low concentrations. The active component(s) of the A. polyphaga CFS is resistant to freezing, but can be inactivated to differing degrees by mechanical disruption and exposure to heat. D. discoideum and its CFS also reduced preformed M. bovis biofilms, whereas V. vermiformis only decreased M. bovis biofilm biomass when amoebae were added. These results highlight the potential of using select amoebae species or their CFS to disrupt preformed bacterial biofilms.

Regulation of Interferon-γ receptor (IFN-γR) expression in macrophages during Mycobacterium tuberculosis infection.

Interferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

PP2Ac Modulates AMPK-Mediated Induction of Autophagy in Mycobacterium bovis-Infected Macrophages.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis in cattle population across the world. Human beings are at equal risk of developing tuberculosis beside a wide range of M. bovis infections in animal species. Autophagic sequestration and degradation of intracellular pathogens is a major innate immune defense mechanism adopted by host cells for the control of intracellular infections. It has been reported previously that the catalytic subunit of protein phosphatase 2A (PP2Ac) is crucial for regulating AMP-activated protein kinase (AMPK)-mediated autophagic signaling pathways, yet its role in tuberculosis is still unclear. Here, we demonstrated that M. bovis infection increased PP2Ac expression in murine macrophages, while nilotinib a tyrosine kinase inhibitor (TKI) significantly suppressed PP2Ac expression. In addition, we observed that TKI-induced AMPK activation was dependent on PP2Ac regulation, indicating the contributory role of PP2Ac towards autophagy induction. Furthermore, we found that the activation of AMPK signaling is vital for the regulating autophagy during M. bovis infection. Finally, the transient inhibition of PP2Ac expression enhanced the inhibitory effect of TKI-nilotinib on intracellular survival and multiplication of M. bovis in macrophages by regulating the host's immune responses. Based on these observations, we suggest that PP2Ac should be exploited as a promising molecular target to intervene in host-pathogen interactions for the development of new therapeutic strategies towards the control of M. bovis infections in humans and animals.

Kallikrein 12 Regulates Innate Resistance of Murine Macrophages against Mycobacterium bovis Infection by Modulating Autophagy and Apoptosis.

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1ß, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-ß expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.

Macrophage infection with combinations of BCG mutants reduces induction of TNF-α, IL-6, IL-1ß and increases IL-4.

Tuberculosis (TB) is the most prevalent infectious disease worldwide, with no fully effective vaccine yet available. Considering that BCG strains devoid of the BCG1416c or BCG1419c genes afforded protection in mice versus highly virulent M. tuberculosis challenge, or in chronic infection models compared to BCG, respectively, we hypothesized that a synergistic effect of these strains might occur and provide enhanced protection against TB. Herein, we evaluated this hypothesis throughout an experimental design approach, where different combinations of these strains were tested for their capacity to induce cytokines in vitro, compared to individual strains. Our results show that mixed-infection of murine macrophages using these strains significantly decreases induction of TNF-α, IL-1ß, IL-6 but increases IL-4 induction compared with individual strains. These results suggest the existence of interaction effects during infection, which reduce induction of pro-inflammatory cytokines, even though individual intracellular replication is not altered when strains are combined. This is the first report of the evaluation of a potential whole-live combined vaccine against tuberculosis, which paradoxically seems to reduce production of pro-inflammatory cytokines while induces IL-4, leading us to further hypothesize that this combination might contribute as a therapeutic vaccine to reduce inflammation in severe TB cases.

The double-edged sword - prosthetic joint infection following BCG treatment for bladder cancer: a case report.

BACKGROUND: Prosthetic joint infections remain a significant cause of morbidity and are frustrating for patients and physicians alike. Unusual causes of infection may be seen in selected circumstances and a high index of suspicion and a careful history are required to ensure an accurate and timely diagnosis can be made. CASE PRESENTATION: We present a case of Mycobacterium bovis prosthetic joint infection secondary to intravesicular Bacillus Calmette-Guérin (BCG) treatment for prior bladder cancer definitively identified by spoligotyping. A favorable clinical outcome was observed following surgical intervention and a 12-month course of anti-mycobacterial therapy. CONCLUSIONS: BCG therapy, a live attenuated strain of M. bovis, has become the mainstay of adjunctive therapy for bladder cancer and infectious complications, including those affecting the musculoskeletal system, may be seen years after initial therapy. An awareness of this complication and appropriate discussions with the institution's microbiology laboratory may allow for an accurate and timely identification.

A Bivalent Recombinant Mycobacterium bovis BCG Expressing the S1 Subunit of the Pertussis Toxin Induces a Polyfunctional CD4+ T Cell Immune Response.

BACKGROUND: A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment. OBJECTIVES: To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice. METHODS: Mycobacterial plasmids were constructed by cloning the s1pt gene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins. FINDINGS: S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γ were observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4+ IFN-γ + and double-positive CD4+ IFN-γ + TNF-α + T cells. CONCLUSIONS: rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4+ T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer.