The ΔfbpAΔsapM candidate vaccine derived from Mycobacterium tuberculosis H37Rv is markedly immunogenic in macrophages and induces robust immunity to tuberculosis in mice.

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.

Live Attenuated Vaccines against Tuberculosis: Targeting the Disruption of Genes Encoding the Secretory Proteins of Mycobacteria.

Tuberculosis (TB), a chronic infectious disease affecting humans, causes over 1.3 million deaths per year throughout the world. The current preventive vaccine BCG provides protection against childhood TB, but it fails to protect against pulmonary TB. Multiple candidates have been evaluated to either replace or boost the efficacy of the BCG vaccine, including subunit protein, DNA, virus vector-based vaccines, etc., most of which provide only short-term immunity. Several live attenuated vaccines derived from Mycobacterium tuberculosis (Mtb) and BCG have also been developed to induce long-term immunity. Since Mtb mediates its virulence through multiple secreted proteins, these proteins have been targeted to produce attenuated but immunogenic vaccines. In this review, we discuss the characteristics and prospects of live attenuated vaccines generated by targeting the disruption of the genes encoding secretory mycobacterial proteins.

Pathogenic mycoplasmas of humans regulate the long noncoding RNAs in epithelial cells.

Non-coding RNAs (ncRNAs), specifically long ncRNAs (lncRNAs), regulate cellular processes by affecting gene expression at the transcriptional, post-transcriptional, and epigenetic levels. Emerging evidence indicates that pathogenic microbes dysregulate the expression of host lncRNAs to suppress cellular defense mechanisms and promote survival. To understand whether the pathogenic human mycoplasmas dysregulate host lncRNAs, we infected HeLa cells with Mycoplasma genitalium (Mg) and Mycoplasma penumoniae (Mp) and assessed the expression of lncRNAs by directional RNA-seq analysis. HeLa cells infected with these species showed up-and-down regulation of lncRNAs expression, indicating that both species can modulate host lncRNAs. However, the number of upregulated (200 for Mg and 112 for Mp) and downregulated lncRNAs (30 for Mg and 62 for Mp) differ widely between these two species. GREAT analysis of the noncoding regions associated with differentially expressed lncRNAs showed that Mg and Mp regulate a discrete set of lncRNA plausibly related to transcription, metabolism, and inflammation. Further, signaling network analysis of the differentially regulated lncRNAs exhibited diverse pathways such as neurodegeneration, NOD-like receptor signaling, MAPK signaling, p53 signaling, and PI3K signaling, suggesting that both species primarily target signaling mechanisms. Overall, the study's results suggest that Mg and Mp modulate lncRNAs to promote their survival within the host but in distinct manners.

Mycobacterium smegmatis secreting methionine sulfoxide reductase A (MsrA) modulates cellular processes in mouse macrophages.

Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme that reduces the oxidized methionine (Met-O) in proteins to methionine (Met). Its pivotal role in the cellular processes has been well established by overexpressing, silencing, and knocking down MsrA or deleting the gene encoding MsrA in several species. We are specifically interested in understanding the role of secreted MsrA in bacterial pathogens. To elucidate this, we infected mouse bone marrow-derived macrophages (BMDMs) with recombinant Mycobacterium smegmatis strain (MSM), secreting a bacterial MsrA or M. smegmatis strain (MSC) carrying only the control vector. BMDMs infected with MSM induced higher levels of ROS and TNF-α than BMDMs infected with MSC. The increased ROS and TNF-α levels in MSM-infected BMDMs correlated with elevated necrotic cell death in this group. Further, RNA-seq transcriptome analysis of BMDMs infected with MSC and MSM revealed differential expression of protein and RNA coding genes, suggesting that bacterial-delivered MsrA could modulate the host cellular processes. Finally, KEGG pathway enrichment analysis identified the down-regulation of cancer-related signaling genes in MSM-infected cells, indicating that MsrA can potentially regulate the development and progression of cancer.

The Identification and Clinical Applications of Mutated Antigens in the Era of Immunotherapy.

The era of personalized cancer therapy is here. Advances in the field of immunotherapy have paved the way for the development of individualized neoantigen-based therapies that can translate into favorable treatment outcomes and fewer side effects for patients. Addressing challenges related to the identification, access, and clinical application of neoantigens is critical to accelerating the development of individualized immunotherapy for cancer patients.

Harnessing the Immune System with Cancer Vaccines: From Prevention to Therapeutics.

Prophylactic vaccination against infectious diseases is one of the most successful public health measures of our lifetime. More recently, therapeutic vaccination against established diseases such as cancer has proven to be more challenging. In the host, cancer cells evade immunologic regulation by multiple means, including altering the antigens expressed on their cell surface or recruiting inflammatory cells that repress immune surveillance. Nevertheless, recent clinical data suggest that two classes of antigens show efficacy for the development of anticancer vaccines: tumor-associated antigens and neoantigens. In addition, many different vaccines derived from antigens based on cellular, peptide/protein, and genomic components are in development to establish their efficacy in cancer therapy. Some vaccines have shown promising results, which may lead to favorable outcomes when combined with standard therapeutic approaches. This review provides an overview of the innate and adaptive immune systems, their interactions with cancer cells, and the development of various different vaccines for use in anticancer therapeutics.

Mycoplasma genitalium and M. pneumoniae Regulate a Distinct Set of Protein-Coding Genes in Epithelial Cells.

Mycoplasma genitalium and M. pneumoniae are two significant mycoplasmas that infect the urogenital and respiratory tracts of humans. Despite distinct tissue tropisms, they both have similar pathogenic mechanisms and infect/invade epithelial cells in the respective regions and persist within these cells. However, the pathogenic mechanisms of these species in terms of bacterium-host interactions are poorly understood. To gain insights on this, we infected HeLa cells independently with M. genitalium and M. pneumoniae and assessed gene expression by whole transcriptome sequencing (RNA-seq) approach. The results revealed that HeLa cells respond to M. genitalium and M. pneumoniae differently by regulating various protein-coding genes. Though there is a significant overlap between the genes regulated by these species, many of the differentially expressed genes were specific to each species. KEGG pathway and signaling network analyses revealed that the genes specific to M. genitalium are more related to cellular processes. In contrast, the genes specific to M. pneumoniae infection are correlated with immune response and inflammation, possibly suggesting that M. pneumoniae has some inherent ability to modulate host immune pathways.

Ethanol in Combination with Oxidative Stress Significantly Impacts Mycobacterial Physiology.

Here, we investigate the mycobacterial response to the combined stress of an organic oxidant (cumene hydroperoxide [CHP]) and a solvent (ethanol). To understand the interaction between the two stressors, we treated Mycobacterium smegmatis cells to a range of ethanol concentrations (2.5% to 10% [vol/vol]) in combination with a subinhibitory concentration of 1 mM CHP. It was observed that the presence of CHP increases the efficacy of ethanol in inducing rapid cell death. The data further suggest that ethanol reacts with the alkoxy radicals to produce ethanol-derived peroxides. These radicals induce significant membrane damage and lead to cell lysis. The ethanol-derived radicals were primarily recognized by the cells as organic radicals, as was evident by the differential upregulation of the ohr-ohrR genes that function in cells treated with the combination of ethanol and CHP. The role of organic peroxide reductase, Ohr, was further confirmed by the significantly higher sensitivity of the deletion mutant to CHP and the combined stress treatment of CHP and ethanol. Moreover, we also observed the sigma factor σB to be important for the cells treated with ethanol alone as well as the aforementioned combination. A ΔsigB mutant strain had significantly higher susceptibility to the stress conditions. This finding was correlated with the σB-dependent transcriptional regulation of ohr and ohrR In summary, our data indicate that the combination of low levels of ethanol and organic peroxides induce ethanol-derived organic radicals that lead to significant oxidative stress on the cells in a concentration-dependent manner.IMPORTANCE Bacterial response to a combination of stresses can be unexpected and very different compared with that of an individual stress treatment. This study explores the physiological and transcriptional response of mycobacteria in response to the combinatorial treatment of an oxidant with the commonly used solvent ethanol. The presence of a subinhibitory concentration of organic peroxide increases the effectiveness of ethanol by inducing reactive peroxides that destroy the membrane integrity of cells in a significantly short time span. Our work elucidates a mechanism of targeting the complex mycobacterial membrane, which is its primary source of intrinsic resistance. Furthermore, it also demonstrates the importance of exploring the effect of various stress conditions on inducing bacterial clearance.

Methionine sulfoxide reductase A (MsrA) modulates cells and protects against Mycoplasma genitalium induced cytotoxicity.

Methionine sulfoxide reductase A (MsrA) is a ubiquitous antioxidant repair enzyme which specifically reduces the oxidized methionine (Met-O) in proteins to methionine (Met). Previous studies have shown that lack of or overexpression of MsrA in cells affects the function of proteins and can lead to altered cellular processes. Interestingly, some pathogenic bacteria secrete and/or carry MsrA on their surface, suggesting some key roles for this enzyme in the modulation of host cellular processes. Therefore, we investigated how exogenously added MsrA affects the ability of the host cells in combating infection by using an in vitroMycoplasma genitalium cytotoxicity model. HeLa cells pretreated with MsrA and infected with M. genitalium showed significantly lower necrosis (cytotoxicity) than untreated cells infected with M. genitalium. Intriguingly, necrotic cell death pathway specific real time RT-PCR revealed that M. genitalium infection upregulates the expression of the TNF gene in HeLa cells and that MsrA pretreatment of the cells downregulates its expression significantly. Consistent with this, enzyme linked immunosorbent assay (ELISA) results showed that HeLa cells pretreated with MsrA secreted reduced levels of TNF-α following M. genitalium infection. Also, our study demonstrates that MsrA treatment of cells affects the phosphorylation status of transcriptional regulators such as NF-кB, JNK and p53 that regulate different cytokines. Further, fluorescent microscopy showed the cellular uptake of exogenously added MsrA fused with red fluorescent protein (MsrA-RFP). Altogether, our results suggest that secreted MsrA may help pathogens to modulate host cellular processes.

An autophagy-inducing and TLR-2 activating BCG vaccine induces a robust protection against tuberculosis in mice.

Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis due to M. tuberculosis (Mtb), which kills millions of people each year. BCG variably protects children, but not adults against tuberculosis. BCG evades phagosome maturation, autophagy, and reduces MHC-II expression of antigen-presenting cells (APCs) affecting T-cell activation. To bypass these defects, an autophagy-inducing, TLR-2 activating C5 peptide from Mtb-derived CFP-10 protein was overexpressed in BCG in combination with Ag85B. Recombinant BCG85C5 induced a robust MHC-II-dependent antigen presentation to CD4 T cells in vitro, and elicited stronger TH1 cytokines (IL-12, IL-1ß, and TNFα) from APCs of C57Bl/6 mice increasing phosphorylation of p38MAPK and ERK. BCG85C5 also enhanced MHC-II surface expression of MΦs by inhibiting MARCH1 ubiquitin ligase that degrades MHC-II. BCG85C5 infected APCs from MyD88 or TLR-2 knockout mice showed decreased antigen presentation. Furthermore, BCG85C5 induced LC3-dependent autophagy in macrophages increasing antigen presentation. Consistent with in vitro effects, BCG85C5 markedly expanded both effector and central memory T cells in C57Bl/6 mice protecting them against both primary aerosol infection with Mtb and reinfection, but was less effective among TLR-2 knockout mice. Thus, BCG85C5 induces stronger and longer lasting immunity, and is better than BCG against tuberculosis of mice.

Utility of OhrR-Ohr system for the expression of recombinant proteins in mycobacteria and for the delivery of M. tuberculosis antigens to the phagosomal compartment.

We have recently reported that in vitro and intracellular organic peroxide stress oxidizes OhrR of Mycobacterium smegmatis and that the oxidized OhrR consequently derepresses the expression of Ohr. Here we demonstrate that the OhrR-Ohr system is highly useful for the expression of recombinant mycobacterial proteins and also for the delivery of Mycobacterium tuberculosis (Mtb) antigens to the phagosomal compartments. Recombinant M. smegmatis strains, which bear plasmid constructs to express Ohr2-T85BCFP and Ohr2-MtrA, showed expression of fusion proteins upon induction with t-butyl hydroperoxide (t-BHP) in a dose dependent manner. The M. smegmatis expressed Ohr2-T85BCFP fusion could be affinity purified by adding a 9x histidine tag to the C-terminal end of the fusion protein. Further, mouse bone marrow derived macrophages (BMDMs) infected with either recombinant M. smegmatis or BCG strains with ohr2-T85BCFP construct showed expression of T85BCFP protein without any exogenously added inducer. In addition, BMDMs infected with either recombinant BCG or Mtb with ohr2-T85BCFP construct could effectively deliver the antigens to T-cells at higher levels than strains bearing the control plasmid alone. Altogether, these results suggest that the OhrR-Ohr system is a novel inducible system to study the biology and pathogenesis of mycobacteria.

Enhanced delivery of Mycobacterium tuberculosis antigens to antigen presenting cells using RVG peptide.

Among the various strategies to improve vaccines against infectious diseases, targeting of antigens to dendritic cells (DCs), which are professional antigen presenting cells (APCs), has received increased attention in recent years. Here, we investigated whether a synthetic peptide region named RVG, originated from Rabies Virus Glycoprotein that binds to the α-7 subunit of the nicotinic acetylcholine receptors (AchR-α7) of APCs, could be used for the delivery of Mycobacterium tuberculosis (Mtb) peptide antigens to DCs and macrophages. Mouse bone marrow derived DCs (BMDCs) and human THP-1 macrophages stimulated with RVG fused peptide epitopes 85B241 and 85B96 (represent Ag85B241-256 and Ag85B96-111, respectively) from antigen 85B (Ag85B) of Mtb showed enhanced antigen presentation as compared to unfused peptide epitopes and BCG. Further, BMDCs stimulated with RVG fused 85B241 showed higher levels of IL-12 positive cells. Consistent with in vitro data, splenocytes of mice immunized with RVG-85B241 showed increased number of antigen specific IFN-γ, IL-2, and TNF-α producing cells in relation to splenocytes from mice immunized with 85B241 alone. These results suggest that RVG may be a promising tool to develop effective alternate vaccines against tuberculosis (TB).

OhrR of Mycobacterium smegmatis senses and responds to intracellular organic hydroperoxide stress.

Organic hydroperoxide reductase regulator (OhrR) in bacteria is a sensor for organic hydroperoxide stress and a transcriptional regulator for the enzyme organic hydroperoxide reductase (Ohr). In this study we investigated, using a GFP reporter system, whether Mycobacterium smegmatis OhrR has the ability to sense and respond to intracellular organic hydroperoxide stress. It was observed that M. smegmatis strains bearing the pohr-gfpuv fusion construct were able to express GFP only in the absence of an intact ohrR gene, but not in its presence. However, GFP expression in the strain bearing pohr-gfpuv with an intact ohrR gene could be induced by organic hydroperoxides in vitro and in the intracellular environment upon ingestion of the bacteria by macrophages; indicating that OhrR responds not only to in vitro but also to intracellular organic hydroperoxide stress. Further, the intracellular expression of pohr driven GFP in this strain could be abolished by replacing the intact ohrR gene with a mutant ohrR gene modified for N-terminal Cysteine (Cys) residue, suggesting that OhrR senses intracellular organic hydroperoxides through Cys residue. This is the first report demonstrating the ability of OhrR to sense intracellular organic hydroperoxides.

Recombinant Bacillus subtilis spores for the delivery of Mycobacterium tuberculosis Ag85B-CFP10 secretory antigens.

Tuberculosis continues to be a great cause of morbidity and mortality in different parts of the world. Unfortunately, the current BCG vaccine being administered is not fully protective against tuberculosis; therefore, there is a great need for alternate vaccines. With an aim to develop such vaccines, we have analyzed the utility of Bacillus subtilis spores for the expression of two major immunodominant antigens of Mycobacterium tuberculosis, Ag85B and CFP10. We created three recombinant B. subtilis strains to express a truncated fusion of Ag85B191-325 and CFP101-70 antigens (T85BCFP), either on the spore coat (MTAG1 strain) or in the cytosol of B. subtilis (MTAG 2 and MTAG 3 strains). Examination of spores isolated from these strains revealed successful expression of T85BCFP antigens on the spore coat of MTAG1 as well as in the cytosol of vegetatively grown cells of MTAG2 and MTAG3, indicating that spores can indeed express M. tuberculosis antigens. In vitro antigen presentation assays with spore-infected mouse bone marrow derived macrophages (BMDM) showed that all three recombinant spores could deliver these antigens to antigen presenting cells (APCs). Mice immunized with recombinant spores displayed significantly higher levels of Ag85B specific IFN-γ producing cells in the spleen than in mice immunized with wild-type (non-recombinant) spores. In addition, these mice showed relatively higher levels of Ag85B specific IgG antibodies in the serum in comparison to mice immunized with non-recombinant spores, thus providing additional evidence that recombinant spores can deliver these antigens in vivo. These results suggest that B. subtilis spores are ideal vehicles for antigen delivery and have great potential in the development of primary and booster vaccines against tuberculosis.

Modulation of Host miRNAs by Intracellular Bacterial Pathogens.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein coding genes of viruses and eukaryotes at the post-transcriptional level. The eukaryotic genes regulated by miRNAs include those whose products are critical for biological processes such as cell proliferation, metabolic pathways, immune response, and development. It is now increasingly recognized that modulation of miRNAs associated with biological processes is one of the strategies adopted by bacterial pathogens to survive inside host cells. In this review, we present an overview of the recent findings on alterations of miRNAs in the host cells by facultative intracellular bacterial pathogens. In addition, we discuss how the altered miRNAs help in the survival of these pathogens in the intracellular environment.

Inactivation of the organic hydroperoxide stress resistance regulator OhrR enhances resistance to oxidative stress and isoniazid in Mycobacterium smegmatis.

The organic hydroperoxide stress resistance regulator (OhrR) is a MarR type of transcriptional regulator that primarily regulates the expression of organic hydroperoxide reductase (Ohr) in bacteria. In mycobacteria, the genes encoding these proteins exist in only a few species, which include the fast-growing organism Mycobacterium smegmatis. To delineate the roles of Ohr and OhrR in defense against oxidative stress in M. smegmatis, strains lacking the expression of these proteins were constructed by deleting the ohrR and ohr genes, independently and together, through homologous recombination. The OhrR mutant strain (MSΔohrR) showed severalfold upregulation of Ohr expression, which could be observed at both the transcript and protein levels. Similar upregulation of Ohr expression was also noticed in an M. smegmatis wild-type strain (MSWt) induced with cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BHP). The elevated Ohr expression in MSΔohrR correlated with heightened resistance to oxidative stress due to CHP and t-BHP and to inhibitory effects due to the antituberculosis drug isoniazid (INH). Further, this mutant strain exhibited significantly enhanced survival in the intracellular compartments of macrophages. In contrast, the strains lacking either Ohr alone (MSΔohr) or both Ohr and OhrR (MSΔohr-ohrR) displayed limited or no resistance to hydroperoxides and INH. Additionally, these strains showed no significant differences in intracellular survival from the wild type. Electrophoretic mobility shift assays (EMSAs) revealed that the overexpressed and purified OhrR interacts with the ohr-ohrR intergenic region with a greater affinity and this interaction is contingent upon the redox state of the OhrR. These findings suggest that Ohr-OhrR is an important peroxide stress response system in M. smegmatis.

Mycoplasma genitalium promotes epithelial crossing and peripheral blood mononuclear cell infection by HIV-1.

BACKGROUND: Mycoplasma genitalium co-infection in HIV-infected individuals has been reported to increase the shedding of HIV in the urogenital region of females. To better understand this relationship, we investigated the influence of M. genitalium on the transmission and replication of HIV using an in vitro model. METHODS: The Transwell co-culture system was employed to assess the crossing of an endocervical cell barrier by HIV-1. Immunocytochemistry and confocal microscopy were used to assess the distribution of the nectin-1 molecule on M. genitalium-infected epithelial cells of the End1/E6E7 endocervical cell line, grown as monolayers in the insert wells. Peripheral blood mononuclear cells (PBMC) were cultured in the bottom wells to assess the effects of M. genitalium, passing through the semipermeable culturing membrane, on subsequent HIV infection of susceptible target cells. RESULTS: Infection of the endocervical cells with the adhesion-positive M. genitalium G37 strain (wild-type) significantly elevated the passage of HIV across the epithelial cell barrier relative to HIV transfer across endocervical cells infected with the adhesion-negative M. genitalium JB1 strain. Immunostaining of the M. genitalium-G37-infected epithelial cells disclosed capping and internalization of the junctional regulatory protein nectin-1, in association with reduced transepithelial resistance (TER) in the cell monolayer. When PBMC were cultured beneath insert wells containing M. genitalium-G37-infected epithelial cell monolayers, we observed significantly enhanced infectivity and replication of HIV added afterward to the cultures. CONCLUSIONS: M. genitalium influences events on both sides of a cultured mucosal epithelial monolayer: (1) by infecting the epithelial cells and reducing the integrity of the barrier itself, and (2) by activating HIV target cells below it, thereby promoting HIV infection and progeny virus production.

A serine/threonine phosphatase encoded by MG_207 of Mycoplasma genitalium is critical for its virulence.

BACKGROUND: Bacterial signal transduction systems like two component system (TCS) and Serine/Threonine kinase (STK) and Serine/Threonine phosphatase (STP) play important roles in the virulence and pathogenesis of bacterial pathogens. Mycoplasma genitalium, a mollicute that causes the urogenital diseases urethritis and cervicitis in men and women, respectively, is a pathogen which lacks TCS but possesses STK/STP. In this study, we investigated the biochemical and virulence properties of an STP protein encoded by the gene MG_207 of this species. RESULTS: We overexpressed MG207 in Escherichia coli overexpression system as a recombinant His10MG207 protein and purified it with affinity chromatography. This recombinant protein readily hydrolyzed the substrate p-nitrophenyl phosphate (pNPP) in a dose-dependent manner. Additional studies using synthetic peptides as substrates revealed that the recombinant protein was able to hydrolyze the threonine phosphate. Further, a transposon insertion mutant strain of M. genitalium (TIM207) that lacks the protein MG207 showed differentially phosphorylated proteins when compared to the wild type G37 strain. Mass spectrometry revealed that some of the key proteins differentially phosphorylated in TIM207 strain were putative cytoskeletal protein encoded by the gene MG_328 and pyruvate dehydrogenase E1 α chain encoded by the gene MG_274. In addition, TIM207 was noticed to be less cytotoxic to HeLa cells and this correlated with the production of less hydrogen peroxide by this strain. This strain was also less efficient in inducing the differentiation of THP-1 cell line as compared to wild type M. genitalium. CONCLUSIONS: The results of the study suggest that MG207 is an important signaling protein of M. genitalium and its presence may be crucial for the virulence of this species.