Proline-glutamic Acid Research Articles

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Rv3539 (PPE63) of Mycobacterium Tuberculosis Promotes Survival of Mycobacterium Smegmatis in Human Macrophages Cell Line via Cell Wall Modulation of Bacteria and Altering Host's Immune Response.

The modulation of host's immune response plays an important role in the intracellular survival of Mycobacterium tuberculosis. The intracellular pathogen counteracts environmental stresses with help of the expression of several genes. The M. tuberculosis genome encodes several immune-modulatory proteins including PE (proline-glutamic acid)/PPE (proline-proline-glutamic acid) superfamily proteins. It is unclear how the unique PE/PPE proteins superfamily contributes to survival under different stress and pathophysiology conditions. Previously, we showed that PPE63 (Rv3539) has C-terminal esterase extension and was localized as a membrane attached and in extracellular compartment. Therefore, the probability of these proteins interacting with the host to modulate the host immune response cannot be ruled out. The physiological role of PPE63 was characterized by expressing the PPE63 in the M. smegmatis, a non-pathogenic strain intrinsically deficient of PPE63. The recombinant M. smegmatis expressing PPE63 altered the colony morphology, lipid composition, and integrity of the cell wall. It provided resistance to multiple hostile environmental stress conditions and several antibiotics. MS_Rv3539 demonstrated higher infection and intracellular survival in comparison to the MS_Vec in the PMA-differentiated THP-1 cells. The decreased intracellular level of ROS, NO, and expression of iNOS was observed in THP-1 cells upon infection with MS_Rv3539 in comparison to MS_Vec. Further, the decrease in expression of pro-inflammatory cytokines like IL-6, TNF-α, and IL-1β and enhanced anti-inflammatory cytokines like IL-10, pointed toward its role in immune modulation. Overall this study suggested the role of Rv3539 in enhanced intracellular survival of M. smegmatis via cell wall modulation and altered immune response of host.

Novel role of PE_PGRS47 in the alteration of mycobacterial cell wall integrity and drug resistance

The proline-glutamic acid and proline-proline-glutamic acid (PE/PPE) family of proteins is widespread in pathogenic mycobacteria and plays different roles in mycobacterial physiology. While several PE/PPE family proteins have been studied, the exact function of most PE/PPE proteins in the physiology of Mycobacterium tuberculosis (Mtb) remains unknown. PE_PGRS47 belongs to the PE/PPE family of proteins reported to help Mtb evade protective host immune responses. In this study, we demonstrate a novel role of PE_PGRS47. Heterologous expression of the pe_pgrs47 gene in a non-pathogenic Mycobacterium smegmatis, intrinsically deficient of PE_PGRS protein, exhibits modulated colony morphology and cell wall lipid profile leading to a marked susceptibility to multiple antibiotics and environmental stressors. Using ethidium bromide/Nile red uptake assays, Mycobacterium smegmatis expressing PE_PGRS47 showed higher cell wall permeability than the control strain. Overall, these data suggested that PE_PGRS47 is cell surface exposed and influences cell wall integrity and the formation of mycobacterial colonies, ultimately potentiating the efficacy of lethal stresses against mycobacteria.

Analysis of the noncoding RNA regulatory networks of H37Rv- and H37Rv△1759c-infected macrophages.

Noncoding RNAs regulate the process of Mycobacterium tuberculosis (M. tb) infecting the host, but there is no simultaneous transcriptional information of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) and the global regulatory networks of non-coding RNA. Rv1759c, a virulence factor, is a member of protein family containing the proline-glutamic acid (PE) in M. tb, which can increase M. tb survival. To reveal the noncoding RNA regulatory networks and the effect of Rv1759c on non-coding RNA expression during M. tb infection, we collected samples of H37Rv- and H37Rv△1759c-infected macrophages and explored the full transcriptome expression profile. We found 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv infection, 356 mRNAs, 433 lncRNAs, 168 circRNAs, and 12 miRNAs differentially expressed during H37Rv△1759c infection. We constructed lncRNA/circRNA-miRNA-mRNA regulatory networks during H37Rv and H37Rv△1759c infection. We demonstrated the role of one of the hubs of the networks, hsa-miR-181b-3p, for H37Rv survival in macrophages. We discovered that the expression changes of 68 mRNAs, 92 lncRNAs, 26 circRNAs, and 3 miRNAs were only related to the deletion of Rv1759c by comparing the transcription profiles of H37Rv and H37Rv△1759c. Here, our study comprehensively characterizes the transcriptional profiles in THP1-derived-macrophages infected with H37Rv and H37Rv△1759c, which provides support and new directions for in-depth exploration of noncoding RNA and PE/PPE family functions during the infection process.

Value of Proline, Glutamic Acid, and Leucine-Rich Protein 1 and GATA Binding Protein 3 Expression in Breast Cancer: An Immunohistochemical study

GATA binding protein 3 was more sensitive than traditional markers such as gross cystic disease fluid protein 15 and mammaglobin for identifying primary and metastatic breast carcinomas, but its significance decreased in triple-negative breast cancer. Recent studies showed a high expression rate of proline glutamic acid and leucine-rich protein in breast cancer and their superiority over GATA3 in triple-negative breast cancer. Our study provided new insights into the diagnostic and prognostic roles of PELP1 and GATA3 in primary and metastatic breast cancer. An immunohistochemical assay was carried out using PELP1 and GATA3 in 60 cases of primary breast cancer and 15 metastatic. Invasive carcinoma of no special type was the predominant type (80%). The majority of cases were grade 3 (68.3%). GATA3 expression was 83.3% positive in primary breast carcinomas and 73.5% positive in metastatic breast carcinomas. In comparison, PELP1 had a 96.7% positive expression rate in primary breast carcinomas and an 86.7% positive expression rate in metastasis. There was a statistically significant agreement between GATA3 and PELP1 in the diagnosis of the cases. PELP1 is a significantly higher proportion of both primary and metastatic breast carcinomas than GATA3. In breast cancer, there was a strong association between favorable prognostic factors and GATA3 expression, with evidence of an inverse association with Ki-67 overexpression.

Computational analysis and prediction of PE_PGRS proteins using machine learning

Mycobacterium tuberculosis genome comprises approximately 10% of two families of poorly characterised genes due to their high GC content and highly repetitive nature. The largest sub-group, the proline-glutamic acid polymorphic guanine-cytosine-rich sequence (PE_PGRS) family, is thought to be involved in host response and disease pathogenicity. Due to their high genetic variability and complexity of analysis, they are typically disregarded for further research in genomic studies. There are currently limited online resources and homology computational tools that can identify and analyse PE_PGRS proteins. In addition, they are computational-intensive and time-consuming, and lack sensitivity. Therefore, computational methods that can rapidly and accurately identify PE_PGRS proteins are valuable to facilitate the functional elucidation of the PE_PGRS family proteins. In this study, we developed the first machine learning-based bioinformatics approach, termed PEPPER, to allow users to identify PE_PGRS proteins rapidly and accurately. PEPPER was built upon a comprehensive evaluation of 13 popular machine learning algorithms with various sequence and physicochemical features. Empirical studies demonstrated that PEPPER achieved significantly better performance than alignment-based approaches, BLASTP and PHMMER, in both prediction accuracy and speed. PEPPER is anticipated to facilitate community-wide efforts to conduct high-throughput identification and analysis of PE_PGRS proteins.

Moonlighting by PPE2 Protein: Focus on Mycobacterial Virulence.

In Mycobacterium tuberculosis, ∼10% of its genome encodes the proline-glutamic acid and proline-proline-glutamic acid (PPE) family of proteins, some of which were recently established to be key players in mycobacterial virulence. PPE2 (Rv0256c) is one among these proteins that we found to have pleiotropic effects during mycobacterial infection. PPE2 weakens the innate immune system by disturbing NO and reactive oxygen species production and myeloid hematopoiesis. We showed that PPE2 is unique for having nuclear localization signal, DNA binding domain, and SRC homology 3 (PXXP) binding domain, which enable it to interfere with the host immune system. Interestingly, PPE2 is a secretary protein, expressed during active tuberculosis (TB) infection, and is involved in facilitating survival of M. tuberculosis Thus, PPE2 could be a valuable drug target for developing effective therapeutics against TB. In this article, we describe possible roles of PPE2 in TB pathogenesis and the importance of PPE2 as a novel therapeutic target against TB.

Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Stimulated by Mycobacterium tuberculosis PPE57 Identifies Characteristic Genes Associated With Type I Interferon Signaling.

Proline-glutamic acid (PE)- and proline-proline-glutamic acid (PPE)-containing proteins are exclusive to Mycobacterium tuberculosis (MTB), the leading cause of tuberculosis (TB). In this study, we performed global transcriptome sequencing (RNA-Seq) on PPE57-stimulated peripheral blood mononuclear cells (PBMCs) and control samples to quantitatively measure the expression level of key transcripts of interest. A total of 1367 differentially expressed genes (DEGs) were observed in response to a 6 h exposure to PPE57, with 685 being up-regulated and 682 down-regulated. Immune-related gene functions and pathways associated with these genes were evaluated, revealing that the type I IFN signaling pathway was the most significantly enriched pathway in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1. These PPE57-related transcriptomic profiles have implications for a better understanding of host global immune mechanisms underlying MTB infection outcomes. However, more studies regarding these DEGs and type I IFN signaling in this infectious context are necessary to more fully clarify the underlying mechanisms that arise in response to PPE57 during MTB infection.

PE_PGRS31-S100A9 Interaction Promotes Mycobacterial Survival in Macrophages Through the Regulation of NF-κB-TNF-α Signaling and Arachidonic Acid Metabolism.

Mycobacterium tuberculosis (M. tb) evades the surveillance of immune responses for survival in macrophages. However, the precise mechanism and toxins/proteins encoded by M. tb involved in the bacterial escape remain elusive. The function of Rv1768 protein (also referred to as PE_PGRS31, belonging to the PE_PGRS family) encoded by the region of deletion 14 (RD-14) in the virulent M. tb H37Rv strain has not, to the best of our knowledge, been reported previously. Here, we found that Rv1768 remarkably promotes bacterial survival in macrophages. Compared to wild type (WT) H37Rv, the Rv1768 deficient strain (H37RvΔ1768) showed significantly decreased colony-forming units in the lungs, spleen, and liver of the murine M. tb infection model. The bacterial burdens of WT H37Rv in WT macrophages and C57BL/6 mice were significantly higher than those in S100A9 deficiency cells and mice, but there were no significant differences for H37RvΔRv1768. Rv1768 binds S100A9 with the proline-glutamic acid domain (PE domain) and blocks the interaction between S100A9 and Toll-like receptor 4 (TLR4), and suppresses TLR4-myeloid differentiation factor 88-nuclear factor-kappa B (NF-κB)-tumor necrosis factor α (TNF-α) signaling in macrophages. Interestingly, Rv1768 binding to S100A9 also disturbs the metabolism of arachidonic acid by activating 5-lipoxygenase, increasing lipotoxin A4, and down-regulating cyclooxygenase-2 and prostaglandin E2 expression, thus, promoting mycobacterial survival. Our results revealed that M. tb Rv1768 promotes mycobacterial survival in macrophages by regulating NF-κB-TNF-α signaling and arachidonic acid metabolism via S100A9. Disturbing the interaction between Rv1768 and S100A9 may be a potential therapeutic target for tuberculosis.

Mycobacterium tuberculosis PE31 (Rv3477) Attenuates Host Cell Apoptosis and Promotes Recombinant M. smegmatis Intracellular Survival via Up-regulating GTPase Guanylate Binding Protein-1.

The Mycobacterium (M.) tuberculosis comprising proline–glutamic acid (PE) subfamily proteins associate with virulence, pathogenesis, and host-immune modulations. While the functions of most of this family members are not yet explored. Here, we explore the functions of “PE only” subfamily member PE31 (Rv3477) in virulence and host-pathogen interactions. We have expressed the M. tuberculosis PE31 in non-pathogenic Mycobacterium smegmatis strain (Ms_PE31) and demonstrated that PE31 significantly altered the cell facet features including colony morphology and biofilm formation. PE31 expressing M. smegmatis showed more resistant to the low pH, diamide, H2O2 and surface stress. Moreover, Ms_PE31 showed higher intracellular survival in macrophage THP-1 cells. Ms_PE31 significantly down-regulated the production of IL-12p40 and IL-6, while up-regulates the production of IL-10 in macrophages. Ms_PE31 also induced the expression of guanylate-binding protein-1 (GBP-1) in macrophages. Further analysis demonstrates that Ms_PE31 inhibits the caspase-3 activation and reduces the macrophages apoptosis. Besides, the NF-κB signaling pathway involves the interplay between Ms_PE31 and macrophages. Collectively, our finding identified that PE31 act as a functionally relevant virulence factor of M. tuberculosis.

Molecular Basis Underlying Host Immunity Subversion by Mycobacterium tuberculosis PE/PPE Family Molecules.

Mycobacterium tuberculosis proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family proteins, with >160 members, are crucial for virulence, cell wall, host cell fate, host Th1/Th2 balance, and CD8+ T cell recognition. Ca2+ signaling is involved in PE/PPE protein-mediated host-pathogen interaction. PE/PPE proteins also function in heme utilization and nitric oxide production. PE/PPE family proteins are intensively pursued as diagnosis biomarkers and vaccine components.

Dissecting the membrane lipid binding properties and lipase activity of Mycobacteriumtuberculosis LipY domains.

The Mycobacteriumtuberculosis LipY protein, a prototype of the proline-glutamic acid (PE) family, exhibits a triacylglycerol (TAG) hydrolase activity that contributes to host cell lipid degradation and persistence of the bacilli. LipY is found either as a full-length intracytosolic form or as a mature extracellular form lacking the N-terminal PE domain. Even though the contribution of the extracellular form in TAG consumption has been partly elucidated, very little information is available regarding the potential interactions of either full-length LipY with the cytoplasmic membrane, or mature form LipY with the outer membrane. Herein, several LipY variants truncated in their N-terminal domain were produced and biochemically characterized in lipid-protein interaction assays, using the monomolecular film technique and FTIR. Comparison of the catalytic activities of these recombinant proteins showed that LipY∆149, corresponding to the extracellular form of LipY lacking the PE domain, is more active than the full-length protein. This confirms previous studies reporting that the PE domain negatively modulates the TAG hydrolase activity of LipY. Lipid-protein interaction studies indicate that the PE domain anchors LipY onto membrane lipids. Consistent with these findings, we show that LipY∆149 is loosely associated with the mycobacterial cell wall, and that this interaction is mediated by the sole lipase domain. Overall, our results bring new information regarding the molecular mechanisms by which LipY either binds and hydrolyses host cell lipids or degrades TAG, the major source of lipids within mycobacterial intracytosolic lipid inclusions.

Vaccine efficacy of a Mycobacterium tuberculosis Beijing-specific proline-glutamic acid (PE) antigen against highly virulent outbreak isolates.

Bacillus Calmette-Guerin vaccine confers insufficient pulmonary protection against tuberculosis (TB), particularly the Mycobacterium tuberculosis (Mtb) Beijing strain infection. Identification of vaccine antigens (Ags) by considering Mtb genetic diversity is crucial for the development of improved TB vaccine. MTBK_20640, a new Beijing genotype-specific proline-glutamic acid-family Ag, was identified by comparative genomic analysis. Its immunologic features were characterized by evaluating interactions with dendritic cells (DCs), and immunogenicity and vaccine efficacy were determined against highly virulent Mtb Beijing outbreak Korean Beijing (K) strain and HN878 strain in murine infection model. MTBK_20640 induced DCs via TLR2 and downstream MAPK and NF-κB signaling pathways, effectively promoting naive CD4-positive (CD4+) T-cell proliferation and IFN-γ production. Different IFN-γ response was observed in mice infected with Mtb K or reference H37Rv strain. Significant induction of T helper type 1 cell-polarized Ag-specific multifunctional CD4+ T cells and a marked Ag-specific IgG2c response were observed in mice immunized with MTBK_20640/glucopyranosyl lipid adjuvant-stable emulsion. The immunization conferred long-term protection against 2 Mtb Beijing outbreak strains, as evidenced by a significant reduction in colony-forming units in the lung and spleen and reduced lung inflammation. MTBK_20640 vaccination conferred long-term protection against highly virulent Mtb Beijing strains. MTBK_20640 may be developed into a novel Ag component in multisubunit TB vaccines in the future.-Kwon, K. W., Choi, H.-H., Han, S. J., Kim, J.-S., Kim, W. S., Kim, H., Kim, L.-H., Kang, S. M., Park, J., Shin, S. J. Vaccine efficacy of a Mycobacterium tuberculosis Beijing-specific proline-glutamic acid (PE) antigen against highly virulent outbreak isolates.

Mycobacterium tuberculosis PPE44 (Rv2770c) is involved in response to multiple stresses and promotes the macrophage expression of IL-12 p40 and IL-6 via the p38, ERK, and NF-κB signaling axis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable threat to global public health. The successful intracellular persistence of M. tuberculosis significantly contributes to the intractability of tuberculosis. Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are mycobacterial exclusive protein families that widely reported to be involved in the bacterial virulence, physiology and interaction with host. Rv2770c (PPE44), a predicted virulence factor, was up-regulated upon the infected guinea pig lungs. To investigate the role of Rv2770c, we heterologously expressed the PPE44 in the nonpathogenic fast-growing M. smegmatis strain. Subcellular location analysis demonstrated that Rv2770c is a cell wall associated protein, suggestive of a potential candidate involved in host-pathogen interaction. The Rv2770c can enhance M. smegmatis survival within macrophages and under stresses such as H2O2, SDS, diamide exposure, and low pH condition. M. smegmatis expressing Rv2770c is more virulent as testified by the increased death of macrophages and the increased expression of interlukin-6 (IL-6) and interlukin-12p40 (IL-12p40). Moreover, Rv2770c altered the secretion of IL-6 and IL-12p40 of macrophages via NF-κB, ERK1/2 and p38 MAPK axis. Taken together, this study implicated that Rv2770c was a virulent factor actively engaged in the interaction with host macrophage.

PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence.

The role of the unique proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins in the pathophysiology and virulence of Mycobacterium tuberculosis is not clearly understood. One of the PE family proteins, PE11 (LipX or Rv1169c), specific to pathogenic mycobacteria is found to be over-expressed during infection of macrophages and in active TB patients. In this study, we report that M. smegmatis expressing PE11 (Msmeg-PE11) exhibited altered colony morphology and cell wall lipid composition leading to a marked increase in resistance against various environmental stressors and antibiotics. The cell envelope of Msmeg-PE11 also had greater amount of glycolipids and polar lipids. Msmeg-PE11 was found to have better survival rate in infected macrophages. Mice infected with Msmeg-PE11 had higher bacterial load, showed exacerbated organ pathology and mortality. The liver and lung of Msmeg-PE11-infected mice also had higher levels of IL-10, IL-4 and TNF-α cytokines, indicating a potential role of this protein in mycobacterial virulence.

Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence

Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.

PPE57 induces activation of macrophages and drives Th1-type immune responses through TLR2

Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are related proteins exclusive to Mycobacteria that play diverse roles in modulating critical innate immune pathways. In this study, we observed that the PPE57 protein is associated with the cell wall and is exposed on the cell surface. PPE57 enhances Mycobacterium spp. entering into macrophages and plays a role in macrophage phagocytosis. To explore the underlying mechanism, we demonstrated that PPE57 is able to recognise Toll-like receptor 2 (TLR2) and further induce macrophage activation by augmenting the expression of several cell surface molecules (CD40, CD80, CD86 and MHC class II) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-12p40) within macrophages. These molecules are involved in the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signalling pathways. We demonstrated that PPE57 effectively polarises T cells to secrete interferon (IFN)-γ and IL-2 and to up-regulate CXCR3 expression in vivo and in vitro, suggesting that this protein may contribute to Th1 polarisation during the immune response. Moreover, recombinant Bacillus Calmette-Guérin (BCG) over-expressing PPE57 could provide better protective efficacy against Mycobacterium tuberculosis challenge compared with BCG. Taken together, our data provides several pieces of evidence that PPE57 may regulate innate and adaptive immunity by interacting with TLR2. These findings indicate that PPE57 protein is a potential antigen for the rational design of an efficient vaccine against M. tuberculosis. PPE57 is located on the cell surface and enhances mycobacterium entry into macrophage. PPE57 interacts directly with TLR2 on macrophages. PPE57 plays a key role in the activation of macrophages in a TLR2-dependent manner. PPE57 induces a Th1 immune response via TLR2-mediated macrophage functions. Recombinant BCG over-expressing PPE57 could improve protective efficacy against M. tuberculosis.

An overview to understand the role of PE_PGRS family proteins in Mycobacterium tuberculosis H37 Rv and their potential as new drug targets.

Tuberculosis has long been the scourge of humanity, claiming millions of lives. The family of PE_PGRS gene has been attributed to the Mycobacterium tuberculosis pathogenesis over the past few decades. The gene of PE_PGRS family proteins are most often clustered in a region of the genome often as overlapping genes and role in cell surface markers, adhesion and invasion of defense cells of the host (macrophage and dendritic cells). The proline-glutamic acid (PE) domain is responsible for the cellular localization of these proteins on bacterial cells. This gene family shows immense genetic variability in terms of multiple insertion-deletions and single-nucleotide polymorphisms as seen in PE_PGRS9, PE_PGRS17, PE_PGRS18, and PE_PGRS33. In spite of variability, there are indications of shared epitopes in these proteins. Few of these gene sequences that have been studied from evolutionary perspective show indication of positive selection and also landmarks of recent evolutionary events. Many of these proteins show calcium-binding motifs and consequently seen to be responsible in inhibition of phagolysosome formation via a calmodulin-kinase-dependent pathway. A number of PE_PGRS genes were tested for its expression with different growth conditions in vitro and in vivo, among which the contrast in expressivity was seen vividly in PE_PGRS16 (upregulated) and PE_PGRS26 (downregulated) in bacteria persisting in macrophages. Similarly, PE_PGRS33 has been indicated in macrophagial necrosis by a tumor necrosis factor-α-induced pathway. These PE_PGRS family genes may be an interesting subject for research and development. Their fibronectin-binding and calcium-binding property may be strongly implicated in immunopathogenesis of virulent M. tuberculosis strain. In this review, an attempt has been made to evaluate and present data for better understanding of in vivo pathogen functions, for understanding the physiological significance of PE_PGRS gene family, and their potential as new drug targets.

Putative roles of a proline–glutamic acid-rich protein (PE3) in intracellular survival and as a candidate for subunit vaccine against Mycobacterium tuberculosis

The proline-glutamic acid (PE) protein family of Mycobacterium tuberculosis (Mtb) plays diverse roles in the pathogenesis and modulation of host immune responses. The uniqueness of conserved regions of PE proteins may be useful to test and validate their corresponding functions. Hence, the present study has been undertaken to demonstrate the role of PE3 (Rv0159c) for persistence, host immune response and immunoprophylaxis. We have expressed Mtb-specific PE3 gene in M. smegmatis (MS) and used the strain to infect J774A.1 macrophage cells and BALB/c mice. It was observed that during the infection, the MS expressing PE3 showed higher bacterial load when compared to infection with wild-type MS. In hypoxic condition, the expression level of PE3 gene was induced in Mtb, which further showed its relevance in the cell survival during hypoxia-induced persistence. The expression level of PE3 in Mtb was markedly induced during chronic stage of murine infection, which reiterated its importance in mycobacterial persistence in the host. The immunization of mice with recombinant PE3 protein stimulated the secretion of TNF, IL-6 and IL-2 cytokines and generated strong protective immunity against challenge with live mycobacteria, which was evidenced by decreased viable bacilli in the lungs, histopathological changes and increased survival of PE3 immunized mice. Conclusively, the results indicated that PE3 plays significant roles in mycobacterial persistence during infection, modulate host immune response and hence could be a prospective candidate for the development of subunit vaccine against tuberculosis.

Abstract 5190: The proto-oncogene Proline Glutamic acid Leucine rich Protein1 (PELP1) is a novel mediator of glioblastoma development.

Abstract Glioblastomas (GBM) are the most common and aggressive tumors of the central nervous system and still remains as one of the deadliest human cancers. Despite tremendous improvement in the standard therapies, the survival of glioblastoma patients is approximately 12-14 months. The development of novel molecular targets and effective therapeutics are urgently needed to curb the progression of GBM. Better understanding of the molecular pathways and mechanisms that contributes to the GBM development is clinically significant and will provide novel therapeutic targets. In the present study, we examined the significance of proto-oncogene PELP1 that functions as a coregulator of many nuclear transcriptional factors. PELP1 expression is common deregulated in hormonal cancers, however, less is known about PELP1 signaling and its significance in glioma progression. Utilizing immunohistochemical analysis of glioma tissue microarrays that contain different grades of astrocytomas, oligodendrogliomas, ependymomas and normal brain tissues, we found that PELP1 is overexpressed in the glial tumors compared to normal brain, with highest expression in GBM, and PELP1 expression levels were positively correlated with histological malignancy. PELP1 expression was also found to be elevated in various established and patient derived GBM cells when compared to normal astrocytes. To establish the functional significance of PELP1 in GBM, PELP1 expression was silenced using PELP1 specific siRNA and shRNA. We observed that silencing of PELP1 has a dramatic effect on cell proliferation, cell migration and colony formation of U87, T98G and LN229 GBM cells. Mechanistic studies showed that PELP1 interacts with β-catenin and silencing of PELP1 resulted in decrease in β-catenin reporter gene activation. Accordingly, PELP1 knockdown dramatically reduced the expression of β-catenin target genes such as Axin2, cyclin D1, c-Myc and MMP-2. Further, we demonstrated that silencing of PELP1 inhibited the accumulation and nuclear translocation of β-catenin. Chromatin-immunoprecipitation assay showed that silencing of PELP1 compromised the recruitment of β-catenin to the target genes promoter. In xenograft based tumorigenecity assays we found that silencing PELP1 dramatically reduced the in vivo tumor growth of U87 GBM cells. Taken together these results provide the first evidence that PELP1 could be a potential therapeutic target for therapeutic intervention in GBM. Citation Format: Gangadhara R. Sareddy, Pratibha Nutalapati, Rajeshwar R. Tekmal, Ratna K. Vadlamudi. The proto-oncogene Proline Glutamic acid Leucine rich Protein1 (PELP1) is a novel mediator of glioblastoma development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5190. doi:10.1158/1538-7445.AM2013-5190 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.