Phagolysosome Maturation Research Articles

Treatment of Mycobacterium tuberculosis infected macrophages with Rifabutin loaded β-glucan microparticles induces macroautophagy mediated bacillary killing

Tuberculosis (TB), attributable to Mycobacterium tuberculosis (M.tb.), constitutes a formidable global health challenge, particularly with the proliferation of multidrug-resistant (MDR-TB) strains. The efficacious clearance of M.tb. from host cells is imperative for mitigating infection and averting disease progression. Autophagy, an intricate cellular mechanism for degrading and recycling biomolecules, plays a pivotal role in the immune response to M.tb. by facilitating the degradation of intracellular pathogen through the formation of autophagosomes and their subsequent fusion with lysosomes. The present study elucidates the therapeutic efficacy of Rifabutin loaded YDGP (DYDGP) microparticles within M.tb.infected macrophage. Our results show that the administration of DYDGP improve the membrane integrity of macrophage infected with H37Rv as well as MDR strains, as compared to that of untreated controls at 30 min, 6 h and 24 h post-exposure time points. DCFHDA staining elucidated that DYDGP treatment significantly enhances intracellular reactive oxygen species (ROS) production compared to blank YDGP, even in the presence of NOX-2 inhibitors. Furthermore, DYDGP promotes the biogenesis of acidic vesicular organelles and phago-lysosomal maturation, as corroborated by acridine orange and Lysotracker Red staining. Immunofluorescence and dansylcadaverine dual staining data evidenced that DYDGP treatment enhances autophagosome formation, autophagy induction and LC3 puncta formation within M.tb. infected macrophage at both 30 min and 24 h post-exposure time points. Further, protein expression analyses demonstrated that DYDGP treatment enhances the expression levels of NOX-2 and LC3, thereby confirming autophagy induction within M.tb. infected macrophage. Antimycobacterial efficacy assessments revealed that DYDGP treatment engendered significant reductions in colony-forming units (CFUs) of H37Rv (64, 40, 19), MDR32420 (44, 35, 18), MDR32422 (44, 39, 21), and MDR32521 (38, 22, 18) after 30 min, 24 h, and 48 h, exposure respectively. These findings accentuate DYDGP's potential to substantially attenuate M.tb. burden, including the addressal of MDR strains, thereby positioning it as a promising adjunctive therapy for augmenting TB treatment.

GATA2 downregulation contributes to pro-inflammatory phenotype and defective phagocytosis of pulmonary macrophages in chronic obstructive pulmonary disease.

Pulmonary macrophages from COPD patients are characterized by lower phagocytic and bactericidal activity whereas there is hypersecretion of pro-inflammatory cytokines. The prominent decline of GATA2 expression in pulmonary macrophages from COPD patients inspired us to figure out its role during COPD development. The expression levels of GATA2 were decreased in alveolar macrophages isolated from cigarette smoke (CS)-induced COPD mice and cigarette smoke extract (CSE)-treated macrophages. In vitro, both CSE and GATA2 knockdown via siRNAs elevated pro-inflammatory cytokines expression whereas inhibiting phagocytosis in macrophages. Integrated analysis of transcriptomics of GATA2-knockdown macrophages and the results of ChIP sequencing of GATA2 together with dual-luciferase reporter assay identified Abca1 and Pacsin1 as functional target genes of GATA2. Mechanistically, ABCA1 mediates the pro-inflammatory secretion phenotype and the dysfunction in early stage of phagocytosis of macrophages through TLR4/MyD88 and MEGF10/GULP1 pathways, respectively. PACSIN1/SUNJ1 partially mediates the disruption effects of GATA2 downregulation on maturation of phagolysosomes in macrophages. Together, our study suggests that GATA2 influences multiple functions of pulmonary macrophages by simultaneous transcriptional regulation of several target genes, contributing to the dysfunctions of pulmonary macrophages in response to CS, which provides an impetus for further investigations of GATA2 or other underappreciated transcription factors as regulatory hubs in COPD pathogenesis.

Mycobacterium tuberculosis hijacks host macrophages-derived interleukin 16 to block phagolysosome maturation for enhancing intracellular growth

ABSTRACT The discovery of promising cytokines and clarification of their immunological mechanisms in controlling the intracellular fate of Mycobacterium tuberculosis (Mtb) are necessary to identify effective diagnostic biomarkers and therapeutic targets. To escape immune clearance, Mtb can manipulate and inhibit the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. In this study, we found that interleukin 16 (IL-16) is elevated in the serum samples of Tuberculosis (TB) patients and can serve as a specific target for treatment TB. There was a significant difference in IL-16 levels among active TB, latent TB infection (LTBI), and non-TB patients. This study first revealed that macrophages are the major source of IL-16 production in response to Mtb infection, and elucidated that IL-16 can promote Mtb intracellular survival by inhibiting phagosome maturation and suppressing the expression of Rev-erbα which can inhibit IL-10 secretion. The experiments using zebrafish larvae infected with M. marinum and mice challenged with H37Rv demonstrated that reducing IL-16 levels resulted in less severe pathology and improved survival, respectively. In conclusion, this study provided direct evidence that Mtb hijacks the host macrophages-derived interleukin 16 to enhance intracellular growth. It is suggesting the immunosuppressive role of IL-16 during Mtb infection, supporting IL-16 as a promising therapeutic target.

TRPV4 Regulates the Macrophage Metabolic Response to Limit Sepsis-induced Lung Injury.

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness-dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for LPS-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by BAL fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. It is interesting that BALF lactate is increased in patients with sepsis compared with healthy control participants, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Phagocytosis.

One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process, named phagocytosis, for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this, he gave us the basis for our modern understanding of inflammation and the innate immune response. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In this chapter, we present a general view of our current knowledge on phagocytosis performed mainly by professional phagocytes through antibody and complement receptors and discuss aspects that remain incompletely understood.

Intracellular iron accumulation facilitates mycobacterial infection in old mouse macrophages

Aging has a significant impact on the immune system, leading to a gradual decline in immune function and changes in the body’s ability to respond to bacterial infections. Non-tuberculous mycobacteria (NTM), also known as atypical mycobacteria or environmental mycobacteria, are commonly found in soil, water, and various environmental sources. While many NTM species are considered opportunistic pathogens, some can cause significant infections, particularly in individuals with compromised immune systems, such as older individuals. When mycobacteria enter the body, macrophages are among the first immune cells to encounter them and attempt to engulf mycobacteria through a process called phagocytosis. Some NTM species, including Mycobacterium avium (M. avium) can survive and replicate within macrophages. However, little is known about the interaction between NTM and macrophages in older individuals. In this study, we investigated the response of bone marrow–derived macrophage (BMMs) isolated from young (5 months) and old (25 months) mice to M. avium serotype 4, one of the main NTM species in patients with pulmonary NTM diseases. Our results demonstrated that BMMs from old mice have an increased level of intracellular iron and are more susceptible to M. avium serotype 4 infection compared to BMMs from young mice. The whole-cell proteomic analysis indicated a dysregulated expression of iron homeostasis–associated proteins in old BMMs regardless of mycobacterial infection. Deferoxamine, an iron chelator, significantly rescued mycobacterial killing and phagolysosome maturation in BMMs from old mice. Therefore, our data for the first time indicate that an intracellular iron accumulation improves NTM survival within macrophages from old mice and suggest a potential application of iron-chelating drugs as a host-directed therapy for pulmonary NTM infection in older individuals.

Metabolic adaptation supports enhanced macrophage efferocytosis in limited-oxygen environments

Apoptotic cell (AC) clearance (efferocytosis) is performed by phagocytes, such as macrophages, that inhabit harsh physiological environments. Here, we find that macrophages display enhanced efferocytosis under prolonged (chronic) physiological hypoxia, characterized by increased internalization and accelerated degradation of ACs. Transcriptional and translational analyses revealed that chronic physiological hypoxia induces two distinct but complimentary states. The first, "primed" state, consists of concomitant transcription and translation of metabolic programs in AC-naive macrophages that persist during efferocytosis. The second, "poised" state, consists of transcription, but not translation, of phagocyte function programs in AC-naive macrophages that are translated during efferocytosis. Mechanistically, macrophages efficiently flux glucose into a noncanonical pentose phosphate pathway (PPP) loop to enhance NADPH production. PPP-derived NADPH directly supports enhanced efferocytosis under physiological hypoxia by ensuring phagolysosomal maturation and redox homeostasis. Thus, macrophages residing under physiological hypoxia adopt states that support cell fitness and ensure performance of essential homeostatic functions rapidly and safely.

Targeting of phagolysosomes containing conidia of the fungus Aspergillus fumigatus with polymeric particles

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens.Key points• Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes.• These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages.• Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.

Postinfluenza Environment Reduces Aspergillus fumigatus Conidium Clearance and Facilitates Invasive Aspergillosis In Vivo.

Aspergillus fumigatus is a human fungal pathogen that is most often avirulent in immunecompetent individuals because the innate immune system is efficient at eliminating fungal conidia. However, recent clinical observations have shown that severe influenza A virus (IAV) infection can lead to secondary A. fumigatus infections with high mortality. Little is currently known about how IAV infection alters the innate antifungal immune response. Here, we established a murine model of IAV-induced A. fumigatus (IAV-Af) superinfection by inoculating mice with IAV followed 6 days later by A. fumigatus conidia challenge. We observed increased mortality in the IAV-Af-superinfected mice compared to mice challenged with either IAV or A. fumigatus alone. A. fumigatus conidia were able to germinate and establish a biofilm in the lungs of the IAV-Af superinfection group, which was not seen following fungal challenge alone. While we did not observe any differences in inflammatory cell recruitment in the IAV-Af superinfection group compared to single-infection controls, we observed defects in Aspergillus conidial uptake and killing by both neutrophils and monocytes after IAV infection. pHrodo Green zymosan bioparticle (pHrodo-zymosan) and CM-H2DCFDA [5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate] staining, indicators of phagolysosome maturation and reactive oxygen species (ROS) production, respectively, revealed that the fungal killing defect was due in part to reduced phagolysosome maturation. Collectively, our data demonstrate that the ability of neutrophils and monocytes to kill and clear Aspergillus conidia is strongly reduced in the pulmonary environment of an IAV-infected lung, which leads to invasive pulmonary aspergillosis and increased overall mortality in our mouse model, recapitulating what is observed clinically in humans. IMPORTANCE Influenza A virus (IAV) is a common respiratory virus that causes seasonal illness in humans, but can cause pandemics and severe infection in certain patients. Since the emergence of the 2009 H1N1 pandemic strains, there has been an increase in clinical reports of IAV-infected patients in the intensive care unit (ICU) developing secondary pulmonary aspergillosis. These cases of flu-Aspergillus superinfections are associated with worse clinical outcomes than secondary bacterial infections in the setting of IAV. To date, we have a limited understanding of the cause(s) of secondary fungal infections in immunocompetent hosts. IAV-induced modulation of cytokine production and innate immune cellular function generates a unique immune environment in the lung, which could make the host vulnerable to a secondary fungal infection. Our work shows that defects in phagolysosome maturation in neutrophils and monocytes after IAV infection impair the ability of these cells to kill A. fumigatus, thus leading to increased fungal germination and growth and subsequent invasive aspergillosis. Our work lays a foundation for future mechanistic studies examining the exact immune modulatory events occurring in the respiratory tract after viral infection leading to secondary fungal infections.

The nature of the fungal cargo induces significantly different temporal programmes of macrophage phagocytosis

Phagocytosis is an essential component of our immune defence against fungal pathogens. Differences in the dynamics of phagocyte migration, recognition, uptake and phagolysosome maturation are dependent on the characteristics of the fungal cargo, and in particular to differences in cell wall composition and cellular morphology. However, studies that have focused on phagocyte interactions with individual fungal species have not enabled comparisons in the kinetics of these interactions to be made between these different species. We therefore used live cell video microscopy to examine the temporal dynamics of phagocytosis for a range of fungal cargoes by thioglycollate–elicited peritoneal macrophages from C57BL/6 mice. Uniform populations of macrophages were challenged at the same time with yeast cells of Candida albicans, Candida glabrata, Saccharomyces cerevisiae and Cryptococcus neoformans (wild–type and an acapsular mutant, cap59Δ), and spores of Aspergillus fumigatus and Mucor circinelloides to enable standardized comparative interactions to be quantified from different stages of phagocytosis. Differences in the rate of uptake of fungal cells varied by up to 26–fold, whilst differences in time to induce phagosome acidification varied by as much as 29–fold. Heat-killing or opsonizing the fungal targets markedly affected the kinetics of the interaction in a species–specific manner. Fungal and macrophage killing assays further revealed cargo–specific differences in phagocytosis and diversity in fungal evasion mechanisms. Therefore, simultaneous assessment of the interaction of macrophages with different fungal pathogens highlighted major differences in the kinetics and growth responses during fungus–phagocyte interactions that are likely to impact on pathogenesis and virulence.

Host P2X7R-p38MAPK axis mediated intra-macrophage leishmanicidal activity of Spergulin-A

Current drugs are inefficient for the treatment of visceral leishmaniasis an immunosuppressive ailment caused by Leishmania donovani. Regrettably, there is no plant-origin antileishmanial drug present. P2X7R is constitutively present on macrophage surfaces and can be a putative therapeutic target in intra-macrophage pathogens with function attributes towards inflammation, host cell apoptosis, altered redox, and phagolysosomal maturation by activating p38MAPK. Here we demonstrated that the initial interaction of Spergulin-A (Sp A), a triterpenoid saponin with RAW 264.7 macrophages was mediated through P2X7R involving the signaling cascade intermediates Ca++, p38MAPK, and NF-κβ. Phospho (P)-p38MAPK involvement is shown to have specific and firm importance in leishmanial killing with increased NF-κβp65. Phago-lysosomal maturation by Sp A also campaigns for another contribution of P2X7R. In vivo evaluation of the anti-leishmanial activity of Sp A was monitored through expression analyses of P2X7R, P-p38MAPK, and NF-κβp65 in murine spleen and bone-marrow macrophages and supported Sp A being a natural compound of leishmanicidal functions which acted through the P2X7R-p38MAPK axis.

Mycobacterium tuberculosis Methyltransferase Rv1515c Can Suppress Host Defense Mechanisms by Modulating Immune Functions Utilizing a Multipronged Mechanism.

Mycobacterium tuberculosis (M. tb) gene Rv1515c encodes a conserved hypothetical protein exclusively present within organisms of MTB complex and absent in non-pathogenic mycobacteria. In silico analysis revealed that Rv1515c contain S-adenosylmethionine binding site and methyltransferase domain. The DNA binding and DNA methyltransferase activity of Rv1515c was confirmed in vitro. Knock-in of Rv1515c in a model mycobacteria M. smegmatis (M. s_Rv1515c) resulted in remarkable physiological and morphological changes and conferred the recombinant strain with an ability to adapt to various stress conditions, including resistance to TB drugs. M. s_Rv1515c was phagocytosed at a greater rate and displayed extended intra-macrophage survival in vitro. Recombinant M. s_Rv1515c contributed to enhanced virulence by suppressing the host defense mechanisms including RNS and ROS production, and apoptotic clearance. M. s_Rv1515c, while suppressing the phagolysosomal maturation, modulated pro-inflammatory cytokine production and also inhibited antigen presentation by downregulating the expression of MHC-I/MHC-II and co-stimulatory signals CD80 and CD86. Mice infected with M. s_Rv1515c produced more Treg cells than vector control (M. s_Vc) and exhibited reduced effector T cell responses, along-with reduced expression of macrophage activation markers in the chronic phase of infection. M. s_Rv1515c was able to survive in the major organs of mice up to 7 weeks post-infection. These results indicate a crucial role of Rv1515c in M. tb pathogenesis.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

Physicochemical and structural characterization, epitope mapping and vaccine potential investigation of a new protein containing Tetratrico Peptide Repeats of Acinetobacter baumannii: An in-silico and in-vivo approach

Acinetobacter baumannii is an opportunistic multidrug-resistant pathogen that causes a significant mortality rate. The proteins containing Tetratrico Peptide Repeats (TPRs) are involved in the pathogenicity and virulence of bacteria and have different roles such as transfer of bacterial virulence factors to host cells, binding to the host cells and inhibition of phagolysosomal maturation. So, in this study, physicochemical properties of a new protein containing TPRs in A. baumannii which was named PcTPRs1 by this study were characterized and its 3D structure was predicted by in-silico tools. The protein B and T cell epitopes were mapped and its vaccine potential was in-silico and in-vivo investigated. Domain analysis indicated that the protein contains the Flp pilus assembly protein TadD domain which has three TPRs. The helix is dominant in the protein structure, and this protein is an outer membrane antigen which, is extremely conserved among A. baumannii strains; thus, has good properties to be applied as a recombinant vaccine. The best-predicted and refined model was applied in ligand-binding sites and conformational epitopes prediction. Based on epitope mapping results, several epitopes were characterized which could stimulate both immune systems. BLAST results showed the introduced epitopes are completely conserved among A. baumannii strains. The in-vivo analysis indicates that a 101 amino acid fragment of the protein which contains the best selected epitope, can produce a good protectivity against A. baumannii as well as the whole TPR protein and thus could be investigated as an effective subunit and potential vaccines.

Flunarizine suppresses Mycobacterium tuberculosis growth via calmodulin-dependent phagosome maturation.

Tuberculosis (TB), an infectious bacterial disease caused by Mycobacterium tuberculosis (Mtb), is a major cause of death worldwide. Multidrug-resistant TB remains a public health crisis and thus novel effective treatments, such as host-directed therapies (HDTs), are urgently required to overcome the challenges of TB infection. In this study, we evaluated 4 calcium modulators for their effects on Mtb growth in macrophages. Only flunarizine enhanced the bactericidal ability of macrophages against Mtb, which was induced by an increase in phosphorylated calcium/calmodulin (CaM)-dependent protein kinase II (pCaMKII) levels. We further discovered that the expression of CaM was decreased in Mtb-infected macrophages and restored following flunarizine treatment; this was associated with phagolysosome maturation and acidification. Consistent with these findings, the anti-TB ability of macrophages was reduced following the silencing of CaM or inhibition of CAMKII activity. In conclusion, our results demonstrated that flunarizine enhanced the bactericidal ability of macrophages and clarified its CaM-pCAMKII-dependent mechanism. Therefore, our findings strongly support further studies of this currently approved drug as an HDT candidate for TB therapy.

Integrative Analysis of Human Macrophage Inflammatory Response Related to Mycobacterium tuberculosis Virulence.

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, kills 1.5 to 1.7 million people every year. Macrophages are Mtb’s main host cells and their inflammatory response is an essential component of the host defense against Mtb. However, Mtb is able to circumvent the macrophages’ defenses by triggering an inappropriate inflammatory response. The ability of Mtb to hinder phagolysosome maturation and acidification, and to escape the phagosome into the cytosol, is closely linked to its virulence. The modulation of the host inflammatory response relies on Mtb virulence factors, but remains poorly studied. Understanding macrophage interactions with Mtb is crucial to develop strategies to control tuberculosis. The present study aims to determine the inflammatory response transcriptome and miRNome of human macrophages infected with the virulent H37Rv Mtb strain, to identify macrophage genetic networks specifically modulated by Mtb virulence. Using human macrophages infected with two different live strains of mycobacteria (live or heat-inactivated Mtb H37Rv and M. marinum), we quantified and analyzed 184 inflammatory mRNAs and 765 micro(mi)RNAs. Transcripts and miRNAs differently modulated by H37Rv in comparison with the two other conditions were analyzed using in silico approaches. We identified 30 host inflammatory response genes and 37 miRNAs specific for H37Rv virulence, and highlight evidence suggesting that Mtb intracellular-linked virulence depends on the inhibition of IL-1β-dependent pro-inflammatory response, the repression of apoptosis and the delay of the recruitment and activation of adaptive immune cells. Our findings provide new potential targets for the development of macrophage-based therapeutic strategies against TB.

Leishmania (V.) braziliensis infection promotes macrophage autophagy by a LC3B-dependent and BECLIN1-independent mechanism

Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.

Crystal Structure and Mechanistic Molecular Modeling Studies of Mycobacterium tuberculosis Diterpene Cyclase Rv3377c.

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.

FasR Regulates Fatty Acid Biosynthesis and Is Essential for Virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world’s leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

Chromosomal segments may explain the antibody response cooperation for canine leishmaniasis pathogenesis

Visceral leishmaniasis (VL) is marked by hyperactivation of a humoral response secreting high quantity of immunoglobulins (Igs) that are inaccessible to intracellular parasites. Here we investigated the contributions of the antibody response to the canine leishmaniasis pathogenesis. Using correlation and genome-wide association analysis, we investigated the relationship of anti-Leishmania infantum immunoglobulin classes levels with parasite burden, clinical response, renal/hepatic biochemical, and oxidative stress markers in dogs from endemic areas of VL. Immunoglobulin G (IgG) and IgA were positively correlated with parasite burden on lymph node and blood. Increased IgG, IgA and IgE levels were associated with severe canine leishmaniasis (CanL) whereas IgM was elevated in uninfected exposed dogs. Correlations of IgM, IgG and IgA with creatinine, urea, AST and ALT levels in the serum were suggested an involvement of those Igs with renal and hepatic changes. The correlogram of oxidative radicals and antioxidants revealed a likely relationship of IgM, IgG and IgA with oxidative stress and lipid peroxidation in the blood, suggested as mechanisms mediating tissue damage and CanL worsening. The gene mapping on chromosomal segments associated with the quantitative variation of immunoglobulin classes identified genetic signatures involved with reactive oxygen species generation, phagolysosome maturation and rupture, free iron availability, Th1/Th2 differenciation and, immunoglobulin clearance. The findings demonstrated the roles of the antibody response as resistance or susceptibility markers and mediators of CanL pathogenesis. In addition we pinpointed candidate genes as potential targets for the therapy against the damage caused by exacerbated antibody response and parasitism in VL.