Gene expression patterns associated with multidrug therapy in multibacillary leprosy.

Multidrug therapy (MDT) has been successfully used in the treatment of leprosy. However, although patients are cured after the completion of MDT, leprosy reactions, permanent disability, and occasional relapse/reinfection are frequently observed in patients. The immune system of multibacillary patients (MB) is not able to mount an effective cellular immune response against M. leprae. Consequently, clearance of bacilli from the body is a slow process and after 12 doses of MDT not all MB patients reduce bacillary index (BI). In this context, we recruited MB patients at the uptake and after 12-month of MDT. Patients were stratified according to the level of reduction of the BI after 12 doses MDT. A reduction of at least one log in BI was necessary to be considered a responder patient. We evaluated the pattern of host gene expression in skin samples with RNA sequencing before and after MDT and between samples from patients with or without one log reduction in BI. Our results demonstrated that after 12 doses of MDT there was a reduction in genes associated with lipid metabolism, inflammatory response, and cellular immune response among responders (APOBEC3A, LGALS17A, CXCL13, CXCL9, CALHM6, and IFNG). Also, by comparing MB patients with lower BI reduction versus responder patients, we identified high expression of CDH19, TMPRSS4, PAX3, FA2H, HLA-V, FABP7, and SERPINA11 before MDT. From the most differentially expressed genes, we observed that MDT modulates pathways related to immune response and lipid metabolism in skin cells from MB patients after MDT, with higher expression of genes like CYP11A1, that are associated with cholesterol metabolism in the group with the worst response to treatment. Altogether, the data presented contribute to elucidate gene signatures and identify differentially expressed genes associated with MDT outcomes in MB patients.

The C-terminal extension of VgrG4 from Klebsiella pneumoniae remodels host cell microfilaments.

Klebsiella pneumoniae is an opportunistic pathogen, which concerns public health systems worldwide, as multiple antibiotic-resistant strains are frequent. One of its pathogenicity factors is the Type VI Secretion System (T6SS), a macromolecular complex assembled through the bacterial membranes. T6SS injects effector proteins inside target cells. Such effectors confer competitive advantages or modulate the target cell signaling and metabolism to favor bacterial infection. The VgrG protein is a T6SS core component. It may present a variable C-terminal domain carrying an additional effector function. Kp52.145 genome encodes three VgrG proteins, one of them with a C-terminal extension (VgrG4-CTD). VgrG4-CTD is 138 amino acids long, does not contain domains of known function, but is conserved in some Klebsiella, and non-Klebsiella species. To get insights into its function, recombinant VgrG4-CTD was used in pulldown experiments to capture ligands from macrophages and lung epithelial cells. A total of 254 proteins were identified: most of them are ribosomal proteins. Cytoskeleton-associated and proteins involved in the phagosome maturation pathway were also identified. We further showed that VgrG4-CTD binds actin and induces actin remodeling in macrophages. This study presents novel clues on the role of K. pneumoniae T6SS in pathogenesis.

Corrigendum: Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

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TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells.

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human ß-defensin-2 (hßD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.

Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis.

Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3ß-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3ß-HSD activity with the 17ß-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicity and point to 3ß-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.

New insights into the pathogenesis of leprosy: contribution of subversion of host cell metabolism to bacterial persistence, disease progression, and transmission.

Chronic infection by the obligate intracellular pathogen Mycobacterium leprae may lead to the development of leprosy. Of note, in the lepromatous clinical form of the disease, failure of the immune system to constrain infection allows the pathogen to reproduce to very high numbers with minimal clinical signs, favoring transmission. The bacillus can modulate cellular metabolism to support its survival, and these changes directly influence immune responses, leading to host tolerance, permanent disease, and dissemination. Among the metabolic changes, upregulation of cholesterol, phospholipids, and fatty acid biosynthesis is particularly important, as it leads to lipid accumulation in the host cells (macrophages and Schwann cells) in the form of lipid droplets, which are sites of polyunsaturated fatty acid-derived lipid mediator biosynthesis that modulate the inflammatory and immune responses. In Schwann cells, energy metabolism is also subverted to support a lipogenic environment. Furthermore, effects on tryptophan and iron metabolisms favor pathogen survival with moderate tissue damage. This review discusses the implications of metabolic changes on the course of M. leprae infection and host immune response and emphasizes the induction of regulatory T cells, which may play a pivotal role in immune modulation in leprosy.

Exploring the Potential Role of Moonlighting Function of the Surface-Associated Proteins From Mycobacterium bovis BCG Moreau and Pasteur by Comparative Proteomic.

Surface-associated proteins from Mycobacterium bovis BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components.

PGL I expression in live bacteria allows activation of a CD206/PPARγ cross-talk that may contribute to successful Mycobacterium leprae colonization of peripheral nerves.

Mycobacterium leprae, an obligate intracellular bacillus, infects Schwann cells (SCs), leading to peripheral nerve damage, the most severe leprosy symptom. In the present study, we revisited the involvement of phenolic glycolipid I (PGL I), an abundant, private, surface M. leprae molecule, in M. leprae-SC interaction by using a recombinant strain of M. bovis BCG engineered to express this glycolipid. We demonstrate that PGL I is essential for bacterial adhesion and SC internalization. We also show that live mycobacterium-producing PGL I induces the expression of the endocytic mannose receptor (MR/CD206) in infected cells in a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent manner. Of note, blocking mannose recognition decreased bacterial entry and survival, pointing to a role for this alternative recognition pathway in bacterial pathogenesis in the nerve. Moreover, an active crosstalk between CD206 and the nuclear receptor PPARγ was detected that led to the induction of lipid droplets (LDs) formation and prostaglandin E2 (PGE2), previously described as fundamental players in bacterial pathogenesis. Finally, this pathway was shown to induce IL-8 secretion. Altogether, our study provides evidence that the entry of live M. leprae through PGL I recognition modulates the SC phenotype, favoring intracellular bacterial persistence with the concomitant secretion of inflammatory mediators that may ultimately be involved in neuroinflammation.

The Essential Role of Cholesterol Metabolism in the Intracellular Survival of Mycobacterium leprae Is Not Coupled to Central Carbon Metabolism and Energy Production.

UNLABELLED: Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. IMPORTANCE: Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies.

Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment.

We recently showed that Mycobacterium leprae (ML) is able to induce lipid droplet formation in infected macrophages. We herein confirm that cholesterol (Cho) is one of the host lipid molecules that accumulate in ML-infected macrophages and investigate the effects of ML on cellular Cho metabolism responsible for its accumulation. The expression levels of LDL receptors (LDL-R, CD36, SRA-1, SR-B1, and LRP-1) and enzymes involved in Cho biosynthesis were investigated by qRT-PCR and/or Western blot and shown to be higher in lepromatous leprosy (LL) tissues when compared to borderline tuberculoid (BT) lesions. Moreover, higher levels of the active form of the sterol regulatory element-binding protein (SREBP) transcriptional factors, key regulators of the biosynthesis and uptake of cellular Cho, were found in LL skin biopsies. Functional in vitro assays confirmed the higher capacity of ML-infected macrophages to synthesize Cho and sequester exogenous LDL-Cho. Notably, Cho colocalized to ML-containing phagosomes, and Cho metabolism impairment, through either de novo synthesis inhibition by statins or depletion of exogenous Cho, decreased intracellular bacterial survival. These findings highlight the importance of metabolic integration between the host and bacteria to leprosy pathophysiology, opening new avenues for novel therapeutic strategies to leprosy.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

P-glycoprotein efflux pump plays an important role in Trypanosoma cruzi drug resistance.

Drug resistance in protozoan parasites has been associated with the P-glycoprotein (Pgp), an energy-dependent efflux pump that transports substances across the membrane. Interestingly, the genes TcPGP1 and TcPGP2 have been described in Trypanosoma cruzi, although the function of these genes has not been fully elucidated. The main goal of this work was to investigate Pgp efflux pump activity and expression in T. cruzi lines submitted to in vitro induced resistance to the compounds 4-N-(2-methoxy styryl)-thiosemicarbazone (2-Meotio) and benznidazole (Bz) and to verify the stability of the resistant phenotypes during the parasite life cycle. We observed that the EC50 values for the treatment of epimastigotes with 2-Meotio or Bz were increased at least 4.7-fold in resistant lines, and this phenotype was maintained in metacyclic trypomastigotes, cell-derived trypomastigotes, and intracellular amastigotes. However, in epimastigotes, 2-Meotio resistance is reversible, but Bz resistance is irreversible. When compared with the parental line, the resistant lines exhibited higher Pgp efflux activity, reversion of the resistant phenotypes in the presence of Pgp inhibitors, cross-resistance with Pgp modulators, higher basal Pgp ATPase activity, and overexpression of the genes TcPGP1 and TcPGP2. In conclusion, the resistance induced in T. cruzi by the compounds 2-Meotio and Bz is maintained during the entire parasite life cycle. Furthermore, our data suggest the participation of the Pgp efflux pump in T. cruzi drug resistance.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages.

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur.

BACKGROUND: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. RESULTS: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. CONCLUSIONS: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Ectonucleotide diphosphohydrolase activity in Crithidia deanei.

In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.

Ecto-phosphatase activity on the cell surface of Crithidia deanei.

In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 +/- 0.47 nmol Pi/h x 10(8) cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent Km for p-NPP was 5.35 +/- 0.89 mM. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl2 and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.