Glycoconjugate coumarins exploiting metabolism-enhanced fluorescence and preferential uptake: New optical tools for tumor cell staining.

The possibility to visually discriminate cells based on their metabolism and capability to uptake exogenous molecules is an important topic with exciting fallback on translational and precision medicine. To this end, probes that combine several complementary features are necessary. The ideal probe is selectively uptaken and activated in tumor cells compared with control ones and is not fluorescent in the extracellular medium. Fluorogenic compounds that combine enzyme-activated pH sensitivity and good cell uptake can be an ideal solution, provided that the sensed enzymes are dysregulated in tumor cells. Here, we present synthesis and in vitro evaluation of a new class of glyco-coumarin based probes that merge all these features. These probes show uptake ratio in tumor vs. control cells up to 3:1, with a cell to background ratio upon administration of the probe up to 5:1. These features make this new family of fluorogenic targeted probes a promising tool in life science.

Hypomagnesemia after heart transplantation or left ventricular assist device implant for end-stage heart failure.

BACKGROUND: Patients with advanced heart failure undergoing heart transplant (HTx) or left ventricular assist device (LVAD) implant are at high risk of magnesium deficiency, that may favor development of diabetes. We aimed to comparatively assess prevalence and correlates of hypomagnesemia during cardiac rehabilitation between 51 HTx and 46 LVAD recipients. METHODS AND RESULTS: We measured serum magnesium and correlated it to clinical and laboratory findings upon admission (T1 ) and at discharge (T2) from cardiac rehabilitation. Among LVAD, magnesium levels increased from admission to discharge. Among HTx, magnesium concentrations were below normal in 33% and 47% at T1 and T2 , respectively, and decreased from admission to discharge. HTx on tacrolimus showed greater decreases in magnesium and increases in glucose levels than those on cyclosporine. Magnesium levels were inversely associated with >15 mg/dL increased glucose concentrations between T2 and T1 (HR 0.373, 95% CI 0.154-0.903, P = .029) after adjustment for pre-existing diabetes, insulin resistance markers, calcineurin inhibitors (cyclosporine/tacrolimus), prednisone doses, and magnesium supplementation. CONCLUSION: Hypomagnesemia is rare in LVAD recipients, but common within 1 month from HTx, worsens during rehabilitation, despite immunosuppression tapering and magnesium supplements, and is independently associated to increasing glucose levels. Studies evaluating whether correcting hypomagnesemia improves outcome are warranted.

Epitaxial Graphene Sensors Combined with 3D-Printed Microfluidic Chip for Heavy Metals Detection.

In this work, we investigated the sensing performance of epitaxial graphene on Si-face 4H-SiC (EG/SiC) for liquid-phase detection of heavy metals (e.g., Pb and Cd), showing fast and stable response and low detection limit. The sensing platform proposed includes 3D-printed microfluidic devices, which incorporate all features required to connect and execute lab-on-chip (LOC) functions. The obtained results indicate that EG exhibits excellent sensing activity towards Pb and Cd ions. Several concentrations of Pb2+ solutions, ranging from 125 nM to 500 µM, were analyzed showing Langmuir correlation between signal and Pb2+ concentrations, good stability, and reproducibility over time. Upon the simultaneous presence of both metals, sensor response is dominated by Pb2+ rather than Cd2+ ions. To explain the sensing mechanisms and difference in adsorption behavior of Pb2+ and Cd2+ ions on EG in water-based solutions, we performed van-der-Waals (vdW)-corrected density functional theory (DFT) calculations and non-covalent interaction (NCI) analysis, extended charge decomposition analysis (ECDA), and topological analysis. We demonstrated that Pb2+ and Cd2+ ions act as electron-acceptors, enhancing hole conductivity of EG, due to charge transfer from graphene to metal ions, and Pb2+ ions have preferential ability to binding with graphene over cadmium. Electrochemical measurements confirmed the conductometric results, which additionally indicate that EG is more sensitive to lead than to cadmium.

Sulfide Species Optical Monitoring by a Miniaturized Silicon Photomultiplier.

The monitoring of water-soluble pollutants is receiving a growing interest from the scientific community. In this context, sulfide anion species S2- and HS- are particularly relevant since they can cause acute and chronic toxicity including neurological effects and at high concentrations, even death. In this study, a new strategy for fast and sensitive optical detection of sulfide species in water samples is described. The method uses an integrated silicon photomultiplier (SiPM) device coupled with the appropriate analytical strategy applied in a plastic microchip with dried reagents on board. More specifically, all sulfide species (H2S, HS- and S2-) in water samples are detected by the fluorescence signal emitted upon the reaction with N,N-dimethyl-phenylenediamine sulfate in the presence of Fe3+, leading to the formation of the fluorescent methylene blue (MB) species. It has been proven that the system herein proposed is able to measure sulfide concentration in a linear range from 0-10 mg L-1 with a sensitivity value of about 6.7 µA mg-1 L and a detection limit of 0.5 mg L-1. A comparison with conventional UV-Vis detection method has been also carried out. Data show a very good linear correlation (R² = 0.98093), proving the effectiveness of the method. Results pave the way toward the development of portable and low-cost device systems for water-soluble sulfide pollutants.

Evaluation of the antibacterial power and biocompatibility of zinc oxide nanorods decorated graphene nanoplatelets: new perspectives for antibiodeteriorative approaches.

BACKGROUND: Nanotechnologies are currently revolutionizing the world around us, improving the quality of our lives thanks to a multitude of applications in several areas including the environmental preservation, with the biodeterioration phenomenon representing one of the major concerns. RESULTS: In this study, an innovative nanomaterial consisting of graphene nanoplatelets decorated by zinc oxide nanorods (ZNGs) was tested for the ability to inhibit two different pathogens belonging to bacterial genera frequently associated with nosocomial infections as well as biodeterioration phenomenon: the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa. A time- and dose-dependent bactericidal effect in cell viability was highlighted against both bacteria, demonstrating a strong antimicrobial potential of ZNGs. Furthermore, the analysis of bacterial surfaces through Field emission scanning electron microscopy (FESEM) revealed ZNGs mechanical interaction at cell wall level. ZNGs induced in those bacteria deep physical damages not compatible with life as a result of nanoneedle-like action of this nanomaterial together with its nanoblade effect. Cell injuries were confirmed by Fourier transform infrared spectroscopy, revealing that ZNGs antimicrobial effect was related to protein and phospholipid changes as well as a decrease in extracellular polymeric substances; this was also supported by a reduction in biofilm formation of both bacteria. The antibacterial properties of ZNGs applied on building-related materials make them a promising tool for the conservation of indoor/outdoor surfaces. Finally, ZNGs nanotoxicity was assessed in vivo by exploiting the soil free living nematode Caenorhabditis elegans. Notably, no harmful effects of ZNGs on larval development, lifespan, fertility as well as neuromuscular functionality were highlighted in this excellent model for environmental nanotoxicology. CONCLUSIONS: Overall, ZNGs represent a promising candidate for developing biocompatible materials that can be exploitable in antimicrobial applications without releasing toxic compounds, harmful to the environment.

Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics.

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

Superior protection against paratuberculosis by a heterologous prime-boost immunization in a murine model.

Vaccination is the best strategy to control Paratuberculosis (PTB), which is a significant disease in cattle and sheep. Previously we showed the humoral and cellular immune response induced by a novel vaccine candidate against PTB based on the Argentinian Mycobacterium avium subspecies paratuberculosis (Map) 6611 strain. To improve 6611 immunogenicity and efficacy, we evaluated this vaccine candidate in mice with two different adjuvants and a heterologous boost with a recombinant modified vaccinia Ankara virus (MVA) expressing the antigen 85A (MVA85A). We observed that boosting with MVA85A did not improve total IgG or specific isotypes in serum induced by one or two doses of 6611 formulated with incomplete Freund's adjuvant (IFA). However, when 6611 was formulated with ISA201 adjuvant, MVA85A boost enhanced the production of IFNγ, Th1/Th17 cytokines (IL-2, TNF, IL-17A) and IL-6, IL-4 and IL-10. Also, this group showed the highest levels of IgG2b and IgG3 isotypes, both important for better protection against Map infection in the murine model. Finally, the heterologous scheme elicited the highest levels of protection after Map challenge (lowest CFU count and liver lesion score). In conclusion, our results encourage further evaluation of 6611 strain + ISA201 prime and MVA85A boost in bovines.

Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. RESULTS: We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. CONCLUSIONS: The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.

Remdesivir administration for SARS-CoV-2 pneumonia in ICU and non-ICU patients: outcome and posttreatment differences - the Italian Military Hospital experience.

BACKGROUND: Four-hundred forty-nine patients affected by Covid-19 were hospitalized at the Rome Military Hospital between March 2020 and July 2022. Depending on the severity of the disease, they were assigned either to the Functional Health Emergency Unit - if suffering from interstitial pneumonia with a clinical manifestation of dyspnea associated with peripheral oxygen saturation < 92%, and oxygen atmospheric pressure therapy - or to the intensive care unit - if the blood gas-lytic index P/F (ratio between partial pressure of arterial O2 and inspired fraction of O2) was below 150. This prospective observation and monocentric study aim to verify the outcome (healing/death) of early use of remdesivir in pneumonia patients. RESULTS: The results highlight the importance of the adoption of remdesivir in the initial stages of infection to prevent the systemic spread and viral multiplication and, in the subsequent phase, a cytokine storm resulting in acute respiratory failure and multiorgan failure. The use of the drug in the most advanced stages of the disease is not associated with a real impact on patient outcomes. Therefore, there is a statistically significant correspondence between the early use of remdesivir in the treatment of SARS-CoV-2 disease - in addition to guidelines therapies - and a favorable clinical outcome. CONCLUSIONS: This work shows therapeutic efficacy in the first 5 days of intravenous administration of remdesivir, following the loading dose. It is also necessary to underline the different behaviors of the drug when administered late in patients undergoing mechanical ventilation, compared to those who only needed low-flow oxygen therapy, whose share of recovery - decidedly relevant - reaches statistical significance.

Glycolytic and non-glycolytic functions of Mycobacterium tuberculosis fructose-1,6-bisphosphate aldolase, an essential enzyme produced by replicating and non-replicating bacilli.

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

On the molecular structure of human neuroserpin polymers.

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85°C, display the same isosbestic points in the Amide I' band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45°C suggesting that the polymerization rate-limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A ß-sheet of neighboring serpin molecule, although with different extents at 45 and 85°C.

Systemic and Mucosal Humoral Immune Response Induced by Three Doses of the BNT162b2 SARS-CoV-2 mRNA Vaccines.

BNT162b2 (BioNTech/Pfizer) was the first SARS-CoV-2 mRNA vaccine approved by the European Medicines Agency. We monitored the long-term humoral responses of healthcare workers (HCWs) who received three vaccine doses. A total of 59 healthcare workers were studied: 47 were never SARS-CoV-2-infected (naïve-HCWs), and 12 (infected-HCWs) recovered from COVID-19 before the first vaccine. Serum and saliva were collected at baseline (before the first dose), just before the second dose, 1, 3, 6, and 9 months after the second dose, and 10 days after the third vaccine. SARS-CoV-2-specific IgG and IgA were evaluated in serum and saliva, respectively, and the presence of neutralizing antibodies (NAb) was analyzed in serum. SARS-CoV-2-specific IgG peaked one month after the second vaccine in naïve-HCWs but right before this timepoint in infected-HCWs. IgG titers significantly decreased during follow-up and at month 9 were still detectable in 50% of naïve-HCWs and 90% of infected-HCWs. NAb were significantly decreased 6 months after the second vaccine in naïve-HCWs and 9 months after this dose in infected-HCWs. Salivary SARS-CoV-2-specific IgA titers were significantly higher in infected-HCWs and were undetectable 9 months after the second vaccine in 43% of the naïve-HCWs alone. The third vaccine greatly increased humoral IgG and mucosal IgA in both groups. Two BNT162b2 doses induced strong systemic and humoral immune responses; to note, these responses weakened over time, although they are more prolonged in individuals who had recovered from COVID-19. The third vaccine dose quickly boosts systemic and mucosal humoral responses.

Genetic diversity of Mycobacterium avium sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of Mycobacterium avium sp. paratuberculosis isolates.

Background: Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates. Method: The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system. Results: Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic. Conclusion: Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.

Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis.

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.

Development of a lateral flow immunochromatography test for the rapid detection of bovine tuberculosis.

Detection of specific antibodies would be a useful test strategy for bovine tuberculosis (bTB) as a complement to the single skin test. We developed a lateral flow immunochromatography (LFIC) test for rapid bTB detection based on the use of a conjugate of gold nanoparticles with a recombinant G protein. After evaluating 3 Mycobacterium bovis (MB) antigens: ESAT-6, CFP-10 and MPB83 for the control line, we selected MPB83 given it was the most specific. The performance of the test was analyzed with 820 bovine sera, 40 sera corresponding to healthy animals, 5 sera from animals infected with Mycobacterium avium subsp. paratuberculosis (MAP) and 775 sera of animals from herds with bTB. All these sera were also submitted to a validated bTB-ELISA using whole-cell antigen from MB. From the 775 sera of animals from herds with bTB, 87 sera were positive by the bTB-ELISA, 45 were positive by LFIC and only 5 animals were positives by skin test (TST). To confirm bTB infection in the group of TST (-), bTB-ELISA (+) and LFIC (+) animals, we performed postmortem examination in 15 randomly selected animals. Macroscopically, these 15 animals had numerous small and large yellow-white granulomas, characteristic of bTB, and the infection was subsequently confirmed by PCR in these tissues with lesions (gold standard). No false positive test result was detected with the developed LFIC either with the sera from healthy animals or from animals infected with MAP demonstrating that it can be a useful technique for the rapid identification of animals infected with bTB.

Evaluation of a virulent strain of Mycobacterium avium subsp. Paratuberculosis used as a heat-killed vaccine.

Bovine paratuberculosis is one of the most important chronic infectious diseases in livestock. This disease is difficult to control because of its inefficient management (test and cull strategy and inadequate biosecurity). Thus, the development of an effective vaccine is essential. In this study, we evaluated a local virulent strain (6611) of Mycobacterium avium subsp. paratuberculosis as an inactivated vaccine in comparison with the Silirum vaccine in mouse model and cattle. Regarding the mice model, only the groups vaccinated with 6611 showed lower colony forming unit (CFU) counts with a lower lesion score in the liver in comparison to the control group at 6 and 12 weeks post-challenge (wpc). The immune response was predominantly humoral (IgG1), although both vaccinated groups presented a cellular response with IFNγ production as well, but the 6611 group had also significant production of IL-2, IL-6, IL-17a, TNF, and IL-10. In cattle, the 6611 vaccinated group was the only one that maintained significant antibody values at the end of the trial, with significant production of IgG2 and IFNγ. No PPDb reactor was detected in the vaccinated animals, according to the intradermal caudal fold tuberculin test. Our results indicate that the 6611 local strain protected mice from challenge with a virulent strain, by inducing a humoral and cellular immune response. In the bovine, the natural host, the evaluated vaccine also induced humoral and cellular immune responses, with higher levels of CD4 + CD25+ and CD8 + CD25+ T cells populations than the commercial vaccine. Despite the encouraging results obtained in this study, an experimental challenge trial in cattle is mandatory to evaluate the efficacy of our candidate vaccine in the main host.

Two latent and two hyperstable polymeric forms of human neuroserpin.

Human neuroserpin (hNS) is a serine protease inhibitor that belongs to the serpin superfamily and is expressed in nervous tissues. The serpin fold is generally characterized by a long exposed loop, termed the reactive center loop, that acts as bait for the target protease. Intramolecular insertion of the reactive center loop into the main serpin ß-sheet leads to the serpin latent form. As with other known serpins, hNS pathological mutants have been shown to accumulate as polymers composed of quasi-native protein molecules. Although hNS polymerization has been intensely studied, a general agreement about serpin polymer organization is still lacking. Here we report a biophysical characterization of native hNS that is shown to undergo two distinct conformational transitions, at 55°C and 85°C, both leading to distinct latent and polymeric species. The latent and polymer hNS forms obtained at 45°C and 85°C differ in their chemical and thermal stabilities; furthermore, the hNS polymers also differ in size and morphology. Finally, the 85°C polymer shows a higher content of intermolecular ß-sheet interactions than the 45°C polymer. Together, these results suggest a more complex conformational scenario than was previously envisioned, and, in a general context, may help reconcile the current contrasting views on serpin polymerization.

Can copper(II) mediate Hoogsteen base-pairing in a left-handed DNA duplex? A pulse EPR study.

Pulse EPR spectroscopy is used to investigate possible structural features of the copper(II) ion coordinated to poly(dG-dC).poly(dG-dC) in a frozen aqueous solution, and the structural changes of the polynucleotide induced by the presence of the metal ion. Two different copper species were identified and their geometry explained by a molecular model. According to this model, one species is exclusively coordinated to a single guanine with the N7 nitrogen atom forming a coordinative bond with the copper. In the other species, a guanine and a cytosine form a ternary complex together with the copper ion. A copper crosslink between the N7 of guanine and N3 of cytosine is proposed as the most probable coordination site. Moreover, no evidence was found for an interaction of either copper species with a phosphate group or equatorial water molecules. In addition, circular dichroism (CD) spectroscopy showed that the DNA of the Cu(II)-poly(dG-dC).poly(dG-dC) adducts resembles the left-handed Z-form. These results suggest that metal-mediated Hoogsteen base pairing, as previously proposed for a right-handed DNA duplex, can also occur in a double-stranded left-handed DNA.

Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis.

Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.

Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system.

BACKGROUND: The exported repetitive protein (erp) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that erp is a virulence-associated factor since the disruption of this gene impairs the growth of Mycobacterium bovis and Mycobacterium tuberculosis in mice. RESULTS: In order to elucidate the function of Erp we searched for Erp-binding proteins from M. tuberculosis by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several Actinomycetales genera, orthologues of Rv2617c are exclusively present in members of the M. tuberculosis complex (MTC). The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from Mycobacterium smegmatis and Mycobacterium leprae were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism. CONCLUSION: We identified interactive partners of Erp, an M. tuberculosis protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.