Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective

IP Multicasting over DVB-T/T2/H and eMBMS using PARPS : Effect of the number of transmitters

With the advancement in the current wireless technology standards such as terrestrial digital video broadcasting systems (DVB-T, DVB-T2, and DVB-H) and the massive usage of the Internet over mobile devices, streaming of television channels in smart phones has become a necessary advancement for mobile users. As, UMTS dominating the entire mobile market globally and with the evolution of LTE, several mobile operators are working on an MBMS framework which will help to launch mobile TV services on respective operators. This paper deals with terrestrial and mobile TV with IP multicasting and broadcasting and is aimed to improve system spectral efficiency.  With the help of IP multicasting, the base station can be able to provide with significantly less spectrum by saving it from the channels which the user is not viewing currently. This case is analysed from several sets, called schemes of resource plan sets. The transmitter scheduling is dealt with by means of a Packet and Resource Plan Scheduling (PARPS) algorithm, and the simulated results are plotted in Matlab which assists in analysing the efficiency in the spectrum management and the coverage probability for the number of transmitters used for each scheme. The schemes are simulated in Matlab for different number of transmitters (2-7) in both the static and random model. The SFN schemes are offering greater coverage probability than MFN schemes, in all cases. Multicasting over Continuous Transmission Dynamic Single Frequency Network (CT-DSFN) offers a 1342% and 582% gain in Multi-user System Spectral Efficiency (MSSE) for 7 transmitters, from Broadcasting over MFN and Broadcasting over SFN respectively.  For 7 transmitters, Multicasting over CT-DSFN offers a 1213% and 428% gain in System Spectral Efficiency (SSE) from Broadcasting over MFN and Broadcasting over SFN respectively

Expression of an antisense hla fragment in Staphylococcus aureus reduces alpha-toxin production in vitro and attenuates lethal activity in a murine model

Isogeneic bacterial strains that differ only in the production of a single microbial factor have been invaluable in studying the pathogenesis of bacterial infections. The targeted, intentional inactivation of a gene encoding a potential virulence determinant generally requires homologous recombination to replace the gene with an inactivated allele. To determine whether the insertion and expression of a fragment of a bacterial gene in an antisense orientation could be used as a rapid alternative to allelic inactivation for producing paired isogeneic isolates, we inverted a 600-bp fragment of the Staphylococcus aureus gene encoding alpha-toxin, hla, behind its native promoter on an Escherichia coli-S. aureus shuttle vector. A transformant of an S. aureus strain carrying the antisense hla fragment produced antisense hla RNA and made 16-fold less alpha-toxin than either its parent or an isogeneic transformant containing vector DNA without hla. Also, intraperitoneal injection of 1.5 x 10(9) CFU of the antisense hla-containing transformant was significantly less lethal in a murine model than that of the parent (1 of 10 versus 7 of 10 mice expired [P &lt; 0.02]) or the transformant without hla (1 of 10 versus 7 of 7 mice expired [P &lt; 0.001]). We conclude that the expression of a fragment of hla in an antisense orientation in S. aureus on a plasmid vector reduces alpha-toxin production and the lethal activity of the strain in a murine model. The antisense strategy for creating isogeneic strains of bacteria may facilitate molecular investigations into the pathogenesis of infection. It also may be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates.</jats:p