Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium Tuberculosis-Induced Apoptosis in Mice

Background: Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. Methodology/Principal Findings: We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. Conclusions/Significance: These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice

Background: Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. Methodology/Principal Findings: We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-alpha, IL-1 beta, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3 beta in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3 beta in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. Conclusions/Significance: These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000274923700001&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Multidisciplinary SciencesSCI(E)11ARTICLE2null

Single nucleotide polymorphisms in efflux pumps genes in extensively drug resistant Mycobacterium tuberculosis isolates from Pakistan

It is challenging to understand mechanisms of drug resistance in Mycobacterium tuberculosis (MTB) due to the large variability in resistance associated genes. Efflux pump genes contribute to drug resistance and thus add to this complexity. Efflux pump gene protein superfamilies have been characterized by genome analysis of drug resistant strains and through invitro transcriptional studies. However, there is limited information regarding efflux pump genes in extensively drug resistant (XDR) tuberculosis (TB) isolates. Whole genome sequencing (WGS) based analysis of 37 extensively drug resistant (XDR) and five drug sensitive (DS) MTB clinical isolates was performed. Single nucleotide polymorphisms (SNPs) in efflux pump genes Rv0194, Rv1217, Rv1218, drrA, drrB, Rv1258, Rv1634, Rv2688, Rv1273, Rv1819, Rv1458, Rv1877 and Rv1250 were determined in the clinical isolates as compared with the H37Rv reference strain. Allele frequencies of SNPs identified in XDR strains were compared with DS strains. Gene expression of Rv0194, Rv2688, Rv1634, drrA and drrB was determined in XDR -TB isolates (n=9), DS-TB strains (n=4) and H37Rv. We identified SNPs in XDR-TB isolates which were either unique or present at very low frequencies in DS strains; Rv0194 G170V; Rv1217 L151R; Rv1258 P369T and G391R; Rv1273 S118G and I175T; Rv1877 I534T; Rv1250 V318X/A and S333A, and Rv2688 P156T. The expression of Rv2688 and drrB was found to be raised in XDR-TB as compared with DS-TB strains. We identified unique SNPs in efflux pump genes which may be associated with increased drug resistance in the isolates. Increased levels of Rv2688 and drrB efflux pump gene expression observed in XDR strains even in the absence of antibiotics suggests that these clinical isolates may be more refractory to treatment. Further studies are required to directly associate these mutations with increased resistance in MTB

Mutations in Efflux Pump Rv1258c (Tap) Cause Resistance to Pyrazinamide, Isoniazid, and Streptomycin in M. tuberculosis

Although drug resistance in Mycobacterium tuberculosis is mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in the possible role of efflux in causing drug resistance. Previously, efflux genes have been shown to be upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance in M. tuberculosis. We constructed point mutations V219A and S292L in Rv1258c in the chromosome of M. tuberculosis and the point mutations were confirmed by DNA sequencing. The susceptibility of the constructed M. tuberculosis Rv1258c mutants to different tuberculosis drugs was assessed using conventional drug susceptibility testing in 7H11 agar in the presence and absence of efflux pump inhibitor piperine. A C14-labeled PZA uptake experiment was performed to demonstrate higher efflux activity in the M. tuberculosis Rv1258c mutants. Interestingly, the V219A and S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs in M. tuberculosis. While V219A point mutation conferred low-level drug resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. The S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance, including PZA resistance, and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance in M. tuberculosis and address their role in affecting treatment outcome in vivo

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents

Energy Dependent Divisible Load Theory for Wireless Sensor Network Workload Allocation

The wireless sensor network (WSN), consisting of a large number of microsensors with wireless communication abilities, has become an indispensable tool for use in monitoring and surveillance applications. Despite its advantages in deployment flexibility and fault tolerance, the WSN is vulnerable to failures due to the depletion of limited onboard battery energy. A major portion of energy consumption is caused by the transmission of sensed results to the master processor. The amount of energy used, in fact, is related to both the duration of sensing and data transmission. Hence, in order to extend the operation lifespan of the WSN, a proper allocation of sensing workload among the sensors is necessary. An assignment scheme is here formulated on the basis of the divisible load theory, namely, the energy dependent divisible load theory (EDDLT) for sensing workload allocations. In particular, the amount of residual energies onboard sensors are considered while deciding the workload assigned to each sensor. Sensors with smaller amount of residual energy are assigned lighter workloads, thus, allowing for a reduced energy consumption and the sensor lifespan is extended. Simulation studies are conducted and results have illustrated the effectiveness of the proposed workload allocation method

Energy Dependent Divisible Load Theory for Wireless Sensor Network Workload Allocation

The wireless sensor network WSN , consisting of a large number of microsensors with wireless communication abilities, has become an indispensable tool for use in monitoring and surveillance applications. Despite its advantages in deployment flexibility and fault tolerance, the WSN is vulnerable to failures due to the depletion of limited onboard battery energy. A major portion of energy consumption is caused by the transmission of sensed results to the master processor. The amount of energy used, in fact, is related to both the duration of sensing and data transmission. Hence, in order to extend the operation lifespan of the WSN, a proper allocation of sensing workload among the sensors is necessary. An assignment scheme is here formulated on the basis of the divisible load theory, namely, the energy dependent divisible load theory EDDLT for sensing workload allocations. In particular, the amount of residual energies onboard sensors are considered while deciding the workload assigned to each sensor. Sensors with smaller amount of residual energy are assigned lighter workloads, thus, allowing for a reduced energy consumption and the sensor lifespan is extended. Simulation studies are conducted and results have illustrated the effectiveness of the proposed workload allocation method

Essential Oils with High Activity against Stationary Phase Bartonella henselae

Bartonella henselae is a fastidious Gram-negative intracellular bacterium that can cause cat scratch disease, endocarditis in humans and animals, as well as other complications, leading to acute or chronic infections. The current treatment for Bartonella infections is not very effective presumably due to bacterial persistence. To develop better therapies for persistent and chronic Bartonella infections, in this study, with the help of SYBR Green I/PI viability assay, we performed a high-throughput screening of an essential oil library against the stationary phase B. henselae. We successfully identified 32 essential oils that had high activity, including four essential oils extracted from Citrus plants, three from Origanum, three from Cinnamomum, two from Pelargonium, and two from Melaleuca, as well as frankincense, ylang-ylang, fir needle, mountain savory (winter), citronella, spearmint, elemi, vetiver, clove bud, allspice, and cedarwood essential oils. The minimal inhibitory concentration (MIC) determination of these 32 top hits indicated they were not only active against stationary phase non-growing B. henselae but also had good activity against log-phase growing B. henselae. The time-kill assay showed 13 active hits, including essential oils of oregano, cinnamon bark, mountain savory (winter), cinnamon leaf, geranium, clove bud, allspice, geranium bourbon, ylang-ylang, citronella, elemi, and vetiver, could eradicate all stationary phase B. henselae cells within seven days at the concentration of 0.032% (v/v). Two active ingredients, carvacrol and cinnamaldehyde, of oregano and cinnamon bark essential oils, respectively, were shown to be very active against the stationary phase B. henselae such that they were able to eradicate all the bacterial cells even at the concentration ≤ 0.01% (v/v). More studies are needed to identify the active components of some potent essential oils, decode their antimicrobial mechanisms, and evaluate their activity against Bartonella infections in animal models

Divisible Load Theory Based Active-Sleep Workload Assignment Schemes for Wireless Sensor Networks

Wireless sensor networks (WSNs) have been widely applied in many monitoring and surveillance processes. Due to the limited onboard energy resources, the operation of a WSN is severely hindered by its shortened lifespan arising from energy depletion. In order to alleviate this problem, and with the possibility of partitioning sensing workloads, the divisible load theory (DLT) can be adopted to derive a proper workload assignment scheme. However, special considerations have to be paid for its generic applicability in feasible workload assignment for WSNs. In this paper, an examination of DLT based WSN operations including the effect of assignment, measurement, and report times is presented, and the problem of negative workloads inherently generated in some schemes is revealed. Furthermore, by making use of the negativity phenomenon, an active-sleep scheme is proposed for the WSN such that sensor energy consumptions can be reduced and consequently extend the WSN operation lifespan. Specifically, sensors with smaller amount of residual energies are put into the sleep mode when the assigned workloads are negative. On the other hand, positive workloads are normalized and reassigned to sensors with larger amount of onboard energies. Simulation studies are carried out to demonstrate the negative workload phenomenon, and satisfactory performances of the proposed active-sleep scheme are verified

Ceftriaxone Pulse Dosing Fails to Eradicate Biofilm-Like Microcolony B. burgdorferi Persisters Which Are Sterilized by Daptomycin/ Doxycycline/Cefuroxime without Pulse Dosing

Although the majority of Lyme disease patients can be cured, at least 10-20% of the patients continue to suffer from persisting symptoms such as fatigue, muscular and joint pain, and neurologic impairment after standard 2-4 week antibiotic treatment. While the causes for this post-treatment Lyme disease symptoms are unclear, one possibility is due to B. burgdorferi persisters that are not effectively killed by current antibiotics such as doxycycline or amoxicillin used to treat Lyme disease. A previous study showed that four rounds of ceftriaxone pulse dosing treatment eradicated B. burgdorferi persisters in vitro using a relatively young late log phase culture (5 day old). In this study, we investigated if ceftriaxone pulse dosing could also eradicate B. burgdorferi persisters in older stationary phase cultures (10 day old) enriched with more resistant microcolony form of persisters. We found that ceftriaxone pulse dosing could only eradicate planktonic log phase B. burgdorferi spirochetal forms and round body forms but not more resistant aggregated biofilm-like microcolony persisters enriched in stationary phase cultures. Moreover, we found that not all drugs are suitable for pulse dosing, with bactericidal drugs ceftriaxone and cefuroxime being more appropriate for pulse dosing than bacteriostatic drug doxycycline and persister drug daptomycin. We also showed that drug combination pulse dosing treatment was more effective than single drug pulse dosing. Importantly, we demonstrate that pulse dosing treatment impaired the activity of the persister drug daptomycin and its drug combination against B. burgdorferi persisters and that the most effective way to kill the more resistant biofilm-like microcolonies is the daptomycin/doxycycline/ceftriaxone triple drug combination without pulse dosing. Our findings indicate pulse dosing may not always work as a general principle but rather depends on the specific drugs used, with cidal drugs being more appropriate for pulse dosing than static or persister drugs, and that drug combination approach with persister drugs is more effective at killing the more resistant microcolony form of persisters than pulse dosing. These observations may have implications for more effective treatment of Lyme disease. Future studies are required to validate these findings in animal models of B. burgdorferi persistence