T-cell release of granulysin contributes to host defense in leprosy

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.     

Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release

Release of cytochrome c from the mitochondrial intermembrane space is critical to apoptosis induced by a variety of death stimuli. Bid is a BH3-only prodeath Bcl-2 family protein that can potently activate this efflux. In the current study, we investigated the mitochondrial localization of Bid and its interactions with mitochondrial phospholipids, focusing on their relationships with Bid-induced cytochrome c release. We found that Bid binding to the mitochondria required only three of its eight helical structures (alpha4-alpha6), but not the BH3 domain, and the binding could not be inhibited by the antideath molecule Bcl-x(L). Membrane fractionations indicated that tBid bound to mitochondrial outer membranes at both contact and noncontact sites. Bid could interact with specific cardiolipin species on intact mitochondria as identified by mass spectrometry. Like the binding to the mitochondria, this interaction could not be blocked by the mutation in the BH3 domain or by Bcl-x(L.) However, a cardiolipin-specific dye, 10-N-nonyl acridine orange, could preferentially suppress Bid binding to the mitochondrial contact site and inhibit Bid-induced mitochondrial cristae reorganization and cytochrome c release. These findings thus suggest that interactions of Bid with mitochondrial cardiolipin at the contact site can contribute significantly to its functions.     

Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme

We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only approximately 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is approximately 50%. More significantly, the processivity of translocation is substantially reduced by approximately 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.     

Synthesis and release of leukotriene C4 by human eosinophils

When human peripheral blood eosinophils isolated to 92.5% +/- 6.9 purity were stimulated with either the calcium ionophore A23187 or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), immunoreactive leukotriene C4 (LTC4) was initially localized intracellularly and was subsequently released to the external medium in kinetically distinguishable steps. Eosinophils were stimulated with 2.5 microM A23187 in the presence of 20 mM L-serine, a hypochlorous acid scavenger that prevents the oxidative metabolism of sulfidopeptide leukotrienes. Total production of immunoreactive LTC4, the sum of intra- and extracellular LTC4, was complete within 5 to 10 min. At 5, 10, and 30 min, 65.9% +/- 15.2, 42.3% +/- 24.3, and 5.5% +/- 3.9, respectively, of the total amount of LTC4 measured remained intracellular as detected after the media and cells were separated and the latter was extracted with methanol. The time course for the intracellular synthesis and extracellular release of immunoreactive LTC4 from eosinophils pretreated with 5 micrograms/ml cytochalasin B and stimulated with 0.5 microM FMLP was like that obtained with ionophore, although the total LTC4 production was only approximately 10%. The identity of the intracellular LTC4 was confirmed by elution with reverse-phase high pressure liquid chromatography followed by scanning UV spectroscopy, radioimmunoassay, and bioassay. Eosinophils that were stimulated with A23187 in the absence of L-serine metabolized newly synthesized LTC4 to 6-trans-LTB4 diastereoisomers and subclass-specific diastereoisomeric sulfoxides that were identified only in the extracellular medium. Thus the response of purified eosinophils to two different stimuli demonstrates a transient intracellular accumulation of biologically active LTC4, the distinct extracellular release, and the apparent limitation of oxidative metabolism to the extracellular location.     

Genetic basis of murine antibacterial defense to streptococcal lung infection

To evaluate the effect of genetic background on antibacterial defense to streptococcal infection, eight genetically diverse strains of mice (A/J, DBA/2J, CAST/Ei, FVB/NJ, BALB/cJ, C57BL/6J, 129/SvImJ, and C3H/HeJ) and tlr2-deficient mice (C57BL/6^tlr2−/−) were infected with three doses of Streptococcus zooepidemicus (500, 5,000, or 50,000 colony-forming units) by alveolar challenge. There was a range of susceptibility between the strains at each dose and time point (6, 24, and 96 h). At the lowest dose, the 129/SvImJ and C3H/HeJ strains had significantly higher bacterial counts at all time points after infection, when compared to A/J, DBA/2J, CAST/Ei, FVB/NJ, which were resistant to infection at the low dose of innoculum. At the medium dose, 129/SvImJ and C3H/HeJ had higher bacterial counts, while A/J, DBA/2J, and BALB/cJ showed reduced streptococcal growth. After the highest dose of Streptococcus, there were minimal differences between strains, suggesting the protective impact of modifier genes can be overcome. TLR2-deficient animals contained increased bacterial load with reduced cytokines after 96 h when compared to C57BL/6J controls suggesting a role of innate immunity in late antibacterial defense. Overall, we identify vulnerable (129/SvlmJ and C3H/HeJ) and resistant (A/J, FVB, and DBA) mouse strains to streptococcal lung infection, which demonstrate divergent genetic expression profiles. These results demonstrate that innate differences in pulmonary host defense to S. zooepidemicus are dependent on host genetic factors.     

Secondary acylation of Klebsiella pneumoniae lipopolysaccharide contributes to sensitivity to antibacterial peptides

Klebsiella pneumoniae is an important cause of nosocomial Gram-negative sepsis. Lipopolysaccharide (LPS) is considered to be a major virulence determinant of this encapsulated bacterium and most mutations to the lipid A anchor of LPS are conditionally lethal to the bacterium. We studied the role of LPS acylation in K. pneumoniae disease pathogenesis by using a mutation of lpxM (msbB/waaN), which encodes the enzyme responsible for late secondary acylation of immature lipid A molecules. A K. pneumoniae B5055 (K2:O1) lpxM mutant was found to be attenuated for growth in the lungs in a mouse pneumonia model leading to reduced lethality of the bacterium. B5055DeltalpxM exhibited similar sensitivity to phagocytosis or complement-mediated lysis than B5055, unlike the non-encapsulated mutant B5055nm. In vitro, B5055DeltalpxM showed increased permeability of the outer membrane and an increased susceptibility to certain antibacterial peptides suggesting that in vivo attenuation may be due in part to sensitivity to antibacterial peptides present in the lungs of BALB/c mice. These data support the view that lipopolysaccharide acylation plays a important role in providing Gram-negative bacteria some resistance to structural and innate defenses and especially the antibacterial properties of detergents (e.g. bile) and cationic defensins.     

Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic pathogen and the leading cause of fungal meningitis. To survive within the host, this organism must be able to protect itself from oxidative stress. Cytochrome c peroxidase (Ccp1) is a mitochondrial antioxidant that catalyzes the degradation of hydrogen peroxide. In the present study, we characterized the contribution of the C. neoformans Ccp1 to antioxidant defense and for virulence. Consistent with studies of Ccp1 function in Sacchromyces cerevisiae, we found that Ccp1 contributes to resistance against exogenous oxidative stress in vitro. However, the oxidative stress phenotype does not diminish the virulence of ccp1 mutant strains in a murine model of C. neoformans disease. These results suggest that Ccp1 is involved in a complex system of protection against exogenous oxidative stress and that the elimination of this component of the antioxidant defense system does not diminish the virulence of C. neoformans.     

Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation– or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.     

IL-17 contributes to cell-mediated defense against pulmonary Yersinia pestis infection

Pneumonic plague is one of the world's most deadly infectious diseases. The causative bacterium, Yersinia pestis, has the potential to be exploited as a biological weapon, and no vaccine is available. Vaccinating B cell-deficient mice with D27-pLpxL, a live attenuated Y. pestis strain, induces cell-mediated protection against lethal pulmonary Y. pestis challenge. In this article, we demonstrate that prime/boost vaccination with D27-pLpxL confers better protection than prime-only vaccination. The improved survival does not result from enhanced bacterial clearance but is associated with increased levels of IL-17 mRNA and protein in the lungs of challenged mice. The boost also increases pulmonary numbers of IL-17-producing CD4 T cells. Interestingly, most of these cells simultaneously produce canonical type 1 and type 17 cytokines; most produce IL-17 and TNF-α, and many produce IL-17, TNF-α, and IFN-γ. Neutralizing IL-17 counteracts the improved survival associated with prime/boost vaccination without significantly impacting bacterial burden. Thus, IL-17 appears to mediate the enhanced protection conferred by booster immunization. Although neutralizing IL-17 significantly reduces neutrophil recruitment to the lungs of mice challenged with Y. pestis, this impact is equally evident in mice that receive one or two immunizations with D27-pLpxL, suggesting it cannot suffice to account for the improved survival that results from booster immunization. We conclude that IL-17 plays a yet to be identified role in host defense that enhances protection against pulmonary Y. pestis challenge, and we suggest that pneumonic plague vaccines should aim to induce mixed type 1 and type 17 cellular responses.     

VPEgamma exhibits a caspase-like activity that contributes to defense against pathogens

BACKGROUND: Caspases are a family of aspartate-specific cysteine proteases that play an essential role in initiating and executing programmed cell death (PCD) in metazoans. Caspase-like activities have been shown to be required for the initiation of PCD in plants, but the genes encoding those activities have not been identified. VPEgamma, a cysteine protease, is induced during senescence, a form of PCD in plants, and is localized in precursor protease vesicles and vacuoles, compartments associated with PCD processes in plants. RESULTS: We show that VPEgamma binds in vivo to a general caspase inhibitor and to caspase-1-specific inhibitors, which block the activity of VPEgamma. A cysteine protease inhibitor, cystatin, accumulates to 20-fold higher levels in vpegamma mutants. Homologs of cystatin are known to suppress hypersensitive cell death in plant and animal systems. We also report that infection with an avirulent strain of Pseudomonas syringae results in an increase of caspase-1 activity, and this increase is partially suppressed in vpegamma mutants. Plants overexpressing VPEgamma exhibit a greater amount of ion leakage during infection with P. syringae, suggesting that VPEgamma may regulate cell death progression during plant-pathogen interaction. VPEgamma expression is induced after infection with P. syringae, Botrytis cinerea, and turnip mosaic virus, and knockout of VPEgamma results in increased susceptibility to these pathogens. CONCLUSIONS: We conclude that VPEgamma is a caspase-like enzyme that has been recruited in plants to regulate vacuole-mediated cell dismantling during cell death, a process that has significant influence in the outcome of a diverse set of plant-pathogen interactions.     

Iron contributes to the antibacterial functions of the haemolymph of Galleria mellonella

Studies with Galleria mellonella larvae and the iron chelating agent EDDA showed that iron was essential for the removal of dead Xenorhabdas nematophila and Bacillus subtilis from the haemolymph. The delay in removal of the bacteria from the iron-restricted haemolymph was attributed to reduced adhesiveness of the haemocytes and prophenoloxidase activity. Iron augmentation returned these activities to control levels. Whereas dead B. subtilis had no effect on the concentration of ferrozine-detectable iron (henceforth iron) in the haemolymph, dead X. nematophila was associated with substantially lower levels of iron as the number of damaged haemocytes increased. Haemocyte lysate lowered the concentrations of iron in both FeCl₃ solutions and deproteinized larval serum independent of serum lipids. Haemocyte lysate added to tryptic soybroth lowered the level of iron and limited the growth of X. nematophila. X. nematophila limited iron availability in the plasma by releasing lipopolysaccharides; such a mechanism may be a means of impairing the antimicrobial defences of the insects.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase

RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP and the ATPase cycle fuel the work performed by helicases is not completely clear. The hepatitis C virus RNA helicase, NS3, is an important model system for this class of enzymes. NS3 binding to a single-/double-strand RNA or DNA junction leads to ATP-independent melting of the duplex and formation of a complex capable of ATP-dependent unwinding by using a spring-loaded mechanism. We have established an RNA substrate for NS3 that can be unwound in a single sub-step. Our studies are consistent with a model in which a single ATP binding and/or hydrolysis event sets the unwinding spring and phosphate dissociation contributes to release of the spring, thereby driving the power stroke used for unwinding.     

GSH-dependent antioxidant defense contributes to the acclimation of colon cancer cells to acidic microenvironment

Due to increased glycolysis and poor local perfusion, solid tumors are usually immersed in an acidic microenvironment. While extracellular acidosis is cytotoxic, cancer cells eventually become acclimated to it. While previous studies have addressed the acute effect of acidosis on cancer cells, little is known about how cancer cells survive chronic acidosis. In this study we exposed colorectal cancer (CRC) cells (HCT15, HCT116 and LoVo) to acidic pH (pH 6.5) continuously for over three months and obtained CRC cells that become acclimated to acidic pH, designated as CRC-acidosis-acclimated or CRC-AA. We unexpectedly found that while acute exposure to low pH resulted in an increase in the level of intracellular reactive oxygen species (ROS), CRC-AA cells exhibited a significantly reduced level of ROS when compared to ancestor cells. CRC-AA cells were found to maintain a higher level of reduced glutathione, via the upregulation of CD44 and glutathione reductase (GSR), among others, than their ancestor cells. Importantly, CRC-AA cells were more sensitive to agents that deplete GSH. Moreover, downregulation of GSR by RNA interference was more deleterious to CRC-AA cells than to control cells. Together, our results demonstrate a critical role of glutathione-dependent antioxidant defense in acclimation of CRC cells to acidic extracellular pH.     

MyD88 dependent signaling contributes to protective host defense against Burkholderia pseudomallei

Background

Toll-like receptors (TLRs) have a central role in the recognition of pathogens and the initiation of the innate immune response. Myeloid differentiation primary-response gene 88 (MyD88) and TIR-domain-containing adaptor protein inducing IFNβ (TRIF) are regarded as the key signaling adaptor proteins for TLRs. Melioidosis, which is endemic in SE-Asia, is a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei. We here aimed to characterize the role of MyD88 and TRIF in host defense against melioidosis.

Methodology and Principal Findings

First, we found that MyD88, but not TRIF, deficient whole blood leukocytes released less TNFα upon stimulation with B. pseudomallei compared to wild-type (WT) cells. Thereafter we inoculated MyD88 knock-out (KO), TRIF mutant and WT mice intranasally with B. pseudomallei and found that MyD88 KO, but not TRIF mutant mice demonstrated a strongly accelerated lethality, which was accompanied by significantly increased bacterial loads in lungs, liver and blood, and grossly enhanced liver damage compared to WT mice. The decreased bacterial clearance capacity of MyD88 KO mice was accompanied by a markedly reduced early pulmonary neutrophil recruitment and a diminished activation of neutrophils after infection with B. pseudomallei. MyD88 KO leukocytes displayed an unaltered capacity to phagocytose and kill B. pseudomallei in vitro.

Conclusions

MyD88 dependent signaling, but not TRIF dependent signaling, contributes to a protective host response against B. pseudomallei at least in part by causing early neutrophil recruitment towards the primary site of infection.     

Increased mitochondrial oxidative damage and oxidative DNA damage contributes to the neurodegenerative process in sporadic amyotrophic lateral sclerosis

To investigate the possibility that mitochondrial oxidative damage or oxidative DNA damage or both contribute to the neurodegenerative process of sporadic amyotrophic lateral sclerosis (sALS), this study used high-performance liquid chromatography with an electrochemical detector to measure the concentrations of the reduced and oxidized forms of coenzyme Q10 (CoQ10) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the cerebrospinal fluid (CSF) of 17 patients with sALS and 17 age-matched controls with no neurological diseases. The percentage of oxidized CoQ10 in the CSF of sALS patients was greater than that in the CSF of controls (p<0.002) and was negatively correlated with the duration of illness (rho=-0.61, p<0.01). The concentration of 8-OHdG in the CSF of sALS patients was greater than that in the CSF of controls (p<0.005) and was positively correlated with the duration of illness (rho=0.53, p<0.005). The percentage of oxidized CoQ10 was correlated with the concentrations of 8-OHdG in the CSF of sALS patients (rho=-0.53, p<0.05). These results suggest that both mitochondrial oxidative damage and oxidative DNA damage play important roles in the pathogenesis of sporadic amyotrophic lateral sclerosis.     

Oxidative damage during chagasic cardiomyopathy development: role of mitochondrial oxidant release and inefficient antioxidant defense

In this study, we evaluated the oxidant status and antioxidant defense capabilities of the heart during the course of Trypanosoma cruzi infection and disease development in a murine model system. Our data show that the extent of protein carbonylation and lipid peroxidation is increased in the heart, but not the skeletal muscle, of infected mice. The level of oxidative injury biomarkers in the myocardium consistently increased with chronic disease severity. The antioxidant defense constituted by catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GSR), and reduced glutathione was increased in murine heart and skeletal tissue in response to the stress of T. cruzi infection. After the initial burst, CAT, GPx, and GSR remained unresponsive to the severity of chronic tissue damage in chagasic hearts. The cardiac level of Mn(2+) superoxide dismutase (MnSOD) was diminished in chagasic mice. Our data suggest that the host responds to acute injuries by activating antioxidant defenses that are of sufficient magnitude to scavenge the reactive oxidants in skeletal tissue. The myocardia of infected mice, however, sustain increased oxidative injuries with disease progression. We surmise that MnSOD deficiencies, resulting in the increased release of mitochondrial free radicals, lead to sustained oxidative stress that exceeds the cardiac antioxidant defense capacity and contribute to persistent oxidative damage in chagasic myocardium.