Stably Luminescent Staphylococcus aureus Clinical Strains for Use in Bioluminescent Imaging

In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.     

Development of a Murine Model with Optimal Routes for Bacterial Infection and Treatment, as Determined with Bioluminescent Imaging in C57BL/6 Mice

Mice intragastrically infected with Listeria monocytogenes EGDe and Staphylococcus aureus Xen 36 showed no visible signs of infection over 48 h. However, high numbers (6.2 × 10⁵ cfu/mg feces) of S. aureus Xen 36 were detected 4 h, and 3.3 × 10⁵ cfu/mg feces of L. monocytogenes EGDe 8 h, after administration. Mice intraperitoneally infected with S. aureus Xen 36 (1 × 10⁷ cfu) developed infection immediately after administration and for at least the following 48 h. Injection with higher cell numbers of S. aureus Xen 36 (2 × 10⁸ cfu) resulted in more intense bioluminescence (infection) of the peritoneal cavity. Injection of S. aureus Xen 36 in the tail and penile veins resulted in localized tissue infection for the first 120 h. Injection of S. aureus Xen 36 into the thigh produced a faint bioluminescent signal for 15 min. Nisin F injected into the peritoneal cavity at the same area of infection led to an immediate statistically significant decrease in infection (from 2 × 10⁶ p/s/cm²/sr to 3 × 10⁵ p/s/cm²/sr) within 2 h. Similar results were recorded when nisin F was injected subcutaneously. Intraperitoneal administration is an optimal administration route for bacterial infection and treatment with antimicrobial peptides.     

A Pre-clinical Animal Model of Trypanosoma brucei Infection Demonstrating Cardiac Dysfunction

African trypanosomiasis (AT), caused by Trypanosoma brucei species, results in both neurological and cardiac dysfunction and can be fatal if untreated. Research on the pathogenesis and treatment of the disease has centred to date on the characteristic neurological symptoms, whereas cardiac dysfunction (e.g. ventricular arrhythmias) in AT remains largely unstudied. Animal models of AT demonstrating cardiac dysfunction similar to that described in field cases of AT are critically required to transform our understanding of AT-induced cardiac pathophysiology and identify future treatment strategies. We have previously shown that T. brucei can interact with heart muscle cells (cardiomyocytes) to induce ventricular arrhythmias in ex vivo adult rat hearts. However, it is unknown whether the arrhythmias observed ex vivo are also present during in vivo infection in experimental animal models. Here we show for the first time the characterisation of ventricular arrhythmias in vivo in two animal models of AT infection using electrocardiographic (ECG) monitoring. The first model utilised a commonly used monomorphic laboratory strain, Trypanosoma brucei brucei Lister 427, whilst the second model used a pleomorphic laboratory strain, T. b. brucei TREU 927, which demonstrates a similar chronic infection profile to clinical cases. The frequency of ventricular arrhythmias and heart rate (HR) was significantly increased at the endpoint of infection in the TREU 927 infection model, but not in the Lister 427 infection model. At the end of infection, hearts from both models were isolated and Langendorff perfused ex vivo with increasing concentrations of the β-adrenergic agonist isoproterenol (ISO). Interestingly, the increased frequency of arrhythmias observed in vivo in the TREU 927 infection model was lost upon isolation of the heart ex vivo, but re-emerged with the addition of ISO. Our results demonstrate that TREU 927 infection modifies the substrate of the myocardium in such a way as to increase the propensity for ventricular arrhythmias in response to a circulating factor in vivo or β-adrenergic stimulation ex vivo. The TREU 927 infection model provides a new opportunity to accelerate our understanding of AT-related cardiac pathophysiology and importantly has the required sensitivity to monitor adverse cardiac-related electrical dysfunction when testing new therapeutic treatments for AT.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

Calflagin Inhibition Prolongs Host Survival and Suppresses Parasitemia in Trypanosoma brucei Infection

African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness, a fatal disease endemic to regions throughout sub-Saharan Africa. Incidence rates of human T. brucei infection have risen dramatically in the past 30 to 50 years, leading to renewed emphasis by the World Health Organization on disease surveillance and control among the millions of people at risk of infection. T. brucei also infects cattle to cause nagana, a disease which renders vast regions unsuitable for livestock and poses a major barrier to economic development in afflicted areas (30).T. brucei is transmitted to its mammalian host via the bite of the infected tsetse fly, when parasites are introduced into the host circulation during a blood meal. To thrive in both the insect vector and the mammalian host, T. brucei has evolved digenetic life cycle stages; the two most commonly studied life cycle stages are the procyclic stage from the fly midgut and the bloodstream form found in the mammalian host. Programmed differentiation between these stages regulates broad aspects of parasite biology, enabling adaptation to either environment. In the mammalian host, avoiding clearance by the humoral immune response is particularly important, and T. brucei has evolved sophisticated mechanisms to this end. Bloodstream form parasites are covered by a thick monolayer of variant surface glycoprotein (VSG) that blocks the access of host antibodies to underlying invariant antigens (2). VSG is highly immunogenic, and VSG-specific antibodies facilitate the clearance of parasites from the blood. However, the parasite undergoes antigenic variation, a process whereby the parasite spontaneously switches from the expression of a single VSG type to that of another of the hundreds in its genomic repertoire. The new parasite clone is resistant to the existing antibodies and persists until antibodies to the new VSG are produced, thus selecting for another antigen variant and propagating the cycle. T. brucei has coevolved with primates for millions of years, and antigenic variation is not its sole means of immune evasion. Additional mechanisms such as host immunosuppression (20), motility-driven internalization and degradation of surface-bound antibodies (13, 22), and the shedding of VSG molecules (7) are each likely to contribute to the survival of T. brucei in the hostile environment of its mammalian host. However, the signaling and genetic pathways by which T. brucei regulates stage-specific adaptations to its environment remain poorly understood.Calcium signaling plays critical roles in virtually every eukaryotic cell type, and trypanosomes are no exception. Regulated changes in intracellular calcium are important for trypanosome replication, differentiation, cell invasion, and virulence (24). The importance of calcium regulation in trypanosomes is further underscored by the presence of a specialized organelle, the acidocalcisome, which contains a major intracellular reservoir of calcium (9). Cellular responses to calcium fluctuations are mediated by calcium-binding proteins. In addition to calmodulin, T. brucei expresses a family of EF-hand proteins named the calflagins. They were discovered as the predominant T. brucei proteins to bind a hydrophobic resin in a calcium-dependent manner (35, 36). These proteins specifically localize to the flagellum, an organelle which, in addition to its obvious role in cellular motility, compartmentalizes signaling proteins, including adenylate cyclases and phosphodiesterases (26, 27). In the flagellum, calflagins associate with lipid raft microdomains, which in many cell types serve as recruitment platforms for signaling molecules (32). We have recently elucidated the role of acylation in calflagin trafficking and raft association and identified the calflagin-specific palmitoyl acyltransferase (11). However, the precise functions of these abundant proteins remain unknown. The calflagins show homology with Trypanosoma cruzi FCaBP, an immunodominant protein with similar flagellar enrichment yet an unidentified biologic function (12). Outside of the common EF-hand domains, the calflagins display only minimal homology to proteins in organisms outside the kinetoplastid lineage.To elucidate the function of T. brucei calflagins, we assessed the consequences of calflagin inhibition to T. brucei in vivo during host infection and in vitro. We can now report for the first time that calflagin expression influences the outcome of parasite infection, as mice infected with calflagin-deficient cells demonstrate a sudden decrease in parasitemia approximately 1 week postinfection. This drop is associated with prolonged host survival and outgrowth of parasites expressing alternative VSG molecules, indicating selective pressure against the initial dominant VSG molecule and suggesting that the primary deficit of calflagin-deficient cells is one of enhanced sensitivity to the host adaptive immune response. We demonstrate that the molecular mechanisms underlying this effect are not due to altered parasite proliferation, motility, or clearance of surface-bound antibodies.     

In Vivo Bioluminescent Imaging of Influenza A Virus Infection and Characterization of Novel Cross-Protective Monoclonal Antibodies

Influenza A virus is a major human pathogen responsible for seasonal epidemics as well as pandemic outbreaks. Due to the continuing burden on human health, the need for new tools to study influenza virus pathogenesis as well as to evaluate new therapeutics is paramount. We report the development of a stable, replication-competent luciferase reporter influenza A virus that can be used for in vivo imaging of viral replication. This imaging is noninvasive and allows for the longitudinal monitoring of infection in living animals. We used this tool to characterize novel monoclonal antibodies that bind the conserved stalk domain of the viral hemagglutinin of H1 and H5 subtypes and protect mice from lethal disease. The use of luciferase reporter influenza viruses allows for new mechanistic studies to expand our knowledge of virus-induced disease and provides a new quantitative method to evaluate future antiviral therapies.     

Disruption of spermatogenesis in boars sub-clinically infected with Trypanosoma brucei brucei

Data from 14 crossbred (Landreace x Large white) boars aged 10-12 months were used to investigate specific germ cells and to what extent Sertoli cells are prone to sub-clinical infection with strain Y58/98 Trypanosome brucei brucei and effects on spermatogenesis. Boars were divided into three groups, A, B and C of 5, 5 and 4 animals, respectively. Groups A and B were infected intraperitoneally with 2.8x10(6) trypanosomes per animal. Group C consisted of intact controls. At stable sub-clinical trypanosomiasis, boars in groups A and B together with two from the controls were weighed, scrotal circumferences were measured and animals were castrated on days 56 and 84 post infection, respectively. Testes were weighed. A portion of a testis was processed for histomorphometric assessment and another portion was used to determine gonadal sperm reserves by haemocytometry. Crude cells were converted to true cells.Sub-clinical trypanosomiasis was characterised by low live and testes weights, reduced scrotal circumference, scanty parasitemia peaks at long intervals and decreased libido. Histomorphometry of animals infected with T. brucei brucei revealed somniferous tubular distortion, denudation and or degeneration of germ cells and Sertoli cells leading to distortion of spermatogenesis. Spermatids and young primary spermatocytes were most prone to, while Sertoli cells and spermatogonia were least affected by sub-clinical trypanosomiasis. There was evidence of regeneration of germ cells from precursor stem cells, resulting in slightly increased gonodal sperm reserves as the post infection period increased. Infected boars may not attain original fertility levels consequently. It was concluded that boars in tropical regions that harbour endemic disease should be maintained under prophylactic conditions.     

Quantitative Ultrastructural Differences between Strains of the Trypanosoma brucei Subgroup During Transformation in Blood*

SYNOPSIS. Differences in the relative and absolute cell organization between strains of the Trypanosoma brucei subgroup were studied during the transformation from slender to stumpy bloodforms. Two pleomorphic and 1 monomorphic T. b. brucei, and 1 pleomorphic T. b. rhodesiense strains were investigated. Volume densities, surface densities and surface to volume ratios showed barely significant differences between the 2 pleomorphic T. b. brucei strains; absolute parameters, however, differ markedly between all the strains investigated. Only the relative parameters of the mitochondrion show notable differences between T. b. brucei and T. b. rhodesiense examined here. During the transformation from slender to stumpy forms the enlargement of the mitochondrial volume in T. b. brucei is achieved by an increase in width of the mitochondrial tube and in T. b. rhodesiense by the formation of a more elaborate network. The ratio of the inner mitochondrial membrane surface area to the mitochondrial matrix volume showed no significant change in all 3 pleomorphic strains examined. Because of their morphometric similarity to slender forms of pleomorphic T. b. brucei strains, it can be assumed that the monomorphic trypanosomes correspond morphologically to slender trypanosomes. Neither pleomorphism nor strain specificity have a significant influence on the relative amount of “vesicles” and lipid inclusions.     

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull^1,2 or thinned-skull ³ preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX₃CR₁ GFP/+ mice ⁴ to visualize microglia with enhanced green fluorescent protein to ≤150 μm beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.     

Curative Effects of the Antipiroplasms Amicarbalide and Imidocarb on Trypanosoma brucei Infection in Mice*

SYNOPSIS The babesicides imidocarb and amicarbalide, which have structural similarities to the antitrypanosomatid diamidines, proved active against Trypanosoma brucei mouse infections: both cured infections when doses were administered daily for 3 days 24 h post-inoculation (curative dose imidocarb, 10 mg/kg; amicarbalide, 25 mg/kg). Mice were considered cured after survival 30 days longer than untreated infected controls, with no trypanosomes present in blood or cerebrospinal fluid smears. Both agents also cured when administered 48 and 72 h after challenge with T. brucei and prolonged the lives of animals 94 h after challenge. The results are discussed in respect to the potential of these carbanilides and their precursors, the antitumor phthalanilides, as lead compounds in chemotherapy of mammalian trypanosomiases.     

Further evidence for the existence of a membrane potential in Trypanosoma brucei brucei

The distribution of 137Cs+, in the presence of valinomycin, has been used to measure the magnitude of the membrane potential (delta psi) in bloodstream forms of Trypanosoma brucei brucei. Values of the delta psi falling in the range -100 mV to -160 mV were observed and the maintenance of this delta psi was sensitive to certain ionophores and protonophores.     

A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport.

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.     

Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei.

Trypanosome tubulin was purified to near homogeneity by chromatography on DEAE-Sephadex, Amicon filtration and assembly-disassembly in vitro. Polymerization of the tubulin in vitro yielded long, structurally normal, microtubules and some sheet structures on addition of GTP and incubation at 37 degrees C, in either the presence or the absence of Mg2+. Tubulin assembly was disrupted by glycerol and a selection of microtubule-reactive drugs. Immunological analysis of the purified tubulin revealed tyrosinated and acetylated alpha-tubulin, in addition to defining the migration characteristics of the alpha- and beta-tubulin on one-dimensional SDS/polyacrylamide gels. This is the first isolation of trypanosome tubulin with the ability to form structurally normal microtubules independent of the addition of taxol or nucleating microtubule fragments. The development of the purification procedure thus provides an important step for subsequent study of microtubule-associated protein-tubulin and plasma-membrane-microtubule cytoskeleton interactions of trypanosomes, and increases the potential for development of tubulin-based anti-trypanosome drugs.     

A proteomic analysis of arsenical drug resistance in Trypanosoma brucei

We have undertaken 2-DE and MS to identify proteins associated with arsenical drug resistance in Trypanosoma brucei. This parasite causes sleeping sickness in humans, and arsenical drug resistance is a significant potential problem. Comparative analysis of approximately 2000 spots resolved by 2-DE in the soluble proteomes of drug-sensitive and drug-resistant isogenic lines of T. brucei identified a protein spot whose absence associated with resistance to the arsenical drug, Cymelarsan. MS matched this protein to an identical pair of tandem genes Tb09.211.0120 and 0130 that encode a putative nascent polypeptide associated complex subunit. This protein also occurs as an isoform located in both resistant and sensitive lines at a similar molecular weight, but different pI. The difference between isogenic lines was confirmed by Western blot using an antibody against recombinant protein. Both genes were identical in sequence between drug-sensitive and drug-resistant lines and both were transcribed as determined by RT-PCR. We postulate that the missing protein isoform arose due to the lack of a PTM.     

Ornithine Decarboxylase in Trypanosoma brucei brucei: Evidence for Selective Toxicity of Difluoromethylornithine1

ABSTRACT. Activity of ornithine decarboxylase, the major rate limiting enzyme of polyamine biosynthesis, was determined in bloodstream trypomastigotes of Trypanosoma brucei brucei. The enzyme required pyridoxal-5′-phosphate, dithiothreitol and EDTA for optimal activity. Several properties of the enzyme were investigated and compared to the mammalian enzyme. Most notably, the parasite enzyme was >60-fold more sensitive to the inhibitor DL-α-difluoromethylornithine than its mammalian counterpart, thus making it an attractive target for chemotherapy.     

PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei,Is Required for Efficient Maturation of Infection

The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T₃₀₅), a modification that depends on the presence of TbMAPK4. Mutation of T₃₀₅ to alanine (T₃₀₅A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T₃₀₅D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein''s structure and the effects of mutation of T₃₀₅ on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite''s decision to divide, differentiate or migrate.