Substrate Preference in a Strain of Megasphaera elsdenii, a Ruminal Bacterium, and Its Implications in Propionate Production and Growth Competition

The NIAH 1102 strain of Megasphaera elsdenii utilized lactate in preference to glucose when the two substrates were present. Even when lactate was supplied to cells fermenting glucose, the cells switched substrate utilization from glucose to lactate and did not utilize glucose until lactate decreased to a low concentration (1 to 2 mM). Since substrate utilization was shifted gradually without intermittence, typical diauxic growth was not seen. The cyclic AMP content did not rise markedly with the shift in substrate utilization, suggesting that this nucleotide is not involved in the regulation of the shift. It was unlikely that propionate was produced from glucose, which was explicable by the fact that lactate racemase activity dropped rapidly with the exhaustion of lactate and cells actively fermenting glucose did not possess this enzyme. A coculture experiment indicated that M. elsdenii NIAH 1102 is overcome by Streptococcus bovis JB1 in the competition for glucose, mainly because M. elsdenii NIAH 1102 is obliged to utilize lactate produced by S. bovis JB1; i.e., glucose utilization by M. elsdenii NIAH 1102 is suppressed by the coexistence of S. bovis JB1.     

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8 g/L vs. 19.4 g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28 g/L·h vs. 0.16 g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53 g/L·h vs. 0.26 g/L·h) and yield (0.32 g/g vs. 0.28 g/g). When the initial total sugar concentration was ~120 g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4 g/L, yield of 0.43 g/g sugar consumed, productivity of 0.87 g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.     

Differentiation of hemagglutinins in tissues infected withChlamydia

Crude, soluble, chlamydial hemagglutinin was prepared from allantoic fluid harvested from embryonated chick eggs and the supernatant fluid of mouse L cells infected with eitherChalamydia psittaci strain 6BC orChlamydia trachomatis strain TW-3. Control nonhemagglutinating specimens of uninfected allantoic fluid and mouse L cells were also prepared. The six preparations were separated by ether-ethanol extraction into lipid-rich and lipid-depleted fractions. Complement-fixing activity was found in the lipid-rich (but not in the lipid-depleted) fraction of infected preparations. In contrast, lipid-rich fractions of infected and uninfected preparations had similar agglutinating activity when sensitive erythrocytes of white Leghorn chickens were used. The lipid-rich fraction of infected and uninfected preparations was separated by thin-layer chromatography (TLC) into seven components with similarR _f values, hemagglutinating patterns, and chemical composition (lipid, protein, and carbohydrate). The highest hemagglutination titers of normal and infected preparations were found in a TLC fraction with similarR _f values and contained lipid, protein, and carbohydrate. This TLC fraction fromC. psittaci 6BC preparations was used in hemagglutination-inhibition studies. The results indicated that chlamydial hemagglutinin extracted by ether-ethanol and separated by TLC contained, in addition to specific hemagglutinin, nonspecific tissue-lipid hemagglutinin(s) identical to that found in normal preparations.     

Multi Locus Sequence Typing of Chlamydia Reveals an Association between Chlamydia psittaci Genotypes and Host Species

Chlamydia comprises a group of obligate intracellular bacterial parasites responsible for a variety of diseases in humans and animals, including several zoonoses. Chlamydia trachomatis causes diseases such as trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Chlamydia pneumoniae is a common cause of community-acquired respiratory tract infections. Chlamydia psittaci, causing zoonotic pneumonia in humans, is usually hosted by birds, while Chlamydia abortus, causing abortion and fetal death in mammals, including humans, is mainly hosted by goats and sheep. We used multi-locus sequence typing to asses the population structure of Chlamydia. In total, 132 Chlamydia isolates were analyzed, including 60 C. trachomatis, 18 C. pneumoniae, 16 C. abortus, 34 C. psittaci and one of each of C. pecorum, C. caviae, C. muridarum and C. felis. Cluster analyses utilizing the Neighbour-Joining algorithm with the maximum composite likelihood model of concatenated sequences of 7 housekeeping fragments showed that C. psittaci 84/2334 isolated from a parrot grouped together with the C. abortus isolates from goats and sheep. Cluster analyses of the individual alleles showed that in all instances C. psittaci 84/2334 formed one group with C. abortus. Moving 84/2334 from the C. psittaci group to the C. abortus group resulted in a significant increase in the number of fixed differences and elimination of the number of shared mutations between C. psittaci and C. abortus. C. psittaci M56 from a muskrat branched separately from the main group of C. psittaci isolates. C. psittaci genotypes appeared to be associated with host species. The phylogentic tree of C. psittaci did not follow that of its host bird species, suggesting host species jumps. In conclusion, we report for the first time an association between C. psittaci genotypes with host species.     

Seroepidemiology of Feline Chlamydiosis by Microimmunofluorescence Assay with Multiple Strains as Antigens

The prevalence of anti-chlamydia antibodies was examined in 232 cat sera collected in 1985 and from 1993 to 1995 from laboratories and veterinary hospitals located in 11 prefectures of Japan. The antibodies were determined by an indirect microimmunofluorescence test using six strains of feline Chlamydia: one strain each of avian- and guinea pig-derived C. psittaci and one strain each of C. pecorum, C. pneumoniae and C. trachomatis. Positive rates of IgG antibodies to chlamydiae were 34.4% in 1985 and 16.5–21.4% from 1993 to 1995. Positive rates of IgM antibodies to chlamydiae were 8.2% in 1985 and 6.6–14.3% from 1993 to 1995. Variations in antibody reactivity to the different feline strains were observed. The results suggest the wide prevalence of chlamydial infection in cats in Japan, and antigenic diversity in the feline strains of C. psittaci.     

Plaque Formation in Chick Embryo Fibroblast Cells by Chlamydia Isolated From Avian and Mammalian Sources

Fifteen strains of chlamydial agents, 2 Chlamydia trachomatis and 13 Chlamydia psittaci, were tested for plaque formation on chick embryo fibroblast cells. C. trachomatis strains did not form plaques; 12 strains of C. psittaci did form plaques and 1 strain did not. The distribution of plaque sizes with C. psittaci strains was studied and it was found that the 12 strains could be tentatively grouped into three plaquing types: small (<0.50 mm), intermediate (0.50 to 0.90 mm), and large (>0.90 mm). Small plaques were predominantly associated with strains M56, ET, CP3, CT4, EBA, CS1, and CT2. Intermediate size plaques were predominantly associated with strains NJ1, VT1, and H81. Large plaques were predominantly associated with strains WC and OG. Plaque size appears to be related to virulence of Chlamydia for laboratory animals.     

Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.     

A New Role of the Complement System: C3 Provides Protection in a Mouse Model of Lung Infection with Intracellular Chlamydia psittaci

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3^−/− mice succumbing to infection after day 9. At a low infective dose, C3^−/− mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3^−/− mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci.     

Lactose utilization in a cryptic strainEscherichia coli ML 35

The permease-negative strainE. coli ML 35 utilized lactose at 30°C 5–6 times more slowly than glucose or permease-positive strains. The rate of utilization could be raised by cultivating the cells in the presence of streptomycin, acriflavine or actinomycin C. Phenethyl alcohol and the basic proteins ribonuclease, protamine or histone stimulated lactose utilization by washed cells of the same strain not precultivated in the presence of these substances. Ribonuclease did not influence lactose utilization by the permease-positive strain ML 308. Under the given experimental conditions, the presence of basic proteins did not cause β-galactosidase to be released into the medium as a result of lysis of the cells. Streptomycin and ribonuclease did not influence TMG transport into the cells. Basic proteins increased the rate of ONPG hydrolysis by intact cells almost to the level observed in the permease-positive strain. Guanidine, spermine, spermidine, putrescine and cadaverine did not influence lactose utilization by strain ML 35. Spermine antagonized the stimulant effect of ribonuclease or histone on lactose utilization. The experiments were supplemented by comparing the effect of temperature on lactose and glucose utilization by a permease-positive and-negative strain ofE. coli.     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37–73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.     

Construction of Recombinant HVT Expressing PmpD,and Immunological Evaluation against Chlamydia psittaci and Marek’s Disease Virus

Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek’s Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek’s disease was developed and examined. The 5''-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek’s Disease virus (MDV) RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.     

Expression of bacterial xylose isomerase in Saccharomyces cerevisiae under galactose supplemented condition

We constructed recombinant Saccharomyces cerevisiae harboring the xylose isomerase (XI) gene isolated from Clostridium phytofermentans to metabolize xylose and use it as a carbon and energy source. In this study, the effect of supplementation using co-substrate such as glucose or galactose on xylose utilization was studied in recombinant S. cerevisiae. Glucose, which is transported with high affinity by the same transport system as is xylose, was not affected by the heterologous expression of XI, thus xylose utilization was not observed in recombinant S. cerevisiae. However, supplemental galactose added to the recombinant S. cerevisiae stimulated xylose utilization as well as the expression of XI protein. Recombinant S. cerevisiae consumed up to 23.48 g/L of xylose when grown in media containing 40 g/L of xylose and supplemented with 20 g/L of galactose. These cells also produced 15.89 g/L of ethanol. Therefore, expression of the bacterial XI in recombinant S. cerevisiae was highly induced by the addition of supplemental galactose as a co-substrate with xylose, and supplemented galactose enabled the yeast strain to grow on xylose and ferment xylose to ethanol.     

Chlamydia buteonis,a new Chlamydia species isolated from a red-shouldered hawk

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHA^T as the type strain.     

The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens

Background

Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.

Results

A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.

Conclusions

This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.     

Glucose and lactic acid trends in suspension cultures of two established mammalian cell strains in chemically defined media

In replicated 30 to 40-ml suspension cultures of rapidly proliferating monkey kidney cells of a comparatively fragile strain, the rates of glucose utilization and lactic acid accumulation averaged about 400 micrograms and 110 micrograms per 10⁶ cells per day respectively, with average molar La/Gl ratios of 0.48. These two rates of glucose utilization and lactic acid accumulation were about 4 × and 10 × as high as the corresponding rates in comparable cultures of the hardier strain 2071-L mouse fibroblasts under the same conditions, with average molar La/Gl ratios of 0.16. In comparable but nonproliferating suspension cultures of the same strain of monkey kidney cells, during about 3 weeks the rates were extremely high, with about 710 micrograms glucose utilized and 445 micrograms lactic acid accumulated per 10⁶ cells per day, with average molar La/Gl ratios of 1.37. The rates of glucose uptake and lactic acid accumulation were higher in the nonproliferating cultures aerated with 5% CO₂ in air than in those aerated with 10% CO₂ in air. This difference was associated with pH, which was higher in the former group. It was concluded that with this fragile strain of monkey Kidney cells(1) in nonproliferating cultures the cells were metabolizing actively but with a marked tendency to higher La/Gl ratios, (2) in the proliferating cultures the high rates of glucose utilization and lactic acid accumulation were definitely not directly correlated with the rate of growth, and (3) in none of the cultures was the amount of glucose remaining in the fluid at fluid changes so low as to have been a limiting factor. Information in the literature concering glucose utilization and lactic acid production by cells vitro is voluminous and in some respects contradictory. In the present study the rates were unexpectedly high for the monkey kidney cells, particularly those in the otherwise apparently inactive nonproliferating cultures. The data seem to be unique, in that an established strain of cells in chemically defined medium in suspension cultures has been characterized for these metabolic parameters in both proliferating cultures and in equivalent nonproliferating cultures under directly comparable conditions. The concept was developed that since these monkey kidney cells are obviously more fragile than the other cells examined, the complex physical stresses imposed upon these cells in agitated cultures can be modified and lessened in order to permit growth. Lessening of such mechanical stress waa brought about in several ways, of which only the smaller flask size seemed to be at least partly effective. Increasing either the concentration or the viscosity type of Methocel waa not effective.     

The cellular ceramide transport protein CERT promotes Chlamydia psittaci infection and controls bacterial sphingolipid uptake

Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9‐mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT‐knockout cells indicating that C. psittaci can exploit CERT‐independent sphingolipid uptake pathways. Moreover, the CERT‐specific inhibitor HPA‐12 strongly diminished sphingolipid transport to inclusions of infected CERT‐knockout cells, suggesting that other HPA‐12‐sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome.     

Neutrophils reduce the parasite burden in Leishmania (Leishmania) amazonensis-infected macrophages

Background

Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L) major, whereas less information is available for L. (L) amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L.) amazonensis (C3H/HePas). In contrast, the susceptible strain (BALB/c) displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L.) amazonensis-infected macrophages in vitro.

Methodology/Principal Findings

Mouse peritoneal macrophages infected with L. (L.) amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1) intracellular parasites were efficiently destroyed in the co-cultures; 2) the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas) or susceptible (BALB/c) to L. (L.) amazonensis; 3) parasite destruction did not require contact between infected macrophages and neutrophils; 4) tumor necrosis factor alpha (TNF-α), neutrophil elastase and platelet activating factor (PAF) were involved with the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.

Conclusions/Significance

The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.