Amoebicidal activity of essential oils and essential oil-based microemulsions of Aloysia citrodora Ortega ex Pers., Cymbopogon winterianus Jowitt ex Bor and Ocimum gratissimum L. against Acanthamoeba polyphaga trophozoites.

AIMS: Evaluate the in vitro efficacy of the essential oils derived from Aloysia citrodora (Verbenaceae), Cymbopogon winterianus (Poaceae), and Ocimum gratissimum (Lamiaceae) against Acanthamoeba polyphaga trophozoites. Additionally, microemulsions formulated with these essential oils, along with their major components, were analyzed. METHODS AND RESULTS: The prepared microemulsions were characterized using polarized light microscopy and rheological techniques. The amoebicidal activity was determined by measuring the inhibitory concentration (IC50). Flow cytometry was employed to detect membrane damage and alterations in trophozoites size. The results revealed transparent and thermodynamically stable microemulsions. The essential oil from O. gratissimum exhibited a lower IC50, with values of 280.66 µg mL-1 and 47.28 µg mL-1 after 24 and 48 hours, respectively. When microemulsions containing essential oils were tested, the IC50 values exhibited a reduction of over 80% after 24 hours. Particularly, eugenol, a constituent of the O. gratissimum essential oil, displayed higher amoebicidal activity. The essential oils also caused damage to the cell membrane, resulting in the subsequent death of the trophozoites. CONCLUSIONS: The EOs of A. citrodora, C. winterianus, and O. gratissimum and their microemulsions showed antiparasitic effect against A. polyphaga trophozoites, representing promising alternatives for the treatment of diseases caused by this protozoan.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Transient internalization of Campylobacter jejuni in Amoebae enhances subsequent invasion of human cells.

The ubiquitous unicellular eukaryote, Acanthamoeba, is known to play a role in the survival and dissemination of Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide and is a major public health problem. The ability of C. jejuni to interact and potentially invade epithelial cells is thought to be key for disease development in humans. We examined C. jejuni grown under standard laboratory conditions, 11168HCBA with that harvested from within Acanthamoeba castellanii (11168HAC/CBA) or Acanthamoeba polyphaga (11168HAP/CBA), and compared their ability to invade different cell lines. C. jejuni harvested from within amoebae had a ~3.7-fold increase in invasiveness into T84 human epithelial cells and a striking ~11-fold increase for re-entry into A. castellanii cells. We also investigated the invasiveness and survivability of six diverse representative C. jejuni strains within Acanthamoeba spp., our results confirm that invasion and survivability is likely host-cell-dependent. Our survival assay data led us to conclude that Acanthamoeba spp. are a transient host for C. jejuni and that survival within amoebae pre-adapts C. jejuni and enhances subsequent cell invasion. This study provides new insight into C. jejuni interactions with amoebae and its increased invasiveness potential in mammalian hosts.

Genomic Signatures Among Acanthamoeba polyphaga Entoorganisms Unveil Evidence of Coevolution.

The definition of a genomic signature (GS) is "the total net response to selective pressure". Recent isolation and sequencing of naturally occurring organisms, hereby named entoorganisms, within Acanthamoeba polyphaga, raised the hypothesis of a common genomic signature despite their diverse and unrelated evolutionary origin. Widely accepted and implemented tests for GS detection are oligonucleotide relative frequencies (OnRF) and relative codon usage (RCU) surveys. A common pattern and strong correlations were unveiled from OnRFs among A. polyphaga's Mimivirus and virophage Sputnik. RCU showed a common A-T bias at third codon position. We expanded tests to the amoebal mitochondrial genome and amoeba-resistant bacteria, achieving strikingly coherent results to the aforementioned viral analyses. The GSs in these entoorganisms of diverse evolutionary origin are coevolutionarily conserved within an intracellular environment that provides sanctuary for species of ecological and biomedical relevance.

Acanthamoeba keratitis in Mexico: Report of a clinical case and importance of sensitivity assays for a better outcome.

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection. The early symptoms include redness, pain, photophobia and intense tearing. Chronic infection usually progresses to stromal inflammation, ring ulcers, corneal opacification and hypopyon. Here we document an AK case in a high myopic 38-year-old woman from Mexico City, with a history of wearing contact lenses while swimming. Corneal scrapes cultures were positive only for amoebae, consequently a treatment including netilmicin 0.3% and oral itraconazole 100 mg/12 h was prescribed. The infection was resolved after 8 months, leaving a slight leucoma outside the visual axis, with a visual acuity of 20/150. In the laboratory, the amoebic isolate was axenized in PYG medium, with an optimal growth at 30 °C, and was identified morphologically as Acanthamoeba polyphaga according to the taxonomic criteria of Page (1988) and placed in the T4 group by genotyping. The virulence of this strain (40%) was determined by intranasal inoculation of 1 × 106/20 µl trophozoites in BALB/c mice recovering from brain, proving their invasion ability and by the interaction with monolayers of epithelial cells of the established MDCK line of canine kidney origin (1:2 ratio of interaction), at 1, 3, 6, 8 and 24 h; trophozoites migrated to cell junctions inducing few lytic zones. In addition to the biological characterization, in vitro drug sensitivity tests were performed using chlorhexidine, itraconazole, netilmicin and voriconazole. Results revealed that voriconazole was the most effective compound. A. polyphaga remains as one of the most frequently isolated species producing AK. The treatment of AK case using netilmicin and oral itraconazole solved the disease, but the healing process was wide-ranging (8 months). The use of voriconazole and chlorhexidine may be an alternative treatment of future AK cases in Mexico.

Fate of internalized Campylobacter jejuni and Mycobacterium avium from encysted and excysted Acanthamoeba polyphaga.

Association of the water- and foodborne pathogen Campylobacter jejuni with free-living Acanthamoeba spp. trophozoites enhances C. jejuni survival and resistance to biocides and starvation. When facing less than optimal environmental conditions, however, the Acanthamoeba spp. host can temporarily transform from trophozoite to cyst and back to trophozoite, calling the survival of the internalized symbiont and resulting public health risk into question. Studies investigating internalized C. jejuni survival after A. castellanii trophozoite transformation have neither been able to detect its presence inside the Acanthamoeba cyst after encystation nor to confirm its presence upon excystation of trophozoites through culture-based techniques. The purpose of this study was to detect C. jejuni and Mycobacterium avium recovered from A. polyphaga trophozoites after co-culture and induction of trophozoite encystation using three different encystation methods (Neff's medium, McMillen's medium and refrigeration), as well as after cyst excystation. Internalized M. avium was used as a positive control, since studies have consistently detected the organism after co-culture and after host excystation. Concentrations of C. jejuni in A. polyphaga trophozoites were 4.5 × 105 CFU/ml, but it was not detected by PCR or culture post-encystation. This supports the hypothesis that C. jejuni may be digested during encystation of the amoebae. M. avium was recovered at a mean concentration of 1.9 × 104 from co-cultured trophozoites and 4.4 × 101 CFU/ml after excystation. The results also suggest that M. avium recovery post-excystation was statistically significantly different based on which encystation method was used, ranging from 1.3 × 101 for Neff's medium to 5.4 × 101 CFU/ml for refrigeration. No M. avium was recovered from A. polyphaga cysts when trophozoites were encysted by McMillen's medium. Since C. jejuni internalized in cysts would be more likely to survive harsh environmental conditions and disinfection, a better understanding of potential symbioses between free-living amoebae and campylobacters in drinking water distribution systems and food processing environments is needed to protect public health. Future co-culture experiments examining survival of internalized C. jejuni should carefully consider the encystation media used, and include molecular detection tools to falsify the hypothesis that C. jejuni may be present in a viable but not culturable state.

CmeABC Multidrug Efflux Pump Contributes to Antibiotic Resistance and Promotes Campylobacter jejuni Survival and Multiplication in Acanthamoeba polyphaga.

Campylobacter jejuni is a foodborne pathogen that is recognized as the leading cause of human bacterial gastroenteritis. The widespread use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter In addition to a role in multidrug resistance (MDR), the Campylobacter CmeABC resistance-nodulation-division (RND)-type efflux pump may be involved in virulence. As a vehicle for pathogenic microorganisms, the protozoan Acanthamoeba is a good model for investigations of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and Acanthamoeba polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga but is dispensable for biofilm formation and motility.IMPORTANCE The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. In this study, we found that Campylobacter jejuni was able to survive and to multiply inside Acanthamoeba polyphaga; since these microorganisms can coexist in the same environment (e.g., on poultry farms), the latter may increase the risk of infection with Campylobacter Our data suggest that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump plays a role in bacterial survival within amoebae. Furthermore, we demonstrated synergistic effects of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role in both the antibiotic resistance and the virulence of C. jejuni, the CmeABC MDR efflux pump could be considered a good target for the development of antibacterial drugs against this pathogen.

Interaction Between Methicillin-Resistant Staphylococcus aureus (MRSA) and Acanthamoeba polyphaga.

The interactions that occur between bacteria and amoebae can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. When these organisms share the same environment, it can result in some changes in the growth of organisms, in adaptation patterns, in morphology, development or even in their ability to synthesize proteins and other substances. In this study, the interaction between Acanthamoeba polyphaga and Staphylococcus aureus (MRSA) was evaluated using a co-culture model at different incubation times. The results showed that 89% of amoebic cells remained viable after contact with the bacteria. The bacterial isolate was visualized inside the amoeba through confocal microscopy and fluorescence for up to 216 h of co-cultivation. The lysate of amoebic culture increased the growth of S. aureus (MRSA), and the effect of supernatant of culture inhibited bacterial growth over the incubation times, suggesting that A. polyphaga produced some metabolite, that inhibited the growth of bacteria. Moreover, the encystment of the A. polyphaga was increased by the bacteria presence. The results show that A. polyphaga and S. aureus interaction may have an important influence on survival of both, and specially indicate a possible effect on the metabolics characteristics each other.

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae.

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

Amebicidal activity of the essential oils of Lippia spp. (Verbenaceae) against Acanthamoeba polyphaga trophozoites.

Amoebic keratitis and granulomatous amoebic encephalitis are caused by some strains of free-living amoebae of the genus Acanthamoeba. In the case of keratitis, one of the greatest problems is the disease recurrence due to the resistance of parasites, especially the cystic forms, to the drugs that are currently used. Some essential oils of plants have been used as potential active agents against this protist. Thus, the aim of this study was to determine the amebicidal activity of essential oils from plants of the genus Lippia against Acanthamoeba polyphaga trophozoites. To that end, 8 × 10(4) trophozoites were exposed for 24 h to increasing concentrations of essential oils from Lippia sidoides, Lippia gracilis, Lippia alba, and Lippia pedunculosa and to their major compounds rotundifolone, carvone, and carvacrol. Nearly all concentrations of oils and compounds showed amebicidal activity. The IC50 values for L. sidoides, L. gracilis L. alba, and L. pedunculosa were found to be 18.19, 10.08, 31.79, and 71.47 µg/mL, respectively. Rotundifolone, carvacrol, and carvone were determined as the major compounds showing IC50 of 18.98, 24.74, and 43.62 µg/mL, respectively. With the exception of oil from L. alba, the other oils evaluated showed low cytotoxicity in the NCI-H292 cell line. Given these results, the oils investigated here are promising sources of compounds for the development of complementary therapy against amoebic keratitis and granulomatous amoebic encephalitis and can also be incorporated into cleaning solutions to increase their amebicidal efficiency.

Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.

A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism.

EVALUATION OF THE CYSTICIDAL ACTIVITY OF CHLOROCRESOL AGAINST ACANTHAMOEBA POLYPHAGA.

Chlorocresol has antibacterial and antifungal properties, yet its effectiveness in eradicating Acanthamoeba spp. remains unexplored. Acanthamoeba species trophozoites are usually sensitive to biocides, whereas cysts tend to be more resistant. This study aimed to evaluate the cysticidal activity of chlorocresol against Acanthamoeba polyphaga. Chlorocresol concentrations of 0.02, 0.04, and 0.08% were prepared and A. polyphaga cysts were incubated at room temperature (28-37 C) for 1, 24, 48, and 72 hr at each concentration. Cyst viability was evaluated using trypan blue staining and the percentage of nonviable cysts was calculated. For qualification assays, treated cysts were cultured on nonnutrient agar medium coated with Escherichia coli, incubated at 30 C, observed under a stereomicroscope for 30 days, and inoculated into peptone-yeast extract-glucose medium at 30 C for 72 hr. The results revealed that the A. polyphaga cysts were susceptible to 0.02, 0.04, and 0.08% chlorocresol. Chlorocresol made a significant difference in viability (P < 0.001) compared with the nontreated control for the same incubation time. This is the first study to examine the efficacy of chlorocresol against A. polyphaga cysts and it was highly effective. Chlorocresol could thus serve as an alternative chemical disinfectant for the eradication of A. polyphaga cysts as well as a prophylactic against transmission of other pathogenic microorganisms for which Acanthamoeba species can act as a carrier.

In Vitro Effectiveness of Soft Contact Lens Solutions Available on the Dutch Market against Acanthamoeba Species.

Acanthamoeba keratitis is almost universally associated with contact lens (CL) use. Until today, however, CL solution manufacturing protocols lack testing of anti-amoebic activity. This study investigates the effectiveness of CL solutions available on the Dutch market against trophozoites and cysts of Acanthamoeba castellanii and Acanthamoeba polyphaga. Sixteen CL solutions were tested: 13 multiple purpose solutions (MPS), 2 hydrogen peroxidase solutions (HPS) and 1 povidone-iodine-based solution (PIS). The Spearman-Karber (SK) log reduction method and an XTT colorimetric assay were used to evaluate the effectiveness at the manufacturer's minimum recommended disinfection time (MMRDT) and after eight hours. At the MMRDT, one MPS showed an SK mean log reduction (MLR) of >3.0 against A. castellanii trophozoites. Two additional MPS and both HPS reached this threshold after eight hours. The SK MLR values for A. polyphaga trophozoites were between 1 and 3 at all time points. Using the XTT colorimetric assay, only HPS 1 showed >99.9% reduction (equivalent to 3 log reduction) in metabolic activity of A. castellanii trophozoites after eight hours. For A. polyphaga, both HPS and PIS showed a metabolic reduction of >99.9% after eight hours. Cysts were resistant against all solutions. We conclude that following the manufacturer's guidelines, few solutions provide sufficient effectiveness against Acanthamoeba trophozoites and none against cysts. The results underline the importance of adequate hygiene when handling CLs.

Virus géants associés aux amibes.

The discovery of Acanthamoeba polyphaga Mimivirus, a giant amoeba-associated virus less than a decade ago, has shattered the definition of what is a virus. With an exceptional size of 500 nm, a genome of more than 1 Mb, a particle containing both DNA and RNA, possibility to be infected by another virus are unusual characteristics that make it immediately exceptional. Since then, several giant viruses have been isolated such as Marseillevirus. It is highly probable that closely related viruses will be isolated as it is now understood that they were not previously isolated because they are not filterable. Environmental metagenomic studies suggest that these viruses are ubiquitous. The discovery of virophages, small viruses able to infect Mimivirus as bacteriophage infect bacteria, fuel the debate about the nature of viruses and their place in the evolution of life. Current works, especially genome sequencing of these new viruses, open new perspectives about evolution and lateral gene transfer with their host but also with bacteria and other viruses. The knowledge about these viruses is only at the first step and increasing interest for it suggests that we are only at the dawn of the understanding of their role in evolution and ecosystems regulation.

Mimiviruses: Giant viruses with novel and intriguing features (Review).

The Mimivirus is a giant virus that infects amoebae and was long considered to be a bacterium due to its size. The viral particles are composed of a protein capsid of ~500 nm in diameter, which is enclosed in a polysaccharide layer in which ~120­140 nm long fibers are embedded, resulting in an overall diameter of 700 nm. The virus has a genome size of 1.2 Mb DNA, and surprisingly, replicates only in the cytoplasm of the infected cells without entering the nucleus, which is a unique characteristic among DNA viruses. Their existence is undeniable; however, as with any novel discovery, there is still uncertainty concerning their pathogenicity mechanisms in humans and the nature of the Mimivirus virophage resistance element system (MIMIVIRE), a term given to describe the immune network of the Mimivirus, which closely resembles the CRISPR­Cas system. The scope of the present review is to discuss the recent developments derived from structural and functional studies performed on the distinctive characteristics of the Mimivirus, and from studies concerning their putative clinical relevance in humans.

Inhibition of Acanthamoeba polyphaga by chlorhexidine-mediated oxidative stress response.

OBJECTIVES: Acanthamoeba keratitis is a severe corneal infection caused by a ubiquitous opportunistic protozoan pathogen known as acanthamoeba. For the last decade, the approach to treating this infection typically includes the use of polyhexamethylene biguanide (0.02%) and/or chlorhexidine (Chx) (0.02%). Although chlorhexidine is reportedly effective, its mode of action towards this type of cell is not clear. The aim of this work was to study the effect of chlorhexidine on the oxidative status of Acanthamoeba polyphaga. METHODS: The effect of chlorhexidine (Chx) on the oxidative state of Acanthamoeba polyphaga was studied using different antiradical methods including ABTS, DPPH and FRAP and measuring the activity of a couple of antioxidant enzyme namely SOD, NADH-FRD and SDH. RESULTS: The chlorhexidine was able to induce oxidative imbalance in cells by over expression of reactive oxygen species and/or inhibiting the antioxidant enzymes. In addition to enhancing the antiradical activity in response to oxidative stress, the present drug was able to reduce the activity of two antioxidant enzymes, superoxide dismutase (SOD) and reduced flavin adenine dinucleotide-fumarate reductase (NADH-FRD), to 30% and 40%, respectively. CONCLUSIONS: We could observe an increase of the antiradical capacity of cell's lysate supernatant, to cope with the overproduction of ROS. The imbalance state The inhibition of both SOD and NADH-FRD activities could have a major role in cell oxidative imbalance.

Synthesis of chalcone derivatives by Claisen-Schmidt condensation and in vitro analyses of their antiprotozoal activities.

Chalcone is a molecule with known biological activities. Based on this, a series of chalcone derivatives bearing methyl, phenyl or furanyl substituents at different positions of A and B rings were synthesised, characterised, and evaluated regarding antiprotozoal activity. Molecules were synthesised via base catalyzed Claisen-Schmidt condensation and characterised by IR and NMR spectral data. Antiprotozoal activity against Phytomonas serpens, Leishmania amazonensis and Acanthamoeba polyphaga was performed. All compounds inhibited more than 50% of the growth of P. serpens while five had this effect on L. amazonensis and all of them no more than 35% of inhibition on A. polyphaga. Remarkably interesting antiprotozoal effects were recorded with compound 5, with IC50 of 1.59 µM for P. serpens and 11.49 µM for L. amazonensis. The addition of a naphthyl group to the B ring can be postulated to be the cause of the 10 times increase observed in its trypanocidal activity.

Waterborne Isolates of Campylobacter jejuni Are Able to Develop Aerotolerance, Survive Exposure to Low Temperature, and Interact With Acanthamoeba polyphaga.

Campylobacter jejuni is regarded as the leading cause of bacterial gastroenteritis around the world. Even though it is generally considered to be a sensitive microaerobic pathogen, it is able to survive in the environment outside of the intestinal tract of the host. This study aimed to assess the impact of selected environmental parameters on the survival of 14 C. jejuni isolates of different origins, including 12 water isolates. The isolates were tested for their antibiotic resistance, their ability to survive at low temperature (7°C), develop aerotolerance, and to interact with the potential protozoan host Acanthamoeba polyphaga. The antibiotic susceptibility was determined by standard disk diffusion according to EUCAST. Out of the 14 isolates, 8 were resistant to ciprofloxacin (CIP) and 5 to tetracycline (TET), while only one isolate was resistant to erythromycin (ERY). Five isolates were resistant to two different antibiotic classes. Tetracycline resistance was only observed in isolates isolated from wastewater and a clinical sample. Further, the isolates were tested for their survival at 7°C under both aerobic and microaerobic conditions using standard culture methods. The results showed that under microaerobic conditions, all isolates maintained their cultivability for 4 weeks without a significant decrease in the numbers of bacteria and variation between the isolates. However, significant differences were observed under aerobic conditions (AC). The incubation led to a decrease in the number of cultivable cells, with complete loss of cultivability after 2 weeks (one water isolate), 3 weeks (7 isolates), or 4 weeks of incubation (6 isolates). Further, all isolates were studied for their ability to develop aerotolerance by repetitive subcultivation under microaerobic and subsequently AC. Surprisingly, all isolates were able to adapt and grow under AC. As the last step, 5 isolates were selected to evaluate a potential protective effect provided by A. polyphaga. The cocultivation of isolates with the amoeba resulted in the survival of about 40% of cells treated with an otherwise lethal dose of gentamicin. In summary, C. jejuni is able to adapt and survive in a potentially detrimental environment for a prolonged period of time, which emphasizes the role of the environmental transmission route in the spread of campylobacteriosis.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Biochemical analysis of a sugar 4,6-dehydratase from Acanthamoeba polyphaga Mimivirus.

The exciting discovery of the giant DNA Mimivirus in 2003 challenged the conventional description of viruses in a radical way, and since then, dozens of additional giant viruses have been identified. It has now been demonstrated that the Mimivirus genome encodes for the two enzymes required for the production of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, namely a 4,6-dehydratase and an aminotransferase. In light of our long-standing interest in the bacterial 4,6-dehydratases and in unusual sugars in general, we conducted a combined structural and functional analysis of the Mimivirus 4,6-dehydratase referred to as R141. For this investigation, the three-dimensional X-ray structure of R141 was determined to 2.05 Å resolution and refined to an R-factor of 18.3%. The overall fold of R141 places it into the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Whereas its molecular architecture is similar to that observed for the bacterial 4,6-dehydratases, there are two key regions where the polypeptide chain adopts different conformations. In particular, the conserved tyrosine that has been implicated as a catalytic acid or base in SDR superfamily members is splayed away from the active site by nearly 12 Å, thereby suggesting that a major conformational change must occur upon substrate binding. In addition to the structural analysis, the kinetic parameters for R141 using either dTDP-d-glucose or UDP-d-glucose as substrates were determined. Contrary to a previous report, R141 demonstrates nearly identical catalytic efficiency with either nucleotide-linked sugar. The data presented herein represent the first three-dimensional model for a viral 4,6-dehydratase and thus expands our understanding of these fascinating enzymes.