Small noncoding RNAs (ncRNAs) are involved in many important physiological functions in pathogenic microorganisms. Previous studies have identified the presence of noncoding RNAs in the major zoonotic pathogen Campylobacter jejuni; however, few have been functionally characterized to date. CjNC110 is a conserved ncRNA in C. jejuni, located downstream of the luxS gene, which is responsible for the production of the quorum sensing molecule autoinducer-2 (AI-2). In this study, we utilized strand specific high-throughput RNAseq to identify potential targets or interactive partners of CjNC110 in a sheep abortion clone of C. jejuni. These data were then utilized to focus further phenotypic evaluation of the role of CjNC110 in motility, autoagglutination, quorum sensing, hydrogen peroxide sensitivity, and chicken colonization in C. jejuni. Inactivation of the CjNC110 ncRNA led to a statistically significant decrease in autoagglutination ability as well as increased motility and hydrogen peroxide sensitivity compared to the wild-type. Extracellular AI-2 detection was decreased in ΔCjNC110; however, intracellular AI-2 accumulation was significantly increased, suggesting a key role of CjNC110 in modulating the transport of AI-2. Notably, ΔCjNC110 also showed a decreased ability to colonize chickens. Complementation of CjNC110 restored all phenotypic changes back to wild-type levels. The collective results of the phenotypic and transcriptomic changes observed in our data provide valuable insights into the pathobiology of C. jejuni sheep abortion clone and strongly suggest that CjNC110 plays an important role in the regulation of energy taxis, flagellar glycosylation, cellular communication via quorum sensing, oxidative stress tolerance, and chicken colonization in this important zoonotic pathogen.
BackgroundSouth American camelids in the United States have rapidly developed into an important agricultural industry in need of veterinary services. Pain management is challenging in camelids because there are no drugs currently approved by the U.S. Food and Drug Administration for use in these species. Dosage regimens used for many therapeutic drugs have been extrapolated from other ruminants; however, the pharmacokinetics, in camelids, may differ from those of other species. Studies investigating the pharmacokinetics of cyclooxygenase-2 (COX-2) selective non-steroidal anti-inflammatory drugs in camelids are deficient in the published literature. Six adult llamas (121- 168 kg) were administered either a 1 mg/kg dose of oral or a 0.5 mg/kg dose of IV meloxicam in a randomized cross-over design with an 11 day washout period between treatments. Plasma samples collected up to 96 hours post-administration were analyzed by high pressure liquid chromatography and mass spectrometry detection (HPLC-MS) followed by non-compartmental pharmacokinetic analysis.ResultsA mean peak plasma concentration (CMAX) of 1.314 μg/mL (Range: 0.826 – 1.776 μg/mL) was recorded at 21.4 hours (Range: 12.0 – 24.0 hours) with a half-life (T ½ λz) of 22.7 hours (Range: 18.0 – 30.8 hours) after oral meloxicam administration. In comparison, a half-life (T ½ λz) of 17.4 hours (Range: 16.2 – 20.7 hours) was demonstrated with IV meloxicam administration. The oral bioavailability (F) of meloxicam (dose normalized) was 76% (Range: 48 – 92%). No adverse effects associated with either treatment modality were observed in the llamas.ConclusionsThe mean bioavailability (F) of oral meloxicam was 76% indicating a high degree of gastrointestinal absorption. Plasma meloxicam concentrations >0.2 μg/mL were maintained for up to 72 h after oral administration; >0.2 μg/mL is considered to be the concentration of meloxicam required for analgesic effects in other species such as the horse. These data suggest that a single dosage of oral meloxicam at 1 mg/kg could potentially maintain therapeutic concentrations in plasma for up to 3 days in adult llamas.
Colonization of the gallbladder by enteric pathogens such as Salmonella typhi, Listeria monocytogenes, and Campylobacter jejuni is thought to play a key role in transmission and persistence of these important zoonotic agents; however, little is known about the molecular mechanisms that allow for bacterial survival within this harsh environment. Recently, a highly virulent C. jejuni sheep abortion (SA) clone represented by the clinical isolate IA3902 has emerged as the dominant cause for sheep abortion in the United States. Previous studies have indicated that the C. jejuni clone SA can frequently be isolated from the gallbladders of otherwise healthy sheep, suggesting that the gallbladder may serve as an important reservoir for infection. To begin to understand the molecular mechanisms associated with survival in the host gallbladder, C. jejuni IA3902 was exposed for up to 24 h to both the natural ovine host in vivo gallbladder environment, as well as ovine bile in vitro. Following exposure, total RNA was isolated from the bile and high throughput deep sequencing of strand specific rRNA-depleted total RNA was used to characterize the transcriptome of IA3902 under these conditions. Our results demonstrated for the first time the complete transcriptome of C. jejuni IA3902 during exposure to an important host environment, the sheep gallbladder. Exposure to the host environment as compared to in vitro bile alone provided a more robust picture of the complexity of gene regulation required for survival in the host gallbladder. A subset of genes including a large number of protein coding genes as well as seven previously identified non-coding RNAs were confirmed to be differentially expressed within our data, suggesting that they may play a key role in adaptation upon exposure to these conditions. This research provides valuable insights into the molecular mechanisms that may be utilized by C. jejuni IA3902 to colonize and survive within the inhospitable gallbladder environment.
Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcB⌬pro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcB⌬pro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacteriumcontaining filopodia. Using an in vivo competitive infection assay, we observed that the plcB⌬pro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcB⌬pro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.
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