The role of apolipoprotein N-acyl transferase, Lnt, in the lipidation of factor H binding protein of Neisseria meningitidis strain MC58 and its potential as a drug target.

BACKGROUND AND PURPOSE: The level of cell surface expression of the meningococcal vaccine antigen, Factor H binding protein (FHbp) varies between and within strains and this limits the breadth of strains that can be targeted by FHbp-based vaccines. The molecular pathway controlling expression of FHbp at the cell surface, including its lipidation, sorting to the outer membrane and export, and the potential regulation of this pathway have not been investigated until now. This knowledge will aid our evaluation of FHbp vaccines. EXPERIMENTAL APPROACH: A meningococcal transposon library was screened by whole cell immuno-dot blotting using an anti-FHbp antibody to identify a mutant with reduced binding and the disrupted gene was determined. KEY RESULTS: In a mutant with markedly reduced binding, the transposon was located in the lnt gene which encodes apolipoprotein N-acyl transferase, Lnt, responsible for the addition of the third fatty acid to apolipoproteins prior to their sorting to the outer membrane. We provide data indicating that in the Lnt mutant, FHbp is diacylated and its expression within the cell is reduced 10 fold, partly due to inhibition of transcription. Furthermore the Lnt mutant showed 64 fold and 16 fold increase in susceptibility to rifampicin and ciprofloxacin respectively. CONCLUSION AND IMPLICATIONS: We speculate that the inefficient sorting of diacylated FHbp in the meningococcus results in its accumulation in the periplasm inducing an envelope stress response to down-regulate its expression. We propose Lnt as a potential novel drug target for combination therapy with antibiotics. LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Global multilocus sequence typing analysis of Mycoplasma bovis isolates reveals two main population clusters.

Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.

Contagious caprine pleuropneumonia: new aspects of an old disease.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae, is a serious OIE-listed disease affecting goats in the Middle East, north and east Africa and Asia. Mortality and morbidity rates can be as high as 60% and 90%, respectively, when the disease first enters a territory, invariably through carrier animals. Recent detections of CCPP in Pakistan and Tajikistan are probably the result of improved diagnosis as the disease has been suspected there for many years, while those in Thrace in 2003 and Mauritius in 2009 represent new outbreaks. CCPP was thought to be highly host specific until recent outbreaks in wildlife species including gazelles and gerenuks show that the causative mycoplasma has broader specificity. Diagnosis was hampered by the fastidiousness of the causative mycoplasma but molecular-based tests like PCR have greatly improved detection. Rapid latex agglutination tests that can be performed at the penside are also available for antibody detection. Clinically affected animals respond to a range of antibiotics although it is unlikely that this results in complete elimination of the mycoplasma. Vaccines consisting of saponized organisms have been shown to be protective but the quality and efficacy may be variable.

Experimental infection of goats with an unusual strain of Mycoplasma mycoides subsp. capri isolated in Jordan.

Goats were infected experimentally with a mycoplasma (the "Irbid" strain) isolated previously from a goat with contagious agalactia in northern Jordan. The strain was unusual in that, although it had been identified by molecular methods as Mycoplasma mycoides subsp. mycoides LC/Mycoplasma mycoides subsp. capri, it showed no inhibition of growth by any of the hyperimmune rabbit antisera conventionally used to speciate members of the Mycoplasma mycoides cluster. Animals were infected either intratracheally or by aerosol and placed "in-contact" with other goats. After 2 weeks, those infected intratracheally became febrile, showing a nasal discharge and slight conjunctivitis, followed a week later by respiratory distress and polyarthritis; lesions seen at necropsy included coagulative necrotic pneumonia, fibrinous pleurisy with pleural exudate, and inflammatory exudates, necrosis and fibrosis in the joints. Animals infected by aerosol showed much milder clinical signs, including nasal discharge and occasional swollen joints. In the "in-contact" goats, seroconversion was first seen after 7 weeks, accompanied by coughing and laboured respiration; lesions in this group consisted of fibrinous pneumonia with focal areas of necrosis and abundant pleural exudate.

Effect of danofloxacin (Advocin A180) on goats affected with contagious caprine pleuropneumonia.

The efficacy of danofloxacin (Advocin A180) was evaluated for the treatment of contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae. Ten healthy Angora goats, confirmed free of CCPP, were exposed to clinically affected animals from a natural outbreak in Thrace, Turkey. After 14 days exposure, 8 goats showed pyrexia ( > or = 41 degrees C). Shortly after, the Angora goats were divided randomly into two groups. Five of these were injected with danofloxacin (6 mg/kg subcutaneously), which was repeated after 48 h; the five remaining animals received saline. Goats were monitored clinically and blood samples were collected for serology. Animals with severe disease were withdrawn from the trial. Goats completing the study were euthanized at day 42. Lung tissue and bronchial fluid were collected for mycoplasma isolation. All danofloxacin-treated goats showed resolution of clinical disease by the end of the trial. Two saline-treated goats failed to complete the study owing to CCPP. Danofloxacin-treated goats showed fewer lung lesions and had significantly lower combined clinical scores than saline controls (p < 0.001). Danofloxacin was found to be highly effective in the treatment of CCPP in goats.