Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occurring after pilus assembly, essential for fibre stability and function

Type IV pili (Tfp) play a critical role in the pathogenic lifestyle of Neisseria meningitidis and N. gonorrhoeae, notably by facilitating bacterial attachment to human cells, but our understanding of their biogenesis, during which the fibres are assembled in the periplasm, then emerge onto the cell surface and are stabilized, remains fragmentary. We therefore sought to identify the genes required for Tfp formation in N. meningitidis by screening a genome-wide collection of mutants for those that were unable to form aggregates, another phenotype mediated by these organelles. Fifteen proteins, of which only seven were previously characterized, were found to be essential for Tfp biogenesis. One novel component, named PilW, was studied in more detail. We found that PilW is an outer-membrane protein necessary for the stabilization of the fibres but not for their assembly or surface localization, because Tfp could be restored on the surface in a pilW mutant by a mutation in the twitching motility gene pilT. However, Tfp-linked properties, including adherence to human cells, were not restored in a pilW/T mutant, which suggests that PilW is also essential for the functionality of the fibres. Together with the finding that PilW is important for the stability of PilQ multimers, our results extend the current model for Tfp biogenesis by suggesting that a multiprotein machinery in the outer-membrane is involved in the terminal stage of Tfp biogenesis during which growing fibres are not only stabilized, but also become perfectly functional.     

Combining inducible protein overexpression with NMR-grade triple isotope labeling in the cyanobacterium Anabaena sp. PCC 7120

The difficulty and expense of preparing protein samples highly enriched in stable isotopes is a bottleneck for structural studies by nuclear magnetic resonance (NMR) spectroscopy. We have developed a new regulatable expression/labeling vector system in the cyanobacterium Anabaena sp. PCC 7120 using the endogenous promoter of the nitrate assimilation nir operon. Standard proteins were overexpressed upon induction with NaNO3, yielding up to 250 mg/L of culture. When the cyanobacteria were grown in the presence of inexpensive 15N-, 13C-labeled mineral salts and 2H2O, the expressed polypeptides were highly (>90%) enriched in stable isotopes. Furthermore, the tight repression of the nir promoter upon induction allowed the production of the toxic oncoprotein E6. In addition, under these conditions, the malE31 protein, while insoluble in Escherichia coli, was found to be soluble in Anabaena. Together, these properties render the described system especially suitable for the production and/or triple labeling of recombinant protein samples. It represents an interesting alternative to conventional protein expression systems used in structural genomics.     

Malaria “epidemic” in Quebec: diagnosis and response to imported malaria

Background

Imported malaria is an increasing problem. The arrival of 224 African refugees presented the opportunity to investigate the diagnosis and management of imported malaria within the Quebec health care system.

Methods

The refugees were visited at home 3–4 months after arrival in Quebec. For 221, a questionnaire was completed and permission obtained for access to health records; a blood sample for malaria testing was obtained from 210.

Results

Most of the 221 refugees (161 [73%]) had had at least 1 episode of malaria while in the refugee camps. Since arrival in Canada, 87 (39%) had had symptoms compatible with malaria for which medical care was sought. Complete or partial records were obtained for 66 of these refugees and for 2 asymptomatic adults whose children were found to have malaria: malaria had been appropriately investigated in 55 (81%); no malaria smear was requested for the other 13. Smears were reported as positive for 20 but confirmed for only 15 of the 55; appropriate therapy was verified for 10 of the 15. Of the 5 patients with a false-positive diagnosis of malaria, at least 3 received unnecessary therapy. Polymerase chain reaction testing of the blood sample obtained at the home visit revealed malaria parasites in 48 of the 210 refugees (23%; 95% confidence interval [CI] 17%– 29%). The rate of parasite detection was more than twice as high among the 19 refugees whose smears were reported as negative but not sent for confirmation (47%; 95% CI 25%– 71%).

Interpretation

This study has demonstrated errors of both omission and commission in the response to refugees presenting with possible malaria. Smears were not consistently requested for patients whose presenting complaints were not “typical” of malaria, and a large proportion of smears read locally as “negative” were not sent for confirmation. Further effort is required to ensure optimal malaria diagnosis and care in such high-risk populations.In many industrialized countries, the incidence of imported malaria is rising because of changing immigration patterns and refugee policies as well as increased travel to malaria-endemic regions.^{1,2,3,4,5,6,7,8,9,10} Imported malaria is not rare in Canada (300–1000 cases per year),³ the United States^2,3,4 or other industrialized countries.^5,6,7,8,9,10 Malaria can be a serious challenge in these countries because of its potentially rapid and lethal course.^11,12,13,14 The task of front-line health care providers is made particularly difficult by the protean clinical presentations of malaria. Classic periodic fevers (tertian or quartan) are seen infrequently.^{9,15,16,17,18,19} Atypical and subtle presentations are especially common in individuals who have partial immunity (e.g., immigrants and refugees from disease-endemic areas) or are taking malaria prophylaxis (e.g., travellers).^9,16,17 Even when malaria is considered, an accurate diagnosis can remain elusive or can be delayed as a result of inadequate or distant specialized laboratory support.^19,20In Quebec, the McGill University Centre for Tropical Diseases collaborates with the Laboratoire de santé publique du Québec to raise awareness of imported malaria, to offer training and quality-assurance testing, and to provide reference diagnostic services. A preliminary diagnosis is typically made by the local laboratory, and smears (with or without staining) are sent to the McGill centre, where they are reviewed within 2–48 hours, depending on the urgency of the request. Initial medical decisions are usually based on local findings and interpretations. Although malaria is a reportable disease, there is no requirement to use the reference service.On Aug. 9, 2000, 224 refugees from Tanzanian camps landed in Montréal aboard an airplane chartered by Canadian immigration authorities. Over the ensuing 5 weeks, the McGill University Centre for Tropical Diseases noted an increase in demand for malaria reference services and an apparent small “epidemic” of imported malaria. This “epidemic” prompted us to investigate the performance of the health care system in the diagnosis and management of imported malaria.     

Bacterial and fungal colonization of burn wounds

A prospective study of fungal and bacterial flora of burn wounds was carried out from February 2004 to February 2005 at the Burns Unit of Hospital Regional da Asa Norte, Brasília, Brazil. During the period of the study, 203 patients were treated at the Burns Unit. Wound swab cultures were assessed at weekly intervals for four weeks. Three hundred and fifty four sampling procedures (surface swabs) were performed from the burn wounds. The study revealed that bacterial colonization reached 86.6% within the first week. Although the gram-negative organisms, as a group, were more predominant, Staphylococcus aureus (28.4%) was the most prevalent organism in the first week. It was however surpassed by Pseudomonas aeruginosa form third week onwards. For S. aureus and P. aeruginosa vancomycin and polymyxin were found to be the most effective drugs. Most of the isolates showed high level resistance to antimicrobial agents. Fungi were found to colonize the burn wound late during the second week postburn, with a peak incidence during the third and fourth weeks. Species identification of fungi revealed that Candida tropicalis was the most predominant, followed by Candida parapsilosis. It is crucial for every burn institution to determine the specific pattern of burn wound microbial colonization, the time-related changes in the dominant flora, and the antimicrobial sensitivity profiles. This would enable early treatment of imminent septic episodes with proper empirical systemic antibiotics, without waiting for culture results, thus improving the overall infection-related morbidity and mortality.     

Canine herpesvirus-1 (CHV-1): clinical, serological and virological patterns in breeding colonies

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with reproductive disorders and neonatal mortality. To advise dog breeders towards an effective management of CHV-1 infected colonies, 27 breeding bitches were studied during one reproductive cycle in field conditions: the effect of cycle stage, kennel size, initial antibody titre, mating and gestation on serologic and viral excretion patterns was evaluated, while the association between reproductive disorders and CHV-1 antibody titres and viral excretion was also analysed. All initially seronegative bitches seroconverted, while 40% of the initially seropositive bitches became seronegative at one or two occasions. No difference in antibody patterns was observed between mated and unmated bitches. Of the mated bitches, 46% experienced infertility, foetal resorption or mummification. No difference in antibody patterns was observed depending on the occurrence of reproductive disorders even if a decrease in antibody titres during early or late-di-oestrus was often present. Significantly higher titres were observed at all cycle stages in large kennels. None of the vaginal and nasal samples or buffy coats tested positive for CHV-1 DNA. The mixed image of clinical and sub-clinical carriage in this study demonstrated CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with vaccination of reproducing bitches in kennels with reproductive disorders should therefore be advised.     

Clinical and environmental isolates of Legionella pneumophila serogroup 1 cannot be distinguished by sequence analysis of two surface protein genes and three housekeeping genes

We used gene sequencing to determine whether clinical (sporadic, epidemic, and endemic) and environmental isolates of Legionella pneumophila serogroup (sg) 1 belong to specific lineages. A total of 178 clinical and environmental L. pneumophila sg 1 isolates, defined by pulsed-field gel electrophoresis and epidemiological data as sporadic, epidemic, or endemic, were analyzed for polymorphisms in five gene fragments. The fragments belonged to three housekeeping genes (coding for aconitase [acn], aspartate-beta-semialdehyde dehydrogenase [asd], and RNA polymerase beta subunit [rpoB]) and two surface protein genes (coding for the macrophage infectivity potentiator [mip] and the major outer membrane protein [mompS]). The phylogenetic tree inferred from sequence polymorphisms of the five genes identified two large clusters, one consisting of 133 poorly differentiated strains and containing two smaller clusters (10 and 2 strains) unrelated to each other and the other consisting of 42 strains. Clinical and environmental isolates could not be distinguished on this basis, and no link between genetic background and epidemiological type was found, suggesting that other factors are responsible for differences in pathogenicity.     

A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase

Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.