Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

Evolutionary history of Mycobacterium leprae in the Pacific Islands

As one of the oldest known human diseases, leprosy or Hansen''s disease remains a public health concern around the world with over 200 000 new cases in 2018. Most human leprosy cases are caused by Mycobacterium leprae, but a small number of cases are now known to be caused by Mycobacterium lepromatosis, a sister taxon of M. leprae. The global pattern of genomic variation in M. leprae is not well defined. Particularly, in the Pacific Islands, the origins of leprosy are disputed. Historically, it has been argued that leprosy arrived on the islands during nineteenth century colonialism, but some oral traditions and palaeopathological evidence suggest an older introduction. To address this, as well as investigate patterns of pathogen exchange across the Pacific Islands, we extracted DNA from 39 formalin-fixed paraffin-embedded biopsy blocks dating to 1992–2016. Using whole-genome enrichment and next-generation sequencing, we produced nine M. leprae genomes dating to 1998–2015 and ranging from 4-63× depth of coverage. Phylogenetic analyses indicate that these strains belong to basal lineages within the M. leprae phylogeny, specifically falling in branches 0 and 5. The phylogeographical patterning and evolutionary dating analysis of these strains support a pre-modern introduction of M. leprae into the Pacific Islands.This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.     

Plasma protein synthesis by isolated rat hepatocytes

A system of preparation of rat hepatocytes with extended viability has been developed to study the role of hormones and other plasma components upon secretory protein synthesis. Hepatocytes maintained in minimal essential medium reduced the levels of all amino acids in the medium except the slowly catabolized amino acids leucine, isoleucine, and valine, which steadily increase as the result of catabolism of liver protein. Although the liver cells catabolize 10-15% of their own protein during a 20-h incubation, the cells continue to secrete protein in a linear fashion throughout the period. The effects of insulin, cortisol, and epinephrine on general protein synthesis, and specifically on fibrinogen and albumin synthesis, have been tested on cells from both normal rats and adrenalectomized rats. Cells from normal animals show preinduction of tyrosine amino transferase (TAT), having at the time of isolation a high level of enzyme which shows only an increase of approximately 60% upon incubation with cortisol. In contrast, cells from adrenalectomized animals initially have a low level of enzyme which increases fourfold over a period of 9 h. The effects of both epinephrine and cortisol on protein synthesis are also much larger in cells from adrenalectomized animals. After a delay of several hours, cortisol increases fibrinogen synthesis sharply, so that at the end of the 20-h incubation, cells treated with hormone have secreted nearly 2.5 times as much fibrinogen as control cells. The effect is specific; cortisol stimulates neither albumin secretion nor intracellular protein synthesis. The combination of cortisol and epinephrine strongly depresses albumin synthesis in both types of cells. Insulin enhances albumin and general protein synthesis but has little effect on fibrinogen synthesis.     

Extraction of fluorescent cell puncta by adaptive fuzzy segmentation

MOTIVATION: The discrimination and measurement of fluorescent-labeled vesicles using microscopic analysis of fixed cells presents a challenge for biologists interested in quantifying the abundance, size and distribution of such vesicles in normal and abnormal cellular situations. In the specific application reported here, we were interested in quantifying changes to the population of a major organelle, the peroxisome, in cells from normal control patients and from patients with a defect in peroxisome biogenesis. In the latter, peroxisomes are present as larger vesicular structures with a more restricted cytoplasmic distribution. Existing image processing methods for extracting fluorescent cell puncta do not provide useful results and therefore, there is a need to develop some new approaches for dealing with such a task effectively. RESULTS: We present an effective implementation of the fuzzy c-means algorithm for extracting puncta (spots), representing fluorescent-labeled peroxisomes, which are subject to low contrast. We make use of the quadtree partition to enhance the fuzzy c-means based segmentation and to disregard regions which contain no target objects (peroxisomes) in order to minimize considerable time taken by the iterative process of the fuzzy c-means algorithm. We finally isolate touching peroxisomes by an aspect-ratio criterion. The proposed approach has been applied to extract peroxisomes contained in several sets of color images and the results are superior to those obtained from a number of standard techniques for spot extraction. AVAILABILITY: Image data and computer codes written in Matlab are available upon request from the first author.     

Osmosensor ProP of Escherichia coli responds to the concentration,chemistry, and molecular size of osmolytes in the proteoliposome lumen

Transporter ProP of Escherichia coli mediates the cellular accumulation of organic zwitterions in response to increased extracellular osmolality. We compared and characterized the osmoregulation of ProP activity in cells and proteoliposomes to define the osmotic shift-induced cellular change(s) to which ProP responds. ProP-(His)(6) activity in cells and proteoliposomes was correlated with medium osmolality, not osmotic shift, turgor pressure, or membrane strain. Both K(M) and V(max) for proline uptake via ProP-(His)(6) increased with increasing medium osmolality, as would be expected if osmolality controls the proportions of transporter with inactive and active conformations. The osmolality yielding half-maximal ProP-(His)(6) activity was higher in proteoliposomes than in cells. The osmolality response of ProP is also attenuated in bacteria lacking soluble protein ProQ. Indeed, the catalytic constant (k(cat)) for ProP-(His)(6) in proteoliposomes approximated that of ProP in intact bacteria lacking ProQ. Thus, the proteoliposome system may replicate a primary osmosensory response that can be further amplified by ProQ. ProP-(His)(6) is designated as an osmosensor because its activity is dependent on the osmolality, but not the composition, of the assay medium to which the cell surface is exposed. In contrast, ProP-(His)(6) activity was dependent on both the osmolality and the composition of the lumen in osmolyte-loaded proteoliposomes. For proteoliposomes containing inorganic salts, glucose, or poly(ethylene glycol) 503, transporter activity correlated with total lumenal cation concentration. In contrast, for proteoliposomes loaded with larger poly(ethylene glycol)s, the osmolality, the lumenal cation concentration, and the lumenal ionic strength at half-maximal transporter activity decreased systematically with poly(ethylene glycol) radius of gyration (range 0.8-1.8 nm). These data suggest that ProP-(His)(6) responds to osmotically induced changes in both cytoplasmic K(+) levels and the concentration of cytoplasmic macromolecules.     

Novel role for low molecular weight plasma thiols in nitric oxide-mediated control of platelet function

Nitric oxide (NO) is a powerful antiplatelet agent, but its notoriously short biological half-life limits its potential to prevent the activation of circulating platelets. Here we used diethylamine diazeniumdiolate (DEA/NO) as an NO generator to determine whether the antiplatelet effects of NO are prolonged by the formation of a durable, plasma-borne S-nitrosothiol reservoir. Preincubation of both platelet rich plasma (PRP) and washed platelets (WP) with DEA/NO (2 microm) for 1 min inhibited collagen-induced platelet aggregation by 82 +/- 5 and 91 +/- 2%, respectively. After 30 min preincubation with DEA/NO, NO was no longer detectable in either preparation, but aggregation remained markedly inhibited (72 +/- 7%) in PRP. In contrast, the inhibitory effect in WP was almost completely lost at this time (5 +/- 3%) but was partially restored (39 +/- 10%) in WP containing human serum albumin (1%) and fully restored by co-incubation with albumin and the low molecular weight (LMW) thiols, glutathione, (5 microm), cysteinyl-glycine (10 microm), or cysteine (10 microm). This NO-mediated effect was not seen with LMW thiols in the absence of albumin and was associated with S-nitrosothiol formation. Our results demonstrate that LMW thiols play an important role in both the formation and activation of an S-nitrosoalbumin reservoir that significantly prolongs the duration of action of NO.