A retrospective study on genetic heterogeneity within Treponema strains: Subpopulations are genetically distinct in a limited number of positions

Pathogenic uncultivable treponemes comprise human and animal pathogens including agents of syphilis, yaws, bejel, pinta, and venereal spirochetosis in rabbits and hares. A set of 10 treponemal genome sequences including those of 4 Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14), 4 T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), 1 T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPLC) were examined with respect to the presence of nucleotide intrastrain heterogeneous sites.The number of identified intrastrain heterogeneous sites in individual genomes ranged between 0 and 7. Altogether, 23 intrastrain heterogeneous sites (in 17 genes) were found in 5 out of 10 investigated treponemal genomes including TPA strains Nichols (n = 5), DAL-1 (n = 4), and SS14 (n = 7), TPE strain Samoa D (n = 1), and TEN strain Bosnia A (n = 5). Although only one heterogeneous site was identified among 4 tested TPE strains, 16 such sites were identified among 4 TPA strains. Heterogeneous sites were mostly strain-specific and were identified in four tpr genes (tprC, GI, I, K), in genes involved in bacterial motility and chemotaxis (fliI, cheC-fliY), in genes involved in cell structure (murC), translation (prfA), general and DNA metabolism (putative SAM dependent methyltransferase, topA), and in seven hypothetical genes.Heterogeneous sites likely represent both the selection of adaptive changes during infection of the host as well as an ongoing diversifying evolutionary process

Transport of Live Cells under Sterile Conditions Using a Chemotactic Droplet

© 2018 The Author(s). 1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated if such droplets can be used as transporters for living cells. We developed a partially hydrophobic alginate capsule as a protective unit that can be precisely placed in a droplet and transported along chemical gradients. Once the droplets with cargo reached a defined final destination, the association of the alginate capsule and decanol droplet was disrupted and cargo deposited. Both Escherichia coli and Bacillus subtilis cells survived and proliferated after transport even though transport occurred under harsh and sterile conditions

Footprint of Positive Selection in Treponema pallidum subsp. pallidum Genome Sequences Suggests Adaptive Microevolution of the Syphilis Pathogen

In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (≤3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify “Chicago-“ or “Nichols -specific” differences. All but one of the 16 SNPs were “Nichols-specific”, with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature

Enzyme histochemistry of corneal wound healing

The usefulness of enzyme histochemical methods for the localization of enzymes as catalysts of molecular interactions in the cells and tissues of healing corneal wounds is shown in rabbits. The current data on corneal wound healing in humans as well as in rabbits with particular reference to serine proteases are reviewed. Some inflammatory mediators are also discussed. Plasmin is a serine protease which is absent (or present only in very low concentration) in the tear fluid, and its activity appears under various pathological conditions in humans or following experimental injuries in rabbits. The role of increasing plasmin activity in the tear fluid in the depending upon the severity of corneal injury is evaluated. Great attention is devoted to conditions leading to long-lasting elevated levels of plasmin activity in the tear fluid correlated with corneal ulceration. The differences between the histochemical pattern of untreated corneas or corneas treated with some serine protease inhibitors are shown, and the efficacy of these drugs is discussed in light of present knowledge

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4- Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear

Changes of superoxide dismutase, catalase and glutathione peroxidase in the corneal epithelium after UVB rays. Histochemical and biochemical study

In this study, the effects of UVA and UVB rays on antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) were examined in the corneal epithelium. The corneas of albino rabbits were irradiated with a UV lamp generating UVA (365 nm wavelength) or UVB rays (312 nm wavelength), 1 X daily for 5 min, from a distance of 0.03 m, over 4 days (shorter procedure) or 8 days (longer procedure). In contrast to UVA rays, which did not evoke significant disturbances, UVB rays changed the activities of antioxidant enzymes. The longer repeated irradiation with UVB rays was performed, the deeper the observed decrease in antioxidant enzymes. The shorter procedure evoked a more profound decrease of glutathione peroxidase and catalase (the enzymes cleaving hydrogen peroxide) than of superoxide dismutase, an enzyme scavenging superoxide radical and producing hydrogen peroxide during the dismutation reaction of a superoxide free radical. This may contribute to an insufficient hydrogen peroxide cleavage at the corneal surface and danger to the cornea from oxidative damage. After the longer procedure (UVB rays), the activities of all antioxidant enzymes were very low or completely absent. In conclusion, repeated irradiation of the cornea with UVB rays evokes a deficiency in antioxidant enzymes in the corneal epithelium, which very probably contributes to the damage of the cornea (and possibly also deeper parts of the eye) from UVB rays and the reactive oxygen products generated by them