Yngve Östberg

An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds

Borrelia burgdorferi is remarkable for its ability to thrive in widely different environments due to its ability to infect various organisms. In comparison to enteric Gram-negative bacteria, these spirochetes have only a few transmembrane proteins some of which are thought to play a role in solute and nutrient uptake and excretion of toxic substances. Here, we have identified an outer membrane protein, BesC, which is part of a putative export system comprising the components BesA, BesB and BesC. We show that BesC, a TolC homolog, forms channels in planar lipid bilayers and is involved in antibiotic resistance. A besC knockout was unable to establish infection in mice, signifying the importance of this outer membrane channel in the mammalian host. The biophysical properties of BesC could be explained by a model based on the channel-tunnel structure. We have also generated a structural model of the efflux apparatus showing the putative spatial orientation of BesC with respect to the AcrAB homologs BesAB. We believe that our findings will be helpful in unraveling the pathogenic mechanisms of borreliae as well as in developing novel therapeutic agents aiming to block the function of this secretion apparatus.

Localization of somatostatin-like immunoreactivity in the pancreatic islets of the hagfish, Myxine glutinosa and the lamprey Lampetra fluviatilis

SummarySomatostatin-like immunofluorescence has been found by immunostaining in cells of the bile duct mucosa and pancreatic islet parenchyma of the hagfish, Myxine glutinosa, and the islet lobules of the lamprey, Lampetra fluviatilis.

P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

ABSTRACT To elucidate antigens present on the bacterial surface ofBorrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis.

Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi

P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in the p13 mutant compared to wild-type B. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions. The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 in B. burgdorferi is an outer membrane protein with porin activity.

Cytochemical, immunofluorescence, and ultrastructural investigations on polypeptide hormone containing cells in the intestinal mucosa of a cyclostome, Myxine glutinosa

Immunofluorescence studies showed that cells in the epithelium of the intestine of the hagfish, Myxine glutinosa, reacted with antisera to porcine glucagon, and to synthetic human gastrin, pentagastrin and caerulein. These cells were argyrophil and stained with lead-haematoxylin, resembling several mammalian polypeptide hormone-producing cells. Ultrastructurally, analogous cells were observed, which contained spherical secretion granules with a diameter of 163–178 nm. The immunoreactive cells extended from the basal lamina to the luminal surface of the gut, and may thus belong to the so-called “open type” of endocrine cells. They were separate from the insulin-containing cells of apparently “closed type” in the bile duct epithelium and from the zymogen cells of the intestine which represent the exocrine pancreas in the hagfish. No cells in the intestinal epithelium reacted with antisera to secretin or insulin. n nAlthough the immunoreactive argyrophil cells resembled mammalian gut endocrine cells in their cytochemical and ultrastructural characteristics, they did not take up and decarboxylate biogenic amine precursors. In this respect they also differed from the insulin-producing B-cells in the hagfish. Nerve fibres were observed in the submucous connective tissue but no close connections between nerve fibres and the endocrine cells were seen.

Ultrastructural Findings in a case of Benign Lymphoepithelial Lesion (Sjögren's Syndrome)

A 71-year-old woman was admitted because of a slowly growing tumour in the left parotid region. Left-sided superficial parotidectomy was performed and light microscopic study of the surgical specimen disclosed a benign lymphoepithelial lesion. The clinical and laboratory examinations showed a sicca syndrome that is a variant of Sjogrens syndrome. Ultrastructural investigation of the resected material confirmed the light microscopic finding of a salivary gland architecture that was destroyed and replaced by lymphoid tissue, cell islands and seemingly proliferating ducts. The duct cells, as well as some of the cells constituting the islands, were characterized by a cytoplasm of low density, rather inconspicuous organelles and occasional delicate, irregular filaments. Other island cells exhibited more electron dense cytoplasm, tonofilaments and prominent desmosomes. In addition, there were cells in the islands that possessed features intermediate between those of the two other kinds of cells. No indetermina...

Pleiotropic Effects of Inactivating a Carboxyl-Terminal Protease, CtpA, in Borrelia burgdorferi

A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization-time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

Cytochemical, immunofluorescence, and ultrastructural investigations on polypeptide hormone localization in the islet parenchyma and bile duct mucosa of a cyclostome, Myxine glutinosa.

Abstract Cells in the islets and bile duct epithelium of the hagfish, Myxine glutinosa , can take up and decarboxylate precursors of biogenic amines. The end product of the process is demonstrated by the freeze-drying and formaldehyde vapor fixation technique. The same cells can be stained by an immunofluorescence reaction for insulin, using guinea pig antiserum to human insulin. Thus, the insulin-producing B-cells of the hagfish are members of the socalled APUD series of polypeptide hormone-producing cells described in mammals. Insulin-containing cells were also observed, forming buds from the bile duct mucosa. Consequently, the islets have obviously been formed from endocrine precursor cells in the bile duct epithelium. Immature extrainsular B-cells of this kind obviously attain APUD characteristics before they show actual insulin production. The content of the characteristic islet cavities was nonimmunoreactive to either anti-human insulin or anti-human C-peptide, and did not take up precursors of biogenic amines. No evidence was obtained of B-cell granule release to the islet cavities. Instead, the secretion granules seemed to be released by emiocytosis to the surrounding connective tissue by secretion of “membrane-release” pattern. Nerve fibers were observed in the connective tissue stroma between the islet lobules but there was no close connection between nerve fibers and B-cells. A pilot study revealed the presence of glucagon-immunoreactive cells in the intestinal epithelium but no cells of this kind were observed in the islet organ or in the bile duct mucosa. No cells in the bile duct mucosa or in the islet parenchyma showed immunoreactivity to gastrin, secretin, or caerulein.

Novel role for a bacterial nucleoid protein in translation of mRNAs with suboptimal ribosome-binding sites

In Escherichia coli, the major nucleoid protein H-NS limits transcription by acting as a repressor or transcriptional silencer, presumably by its ability to close the looped chromosome domains in the nucleoid through DNA-protein-DNA bridging. Here, we demonstrate the direct involvement of H-NS as a positive factor stimulating translation of the malT mRNA. In vitro studies showed that H-NS facilitates a repositioning of the 30S preinitiation complex on the malT mRNA. H-NS stimulation of translation depended on the AU-rich -35 to -40 region of the mRNA. Several additional examples were found demonstrating a novel function for H-NS in translation of genes with suboptimal ribosome-binding sequences.

The Etiological Agent of Lyme Disease, Borrelia burgdorferi, Appears To Contain Only a Few Small RNA Molecules

Small regulatory RNAs (sRNAs) have recently been shown to be the main controllers of several regulatory pathways. The function of sRNAs depends in many cases on the RNA-binding protein Hfq, especially for sRNAs with an antisense function. In this study, the genome of Borrelia burgdorferi was subjected to different searches for sRNAs, including direct homology and comparative genomics searches and ortholog- and annotation-based search strategies. Two new sRNAs were found, one of which showed complementarity to the rpoS region, which it possibly controls by an antisense mechanism. The role of the other sRNA is unknown, although observed complementarities against particular mRNA sequences suggest an antisense mechanism. We suggest that the low level of sRNAs observed in B. burgdorferi is at least partly due to the presumed lack of both functional Hfq protein and RNase E activity.