Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Gaze Holding in Healthy Subjects

Eccentric gaze in darkness evokes minor centripetal eye drifts in healthy subjects, as cerebellar control sufficiently compensates for the inherent deficiencies of the brainstem gaze-holding network. This behavior is commonly described using a leaky integrator model, which assumes that eye velocity grows linearly with gaze eccentricity. Results from previous studies in patients and healthy subjects suggest caution when this assumption is applied to eye eccentricities larger than 20 degrees. To obtain a detailed characterization of the centripetal gaze-evoked drift, we recorded horizontal eye position in 20 healthy subjects. With their head fixed, they were asked to fixate a flashing dot (50 ms every 2 s)that was quasi-stationary displacing(0.5 deg/s) between ±40 deg horizontally in otherwise complete darkness. Drift velocity was weak at all angles tested. Linearity was assessed by dividing the range of gaze eccentricity in four bins of 20 deg each, and comparing the slopes of a linear function fitted to the horizontal velocity in each bin. The slopes of single subjects for gaze eccentricities of ±0−20 deg were, in median,0.41 times the slopes obtained for gaze eccentricities of ±20−40 deg. By smoothing the individual subjects'' eye velocity as a function of gaze eccentricity, we derived a population of position-velocity curves. We show that a tangent function provides a better fit to the mean of these curves when large eccentricities are considered. This implies that the quasi-linear behavior within the typical ocular motor range is the result of a tuning procedure, which is optimized in the most commonly used range of gaze. We hypothesize that the observed non-linearity at eccentric gaze results from a saturation of the input that each neuron in the integrating network receives from the others. As a consequence, gaze-holding performance declines more rapidly at large eccentricities.     

Partitioning of gonococcal LPS into phenol and water: Different LPS composition of in vivo and in vitro selected variants

Abstract Depending on the culture conditions, Pyrodictium occultum cells revealed two different types of fibers with significant differences in their width in the electron microscope. During growth on elemental sulfur preferentially fibres with a diameter of about 23 nm (type I) were produced. When elemental sulfur was substituted by thiosulfate fibers with a diameter of around 15 nm (type II), were the main appendages. Both types form hollow cylinders consisting of helically arranged sub-units with a wall thickness of 2–3 nm. A triple- layered unit membrane could not be found.     

The Neisseria gonorrhoeae lpxLII gene encodes for a late-functioning lauroyl acyl transferase, and a null mutation within the gene has a significant effect on the induction of acute inflammatory responses

LPS is a fundamental constituent of the outer membrane of all Gram-negative bacteria, and the lipid A domain plays a central role in the induction of inflammatory responses. We identified genes of the Neisseria gonorrhoeae lipid A biosynthetic pathway by searching the complete gonococcal genome sequence with sequences of known enzymes from other species. The lpxLII gene was disrupted by an insertion-deletion in an attenuated aroA mutant of the gonococcal strain MS11. Lipopolysaccharide (LPS) and lipid A analysis demonstrated that the lpxLII mutant had synthesized an altered LPS molecule lacking a single lauric fatty acid residue in the GlcN II of the lipid A backbone. LPS of the lpxLII mutant had a markedly reduced ability to induce the proinflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human macrophages and IL-8 from polymorphonuclear cells. This study demonstrates that the lpxLII gene in gonococci encodes for a late-functioning lauroyl acyl transferase that adds a lauric acid at position 2' in the lipid A backbone. The presence of lauric acid at such a position appears to be crucial for the induction of full inflammatory responses by N. gonorrhoeae LPS.     

Neo-epitopes emerging in the degenerative hippocampal granules of aged mice can be recognized by natural IgM auto-antibodies

Background

Degenerative granular structures appear progressively with age in the hippocampus of most mouse strains. We recently reported that these granules contain a neo-epitope that is recognised by IgM antibodies present as contaminants in many commercial antibodies obtained from mouse ascites and mouse or rabbit serum. We hypothesise that these anti-neo-epitope IgMs are in fact natural auto-antibodies that are generated spontaneously during the foetal stage without previous contact with external antigens and whose repertoire and reactivity pattern have been determined through evolution, being remarkably stable within species and even between species.

Findings

In the present work we found that mice from the ICR-CD1, BALB/C and SAMP8 strains have anti-neo-epitope IgM antibodies in their plasma at all ages tested and even when maintained under specific opportunistic pathogen-free conditions. Moreover, we determined that these anti-neo-epitope IgMs are also present in rabbit, goat and rat serum. We also found that, in each mouse that presented hippocampal granules, the anti-neo-epitope IgMs contained in its plasma recognised the neo-epitopes in its own granules.

Conclusions

This study led to the conclusion that anti-neo-epitope IgMs are widespread natural auto-antibodies contained in the plasma of mice and other species. The presence of these natural auto-antibodies not only explains why they are frequently found as contaminants in commercial antibodies, but also paves the way for a new approach to a treatment and diagnosis of pathological brain processes based on natural IgMs and neo-epitopes.

     

The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBΔN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBΔN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.     

Role of hns in the virulence phenotype of pathogenic salmonellae

A TnphoA-generated mutant C5060, attenuated for virulence, was derived from the mouse-virulent Salmonella typhimurium strain C5. This mutation, designated hns-112::TnphoA, harbours the transposon in the 3 end of hns, with the alkaline phosphatase open reading frame in the opposite orientation to that of hns. Bacterial strains harbouring hns-112::TnphoA were mucoid and had altered levels of DNA supercoiling, as monitored using pUC18 as a reporter plasmid. Transduction of hns-112::TnphoA into mouse virulent strains, including S. typhimurium SL1344 and Salmonella enteritidis Se795, resulted in attenuation. When an independent hns mutation, harbouring a kanamycin-resistance cassette inserted into the Kpnl site at base pair 237 of the hns gene, was introduced into S. typhimurium C5, the isolates were also attenuated. S. typhimurium C5 isolates harbouring the multicopy plasmid pGB651, which encodes the Escherichia coli hns gene, were partially attenuated in mice. Transductional analysis, using Tn10 insertions located close to the hns gene, showed that virulence could be restored In genetic crosses that eliminated the resident hns mutations. However, some hns⁺ transductants were stilt attenuated, suggesting that secondary attenuating lesions can accumulate in hns-deficient strains. These studies show that the hns locus plays a role in Salmonella virulence.     

Involvement of lipid droplets in hepatic responses to lipopolysaccharide treatment in mice

Infection and inflammation induce important changes in lipid metabolism, which result in increased free fatty acids and triacylglycerol in plasma and altered high density lipoprotein (HDL) metabolism. Our aim was to elucidate whether hepatic lipid droplets (LDs) are involved in the adaptations of lipid metabolism to endotoxemia. We characterized the lipid content and several enzymatic activities in subcellular fractions and subpopulations of LDs from livers of mice 24 h after lipopolysaccharide (LPS) treatment and analyzed the expression of key genes involved in lipid management. Endotoxemic mice showed lower lipid content in LDs with decreased molar fraction of cholesteryl ester and higher diacylglycerol/triacylglycerol ratio as compared to their controls. They also showed a decrease in cytosolic triacylglycerol hydrolase activity, specifically in dense LDs, and in microsomal and cytosolic diacylglycerol hydrolase activity; concomitantly neutral lipid biosynthetic capacity and triacylglycerol levels in plasma lipoproteins increased. Together with the overexpression of genes involved in lipogenesis and HDL formation our results suggest that altered hepatic management of LD lipids in LPS-treated mice might be related to the channeled mobilization of triacylglycerol for very low density lipoprotein assembly and to the induction of cholesterol export.