- Volume 152, Issue 5, 2006

Listeria monocytogenes is a facultative intracellular bacterial pathogen responsible for severe opportunistic infections in humans and animals. The secreted cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates phagosomal escape and allows bacterial growth in the cytosol of infected cells. In order to identify new LLO determinants participating in bacterial pathogenesis, this study focused on a major target of LLO proteolytic cleavage in vitro, the CTL epitope region (residues 91–99). Mutations were generated by site-directed mutagenesis in the epitope or in the two clusters of positive charges flanking the epitope. Two LLO mutants (a single mutation K103A and a double mutation R89G, K90G) were normally and stably secreted by L. monocytogenes. In contrast, a mutant carrying four amino acid substitutions in the epitope itself (Y92K, D94A, E97K, Y98F) was highly susceptible to proteolytic degradation. While these three LLO mutant proteins showed a reduced haemolytic activity, they all promoted efficient phagosomal escape and intracellular multiplication in different cell types, and were non-cytotoxic. The deletion of the epitope (Δ91–99), as well as the substitution of two, three or four of the four lysine residues (K103 to K106) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91–99 region in the production of a stable and functional LLO. LD50 determinations in the mouse model suggest a possible link between LLO stability and virulence.

Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues 55DKSRYGGLG63 of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis.

The Helicobacter pylori vacuolating cytotoxin VacA shares homology in its C-terminal domain with many autotransporter proteins, suggesting a similar mechanism of secretion. Like most autotransporters, VacA contains a single pair of cysteine residues located near the C-terminus of the passenger domain. This study aimed to investigate the role of these conserved cysteine residues. This involved changing each cysteine in the VacA passenger domain to serine, quantifying the effect on VacA levels and assessing toxin activity in H. pylori. It was shown that both cysteine residues were required for high VacA levels, although mutation of each cysteine reduced toxin amounts to differing extents, implying that their importance was not simply for intramolecular disulphide bond formation. Although less VacA was observed for the cysteine mutants, vacuolating activity was detected, showing that the cysteines were not required for VacA function.

It was previously shown that α1-antitrypsin (AAT) interacts with the type III secreted (T3S) EspB and EspD proteins of enteropathogenic Escherichia coli (EPEC), resulting in reduced functionality of the proteins. To determine if AAT is also able to interact with T3S proteins of other pathogens, the binding of AAT to Yop proteins of Yersinia enterocolitica was analysed. AAT did not interact with YopB or YopD, which have functions in type III translocation similar to EspB and EspD in EPEC, but specifically interacts with YopM, a member of the leucine-rich repeat (LRR) family of proteins, in overlay and pull-down assays. To determine regions of YopM involved in AAT binding, various N- and C-terminally truncated versions of YopM were recombinantly expressed, and their ability to interact with AAT analysed. All versions tested were able to bind AAT, indicating that at least eight LRR of YopM are sufficient for AAT interaction. The main physiological role of AAT is to inhibit neutrophil elastase; however, elastase was efficiently inhibited by AAT in the presence and absence of YopM, indicating that YopM does not interfere with the anti-protease inhibition activity of AAT, and that the domain of AAT interacting with YopM is not identical to AAT's protease interaction domain. Furthermore, it was shown that elastase efficiently degrades YopM and other Yop proteins. The data suggest that AAT has additional functions in the host response against bacterial infections that are not related to its anti-protease activity.

Salmonella enterica serovar Enteritidis has emerged during the last 20 years as the major causative agent of food-borne gastroenteritis in humans and as the major infectious agent on poultry farms, replacing Salmonella enterica serovar Typhimurium as the dominant pathogenic serovar. Because adhesion to gut tissues and colonization of the alimentary tract, mediated in large part by the FimH adhesins located on type 1 fimbriae, is an important stage in the pathogenesis of both serovars, the binding properties of the FimH adhesins from these two enteropathogens were compared. Salmonella Enteritidis FimH protein and the Salmonella Typhimurium low-adhesive variant of this adhesin were expressed in Escherichia coli and the recombinant proteins were analysed for their ability to bind glycoproteins carrying different oligomannosidic structures and different types of eukaryotic cells. In static binding assays (ELISA and Western blotting) both FimH proteins bound equally well to all three tested glycoproteins (RNase B, horseradish peroxidase and mannan-BSA). In addition, no differences were found in the binding specificity of the FimH proteins and intact cells of Salmonella Enteritidis and Salmonella Typhimurium to human colon carcinoma or bladder cancer cells. The presence of the same amino acid residues at positions 61 (glycine) and 118 (phenylalanine) and the similar binding properties of these two adhesins suggest that the newly described FimH protein of Salmonella Enteritidis represents the low-adhesive variant found in Salmonella Typhimurium. To study the binding specificity of Salmonella Enteritidis FimH protein further, direct kinetic analysis using surface plasmon resonance was performed. With this method it was found that Salmonella Enteritidis FimH adhesin bound with the highest K d value to high-mannose type N-glycans carried by RNase B; about 100 times lower K d values were obtained in the interactions with mannan-BSA and horseradish peroxidase.

Polyunsaturated fatty acids (PUFAs), including linoleic acid (C18 : 2) and α-linolenic acid (C18 : 3), are major components of membranes. PUFAs are produced from monounsaturated fatty acids by several fatty acid desaturases (FADs) in many fungi, but Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans do not have these enzymes. Although the fungal pathogen Candida albicans produces C18 : 2 and C18 : 3, the enzymes that synthesize them have not yet been investigated. In this report, two ORFs, CaFAD2 and CaFAD3, were identified based on their homology to other yeast FADs, and CaFAD2 and CaFAD3 gene disruptants were constructed. Cafad2Δ and Cafad3Δ lost their ability to produce C18 : 2 and C18 : 3, respectively. Furthermore, S. cerevisiae cells expressing CaFad2p converted palmitoleic acid (C16 : 1) and C18 : 1 to hexadecadienoic acid (C16 : 2) and C18 : 2, respectively, and CaFad3p-expressing cells converted C18 : 2 to C18 : 3. These results strongly supported that CaFAD2 encodes the Δ12 FAD and that CaFAD3 encodes the ω3 FAD. However, phenotypic analysis demonstrated that the presence of these PUFAs did not affect the virulence to mice, or morphogenesis in the culture media used to induce morphological change of C. albicans.

Glycine betaine (GB) and its immediate precursors choline and carnitine, dimethylsulfonioacetate, dimethylsulfoniopropionate, ectoine and proline were effective osmoprotectants for Pseudomonas aeruginosa, but pipecolate, trehalose and sucrose had no osmoprotective effect. GB was accumulated stably or transiently when succinate or glucose, respectively, was used as a carbon and energy source. The catabolite repression mediated by succinate occurred at both low and high salinities, and it did not involve the global regulators Vfr and Crc. A proteomic analysis showed that at least 21 proteins were induced when GB was used as a carbon and energy source, and provided evidence that succinate repressed the synthesis of all these proteins. Many of the proteins induced by GB (sarcosine oxidase, serine hydroxymethyltransferase and serine dehydratase) are involved in GB catabolism. In addition, GB uptake was stimulated at high medium osmolalities but it was insensitive to catabolite repression by succinate. Despite its ability to inhibit betaine catabolism, succinate did not allow any better growth of P. aeruginosa cells under hyperosmotic constraint. Conversely, as observed for cells supplied with glucose, a transient accumulation of GB was sufficient to provide a significant cell osmoprotection.

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO. It was found that cyanide is produced in P. aeruginosa at similar levels irrespective of its complement of CIO, indicating that the CIO is not an obligatory electron sink for cyanide synthesis. However, MICs for cyanide and growth in its presence demonstrated that the CIO provides P. aeruginosa with protection against the effects of exogenous cyanide. Nevertheless, the presence of cyanide did not affect the viability of cio mutant strains compared to the wild-type during prolonged incubation in stationary phase. The detection of the fermentation end products acetate and succinate in stationary-phase culture supernatants suggests that P. aeruginosa, irrespective of its CIO complement, may in part rely upon fermentation for energy generation in stationary phase. Furthermore, the decrease in cyanide levels during incubation in sealed flasks suggested that active breakdown of HCN by the culture was taking place. To investigate the possibility that the CIO may play a role in pathogenicity, wild-type and cio mutant strains were tested in the paralytic killing model of Caenorhabditis elegans, a model in which cyanide is the principal toxic agent leading to nematode death. The CIO mutant had delayed killing kinetics, demonstrating that the CIO is required for full pathogenicity of P. aeruginosa in this animal model.

Lactose (1,4-O-β-d-galactopyranosyl-d-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on d-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that d-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with d-galactose, lactose, galactitol and d-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that d-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0·015 h−1) but not at higher dilution rates. At the same dilution rate, growth on d-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on d-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0·015 h−1 than d-galactose and induced cellulases even at growth rates up to 0·042 h−1. Results of chemostats with an equimolar mixture of d-galactose and d-glucose essentially mimicked the behaviour on d-galactose alone, whereas an equimolar mixture of d-galactose and galactitol, the first intermediate of a recently described second pathway of d-galactose catabolism, led to cellulase induction at D=0·030 h−1. It is concluded that d-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

The importance of aquaporin expression in water permeability in Saccharomyces cerevisiae was assessed by measuring the osmotic water permeability coefficient (P f) and the activation energies (E a) from both hypo- and hypertonic experiments performed with whole protoplasts from four strains differing in aquaporin level of expression: parental, double-deleted and overexpressing AQY1 or AQY2. Double-deleted (lower P f) and AQY1-overexpressing strains (higher P f) presented linear Arrhenius plots with E a consistent with fluxes mainly through the lipids [16·3 kcal mol−1 (68·2 kJ mol−1)] and with a strong contribution of channels [9·6 kcal mol−1 (40·2 kJ mol−1)], respectively. The Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 °C. The E a values for these strains, calculated for temperatures ranging from 7 to 23 °C, were lower [9·5 kcal mol−1 (39·7 kJ mol−1)] than the values obtained from 23 to 38 °C [17 kcal mol−1 (71·1 kJ mol−1)]. This behaviour indicates that only in the lower temperature range did the water fluxes occur predominantly via the water channels. The permeabilities for each strain relative to the deletion strain show that an increase in permeability due to the presence of aquaporins was more relevant at low temperatures. Following our results, we propose that water channels play an important role for osmotic adjustment of yeast cells at low temperature.