- Volume 144, Issue 2, 1998

Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish, causing motile aeromonad septicaemia. It can enter (invade) fish cells, and survive as an intracellular parasite. The host-pathogen interaction and signal transduction pathway were studied by screening signal transduction inhibitors using carp epithelial cells and a virulent strain of the bacterium, PPD134/91. Genistein, a tyrosine kinase inhibitor, postponed internalization of A. hydrophila into host cells, suggesting that tyrosine phosphorylation plays a role in internalization. In contrast, staurosporine, a protein kinase C inhibitor, and sodium orthovanadate, a protein tyrosine phosphatase inhibitor, accelerated internalization of PPD134/91. Other virulent strains of A. hydrophila were also examined and it is likely that all strains, irrespective of serogroup, use the same signalling pathway to facilitate bacterial uptake.

The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry. P. brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin. Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P. brasiliensis cell wall. Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P. brasiliensis surface. Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal βD-galactopyranosyl residues. The sialic acid linkages to galactose are α2,6 and α2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins. The α2,6 linkage clearly predominated in both experiments. Flow cytofluorimetry analysis revealed the heterogenicity of P. brasiliensis yeast cell populations, which comprised young and mature budding yeasts. Both express binding sites to LFA and Limulus polyphemus agglutinin.

The emergence and rapid rise to dominance of Vibrio cholerae O139 in India and Bangladesh in 1992 led to the consideration that choleraphage might serve as both a selective mechanism and a means for horizontal transmission of genetic information. A filamentous phage ‘493′ from O139 strain AJ27-493 has been purified and partially characterized. The phage was inactive on classical biotype V. cholerae 01 but it was active on El Tor biotype strains isolated prior to 1994 when El Tor re-emerged in Bangladesh. More recent El Tor isolates were all resistant to the phage. The phage was also active on O139 strains. Unlike the filamentous ctxφ, the receptor for 493 is not TcpA. The phage genome was a 9.3 kb closed circular single-stranded molecule containing a 0.4 kb double-stranded stem supporting a 2 kb single-stranded loop. A 283 bp fragment was cloned and used as a probe in Southern hybridization, in parallel with total phage 493 DNA. These probes hybridized both chromosomally and extrachromosomally with most O139 strains, but not with O1 strains. Infection of hybridization-negative El Tor or O139 strains resulted in the presence of hybridizing loci (both plasmid and chromosomal), in the appearance of an 18 kDa protein, and in marked alterations in colonial morphology. Phage 493 is clearly distinct from other O139 choleraphages which have been described. Phage 493 DNA hybridized with an encapsulated non-O1 (O31) strain (NRT36S) which was isolated before O139 was recognized. NRT36S also produces a phage which can infect El Tor strains with low efficiency. Further studies may reveal whether bacteriophage play a role in the emergence and the territoriality of new choleragenic vibrios.

Streptococcus suis serotype 2 is responsible for a wide variety of porcine infections. In addition, it is considered a zoonotic agent. Knowledge about the virulence factors for this bacterium is limited but its polysaccharide capsule is thought to be one of the most important. Transposon mutagenesis with the self-conjugative transposon Tn916 was used to obtain acapsular mutants from the virulent S. suis type 2 reference strain S735. Clones were screened by colony-dot ELISA with a monoclonal antibody specific for a type 2 capsular epitope and clones that failed to react with the antibody were characterized. Two mutants, 2A and 79, having one and two Tn916 insertions respectively, were chosen for further characterization. Absence of capsule was confirmed by coagglutination, capillary precipitation and capsular reaction tests and by transmission electron microscopy. Absence of capsular polysaccharides correlated with increased hydrophobicity and phagocytosis by both murine macrophages and porcine monocytes compared to the wild-type strain. Furthermore, both mutants were shown to be avirulent in murine and pig models of infection. Finally, mutant 2A was readily eliminated from circulation in mice compared to the wild-type strain, which persisted more than 48 h in blood. Thus, isogenic mutants defective in capsule production demonstrate the importance of capsular polysaccharides as a virulence factor for S. suis type 2.

A panel of ten site-directed mutants of Clostridium perfringens ε-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native ε-toxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated ε-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant ε-toxin.

Uropathogenic Escherichia coli strains express chromosomal and plasmid-encoded virulence-associated factors such as specific adhesins, toxins and iron-uptake systems. A ColV plasmid (pRK100) of a uropathogenic strain and its host KS533 were studied. The host strain encodes the K1 capsule, and P and S fimbriae, but neither haemolysin nor the cytotoxic-necrotic factor CNF1, indicating that this strain does not harbour a larger pathogenicity island. A restriction map of pRK100 was constructed on the basis of hybridization experiments and nucleotide sequencing. pRK100 harbours ColV, the conserved replication region RepFIB, the aerobactin-uptake system, a RepFIC replicon and additionally Colla as well as transposon Tn5431. The location of the RepFIC replicon was similar to that in plasmid F. ColV plasmids and F thus share a region spanning more than half the length of plasmid F. Even though their replication and transfer regions are homologous, ColV plasmids are found only in E. coli strains. Among the four other species tested, conjugal transfer of pRK100 was demonstrated, with low frequency, only to Klebsiella pneumoniae, suggesting that a natural barrier effectively bars transfer. In vitro stability of the plasmid with integration into the chromosome to ensure maintenance in the presence of an incompatible plasmid was demonstrated.

To evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92%) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.

Previous results indicated that molar growth yields are reduced when Clostridium cellulolyticum is cultured in media containing cellobiose concentrations greater than 1 g I−1. Continuous cultures were examined to determine the physiological basis of these poor growth yields. Acetate was the main product of C. cellulolyticum metabolism, whereas the production of reduced compounds such as ethanol or lactate was low. Such patterns of product formation were accompanied by a 12-fold increase in intracellular NADH concentration when the cellobiose flow was increased. Catabolic enzymic activities were measured in vitro. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acetate kinase and phosphoroclastic activities were found at similar levels as in cells metabolizing higher substrate concentrations. In contrast, lactate dehydrogenase activity was low and correlated with the rate of lactate production. Furthermore, an inhibition of GAPDH activity by high NADH/NAD+ ratios was established. These results suggested that a decreased NADH reoxidation could be responsible for limiting C. cellulolyticum growth. Lactate and ethanol production were not sufficient to balance out the NADH produced in the GAPDH step of glycolysis. One consequence of poor NADH reoxidation would be an increase in intracellular concentration of NADH, which in turn could inhibit GAPDH activity.

Polyethyleneimine (PEI), a polycationic polymer substance used in various bioprocesses as a flocculating agent and to immobilize enzymes, was recently shown to make Gram-negative bacteria permeable to hydrophobic antibiotics and to detergents. Because this suggests impairment of the protective function of the outer membrane (OM), the effect of PEI on the ultrastructure of Salmonella typhimurium was investigated. Massive alterations in the OM of PEI-treated and thin-sectioned bacteria were observed by electron microscopy. Vesicular structures were seen on the surface of the OM, but no liberation of the membrane or its fragments was evident. Since a potential mechanism for the action of PEI could be its binding to anionic LPSs on the OM surface, the interaction of PEI with isolated LPSs was assayed in vitro. The solubility of smooth-type LPSs of Salmonella, regardless of the sugar composition of their O-specific chains, was not affected by PEI, nor was that of Ra-LPS (lacking O-specific chains but having a complete core oligosaccharide). PEI strongly decreased the solubility of rough-type LPSs of the chemotypes Rb2 and Re, whereas it had only a weak effect on the abnormally cationic Rb2-type pmrA mutant LPS, suggesting that the negative charge to mass ratio of LPS plays a critical role in the interaction.

Candida albicans has three genes encoding chitin synthase enzymes. In wild-type strains, the expression of CHS2 and CHS3 peaked 1-2 h after the induction of hyphal growth, whilst mRNA levels in a non-germinative strain, CA2, remained low under the same conditions. CHS1 gene expression did not peak during germ tube formation but remained at low levels in both yeast and hyphal growth. The pattern of gene expression did not predict the changes in measured chitin synthase activities or changes in chitin content during dimorphic transition. Chitin synthase activity increased steadily, and did not peak shortly after germ tube induction, and activity profiles were similar in germ-tube-competent and germ-tube-negative strains. The phenotype of a Δchs2 null mutant suggested that CHS2 encoded the major enzyme activity in vitro and was largely responsible for elevated chitin synthase activities in microsomal preparations from hyphal cells compared to yeast cells. However, CaChs3p was responsible for synthesis of most chitin in both yeast and hyphae. Three independent chitin assays gave markedly different estimates of the relative chitin content of yeast and hyphae and wild-type and chs mutants. Only one of the methods gave a significantly higher chitin content for hyphal compared to yeast cell walls and a lower chitin content for hyphae of the Δchs2 null mutant compared to the parental strain.

The A subunit of DNA gyrase in mycobacteria is frequently subjected to splicing events as its gene, gyrA, harbours an insertion encoding an intein. Investigation of a number of different isolates of Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium ulcerans and Mycobacterium xenopi demonstrated that the presence of GyrA inteins is not random but a taxonomic character specific for a given taxon at a species or subspecies level.

Campylobacter jejuni is recognized as the major cause of food-borne gastrointestinal disease in the developed world. To facilitate the molecular genetic analysis of this pathogen, an approximately 18-fold redundant Tropist3 cosmid library was constructed from C. jejuni NCTC 11168 genomic DNA. The cosmid library was partially ordered by hybridization to 15 pulsed-field electrophoresis (PFGE) restriction fragments. This analysis confirmed the estimated size of the genome to be 1730 kb, but suggested discrepancies in some regions of the published physical map. The precise locations of two of the three rRNA gene clusters were mapped using a combination of restriction fingerprinting, sample sequencing and riboprobing. Additionally, 15 further genes were located on the rev-recd map. A more detailed physical and genetic map of C. jejuni NCTC 11168 is presented.

A genome map of Staphylococcus carnosus TM300, an important micro-organism in the food industry and long used as a starter culture, was constructed by pulsed-field gel electrophoresis of DNA fragments obtained after digestion with NotI, SfiI and ApaI. The size of the chromosome was estimated to be 2590 kb. The fragments were assembled into a physical map using a combination of complementary methods including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. Fifteen genes or gene clusters were positioned on the physical map by Southern hybridization analysis. The map provides a basis for further analysis of the S. carnosus chromosome.