- Volume 149, Issue 7, 2003

The cell envelope of pathogenic mycobacteria is highly distinctive in that it contains a large number of structurally related very long multiple methyl-branched fatty acids. These complex molecules are thought to play important roles in cell envelope organization and virulence. The genetic and enzymic characterization of the polyketide synthase Mas, which is responsible for the synthesis of one such family of fatty acids (the mycocerosic acids), paved the way towards the identification of other enzymes involved in the synthesis of methyl-branched fatty acids in M. tuberculosis. In an effort to elucidate the origin of these complex fatty acids and their possible involvement in pathogenesis, the two mas-like polyketide genes pks5 and pks7 were disrupted in M. tuberculosis and the effects of their inactivation on fatty acid composition and virulence were analysed. While the disruption of pks7 resulted in a mutant deficient in the production of phthiocerol dimycocerosates, the cell envelope composition of the pks5 mutant was found to be identical to that of the wild-type parental strain M. tuberculosis H37Rv. Interestingly, both the pks5 and pks7 mutants displayed severe growth defects in mice.

Meningococcal FetA (FrpB), an iron-regulated outer-membrane protein and vaccine component, was shown to be highly diverse: a total of 60 fetA alleles, encoding 56 protein sequences, were identified from 107 representative Neisseria meningitidis isolates. Phylogenetic analysis established that the allelic variants had been generated by both point mutation and horizontal genetic exchange. Nucleotide substitution was unevenly distributed in the gene, which contained both conserved and variable sequence regions. The most conserved region of the translated peptide sequence corresponded to an amino-terminal domain of the protein and the most diverse region to a previously identified variable region (VR). A nomenclature system for the peptides encoded by the VR was devised which classified 24 variants into 5 FetA variant families. On the basis of these data, murine polyclonal sera specific for four FetA variants were generated. The reactivities of these sera in whole-cell ELISA experiments were consistent with the hypothesis that the VR encoded an immunodominant epitope and indicated that the sera reacted mainly with variants against which they were raised. The diversity of this protein is likely to limit its effectiveness as a vaccine component.

Neisseria meningitidis is a Gram-negative bacterium which is an important causative agent of septicaemia and meningitis. LuxS has been shown to be involved in the biosynthesis of a quorum sensing molecule, autoinducer-2 (AI-2), known to play a role in virulence in Escherichia coli, as well as other bacteria. Evidence that serogroup B of N. meningitidis produces AI-2, along with the observation that a luxS mutant of this strain had attenuated virulence in an infant rat model of bacteraemia, led to further investigation of the role of this quorum sensing molecule in N. meningitidis. In this study, it is demonstrated that AI-2 is not involved in regulating growth of meningococci, either in culture or in contact with epithelial cells. Furthermore, transcriptional profiling using DNA microarrays shows an absence of the concerted regulation seen in other bacteria. Taken together, these data suggest that in N. meningitidis, AI-2 may be a metabolic by-product and not a cell-to-cell signalling molecule.

The AIDA-I autotransporter adhesin, as a prototype of the AIDA adhesin family, represents a tripartite antigen consisting of the functional adhesin AIDA-I (α-domain), which mediates the specific attachment of bacteria to target cells, and a two-domain translocator (AIDAc) organized in the β 1- and β 2-domains. Cellular receptor moieties for the adhesin AIDA-I have not been identified. Here, it is demonstrated that the purified adhesin binds specifically to a high-affinity class of receptors on HeLa cells. Additionally, the adhesin was found to bind to a variety of mammalian cell types, indicating a broad tissue distribution of the receptor moiety. By using complementary techniques, including co-immunoprecipitation and one- and two-dimensional gel electrophoresis, the AIDA-I binding protein on HeLa cells was identified as a surface glycoprotein of about 119 kDa (gp119). The gp119 AIDA-I cellular receptor protein was characterized biochemically and found to be an integral N-glycosylated membrane protein with a pI of 5·2.

Vibrio cholerae of both biotypes produce a soluble Zn2+-dependent metalloprotease: haemagglutinin/protease (Hap), encoded by hapA. Hap has been shown to have mucinolytic and cytotoxic activity. These activities are likely to play an important role in the pathogenesis of cholera and the reactogenicity of attenuated vaccine strains. Production of Hap requires transcriptional activation by the HapR regulator and is repressed by glucose. The present study shows that mucin purified from two sources, bile salts, and growth at 37 °C enhanced Hap protease production. Analysis of hapA and hapR promoter fusions with the lacZ gene showed both promoters to be activated in a cell-density-dependent pattern. Glucose repressed and mucin induced the hapA promoter by a HapR-independent mechanism. Bile had no effect on either hapR or hapA promoter activity. Expression of hapA was required for vibrios to translocate through a mucin-containing gel. These results suggest Hap to play an important role in cholera pathogenesis by promoting mucin gel penetration, detachment and spreading of infection along the gastrointestinal tract.

Conidiobolus coronatus (Zygomycota) is a facultative saprobe that is a pathogen of many insect species. Almost 2000 expressed sequence tag (EST) cDNA clones were sequenced to analyse gene expression during growth on insect cuticle. Sixty percent of the ESTs that could be clustered into functional groups (E⩽10−5) had their best blast hits among fungal sequences. These included chitinases and multiple subtilisins, trypsin, metalloprotease and aspartyl protease activities with the potential to degrade host tissues and disable anti-microbial peptides. Otherwise, compared to the ascomycete entomopathogen Metarhizium anisopliae, Con. coronatus produced many fewer types of hydrolases (e.g. no phospholipases), antimicrobial agents, toxic secondary metabolites and no ESTs with putative roles in the generation of antibiotics. Instead, Con. coronatus produced a much higher proportion of ESTs encoding ribosomal proteins and enzymes of intermediate metabolism that facilitate its rapid growth. These results are consistent with Con. coronatus having adapted a modification of the saprophytic ruderal-selected strategy, using rapid growth to overwhelm the host and exploit the cadaver before competitors overrun it. This strategy does not preclude specialization to pathogenicity, as Con. coronatus produces the greatest complexity of proteases on insect cuticle, indicating an ability to respond to conditions in the cuticle.

Studies with the mouse-adapted Helicobacter pylori strain SS1 had supported an idea that infections by this pathogen start in the gastric antrum and spread to the corpus after extensive mucosal damage. This paper shows that the unrelated strain X47 colonizes the corpus preferentially. Differences between strains in preferred gastric region were detected by co-inoculating mice with a mixture of SS1 and X47, and genotyping H. pylori recovered after 2–8 weeks of infection by vacA s allele PCR and RAPD fingerprinting. Mixed infections were found in each of 59 co-inoculated young C57BL/6J mice. On average, however, SS1 was fourfold more abundant than X47 in the antrum and X47 was threefold more abundant than SS1 in the corpus. Similar results were obtained in mice inoculated first with one strain and then the other strain 2 weeks later. SS1 was even more abundant in the antrum of elderly (>1 year old) mice (97 % of isolates). Qualitatively similar SS1 and X47 tissue distributions were seen using unrelated mouse lines (AKR/J, A/J, DBA/2J, BALB/cJ, LG/J, SM/J), but with significantly different SS1 : X47 ratios in some cases. These results suggest the existence of at least two distinct gastric niches whose characteristics may be affected by host genotype and age (physiology), and indicate that strains differ in how effectively they colonize each niche. Differences among gastric regions and the mixed infections that these allow may contribute to H. pylori diversity and genome evolution.

Quorum sensing systems serve as a means of ‘census taking’ of conspecific and non-conspecific bacteria in the near vicinity. The acylated homoserine lactone (AHL) quorum sensing system has been proposed to be primarily an intra-specific communication system, while the AI-2 autoinducer signalling system is proposed to be an interspecific communication system. Here it is shown that AI-2-like signalling in two marine Vibrio species, Vibrio vulnificus and ‘Vibrio angustum’ S14, induces the core response phenotypes of starvation adaptation and stress resistance, and that a signal antagonist can competitively inhibit these phenotypes. Furthermore, the signals produced by a range of Vibrio species have the ability to induce these phenotypes in V. vulnificus and ‘V. angustum’ S14, indicating that, at least in Vibrio species, AI-2-like signalling systems function as interspecies communication systems capable of ‘cross-talk’ and of regulating environmentally relevant phenotypes.

The physiological behaviour of Lactococcus lactis subsp. cremoris MG 1363 was characterized in continuous culture under various acidic conditions (pH 4·7–6·6). Biomass yield was diminished in cultures with low pH and the energy dedicated to maintenance increased due to organic acid inhibition and cytoplasmic acidification. Under such acidic conditions, the specific rate of glucose consumption by the bacterium increased, thereby enhancing energy supply. This acceleration of glycolysis was regulated by both an increase in the concentrations of glycolytic enzymes (hierarchical regulation) and the specific modulation of enzyme activities (metabolic regulation). However, when the inhibitory effect of intracellular pH on enzyme activity was taken into account in the model of regulation, metabolite regulation was shown to be the dominant factor controlling pathway flux. The changes in glycolytic enzyme concentrations were not correlated directly to modifications in transcript concentrations. Analyses of the relative contribution of the phenomena controlling enzyme synthesis indicated that translational regulation had a major influence compared to transcriptional regulation. An increase in the translation efficiency was accompanied by an important decrease of total cellular RNA concentrations, confirming that the translation apparatus of L. lactis was optimized under acid stress conditions.

Cryptococcus neoformans is a pathogenic fungus that produces melanin when incubated in the presence of certain phenolic substrates such as l-3,4-dihydroxyphenylalanine (l-dopa). Melanin is an enigmatic polymer that is deposited in the cell wall and contributes to virulence. Substantial progress has been made in understanding the synthesis of melanin and the mechanisms by which it contributes to virulence, but relatively little is known about how melanin is rearranged during growth and budding. In this study we used transmission and scanning electron microscopy and immunofluorescence of melanized cells and melanin ‘ghosts' to study the process of melanization during replication. Budding in melanized C. neoformans results in focal disruption of cell-wall melanin at the bud site. In the presence of l-dopa, bud-related melanin defects are repaired and daughter cells are melanized. However, in the absence of substrate, mother cells cannot repair their melanin defects and daughter cells are non-melanized. Hence, melanin in the parent cell is not carried to the daughter cells, but rather is synthesized de novo in buds. These results imply that melanin remodelling occurs during cell growth in a process that involves degradation and synthesis at sites of budding.