- Volume 152, Issue 6, 2006

The complete structures of LPS core types 1 and 2 from Klebsiella pneumoniae have been described by other authors. They are characterized by a lack of phosphoryl residues, but they contain galacturonic acid (GalA) residues, which contribute to the necessary negative charges. The presence of a capsule was determined in core-LPS non-polar mutants from strains 52145 (O1 : K2), DL1 (O1 : K1) and C3 (O8 : K66). O-antigen ligase (waaL) mutants produced a capsule. Core mutants containing the GalA residues were capsulated, while those lacking the residues were non capsulated. Since the proteins involved in the transfer of GalA (WabG) and glucosamine residues (WabH) are known, the chemical basis of the capsular-K2–cell-surface association was studied. Phenol/water extracts from K. pneumoniae 52145ΔwabH waaL and 52145ΔwaaL mutants, but not those from from K. pneumoniae 52145ΔwabG waaL mutant, contained both LPS and capsular polysaccharide, even after hydrophobic chromatography. The two polysaccharides were dissociated by gel-filtration chromatography, eluting with detergent and metal-ion chelators. From these results, it is concluded that the K2 capsular polysaccharide is associated by an ionic interaction to the LPS through the negative charge provided by the carboxyl groups of the GalA residues.

There is increasing evidence that Staphylococcus aureus may colonize the intestinal tract, especially among hospitalized patients. As Staph. aureus has been found to be associated with certain gastrointestinal diseases, it has become important to study whether this bacterium can colonize the intestinal tract and if so, whether it is possible to prevent colonization. Adhesion is the first step in colonization; this study shows that Staph. aureus adheres to mucus from resected human intestinal tissue. Certain lactic acid bacteria (LAB), mainly commercial probiotics, were able to reduce adhesion and viability of adherent Staph. aureus. In displacement assays the amount of adherent Staph. aureus in human intestinal mucus was reduced 39–44 % by Lactobacillus rhamnosus GG, Lactococcus lactis subsp. lactis and Propionibacterium freudenreichii subsp. shermanii. Moreover, adherent Lactobacillus reuteri, Lc. lactis and P. freudenreichii reduced viability of adherent Staph. aureus by 27–36 %, depending on the strain, after 2 h incubation. This was probably due to the production of organic acids and hydrogen peroxide and possibly in the case of L. reuteri to the production of reuterin. This study shows for the first time that Staph. aureus can adhere to human intestinal mucus and adherent bacteria can be displaced and killed by certain LAB strains via in situ production of antimicrobial substances.

Listeria monocytogenes σ B and PrfA are pleiotropic regulators of stress response and virulence gene expression. Quantitative RT-PCR (qRT-PCR) was used to measure transcript levels of σ B- and PrfA-dependent genes in exponential-phase L. monocytogenes wild-type and ΔsigB strains as well as in bacteria exposed to environmental stresses (0.3 M NaCl or growth to stationary phase) or present in the vacuole or cytosol of human intestinal epithelial cells. Stationary-phase or NaCl-exposed L. monocytogenes showed σ B-dependent increases in opuCA (10- and 17-fold higher, respectively) and gadA transcript levels (77- and 14-fold higher, respectively) as compared to non-stressed, exponential-phase bacteria. While PrfA activity, as reflected by plcA transcript levels, was up to 95-fold higher in intracellular L. monocytogenes as compared to non-stressed bacteria, σ B activity was only slightly higher in intracellular than in non-stressed bacteria. Increased plcA transcript levels, which were similar in both host cell vacuole and cytosol, were associated with increases in both prfA expression and PrfA activity. qRT-PCR assays were designed to measure expression of prfA from each of its three promoter regions. Under all conditions, readthrough transcription from the upstream plcA promoter was very low. The relative contribution to total prfA transcription from the σ A-dependent P1prfA promoter ranged from ∼17 % to 30 %, while the contribution of the P2prfA region, which appears to be transcribed by both σ A and σ B, ranged from ∼70 % to 82 % of total prfA transcript levels. In summary (i) σ B is primarily activated during environmental stress and does not contribute to PrfA activation in intracellular L. monocytogenes and (ii) the partially σ B-dependent P2prfA promoter region contributes the majority of prfA transcripts in both intra- and extracellular bacteria.

The lysine- and arginine-specific gingipains (Kgp, and RgpA and RgpB) are the major proteinases produced by the black-pigmented periodontopathogen Porphyromonas gingivalis. They play a role in degrading host proteins, including haemoglobin, from which is formed the μ-oxo bishaem complex of iron(III) protoporphyrin IX, [Fe(III)PPIX]2O, the major haem component of the black pigment. Kgp and RgpA bind haem and haemoglobin via the haemagglutinin-adhesin 2 (HA2) domain, but the role of this domain in the formation of μ-oxo bishaem-containing pigment is not known. UV-visible spectroscopy was used to examine the interaction of iron(III) protoporphyrin IX monomers [Fe(III)PPIX.OH] with recombinant HA2 and purified HRgpA, Kgp and RgpB gingipains. The HA2 domain reacted with Fe(III)PPIX.OH to form μ-oxo bishaem, the presence of which was confirmed by Fourier transform infrared spectroscopy. Both HRgpA and Kgp, but not RgpB, also mediated μ-oxo bishaem formation and aggregation. It is concluded that the Arg- and Lys-gingipains with HA2 haemagglutinin domains may play a crucial role in haem-pigment formation by converting Fe(III)PPIX.OH monomers into [Fe(III)PPIX]2O and promoting their aggregation.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The ΔascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or ΔascC. The experimental infection by immersion with ΔaopO was indistinguishable from that of the parental strain, that of ΔaexT was delayed, whilst the virulence of ΔaopH was reduced significantly but not abolished. By i.p. injection, ΔaexT, ΔaopH and ΔaopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The ΔascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.

This study investigated the subcellular localization of key enzymes of the glyoxylate cycle, i.e. isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (EC 2.3.3.9), that function constitutively in coordination with oxalate biosynthesis of glucose-grown Fomitopsis palustris. The ICL purified previously from F. palustris is termed FPICL1. Subcellular fractionation analysis of the cell homogenate by the sucrose density-gradient method showed that both key enzymes were present in peroxisomes, whereas acetyl-CoA synthase (EC 6.2.1.1) and oxalate-producing oxaloacetate acetylhydrolase (EC 3.7.1.1) were cytosolic. The peroxisomal localization of FPICL1 was further confirmed by electron microscopic and immunocytochemical analysis with anti-FPICL1 antibody. In addition, the peroxisomal target signal, composed of SKL at the C terminus of the cDNA encoding FPICL1, was found, which also suggests that FPICL1 is peroxisomal. Accordingly, it is postulated that transportation of succinate from peroxisomes to mitochondria, and vice versa, for the transportation of isocitrate or citrate, occurs in glucose-grown F. palustris for the constitutive metabolic coordination of the TCA and glyoxylate cycles with oxalate biosynthesis.

Fusarium graminearum strains responsible for causing the plant disease Fusarium head blight vary greatly in their ability to cause disease and produce mycotoxins on wheat. With the goal of understanding fungal gene expression related to pathogenicity, three cDNA libraries were created by suppression subtractive hybridization using wheat heads inoculated with a highly aggressive strain and either water or a less aggressive strain of this pathogen. Eighty-four fungal genes expressed during initial disease development were identified. The probable functions of 49 of these genes could be inferred by bioinformatic analysis. Thirty-five ESTs had no known homologues in current databases and were not identified by ab initio gene prediction methods. These ESTs from infected wheat heads probably represent F. graminearum genes that previously were not annotated. Four genes represented in one of these libraries were selected for targeted gene replacement, leading to the characterization of a two-component response regulator homologue involved in pathogenicity of the fungus. The mutants for this gene showed reduced sporulation and delayed spread of Fusarium head blight on wheat.