- Volume 148, Issue 12, 2002

Primary infection by group A streptococci (GAS) takes place at either the throat or skin of the human host, often leading to pharyngitis or impetigo, respectively. Many GAS strains differ in their preference for throat and skin tissue sites. Previous epidemiological findings show that many of the strains displaying strong tropism for the skin have a high-affinity binding site for plasminogen, located within M protein (PAM), a prominent surface fibril. Plasminogen bound by PAM interacts with streptokinase, a plasminogen activator secreted by GAS, to yield bacterial-bound plasmin activity. In this study, PAM and streptokinase were tested for their roles in infection using an experimental model that closely mimics human impetigo. Inactivation of genes encoding either PAM or streptokinase led to a partial, but significant, loss of virulence in vivo, as measured by net growth of the bacteria and pathological alterations. The relative loss in virulence in vivo was greater for the streptokinase mutant than for the PAM mutant. However, the PAM mutant, but not the streptokinase mutant, displayed a partial loss in resistance to phagocytosis in vitro. The combined experimental and epidemiological data provide evidence that PAM and streptokinase play a key role in mediating skin-specific infection by GAS. In addition, secreted cysteine proteinase activity due to SpeB leads to degradation of streptokinase in stationary phase broth cultures. Since SpeB is also a determinant of tissue-specific GAS infection at the skin, direct interactions between these two proteolytic pathways may constitute an important pathogenic mechanism. An integrated model for superficial infection at the skin is presented.

The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P499), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P499 is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 0·1% activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C492) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C492, which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.

Chlamydial infection has been associated with myocarditis in animals and humans. However, the mechanism resulting in myocarditis following infection is not known. Here, evidence is presented that both Chlamydia trachomatis and Chlamydia pneumoniae can infect and replicate in myocytes isolated from neonate rats. The infected myocytes contained chlamydial inclusions, indicative of chlamydial growth, and infectious particles were recovered from the infected myocytes. It was also found that chlamydial infection at a late stage induced significant damage to the infected myocytes, as evidenced by an increased lactate dehydrogenase release, reactive oxygen species production and a reduced ATP level. However, no nuclear apoptosis was detected in the infected myocytes. Collectively, these observations have demonstrated that Chlamydia spp. are able to both infect and damage myocytes, suggesting a potential role of chlamydial infection in myocarditis.

Hydrogen sulfide is highly toxic to mammalian cells. It has also been postulated that hydrogen sulfide modifies haemoglobin resulting in haemolysis. The enzyme that produces hydrogen sulfide from L-cysteine was purified from Streptococcus anginosus. Using the N-terminal amino acid sequence of the purified enzyme, the lcd gene encoding L-cysteine desulfhydrase was cloned; the recombinant protein was then purified to examine its enzymic and biological characteristics. This L-cysteine desulfhydrase had the Michaelis–Menten kinetics K m=0·62 mM and V max=163 μmol min−1 mg−1. DL-Cystathionine, L-cystine, S-(2-aminoethyl)-L-cysteine, 3-chloro-DL-alanine and S-methyl-L-cysteine were substrates for the enzyme, whereas D-cysteine, DL-homocysteine, L-methionine, DL-serine, DL-alanine, L-cysteine methyl ester, L-tryptophan, L-tyrosine and L-phenylalanine were not. These findings suggest that this L-cysteine desulfhydrase is a C-S lyase that catalyses the α,β-elimination (αC-N and βC-S) reaction. In addition, it is demonstrated that the hydrogen sulfide produced by this enzyme caused the modification and release of haemoglobin in sheep erythrocytes.

The spores of Bacillus subtilis have characteristic properties and consist of complex structures including various types of proteins. To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of B. subtilis. A total of 154 proteins were identified, and 69 of them were novel. The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells. The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a lacZ reporter gene. The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely σF, σE, σG and σK. In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of B. subtilis, is an effective tool for the analysis of complicated cellular structures.

The chromosomal arsenic-resistance (ars) operon of Acidithiobacillus ferrooxidans is atypical in that it is divergent, with its arsCR and arsBH genes transcribed in opposite directions. Furthermore, the amino-acid sequence of the putative ArsR-like regulator of the ars operon is not conserved in regions that have been shown to be responsible for binding to arsenic. Instead, the ArsR-like protein of At. ferrooxidans is related to a group of unstudied ArsR-like proteins that have been found to be associated with chromosomal ars-like operons identified during genome-sequencing projects. Using arsB–lacZ, arsR–lacZ and arsC–lacZ fusions, it was shown that the ArsR-like protein of At. ferrooxidans is a repressor of the arsBH and arsRC genes of this organism, and that induction of gene expression took place when either AsIII (arsenite) or AsV (arsenate) were added. Deletion of 19 aa from the C terminus of the 118 aa ArsR protein did not affect the regulation of its activity, but deletion of an additional 28 aa inactivated ArsR. Northern-blot hybridization suggested that on induction of expression, the arsRC genes were transcribed in greater quantities than the arsBH genes, but that the level of induction was not affected by the form of arsenic added (AsIII or AsV).

To get a better insight into the physiology of the high-toxic JP2 clone of Actinobacillus actinomycetemcomitans serotype b, which is strongly associated with juvenile periodontitis in adolescents of African descent, the modes of iron acquisition in this clone were examined and compared to those of other strains of the species. None of the strains examined could utilize human transferrin as a source of iron. This was in accordance with the presence of a non-functional tbpA gene, which normally encodes the A subunit of the transferrin-binding-protein complex. Southern blot analysis indicated that functional duplications of tbpA were not present in the genome. Thus, A. actinomycetemcomitans seems to be in a process of evolution, in which iron acquisition from host transferrin is not essential as in many other members of the Pasteurellaceae. All strains could utilize haem as a source of iron. All 11 A. actinomycetemcomitans strains examined harboured a single genomic sequence with homology to the hgpA gene encoding haemoglobin-binding protein A in Haemophilus influenzae. However, in all three strains belonging to the JP2 clone and in one serotype e strain hgpA was a pseudogene. Seven other strains possessed a functional hgpA gene which, according to insertion mutagenesis experiments, was responsible for the ability of these strains to utilize haemoglobin as a source of iron. Thus, the presence of an hgpA pseudogene and the inability to use human haemoglobin as an iron source discriminate the high-toxic JP2 clone from low-toxic serotype b strains and most other strains of A. actinomycetemcomitans.

The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. The native Ple. sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic. pastoris. The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4·38. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3·3, 6, 6·5 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 °C and pH 3·5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.

Erwinia pyrifoliae is a novel bacterial pathogen, which causes Asian pear blight and is related to Erwinia amylovora, the causative agent of fire blight. E. pyrifoliae produces exopolysaccharide (EPS) related to amylovoran in its sugar composition and sugar linkages. This was shown by degradation of the EPS with a viral depolymerase, and by methylation analysis and ESI/MS. The structure of the repeating units was confirmed by 1H-NMR spectra. The EPS of E. pyrifoliae carried side chains, which were mainly terminated by acetyl and pyruvyl residues as found previously for amylovoran. On the other hand, a second side chain with glucose found for up to 65% of the repeating units of amylovoran was completely absent. The nucleotide sequences of five genes of the cps cluster of E. pyrifoliae encoding proteins for EPS synthesis were characterized and displayed a high homology with the corresponding ams genes. Similar functions of the gene products are assumed. As for ams mutants of E. amylovora, a cpsB mutant of E. pyrifoliae did not synthesize EPS and did not produce ooze on slices of immature pears or symptoms on pear seedlings. The cps mutant was complemented for EPS synthesis and virulence on pear slices with a gene cluster of E. amylovora that included amsB.

In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence obtained for csmA indicated that it had high similarity to class V chitin synthases. chsB and csmA disruption strains and a strain in which chsB transcription was controlled were constructed using the nitrite reductase (niiA) promoter. The strains were examined during hyphal growth by Northern analysis, analysis of the cell-wall composition and growth in the presence of Calcofluor white (CFW). The chsB disrupted strain and the uninduced p niiA –chsB strain exhibited hyperbranching, they had a lower level of conidiation than the wild-type and were sensitive to CFW at 50 mg l−1. When chsB transcription was induced in the strain containing the p niiA –chsB construct, the strain displayed wild-type morphology on solid medium and at sub-maximum growth rates but the wild-type morphology was not fully restored during rapid growth in batch cultivation. The csmA disruption strain displayed morphological abnormalities, such as ballooning cells, intrahyphal hyphae and conidial scars. The growth was severely inhibited in the presence of 10 mg CFW l−1. In none of the constructed strains did the cell-wall composition differ from the wild-type. Northern analysis indicated no change in the transcription of the chitin synthase genes csmA and chsC when chsB expression was altered, and there was no change in the transcription of chsB and chsC when csmA was disrupted.

KIC1 encodes a PAK kinase that is involved in morphogenesis and cell integrity. Both over- and underexpressing conditions of KIC1 affected cell wall composition. Kic1-deficient cells were hypersensitive to the cell wall perturbing agent calcofluor white and had less 1,6-β-glucan. When Kic1-deficient cells were crossed with various kre mutants, which also have less 1,6-β-glucan in their wall, the double mutants displayed synthetic growth defects. However, when crossed with the 1,3-β-glucan-deficient strain fks1Δ, no synthetic growth defect was observed, supporting a specific role for KIC1 in regulating 1,6-β-glucan levels. Kic1-deficient cells also became highly resistant to the cell wall-degrading enzyme mixture Zymolyase, and exhibited higher transcript levels of the cell wall protein-encoding genes CWP2 and SED1. Conversely, overexpression of KIC1 resulted in increased sensitivity to Zymolyase and in a higher level of 1,6-β-glucan. Multicopy suppressor analysis of a Kic1-deficient strain identified RHO3. Consistent with this, expression levels of RHO3 correlated with 1,6-β-glucan levels in the cell wall. Interestingly, expression levels of KIC1 and the MAP kinase kinase PBS2 had opposite effects on Zymolyase sensitivity of the cells and on cell wall 1,6-β-glucan levels in the wall. It is proposed that Kic1 affects cell wall construction in multiple ways and in particular in regulating 1,6-β-glucan levels in the wall.

In this study, cDNA and genomic clones encoding a homologue of the yeast gene anti-oxidant 1 (ATX1) from the white-rot fungus Trametes versicolor, a basidiomycete known to produce several laccase isoenzymes involved in lignin degradation, were identified. This gene, named Trametes ATX homologue (tahA), encodes a protein of 7·9 kDa with 56% identity to the yeast Atx1p sequence. Two different alleles of tahA were obtained that differed mainly in their intervening sequences and in a 425 nt insertion located 183 nt upstream of the transcription start site. tahA is present as one copy per haploid nucleus in T. versicolor, as shown by Southern analysis. Expression of tahA cDNA restored high-affinity iron uptake in a Δatx1 yeast strain and oxygen sensitivity in a Δsod1 Δsod2 yeast strain, showing that tahA is also a functional homologue of ATX1. The inability of tahA to rescue the Δsod1 phenotype on copper-deficient medium indicated that tahA function is copper-dependent. Sequence analysis of the tahA promoter revealed several motifs that were similar to the conserved motifs found in the copper-regulated metallothionein and Cu, Zn superoxide dismutase genes, CUP1 and SOD1, of Saccharomyces cerevisiae, Neurospora crassa and Candida glabrata. In contrast to its yeast homologue ATX1, tahA is induced under elevated copper concentrations in the medium (>0·25 μM CuSO4) and repressed under copper starvation. The transcription of tahA was analysed in response to copper and iron, and after adding xenobiotica. The results are discussed in relevance to laccase expression.

Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Fe-mediated gene regulation. Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media. Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N2 fixation.