Reinvestigation of the virulence of Rhodococcus equi isolates from patients with and without AIDS

Rhodococcus equi emerged as a zoonotic pathogen of human immunodeficiency virus-infected patients over the last three decades. Two virulence plasmid types of R. equi, pVAPA and pVAPB associated with equine and porcine isolates, have been recognized, and more recently, pVAPN, a novel host-associated virulence plasmid in R. equi, was found in bovine and caprine isolates. We reinvestigated 39 previously reported isolates of R. equi from patients with and without acquired immunodeficiency syndrome (AIDS) by detecting vapA, vapB and vapN using PCR and plasmid profiling. After excluding one isolate that could not be cultured from frozen storage, eight isolates carried a virulence plasmid encoding vapA (pVAPA), 10 carried a virulence plasmid encoding vapB (pVAPB), seven carried a virulence plasmid encoding vapN (pVAPN) and 13 were negative for those genes. Of the 29 isolates from patients with AIDS, 7, 10 and 5 harboured pVAPA, pVAPB and pVAPN respectively. Among nine isolates from patients without AIDS, one and two harboured pVAPA and pVAPN respectively. This study demonstrated that pVAPN-positive R. equi existed in human isolates before 1994 and reaffirmed that equine-associated pVAPA-positive, porcine-associated pVAPB-positive and bovine- or caprine-associated pVAPN-positive R. equi are widely spread globally. Because domestic animals might be major sources of human infection, further research is needed to reveal the prevalence of pVAPN-positive R. equi infection in cattle and goats.     

Molecular and infection biology of the horse pathogen Rhodococcus equi

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi -containing vacuoles inside host cells, factors involved in virulence and host resistance and on host–pathogen interaction on organismal and cellular levels.     

Pathogenesis of Rhodococcus equi infection in mice: Roles of virulence plasmids and granulomagenic activity of bacteria

Abstract Virulence of Rhocococcus equi ATCC 33701 and its plasmid-cured derivative ATCC 33701P⁻ was compared in BALB/c and C3H/HeJ mice in terms of bacterial growth kinetics and histological changes in the liver, spleen and lungs, and humoral immune responses. Injection with a sublethal dose of 10⁶ ATCC 33701 in mice resulted in microabscess formation after rapid multiplication in the liver and spleen by day 4, and then the bacteria were gradually eliminated with the formation of granuloma and the production of specific antibodies against 15- to 17-kDa antigens of the virulent bacteria. By contrast, ATCC 33701P⁻ was avirulent as shown by early elimination of viable bacteria and no evidence of net multiplication in the organs. Histopathological changes consisted of only slight, transient infiltration of neutrophils and macrophages in the liver. Although live ATCC 33701P⁻ did not evoke any humoral or histological responses in the mice, a large inoculum (10⁸) of killed ATCC 33701 and ATCC 33701P⁻ resulted in the formation of granuloma in the liver and accelerated extramedullary hemopoiesis in the spleen. These results suggest that the pathogenesis of R. equi infection involves at least two important virulence determinants, both of which play critical roles in the disease: one is the virulence plasmid, which is required for R. equi to resist and grow within host cells; and the other is the granulomagenic activity that is related to the lipids and nature of the cell wall of the species, which induces the characteistic pathological changes.     

Virulence‐associated protein A from Rhodococcus equi is an intercompartmental pH‐neutralising virulence factor

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane‐bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram‐positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid‐encoded and secreted virulence‐associated protein A (VapA) participates in exclusion of the proton‐pumping vacuolar‐ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH‐neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid‐less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH‐neutral and hence growth‐promoting intracellular niche. VapA represents a new type of Gram‐positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton‐pumping ATPase, and consequently disarming host defences.     

Structural relationship among the members of a multigene family coding for the sweet potato tuberous root storage protein

Sporamin, the major soluble protein of the sweet potato tuberous root, is coded for by a multigene family. Fourty-nine essentially full-length sporamin cDNAs isolated from tuberous root cDNA library have been classified by cross hybridization, restriction endonuclease cleavage pattern and ribonuclease cleavage mapping. All the cDNAs fall into one of the two distinct homology groups, subfamilies A and B, which correspond to the polypeptide classes sporamin A and B, respectively. At least 5 different sequences are detected in both of the 22 sporamin A and 27 sporamin B cDNAs. Comparison of the nucleotide sequences of the coding region of three each of sporamin A and B subfamily members, four from cDNAs and two from genomic clones, indicates that intra-subfamily homologies (94 to 98%) are much higher than inter-subfamily homologies (82 to 84%), and there are deletions or insertions of one or two codons at three locations which characterize each subfamily. Large portions of base substitutions in the coding region accompany amino acid substitutions. In contrast to the coding region, most of the structural differences among the members in the 5 and 3 noncoding regions are deletions or insertions.     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica. Received: 15 April 1997 / Accepted: 13 June 1997     

MarR家族转录因子HpaR和XC0449协同调控野油菜黄单胞菌致病性

MarR家族转录因子广泛存在于细菌及古生菌中,并灵活、精细地调控多种毒力、抗胁迫及抗生素相关的生理生化途径。在野油菜黄单胞菌中,MarR家族转录因子HpaR (XC2827)的失活会显著降低细菌对于寄主甘蓝的致病力,同时会导致胞外蛋白酶的过量表达。本研究进一步发现,Xcc 8004基因组一共编码9个MarR家族转录因子。表达并纯化其中的HpaR (XC2827)和XC0449,体外微量热泳动(MST)实验及Pull-down实验证明二者可以在体外特异性结合。同时,表型检测发现XC0449突变会导致细菌致病力显著下降。通过体外凝胶迁移阻滞试验(EMSA)、体内qRT-PCR和GUS检测证明,XC0449和HpaR均作为转录激活子协同调控下游致病相关基因XC0705的表达,最终调控细菌毒力及胞外酶合成。     

Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus

In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome.In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms.Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products.In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.     

Phylogeny and evolution of the major intrinsic protein family

BACKGROUND INFORMATION: MIPs (major intrinsic proteins) form channels across biological membranes that control recruitment of water and small solutes such as glycerol and urea in all living organisms. Because of their widespread occurrence and large number, MIPs are a sound model system to understand evolutionary mechanisms underlying the generation of protein structural and functional diversity. With the recent increase in genomic projects, there is a considerable increase in the quantity and taxonomic range of MIPs in molecular databases. RESULTS: In the present study, I compiled more than 450 non-redundant amino acid sequences of MIPs from NCBI databases. Phylogenetic analyses using Bayesian inference reconstructed a statistically robust tree that allowed the classification of members of the family into two main evolutionary groups, the GLPs (glycerol-uptake facilitators or aquaglyceroporins) and the water transport channels or AQPs (aquaporins). Separate phylogenetic analyses of each of the MIP subfamilies were performed to determine the main groups of orthology. In addition, comparative sequence analyses were conducted to identify conserved signatures in the MIP molecule. CONCLUSIONS: The earliest and major gene duplication event in the history of the MIP family led to its main functional split into GLPs and AQPs. GLPs show typically one single copy in microbes (eubacteria, archaea and fungi), up to four paralogues in vertebrates and they are absent from plants. AQPs are usually single in microbes and show their greatest numbers and diversity in angiosperms and vertebrates. Functional recruitment of NOD26-like intrinsic proteins to glycerol transport due to the absence of GLPs in plants was highly supported. Acquisition of other MIP functions such as permeability to ammonia, arsenite or CO2 is restricted to particular MIP paralogues. Up to eight fairly conserved boxes were inferred in the primary sequence of the MIP molecule. All of them mapped on to one side of the channel except the conserved glycine residues from helices 2 and 5 that were found in the opposite side.     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Nucleotide polymorphism in the chalcone synthase-A locus and evolution of the chalcone synthase multigene family of common morning glory Ipomoea purpurea

Chalcone synthase (CHS) is a small multigene family with at least four members (CHS-A, B, C and PS) in common morning glory Ipomoea purpurea ROTH. The chalcone synthase enzyme performs the initial condensation reaction that results in the 15-carbon three-ring structure that is the backbone of flavonoid biosynthesis. The biochemical pathway that commences with CHS is important in plant disease defence, pigment biosynthesis and UV protection. Accordingly, it is of substantial interest to characterize levels and patterns of molecular diversity for genes that encode this important enzyme. We report the sequence of 19 CHS-A alleles from Mexican and American populations of common morning glory. American populations of this annual self-compatible vine are believed to have been introduced from Mexico, where the species is native. Individual plants were sampled from populations of common morning glory throughout Mexico and the south-eastern USA. Four American alleles were sequenced and these, together with one allele from Mexico City, were identical in primary nucleotide sequence. These data suggest a restricted origin for the American population, probably as a consequence of selection for domestication by pre-Columbian peoples. Additionally the Mitontic (Chiapas, Mexico) population is significantly more homogeneous than expected by chance indicating that this population may also have experienced a recent population bottleneck. Estimates of nucleotide diversity from the Mexican CHS-A alleles were high. We present evidence that these estimates may, in part, result from low to moderate levels of interlocus recombination/gene conversion. We also present evidence that the ancient duplication of the CHS gene family, preceding the origin of the genus Ipomoea, was associated with heterogeneity in the rate of substitution between the resulting gene family members. The group of gene family members whose sequences possess a signature amino acid of the closely related Stilbene synthase exhibit a significantly faster proportional rate of nonsynonymous substitution.