Simultaneous identification of bacterial virulence genes by negative selection.

An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase.

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

Insertional mutagenesis of pathogenic fungi.

Screening insertional mutants for loss of virulence is an effective method for investigating the molecular genetic basis of bacterial pathogenesis, but has only recently been applied to fungal pathogens. For many pathogenic fungi transformation with heterologous plasmid DNA results in complex integration events. This problem can now be circumvented for some species using restriction enzyme mediated integration. Insertional mutagenesis of Fusarium oxysporum using the naturally occurring fungal transposon impala has been described, but transposon tagging for other fungi has yet to be developed. Although insertional mutagenesis has recently identified important virulence determinants of fungal phytopathogens, the lack of suitable screening strategies has so far limited its applicability for fungal pathogens of humans.

The Ustilago maydis pyr3 gene: sequence and transcriptional analysis.

The pyr3 gene of Ustilago maydis encodes a 391-amino acid (aa) polypeptide. The sequence has identifies with dihydro-orotases (DHOases) from other organisms, but is most related to sequences of other monofunctional enzymes. The polypeptide contains the three domains conserved in other DHOases. The polypeptide encoded by the pyr3-1 allele has an aa change seven residues away from the C-terminal conserved domain. Transcription start point (tsp) is 58 nucleotides upstream from the start codon, and is in a region characterised by CTTT and CATC motifs. In the absence of TATA and CAAT boxes, these motifs might be important in transcriptional regulation. Gene disruption experiments suggest that the pyr3 gene product might have another function in addition to that in pyrimidine biosynthesis.

Evolution of transcription-regulating proteins: caveat lector!

The paper of Hawkins et al. [Gene 146 (1994) 145-158] reports incorrect descriptions of mutant phenotypes, omits mention of the absence of a highly relevant glutamine-binding site and contains sequence alignments which might mislead the reader. Extensive sequence analysis reveals as untenable a central hypothesis of the paper concerning a possible evolutionary relationship between anthranilate synthetases and the transcription factors mediating nitrogen metabolite repression in fungi.

The Ustilago maydis nar1 gene encoding nitrate reductase activity: sequence and transcriptional regulation.

The nar1 gene was cloned from Ustilago maydis and the 908-amino-acid (aa) sequence of the encoded protein found to have strong identities with other nitrate reductases from fungi and plants. This was especially so in three domains which define enzyme cofactor-binding sites. The gene was isolated alone and in association with the nir1 gene, suggesting that the two genes are closely linked on the chromosome. The phenotype of a strain in which nar1 had been disrupted was consistent with the only role of nar1 being in nitrate reduction. Nitrate ions induced a 90-fold increase in nar1 transcript levels, while ammonium ions repressed transcript levels.

Isolation of metabolic genes and demonstration of gene disruption in the phytopathogenic fungus Ustilago maydis.

A cDNA library was constructed in the yeast expression vector pYcDE8 using mRNA from the phytopathogenic fungus Ustilago maydis and cDNAs capable of complementing mutations in three yeast genes, URA3, LEU2 and TPI1, were identified. Nucleotide sequence analysis indicated that the cDNA clone, which complemented the yeast ura3 mutation, carries the pyr6 gene encoding orotidine-5'-phosphate decarboxylase. The genomic copy of the pyr6 gene was isolated by hybridization with the cDNA and used to complement a pyr- mutant of U. maydis. One-step gene disruption was demonstrated by transforming U. maydis with a copy of the pyr6 gene interrupted in the coding region by a selectable marker for resistance to hygromycin B.

Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo.

In the Salmonella-mouse model of systemic infection, high dose inoculation results in the multiplication of many of the cells present in the inoculum, rather than the clonal amplification of a small number. This characteristic has allowed the development of methods to screen multiple strains for either virulence attenuation or gene expression within the same animal. Mixed infections with mutant and wild-type strains are used to provide a sensitive measure of virulence attenuation referred to as the competitive index. We have recently used a variation of this method, involving mixed infections of single and double mutant strains, to study virulence gene interaction in vivo.

Cloning and characterization of chsD, a chitin synthase-like gene of Aspergillus fumigatus.

A chitin synthase-like gene (chsD) was isolated from an Aspergillus fumigatus genomic DNA library. Comparisons with the predicted amino acid sequence from chsD reveals low but significant similarity to chitin synthases, to other N-acetylglucosaminyltransferases (NodC from Rhizopus spp., HasA from Streptococcus spp. and DG42 from vertebrates. A chsD- mutant strain constructed by gene disruption has a 20% reduction in total mycelial chitin content; however, no differences between the wild-type strain and the chsD- strain were found with respect to morphology, chitin synthase activity or virulence in a neutropenic murine model of aspergillosis. The results show that the chsD product has an important but inessential role in the synthesis of chitin in A. fumigatus.

RrgA is a pilus-associated adhesin in Streptococcus pneumoniae.

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.

A pneumococcal pilus influences virulence and host inflammatory responses.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality world-wide. The initial event in invasive pneumococcal disease is the attachment of encapsulated pneumococci to epithelial cells in the upper respiratory tract. This work provides evidence that initial bacterial adhesion and subsequent ability to cause invasive disease is enhanced by pili, long organelles able to extend beyond the polysaccharide capsule, previously unknown to exist in pneumococci. These adhesive pili-like appendages are encoded by the pneumococcal rlrA islet, present in some, but not all, clinical isolates. Introduction of the rlrA islet into an encapsulated rlrA-negative isolate allowed pilus expression, enhanced adherence to lung epithelial cells, and provided a competitive advantage upon mixed intranasal challenge of mice. Furthermore, a pilus-expressing rlrA islet-positive clinical isolate was more virulent than a nonpiliated deletion mutant, and it out-competed the mutant in murine models of colonization, pneumonia, and bacteremia. Additionally, piliated pneumococci evoked a higher TNF response during systemic infection, compared with nonpiliated derivatives, suggesting that pneumococcal pili not only contribute to adherence and virulence but also stimulate the host inflammatory response.

Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes.

Methods to detect "native" proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.

Proteins in intercellular washing fluid from noninoculated and rust-affected leaves of wheat and barley.

Proteins in intercellular washing fluid (IWF) from wheat (Triticum aestivum) and barley (Hordeum vulgare) leaves were separated by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB) or silver. Intracellular protein from the cut ends of leaves accounted for only a small proportion of total protein in IWF from wheat leaves. When these were heavily infected with the stem rust fungus (Puccinia graminis f. sp. tritici) and grown at 19 degrees C, four infection-related CBB-stainable proteins were detected in IWF.To compare IWF proteins from wheat and barley leaves infected with the same pathogen, conditions were established that permitted luxuriant growth of stem rust of wheat in barley (exposure to chloroform before inoculation and maintenance at 25 degrees C thereafter). Under these conditions, at least 10 infection-related silver-stainable proteins were detected in IWF from infected wheat in addition to the more than 50 that were of host origin. The electrophoretic properties of 8 of the infection-related proteins were the same as those of 8 infection-related proteins in IWF from barley.IWF from wheat and barley grown under these conditions was analyzed for Concanavalin A-binding glycoproteins immobilized on nitrocellulose membrane replicas made from gels. Of the many infection-related glycoproteins that were detected in IWF from stem rust-affected wheat, approximately 20 occupied the same positions as those from stem rust-affected barley. The glycoprotein pattern of IWF prepared from wheat leaves grown at 19 degrees C and infected with the leaf rust fungus (P. recondita f. sp. tritici) was markedly different to that of IWF from the same host infected with the stem rust fungus. We conclude that IWF from rust-affected cereal leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium.

Peroxidases and glycosidases in intercellular fluids from noninoculated and rust-affected wheat leaves : isozyme assay on nitrocellulose blots from two-dimensional gels.

Proteins in intercellular washing fluid (IWF) from noninoculated and stem rust-affected wheat leaves were separated by isoelectric focusing and polyacrylamide gel electrophoresis under nondenaturing conditions, transferred to nitrocellulose membranes, and assayed in situ for peroxidase and glycosidase activity.Two infection-related peroxidase isozymes were detected in addition to more than ten that were present only in noninoculated plants. One other peroxidase isozyme was present in much higher concentration in IWF from infected leaves than in IWF from noninoculated leaves.IWF contained many polymorphic glycosidases. A new method is described to localize the glycosidase isozymes accurately on nitrocellulose blots for evaluation of their substrate specificities: each blot was developed with the appropriate p-nitrophenyl glycoside to reveal glycosidase activity, then reprobed for concanavalin A-binding glycoproteins to serve as an internal reference frame for blot-to-blot comparisons. This procedure also provided information on glycosylation of the isozymes.The locations of at least 15 (out of 37) isozymes were coincident with concanavalin A binding, including those of all 10 alpha-d-mannosidases, and of 6 beta-d-xylosidases. On five areas of the blots there was coincidence of beta-d-xylosidase and alpha-l-arabinosidase activity. Three of these areas corresponded to three of the most prominent Coomassie brilliant blue-stainable IWF proteins in gels. Isozymes that could convert p-nitrophenyl-beta-d-glucoside, -beta-d-galactoside, and/or -beta-d-fucoside revealed a complex pattern of partially overlapping substrate specificities: two isozymes utilized both glucose and fucose derivatives, one utilized all three derivatives, and several others converted only one of the three substrates. No enzymes were detected with activity on p-nitrophenyl-beta-d-galactosaminide, -beta-l-fucoside, or -alpha-d-galactoside. No additional glycosidases were detected in IWF from stem rust-affected leaves.

Identification and analysis of bacterial virulence genes in vivo.

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

Linking approaches in the study of fungal pathogenesis: a commentary.

Thus far, it has not been possible to demonstrate that fungal pathogens possess specific extracellular enzymes with important roles in pathogenesis. In contrast, candidate gene knock-out experiments have revealed regulatory networks that appear to be specific to pathogenesis. The tracings of these networks may have already been described in the well-known cell differentiation pathways of many nonpathogens. The challenge for fungal pathologists will be to link these signaling pathways to the host environment and to the expression of determinants that advance the disease state.

Mutation in a heat-regulated hsp70 gene of Ustilago maydis.

Four genes (ums1, ums2, ums3 and ums4) representing an hsp70-related gene family were isolated from a genomic library of Ustilago maydis. All four genes are transcriptionally active during normal growth. Following a heat shock, the mRNA levels of ums1 and ums2 increase by approximately 5-fold, whereas the ums3 transcript becomes less abundant. The amount of ums4 mRNA remains relatively unchanged after heat treatment. The nucleotide sequence of the 5' non-coding and a portion of the ums2 coding region was determined. The sequence encoding the first 90 amino acids is 73% identical to corresponding regions of the Drosophila and yeast (SSA1) hsp70 genes. To investigate the effect of a mutation in ums2, a plasmid was constructed in which most of the transcriptional unit of ums2 was deleted and substituted with the Escherichia coli hygromycin B (hygB) phosphotransferase gene. Transcription of this gene is controlled by the ums2 promoter, allowing the expression of hygB resistance in Ustilago. The marker was introduced into diploid cells as a linear sequence with termini homologous to the 5' and 3' flanking regions of ums2. In approximately 50% of transformants examined, one of the two wild-type ums2 alleles had been replaced by the mutated sequence, demonstrating the feasibility of using one-step gene disruption to create heterozygous diploids in Ustilago. The ums2/ums2::hygBr heterozygote produced teliospores after injection into corn plants, but only cells carrying functional ums2 were found among their meiotic progeny. Therefore ums2::hygBr segregates as a recessive lethal, which strongly suggests that ums2 is essential for growth in Ustilago.

Gene transfer system for the phytopathogenic fungus Ustilago maydis.

A selectable marker for transformation was constructed by transcriptional fusion of a Ustilago maydis heat shock gene promoter with the hygromycin B phosphotransferase gene of Escherichia coli. U. maydis was transformed to hygromycin B resistance by polyethylene glycol-induced fusion of spheroplasts following exposure to plasmid DNA that carried the marker gene. Transformation frequencies of 50 and 1000 transformants per microgram of DNA per 2 x 10(7) spheroplasts were obtained for circular and linear vector DNA, respectively. In the majority of transformants, the vector was integrated at a single chromosomal site, in either single copy or tandem duplication, as determined by Southern hybridization analysis of electrophoretically separated chromosomes and of restriction-endonuclease-cleaved DNA. The predominant form (82%) of vector integration was by nonhomologous recombination; the remainder carried the plasmid at the homologous heat shock gene locus. No evidence for gene conversion or gene replacement was obtained in 28 transformants. Hygromycin B phosphotransferase activity and resistance to hygromycin B were roughly correlated with the copy number of the integrated vector at the homologous location. Transforming DNA was stably maintained during mitosis and meiosis. This transformation procedure and associated vector should permit the cloning of genes by direct complementation in U. maydis.

Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus.

Forty-four oligonucleotide decamers were tested for their abilities to generate randomly amplified polymorphic DNA (RAPD) markers from genomic DNAs of three different isolates of Aspergillus fumigatus. Seven primers generated RAPDs that allowed the three isolates to be differentiated; one of the primers also yielded a unique RAPD pattern in each of an additional six fungal isolates, demonstrating the utility of this technique for distinguishing between A. fumigatus isolates.