Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis

P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets.     

Staphylococcus aureus virulence factors identified by using a high-throughput Caenorhabditis elegans-killing model

Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.     

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species

Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.     

Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature

Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 degrees C. Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes. Only 12 of those genes were previously known to be required for salt-tolerance. Among the 61 insertions, three caused general osmo-sensitivity. We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock. A total of 31 insertions caused failure to grow at low temperature. Those identified ten different genes, three of which had previously been reported to affect cold-tolerance. Four genes were identified in both the salt and the cold-sensitivity screen. We found several unusual insertion mutations: (1) insertions in or close to essential genes, (2) insertion in an intergenic region and (3) insertions causing stress-sensitivity in W303-1A, while the deletion mutant in BY4741 did not show such a phenotype. Surprisingly, our mutant set and that reported in the large-scale transposon insertion project (TRIPLES, http://ygacmed.yale.edu/triples/triples.htm) only marginally overlap. We discuss some of the features of transposon mutagenesis in light of the availability of the complete set of yeast deletion mutants and we discuss the possible roles of the genes we identified.     

Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model

Actinobacillus pleuropneumoniae is a significant respiratory pathogen of swine causing a severe and often fatal fibrinous hemorrhagic bronchopneumonia with significant economic losses resulting from chronic as well as acute infections. This study describes the application of a signature-tagged mutagenesis (STM) system to identify in vivo critical genes of A. pleuropneumoniae. Twenty pools representing over 800 A. pleuropneumoniae mutants were screened in a natural-host porcine infection model and presumptive attenuated mutants were selected. The identity of the disrupted gene in each mutant was determined using an inverse PCR approach to amplify DNA sequences adjacent to the transposon insertion, followed by sequencing of the PCR product and comparison to bacterial databases. In vitro and in vivo competitive indices were determined for each unique mutant, and a total of 20 unique, attenuating gene disruptions were identified including insertions into homologues of genes involved in biosynthesis, virulence determinants, regulation, translation and unknown functions. Three of the genes required for virulence of A. pleuropneumoniae in this study were also identified in a previous STM study of Pasteurella multocida. Seven of the STM-derived mutants were also evaluated for their potential as live vaccine strains and provided good protection against homologous challenge.     

Tn7-based genome-wide random insertional mutagenesis of Candida glabrata

We describe and characterize a method for insertional mutagenesis of the yeast pathogen Candida glabrata using the bacterial transposon Tn7. Tn7 was used to mutagenize a C. glabrata genomic fosmid library. Pools of random Tn7 insertions in individual fosmids were recovered by transformation into Escherichia coli. Subsequently, these were introduced by recombination into the C. glabrata genome. We found that C. glabrata genomic fragments carrying a Tn7 insertion could integrate into the genome by nonhomologous recombination, by single crossover (generating a duplication of the insertionally mutagenized locus), and by double crossover, yielding an allele replacement. We were able to generate a highly representative set of approximately 10(4) allele replacements in C. glabrata, and an initial characterization of these shows that a wide diversity of genes were targeted in the mutagenesis. Because the identity of disrupted genes for any mutant of interest can be rapidly identified, this method should be of general utility in functional genomic characterization of this important yeast pathogen. In addition, the method might be broadly applicable to mutational analysis of other organisms.     

Role of ornibactin biosynthesis in the virulence of Burkholderia cepacia: characterization of pvdA, the gene encoding L-ornithine N(5)-oxygenase.

Burkholderia cepacia is a frequent cause of respiratory infections in cystic fibrosis patients. B. cepacia has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn5-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of Pseudomonas aeruginosa. I117 contained an insertion in a pvdA homolog, the gene for the enzyme L-ornithine N(5)-oxygenase, which catalyzes the hydroxylation of L-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor L-N(5)-OH-Orn was added to the culture medium. T10 contained an insertion in a pvdD homolog, which is a peptide synthetase involved in pyoverdine synthesis. beta-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn5-OT182 contains a lacZ gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The B. cepacia pvdA homolog was approximately 47% identical and 59% similar to L-ornithine N(5)-oxygenase from P. aeruginosa. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of (59)Fe-ornibactin uptake in I117. A chromosomal pvdA deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The pvdA mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional pvdA gene appears to be required for effective colonization and persistence in B. cepacia lung infections.     

Genetic analysis of Pseudomonas aeruginosa adherence: distinct genetic loci control attachment to epithelial cells and mucins.

Infection of mucosal tissues by the opportunistic pathogen Pseudomonas aeruginosa is initiated by attachment of the bacterium to host tissues. To gain a better understanding of this interaction, we used two methods to isolate mutants of P. aeruginosa with altered adherence to cultured A549 cells and to mucins. First, from a population of nonpiliated mutants of P. aeruginosa mutagenized with transposon Tn5G, we have isolated variants that are defective in binding to both A549 cells and respiratory mucins. Using a cloned transposon plus flanking DNA from one such mutant as a DNA probe, we have isolated plasmids from a cosmid bank, which, upon reintroduction to the original mutants, restored adhesion to both A549 cells and mucin. The second strategy to identify genes involved in adhesion used mutagenesis of P. aeruginosa N1G, an rpoN mutant which is unable to bind to either A549 cells or mucin, with transposon Tn5 containing an outward-directed promoter. From this bank of mutagenized P. aeruginosa N1G, two classes of adhesion variants were isolated; one class attached to A549 cells and to mucin, and the other class restored binding of the rpoN mutant to mucin but not to A549 cells. These findings suggest that P. aeruginosa can express at least two adhesins distinct from pili, one recognizing receptors shared by epithelial cells and mucins and the other recognizing mucins alone.     

Transposon mutagenesis of Mycobacterium marinum identifies a locus linking pigmentation and intracellular survival

Pathogenic mycobacteria survive and replicate within host macrophages, but the molecular mechanisms involved in this necessary step in the pathogenesis of infection are not completely understood. Mycobacterium marinum has recently been used as a model for aspects of the pathogenesis of tuberculosis because of its close genetic relationship to Mycobacterium tuberculosis and because of similarities in the pathology and course of infection caused by this organism in its natural hosts, fish and frogs, with tuberculosis in humans. In order to advance the utility of the M. marinum model, we have developed efficient transposon mutagenesis of the organism by using a Drosophila melanogaster mariner-based transposon. To determine the efficiency of transposition, we have analyzed pigmentation mutants from the transposon mutant library. In addition to insertions in four known genes in the pathway of pigment biosynthesis, two insertions in novel genes were identified in our mutant library. One of these is in a putative inhibitor of the carotenoid biosynthesis pathway. The second unexpected insertion is in an intergenic region between two genes homologous to Rv2603c and Rv2604c of M. tuberculosis. In addition to a pigmentation defect, this mutant showed increased susceptibility to singlet oxygen and grew poorly in murine macrophages. Complementation with M. tuberculosis genomic DNA encompassing Rv2603c to Rv2606c corrected the pigmentation and growth defects of the mutant. These data demonstrate the utility of mariner-based transposon mutagenesis of M. marinum and that M. marinum can be used to study the function of M. tuberculosis genes involved in intracellular survival and replication.     

In vitro transposon mutagenesis of an equine herpesvirus 1 genome cloned as a bacterial artificial chromosome

Summary. The 150-kbp genome of the alphaherpesvirus equine herpesvirus 1 (EHV-1) strain HVS25A was cloned as a bacterial artificial chromosome (EHV-1 BAC), with mini F plasmid sequences inserted between genes 62 and 63. Transfection of EHV-1 BAC DNA purified from E. coli gave rise to progeny virus that had a similar growth rate and yield in mammalian cell culture to those of parental wild-type EHV-1. Using in vitro mutagenesis with a Mu transposon, a large library of EHV-1 BAC mutants was generated, and sequence analysis indicated that insertions were dispersed randomly across the EHV-1 genome. Following transfections of a pilot sample of mutant EHV-1 BAC DNAs into mammalian cells, no CPE was observable by light microscopy for mutants carrying insertions in genes for the major capsid protein, large tegument protein, glycoprotein K, catalytic subunit of DNA polymerase, or single-stranded DNA-binding protein. Mutants that were able to produce CPE similar to wild-type EHV-1 included those with interruptions in ORFs of several tegument proteins. Analysis of several glycoprotein gene mutants indicated that those carrying insertions near the start of genes encoding glycoproteins E and I were viable, but showed markedly diminished plaque areas. These results were supported by confocal microscopy of transfected or infected cultures. Electron microscopy of cells infected with a gE mutant revealed accumulations of particles within cytoplasmic vesicles, consistent with a partial obstruction of maturation. The transposon library is a resource for comprehensive functional analysis of the HVS25A genome, with multiple mutants available in any of the predicted genes of EHV-1.     

Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.     

RpmA is required for nonopsonic phagocytosis of Pseudomonas aeruginosa

Pseudomonas aeruginosa causes severe respiratory tract infections in patients with cystic fibrosis (CF). We have been examining nonopsonic phagocytosis of P. aeruginosa by macrophages. To study the P. aeruginosa-macrophage interaction at the molecular level, we have constructed a transposon Tn5G bank in a clinical isolate of P. aeruginosa (strain 4020) and identified mutants resistant to nonopsonic phagocytosis. Phagocytosis-resistant mutants were enriched by passaging the transposon bank over 18 macrophage monolayers. Of 900 individual mutants isolated from this enriched pool in a nonopsonic phagocytosis assay, we identified 85 putative mutants that were resistant to phagocytosis. In this study, we have characterized one of these transposon mutants, P. aeruginosa 4020 H27A, which was poorly ingested. H27A possessed a Tn5G insertion in a gene encoding a protein with homology to the MotA proteins of several species of bacteria. We have called this gene rpmA for required for phagocytosis by macrophages. RpmA is one of two MotA paralogs in P. aeruginosa. This rpmA::Tn5G mutant was motile both on agar plates and in visual examination of wet mounts. The phagocytosis defect was partially complemented by providing the rpmA gene in trans and fully complemented when both rpmA and rpmB were provided. A rpmA null mutant was ingested by macrophages similar to the H27A transposon mutant. These data suggest that the rpmA and rpmB gene products are required for the efficient ingestion of P. aeruginosa by macrophages.     

Identification of Pseudomonas aeruginosa genes involved in virulence and anaerobic growth

Pseudomonas aeruginosa is a gram-negative, opportunistic pathogen and a significant cause of acute and chronic infections in patients with compromised host defenses. Evidence suggests that within infections P. aeruginosa encounters oxygen limitation and exists in microbial aggregates known as biofilms. However, there is little information that describes genes involved in anaerobic growth of P. aeruginosa and their association with virulence of this pathogen. To identify genes required for anaerobic growth, random transposon (Tn) mutagenesis was used to screen for mutants that demonstrated the inability to grow anaerobically using nitrate as a terminal electron acceptor. Of approximately 35,000 mutants screened, 57 mutants were found to exhibit no growth anaerobically using nitrate. Identification of the genes disrupted by the Tn revealed 24 distinct loci required for anaerobic growth on nitrate, including several genes not previously associated with anaerobic growth of P. aeruginosa. Several of these mutants were capable of growing anaerobically using nitrite and/or arginine, while five mutants were unable to grow anaerobically under any of the conditions tested. Three mutants were markedly attenuated in virulence in the lettuce model of P. aeruginosa infection. These studies have identified novel genes important for anaerobic growth and demonstrate that anaerobic metabolism influences virulence of P. aeruginosa.     

Transposon-mediated mutagenesis of a baculovirus

Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed. These mutants produce few polyhedra in infected cells and have insertions of host cell DNA. All the insertions mapped to two adjacent sites in the genome. The junctions between two host insertions and the viral DNA were sequenced. One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion. Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.     

Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.

We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP-positive F0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole-mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F0 EGFP-positive founders have been genetically analyzed up to the F2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts.     

Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis

Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine, p-aminobenzoic acid, and a mutant with an insertion in aroB causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the aroB mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.     

Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants

Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases.