Identification of Mycobacterium marinum macrophage infection mutants

Mycobacterium marinum is an important pathogen of humans, amphibians and fish. Most pathogenic mycobacteria, including M. marinum, infect, survive and replicate primarily intracellularly within macrophages. We constructed a transposon mutant library in M. marinum using Tn5367 delivered by phage transduction in the shuttle phasmid phAE94. We screened 529 clones from the transposon library directly in macrophage infection assays. All clones were screened for their ability to initially infect macrophages as well as survive and replicate intracellularly. We identified 19 mutants that fit within three classes: class I) defective for growth in association with macrophages (42%), class II) defective for macrophage infection (21%) and class III) defective for infection of and growth in association with macrophages (37%). Although 14 of the macrophage infection mutants (Mim) carry insertions in genes that have not been previously identified, five are associated with virulence of mycobacteria in animal models. These observations confirm the utility of mutant screens directly in association with macrophages to identify new virulence determinants in mycobacteria. We complemented four of the Mim mutants with their M. tuberculosis homologue, demonstrating that secondary mutations are not responsible for the observed defect in macrophage infection. The genes we identified provide insight into the molecular mechanisms of macrophage infection by M. marinum.     

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.     

New putative virulence factors of Streptococcus suis involved in invasion of porcine brain microvascular endothelial cells

Streptococcus suis serotype 2 is an important pathogen causing a wide range of infections in swine, the most important being meningitis. Few virulence factors have been identified and the pathogenesis of infection is not well understood. Recently, we demonstrated the ability of S. suis to adhere to and invade porcine brain microvascular endothelial cells (PBMEC) forming the blood-brain barrier. In this paper we describe the screening of a mutant library, produced by insertion of transposon Tn917 into the chromosome of S. suis strain P1/7, for mutants that are less able to interact with PBMEC. Both qualitative and quantitative screening assays were used to identify poorly invasive mutants. Tn917 insertion sites from nineteen poorly invasive mutants were sequenced and characterized. Five mutants were selected and their virulence was assessed in a mouse model of infection. Two out of these five mutants were attenuated as measured by decreased colonization of organs, as well as reduced mortality and morbidity. When tested in swine these two attenuated mutants led to decreased bacterial loads in blood, less severe and delayed clinical signs, and lower plasma IL-6 levels than did infection with the wild-type strain. Overall, our results suggest that these two genes may contribute to the virulence of S. suis.     

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.     

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.     

Identification of virulence genes of Helicobacter pylori by random insertion mutagenesis

The complete genome of the gram-negative bacterial pathogen Helicobacter pylori, an important etiological agent of gastroduodenal disease in humans, has recently been published. This sequence revealed that the putative products of roughly one-third of the open reading frames (ORFs) have no significant homology to any known proteins. To be able to analyze the functions of all ORFs, we constructed an integration plasmid for H. pylori and used it to generate a random mutant library in this organism. This integration plasmid, designated pBCalpha3, integrated randomly into the chromosome of H. pylori. To test the capacity of this library to identify virulence genes, subsets of this library were screened for urease-negative mutants and for nonmotile mutants. Three urease-negative mutants in a subset of 1,251 mutants (0.25%) and 5 nonmotile mutants in a subset of 180 mutants (2.7%) were identified. Analysis of the disrupted ORFs in the urease-negative mutants revealed that two had disruptions of genes of the urease locus, ureB and ureI, and the third had a disruption of a unrelated gene; a homologue of deaD, which encodes an RNA helicase. Analysis of the disrupted ORFs in the nonmotile mutants revealed one ORF encoding a homologue of the paralyzed flagellar protein, previously shown to be involved in motility in Campylobacter jejuni. The other four ORFs have not been implicated in motility before. Based on these data, we concluded that we have generated a random insertion library in H. pylori that allows for the functional identification of genes in H. pylori.     

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.     

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.     

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.     

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.     

Isolation of Citrobacter sp. mutants defective in decolorizing malachite green

To identify genes involved in the decolorization of malachite green, random mutants generated by transposon insertion in the malachite green-decolorizing bacterium, Citrobacter sp. were isolated. The resulting mutant bank yielded 24 mutants with complete defects in their abilities to decolorize malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants, which appeared to have insertions at different sites of the chromosome. The Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. Based on a sequence database, the putative protein products encoded by the mg genes were identified as follows. mg3, an ABC transporter homolog; mg6, a LysR-type regulatory protein; m11, an oxidoreductase; mg17, a MalG protein in the maltose transport system; and mg21, a sugar kinase. The deduced sequences from two mg genes (mg7 and mg18) showed no significant similarity to any protein with a known function, suggesting that these two mg genes encode unidentified proteins that are responsible for the decolorization of malachite green.     

Exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii is related to various metabolic pathways

Rhizobium leguminosarum bv. trifolii synthesizes extracellular polysaccharide (EPS) that is postulated to be a biologically active signalling molecule in clover symbiosis. A group of seven exopolysaccharide-deficient (Exo), non-nitrogen-fixing mutants of R. leguminosarum bv. trifolii strain 24.1 isolated by transposon mutagenesis were complemented to mucoid phenotype by a low-copy plasmid carrying the pssA gene encoding the first glucosyl-IP-transferase. Some of these mutants were not corrected in their symbiotic defect by the pssA gene. Precise localization of Tn5 insertion sites by subcloning and sequencing the adjacent genomic DNA in the Exo mutants identified the disrupted genes and their possible functions. Only one mutant (Rt74) was mutated in pssA gene; others were mutated in diverse genes that were not directly involved in EPS biosynthesis. The suppression of EPS deficiency in these mutants by additional copies of pssA indicated a possible connection between exopolysaccharide biosynthesis and various metabolic pathways.     

Salmonella typhimurium loci involved in survival within macrophages.

A set of Tn10 mutants of Salmonella typhimurium which have a diminished capacity to survive in murine macrophages and decreased virulence in mice has been described previously. In this study, we characterized 30 of these mutants and determined map locations of Tn10 insertions for 23 of these strains. In addition, short fragments of transposon-flanking DNA were cloned, and the nucleotide sequence was determined for 23 mutants. Seven mutants carried transposon insertions in known genes, representing six loci: htrA, prc, purD, fliD, nagA, and smpB. The possible roles of these genes in Salmonella virulence are discussed. One insertion was found to be in an unknown gene which shared homology with the open reading frames Bv' and Bv located in the pin inversion system of Shigella boydii. In one mutant, Tn10 was found to be inserted in a gene with significant homology to adhE of Escherichia coli and Clostridium acetobutylicum. The map location and degree of homology indicate that the Salmonella gene encodes a related, but different, dehydrogenase. In 14 of the mutants analyzed, Tn10 was inserted into genes which had no significant homologies to entries in the DNA and protein data bases. In conclusion, 16 insertions define loci, termed ims for impaired macrophage survival, which have not yet been described in S. typhimurium but have been shown previously to be necessary for full virulence in mice. Although most ims loci are distributed randomly throughout the genome, a cluster was found between 75 and 78 min on the Salmonella chromosome.     

Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes.

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.     

Identification of genes required for chronic persistence of Brucella abortus in mice

The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB of Mycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.     

Signature Tagged Mutagenesis of Haemophilus influenzae identifies genes required for in vivo survival

The pathogenic bacterium Haemophilus influenzae causes meningitis, epiglottitis, pneumonia, otitis media and other infections. To further understand the genetic basis of invasive disease and to inform about the bacterium's requirements in an in vivo environment, we analysed a library of 1632 insertional Tn1545 -Delta3 transposon mutants for their capacity to cause systemic infection in an animal model. We identified 25 genes that are potentially essential for H. influenzae invasive disease, and are candidates for further exploratory research. Seven of the genes encode hypothetical proteins, the function of six of which could be tentatively assigned on the basis of functional motifs and low homology to other bacterial genes. Eleven genes encode central metabolic enzymes or transporters; eight encode proteins that interact with DNA or modify other proteins; and four encode enzymes involved in the elaboration of classical virulence determinants. Two genes have no known function. Independent mutagenesis of six of the 25 genes and determination of the competitive index confirmed that these genes are important or essential to the organism in an in vivo environment. This genome-wide analysis has identified metabolic and other genes required during invasive disease, and the findings may lead to new interventions to prevent and treat H. influenzae infections.     

Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae.

A library of Tn916-generated, tetracycline-resistant (Tc) mutants of the group B Neisseri meningitidis strain NMB was screened by using monoclonal antibodies (MAbs) that recognize structural differences in neisserial lipooligosaccharide (LOS). The LOS of parental strain NMB had a relative molecular mass of 4.5 kDa, reacted with MAbs 3F11 and 6B4 but not with MAb 4C4 or 6E4, and contained a lacto-N-neotetrose unit. Two phenotypically stable mutants, SS3 and R6, altered in LOS, were identified by colony immunoblots, electrophoresis, and Western immunoblots. The LOS of mutant SS3 was 3.4 kDa and reacted with MAbs 4C4 and 6E4 but not MAb 3E11 or 6B4. The LOS of mutant R6 was 3.1 to 3.2 kDa and reacted with MAb 6E4 but not MAb 3F11, 6B4, or 4C4. Thus, the LOSs of the R6 and SS3 mutants were predicted to contain different truncations of the core oligosaccharide. The LOS phenotype of each mutant was linked to Tc(r), as determined by transformation of the parent strain with DNA from the mutant. Southern hybridizations and single-specific-primer PCR revealed in each mutant a single truncated tn916 insertion which had lost genes required for mobilization. Tn916 mutagenesis was used to identify two distinct genetic sites in the meningococcal chromosome involved in biosynthesis of the oligosaccharide chain of LOS and to create genetically defined LOS mutants of N. meningitidis and Neisseria gonorrhoeae.     

A new assay for invasion of HeLa 229 cells by Bordetella pertussis: effects of inhibitors, phenotypic modulation, and genetic alterations.

Invasion and intracellular survival of Bordetella pertussis in HeLa 229 cells was studied by a new assay that utilizes polymyxin B instead of gentamicin to rapidly kill extracellular organisms. Invasion measured by this assay was time and temperature dependent and was inhibited by the microfilament drug cytochalasin D. The invasion process was also dependent on a functional vir locus (also known as bvg), the positive regulator of virulence gene expression in B. pertussis. Four spontaneous Vir- phase variants of B. pertussis and a mutant with a transposon insertion mutation in the vir locus did not invade. Cells that were environmentally modulated and thus did not express virulence determinants also did not invade. Two Vir- mutants, a vir-directed plasmid insertion mutant and a UV-light-induced mutant, were capable of invasion, although they did not produce other known virulence factors such as pertussis toxin and hemolysin but did produce small amounts of filamentous hemagglutinin (FHA) and the 69-kilodalton outer membrane protein. None of 70 Tn5 IS50L::phoA (TnphoA) insertion mutants of strain Bp18323 (including three mutants defective in FHA) tested showed any reproducible defect in invasion. A mutant carrying a site-directed deletion mutation in FHA was also capable of invasion in our assay. These data suggest that there is redundancy in the invasion functions of B. pertussis and that one or more of these are coordinately regulated with FHA and the 69-kilodalton outer membrane protein more tightly than with other vir-activated gene products.