Investigation of the mechanisms by which Listeria monocytogenes grows in porcine gallbladder bile

The food-borne pathogen Listeria monocytogenes is known to colonize the lumen of the gallbladder in infected mice and to grow rapidly in this environment (J. Hardy et al., Science 303:851-853, 2004). However, relatively little is known about the mechanisms utilized by the pathogen to survive and grow in this location. We utilized gallbladder bile (GB bile) isolated directly from porcine gallbladders as an ex vivo model of gallbladder growth. We demonstrate that GB bile is generally nontoxic for bacteria and can readily support growth of a variety of bacterial species including L. monocytogenes, Lactococcus lactis, Salmonella enterica serovar Typhimurium, and Escherichia coli. Significantly, L. monocytogenes grew at the same rate as the nonpathogenic species Listeria innocua, indicating that the pathogen does not possess specialized mechanisms that enable growth in this environment. However, when we reduced the pH of GB bile to pH 5.5 in order to mimic the release of bile within the small intestine, the toxicity of GB bile increased significantly and specific resistance mechanisms (Sigma B, BSH, and BilE) were essential for survival of the pathogen under these conditions. In order to identify genetic loci that are necessary for growth of L. monocytogenes in the gallbladder, a mariner transposon bank was created and screened for mutants unable to replicate in GB bile. This led to the identification of mutants in six loci, including genes encoding enzymes involved in purine metabolism, amino acid biosynthesis, and biotin uptake. Although GB bile does not represent a significant impediment to bacterial growth, specific metabolic processes are required by L. monocytogenes in order to grow in this environment.     

Identification of Actinobacillus pleuropneumoniae genes important for survival during infection in its natural host

Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found--an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.     

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.     

Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.     

Identification of the agr locus of Listeria monocytogenes: role in bacterial virulence

Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.     

Enhanced Priming of Adaptive Immunity by Mycobacterium smegmatis Mutants with High-Level Protein Secretion

Mycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growing Mycobacterium smegmatis can express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity of M. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion ("high secretors") and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8(+) T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV) gag gene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8(+) T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8(+) T cells in vivo. The data also suggest that these M. smegmatis transposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses.     

Legionella dumoffii DjlA, a member of the DnaJ family, is required for intracellular growth

Legionella dumoffii is one of the common causes of Legionnaires' disease and is capable of replicating in macrophages. To understand the mechanism of survival within macrophages, transposon mutagenesis was employed to isolate the genes necessary for intracellular growth. We identified four defective mutants after screening 790 transposon insertion mutants. Two transposon insertions were in genes homologous to icmB or dotC, within dot/icm loci, required for intracellular multiplication of L. pneumophila. The third was in a gene whose product is homologous to the 17-kDa antigen forming part of the VirB/VirD4 type IV secretion system of Bartonella henselae. The fourth was in the djlA (for "dnaj-like A") gene. DjlA is a member of the DnaJ/Hsp40 family. Transcomplementation of the djlA mutant restored the parental phenotype in J774 macrophages, A549 human alveolar epithelial cells, and the amoeba Acanthamoeba culbertsoni. Using confocal laser-scanning microscopy and transmission electron microscopy, we revealed that in contrast to the wild-type strain, L. dumoffii djlA mutant-containing phagosomes were unable to inhibit phagosome-lysosome fusion. Transmission electron microscopy also showed that in contrast to the virulent parental strain, the djlA mutant was not able to recruit host cell rough endoplasmic reticulum. Furthermore, the stationary-phase L. dumoffii djlA mutants were more susceptible to H2O2, high osmolarity, high temperature, and low pH than was their parental strain. These results indicate that DjlA is required for intracellular growth and organelle trafficking, as well as bacterial resistance to environmental stress. This is the first report demonstrating that a single DjlA-deficient mutant exhibits a distinct phenotype.     

Nonhemolytic Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture: evidence for coordinate regulation of virulence.

We identified nonhemolytic mutants of Listeria monocytogenes that were severely deficient in their ability to invade mammalian nonprofessional phagocytes. These mutants were generated spontaneously or by means of transposon Tn916 mutagenesis. In terms of their extracellular proteins, the noninvasive mutants were deficient not only in the sulfhydryl-activated hemolysin (listeriolysin) but also in an antigenically unrelated extracellular protein with an apparent molecular weight of 32,000 which could induce opacity in egg yolk and is considered to be a phospholipase. Our results suggest the existence of a common genetic control between the expression of listeriolysin and that of other determinants, including a phospholipase and determinants involved in the ability of L. monocytogenes to enter mammalian cells.     

Use of gene dosage effects for a whole-genome screen to identify Mycobacterium marinum macrophage infection loci

We recently identified two loci, mel1 and mel2, that affect macrophage infection by Mycobacterium marinum. The ability of these loci to confer enhanced infection in trans is presumably due to gene dosage effects since their presence on plasmids increases expression from five- to eightfold. Reasoning that this phenomenon would allow identification of other mycobacterial genes involved in macrophage infection, we conducted a screen of an M. marinum DNA library that provides 2.6-fold coverage of the entire genome for clones that affect macrophage infection. Our preliminary screen identified 76 plasmids that carry loci affecting macrophage infection. We eliminated plasmids that do not confer the expected phenotype when retransformed (70%), that have identical physical maps (5%), or that carry either of the mel1 or mel2 loci (14%) from further consideration. Four loci that confer enhanced infection (mel) and four that confer repressed infection (mrl) of macrophages were identified, and two of each group were chosen for detailed analysis. Saturating transposon mutagenesis was used to identify the loci responsible, and M. marinum mutants were constructed in the genes involved. We expect these genes to provide insight into how mycobacteria parasitize macrophages, an important component of innate immunity.     

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.     

Loss of catalase activity in Tn1545-induced mutants does not reduce growth of Listeria monocytogenes in vivo.

Two catalase-negative mutants of Listeria monocytogenes were obtained by chromosomal insertions of the conjugative transposon Tn1545. The loss of catalase activity did not reduce the level of virulence of these mutants in mice. Indeed, both mutants were capable of growing in host tissues at the same rate as the parental catalase-positive strain. These results favor the view that catalase does not play a critical role in the resistance of L. monocytogenes to macrophage killing.     

Isolation and characterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient in internalization and intracellular growth in HeLa cells

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.     

Mutagenesis of murine cytomegalovirus using a Tn3-based transposon

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals.     

Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis

Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of Brucella virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D(3)-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the virB operon and manB), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of Brucella for the different factors identified are discussed.     

Identification of gene products involved in biofilm production by Moraxella catarrhalis ETSU-9 in vitro

Moraxella catarrhalis ETSU-9 was subjected to random transposon insertion mutagenesis to identify genes encoding products involved in the ability of the organism to form biofilms in vitro. Screening of approximately 3,000 transposon insertion mutants in the crystal violet-based biofilm assay system yielded six mutants that exhibited greatly reduced abilities to form biofilms. Three of these mutants had transposon insertions in the uspA2H gene, which encodes a surface protein previously shown to be involved in the ability of M. catarrhalis to both attach to human cell lines in vitro and resist killing by normal human serum. Random insertion mutagenesis of the uspA2H gene, involving the introduction of a 15-nucleotide fragment encoding 5 amino acids, was used to attempt to identify the domain(s) necessary for biofilm formation. Most of these insertions adversely affected biofilm formation, whereas the abilities of these same mutants to attach to Chang conjunctival epithelial cells in vitro were usually not reduced. Gain-of-function experiments showed that introduction of the M. catarrhalis ETSU-9 uspA2H gene into Escherichia coli conferred biofilm formation ability on this recombinant strain. Two of the other three M. catarrhalis ETSU-9 transposon insertion mutants that had greatly reduced abilities to form biofilms were shown to have insertions in genes encoding products predicted to be directly or indirectly involved in cell wall metabolism.     

Identification of Burkholderia cenocepacia genes required for bacterial survival in vivo

Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.     

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.