Jonathan M. Urbach

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

BackgroundPseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.ResultsThe PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.ConclusionGenes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.

Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates.

The genetically tractable model organism Caenorhabditis elegans was first used to model bacterial virulence in vivo a decade ago. Since then, great strides have been made in identifying the host response pathways that are involved in its defence against infection. Strikingly, C. elegans seems to detect, and respond to, infection without the involvement of its homologue of Toll-like receptors, in contrast to the well-established role for these proteins in innate immunity in mammals. What, therefore, do we know about host defence mechanisms in C. elegans and what can they tell us about innate immunity in higher organisms?

A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae

Defined mutant libraries allow for efficient genome-scale screening and provide a convenient collection of mutations in almost any nonessential gene of interest. Here, we present a near-saturating transposon insertion library in Vibrio cholerae strain C6706, a clinical isolate belonging to the O1 El Tor biotype responsible for the current cholera pandemic. Automated sequencing analysis of 23,312 mutants allowed us to build a 3,156-member subset library containing a representative insertion in every disrupted ORF. Because uncharacterized mutations that affect motility have shown utility in attenuating V. cholerae live vaccines, we used this genome-wide subset library to define all genes required for motility and to further assess the accuracy and purity of the library. In this screen, we identified the hypothetical gene VC2208 (flgT) as essential for motility. Flagellated cells were very rare in a flgT mutant, and transcriptional analysis showed it was specifically stalled at the class III/IV assembly checkpoint of the V. cholerae flagellar regulatory system. Because FlgT is predicted to have structural homology to TolB, a protein involved in determining outer membrane architecture, and the sheath of the V. cholerae flagellum appears to be derived from the cells outer membrane, FlgT may play a direct role in flagellar sheath formation.

Genome-Wide Identification of Pseudomonas aeruginosa Virulence-Related Genes Using a Caenorhabditis elegans Infection Model

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

Construction of an Enterococcus faecalis Tn917-mediated-gene-disruption library offers insight into Tn917 insertion patterns.

Sequencing the insertion sites of 8,865 Tn917 insertions in Enterococcus faecalis strain OG1RF identified a hot spot in the replication terminus region corresponding to 6% of the genome where 65% of the transposons had inserted. In E. faecalis, Tn917 preferentially inserted at a 29-bp consensus sequence centered on TATAA, a 5-bp sequence that is duplicated during insertion. The regional insertion site preference at the chromosome terminus was not observed in another low-G+C gram-positive bacterium, Listeria monocytogenes, although the consensus insertion sequence was the same. The 8,865 Tn917 insertion sites sequenced in E. faecalis corresponded to only approximately 610 different open reading frames, far fewer than the predicted number of 2,400, assuming random insertion. There was no significant preference in orientation of the Tn917 insertions with either transcription or replication. Even though OG1RF has a smaller genome than strain V583 (2.8 Mb versus 3.2 Mb), the only E. faecalis strain whose sequence is in the public domain, over 10% of the Tn917 insertions appear to be in a OG1RF-specific sequence, suggesting that there are significant genomic differences among E. faecalis strains.

Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf.

Thiol dependent isomerization of all-trans-retinoic acid to 9-cis-retinoic acid

The important biological effector 9‐cis‐retinoic acid can be generated by liver microsomes or by bovine serum albumin in detergent. The mechanism of this isomerization reaction is the subject of these studies. The protein mediated isomerization process is shown to be thiol‐ and pH‐dependent. Moreover, the retinoic acids are also isomerized by 1‐dodecanethiol in the presence of detergents. This isomerization process is pH‐dependent as well, with isomerization rates increasing with pH. The isomerization reactions are quenched with free radical traps, such as α‐tocopherol and ascorbic acid, suggesting that a thiol radical mechanism, rather than a thiolate anion‐dependent mechanism, is implicated here. The pH dependence can be understood in terms of a thiol radical mechanism, because thiol radicals are produced from thiolate anions in the presence of oxygen. The facile thiol‐mediated isomerization of the retinoic acids suggests that this could be a physiologically relevant mechanism for the formation of 9‐cis‐retinoic acid from all‐trans‐retinoic acid.

The NBS-LRR architectures of plant R-proteins and metazoan NLRs evolved in independent events

Significance Instances of convergent evolution in innate immune systems can reveal fundamental principles of immunity. We use phylogenetic reconstruction and tests of alternative evolutionary hypotheses combined with ancestral state reconstruction and an analysis of the phylogenetic distribution of the nucleotide binding site–leucine rich repeat (NBS-LRR) architecture to demonstrate that most likely, two distinct and separate evolutionary events gave rise to the NBS-LRR architecture in plant resistance proteins (R-proteins) and metazoan NOD-like receptors (NLRs), and furthermore, the STAND NTPase of both protein families was likely inherited from ancestral proteins with an NBS–tetratricopeptide (TPR) architecture. The implication is that the NBS-LRR architecture has special functionality for innate immune receptors. There are intriguing parallels between plants and animals, with respect to the structures of their innate immune receptors, that suggest universal principles of innate immunity. The cytosolic nucleotide binding site–leucine rich repeat (NBS-LRR) resistance proteins of plants (R-proteins) and the so-called NOD-like receptors of animals (NLRs) share a domain architecture that includes a STAND (signal transduction ATPases with numerous domains) family NTPase followed by a series of LRRs, suggesting inheritance from a common ancestor with that architecture. Focusing on the STAND NTPases of plant R-proteins, animal NLRs, and their homologs that represent the NB-ARC (nucleotide-binding adaptor shared by APAF-1, certain R gene products and CED-4) and NACHT (named for NAIP, CIIA, HET-E, and TEP1) subfamilies of the STAND NTPases, we analyzed the phylogenetic distribution of the NBS-LRR domain architecture, used maximum-likelihood methods to infer a phylogeny of the NTPase domains of R-proteins, and reconstructed the domain structure of the protein containing the common ancestor of the STAND NTPase domain of R-proteins and NLRs. Our analyses reject monophyly of plant R-proteins and NLRs and suggest that the protein containing the last common ancestor of the STAND NTPases of plant R-proteins and animal NLRs (and, by extension, all NB-ARC and NACHT domains) possessed a domain structure that included a STAND NTPase paired with a series of tetratricopeptide repeats. These analyses reject the hypothesis that the domain architecture of R-proteins and NLRs was inherited from a common ancestor and instead suggest the domain architecture evolved at least twice. It remains unclear whether the NBS-LRR architectures were innovations of plants and animals themselves or were acquired by one or both lineages through horizontal gene transfer.

Combining Genomic Tools to Dissect Multifactorial Virulence in Pseudomonas aeruginosa

The growing number of sequenced bacterial genomes, including those of important pathogenic isolates, has made a significant impact on the field of bacterial pathogenesis (Raskin et al., 2006). Combined with other technical advances in the laboratory, this wealth of information has made possible the development and widespread adoption of genomic tools for the study of infectious disease. This chapter focuses on the sequencing and functional analysis of PA14, a clinical isolate of the ubiquitous environmental bacterium and important opportunistic human pathogen, Pseudomonas aeruginosa. By comparing PA14 to the sequence of the less virulent P. aeruginosa isolate, PAO1, we have identified genomic sequences absent in one or the other strain in order to examine the relationship between genomic content and pathogenicity. To assess the importance of strain-specific genes and virulence, we tested 20 diverse P. aeruginosa strains, including PA14 and PAO1, in a Caenorhabdidits elegans pathogenesis model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of genes present in PA14 and absent in PAO1 did not correlate with the virulence of these strains. To further examine the roles in virulence of PA14 genes absent in PAO1, we utilized a full-genome non-redundant mutant library of PA14 to identify five ORFs (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans. Genes required for pathogenicity in one strain are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is a complex process that is both multifactorial and combinatorial. Not only are multiple pathogenicity determinants acting in parallel within a given strain, but also, when comparing different strains, different combinations of pathogenicity factors may be selected to determine the ultimate virulence

Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.

Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.