Kimberly N. Cowles

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila

Xenorhabdus nematophila is a Gram‐negative bacterium that leads both pathogenic and mutualistic lifestyles. In this study, we examine the role of Lrp, the leucine‐responsive regulatory protein, in regulating both of these lifestyles. lrp mutants have attenuated virulence towards Manduca sexta insects and are defective in suppression of both cellular and humoral insect immunity. In addition, an lrp mutant is deficient in initiating colonization of and growth within mutualistic host nematodes. Furthermore, nematodes reared on lrp mutant lawns exhibit decreased overall numbers of nematode progeny. To our knowledge, this is the first demonstration of virulence attenuation associated with an lrp mutation in any bacterium, as well as the first report of a factor involved in both X. nematophila symbioses. Protein profiles of wild‐type and mutant cells indicate that Lrp is a global regulator of expression in X. nematophila, affecting ∼65% of 290 proteins. We show that Lrp binds to the promoter regions of genes known to be involved in basic metabolism, mutualism and pathogenesis, demonstrating that the regulation of at least some host interaction factors is likely direct. Finally, we demonstrate that Lrp influences aspects of X. nematophila phenotypic variation, a spontaneous process that occurs during prolonged growth in stationary phase.

Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects

As an insect pathogen, the γ‐proteobacterium Xenorhabdus nematophila likely possesses an arsenal of virulence factors, one of which is described in this work. We present evidence that the X . nematophilahaemolysin XhlA is required for full virulence towards Manduca sexta larvae. Lrp (leucine‐responsive regulatory protein), FlhDC (regulator of flagella synthesis), and iron (II) limitation positively influenced xhlA transcript levels, suggesting XhlA expression is linked with nutrient acquisition and motility regulons. To help understand the role of XhlA in virulence, we examined its cellular targets and found that XhlA was a cell‐surface associated haemolysin that lysed the two most prevalent types of insect immune cells (granulocytes and plasmatocytes) as well as rabbit and horse erythrocytes. Taken together, the need for xhlA for full virulence and XhlA activity towards insect immune cells suggest this haemolysin functions in X. nematophila immune evasion during infection. Analysis of a gene located immediately upstream of the xhlA locus, hcp (haemolysin co‐regulated protein) revealed that its transcript levels were elevated during iron (III) limitation and its expression was Lrp‐dependent. Further characterization of xhlA, hcp, and lrp will clarify their regulatory and functional relationships and their individual roles during the infectious process.

Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity.

Virulence of the insect pathogen Xenorhabdus nematophila is attributed in part to its ability to suppress immunity. For example, X. nematophila suppresses transcripts encoding several antimicrobial proteins, even in the presence of Salmonella enterica, an inducer of these transcripts. We show here that virulence and immune suppression phenotypes can be lost in a subpopulation of X. nematophila. Cells that have undergone ‘virulence modulation’ (vmo) have attenuated virulence and fail to suppress antimicrobial transcript levels, haemocyte aggregation and nodulation in Manduca sexta insects. When plated on certain media, vmo cells have a higher proportion of translucent (versus opaque) colonies compared with non‐vmo cells. Like vmo strains, translucent colony isolates are defective in virulence and immune suppression. The X. nematophila genome encodes two ‘opacity’ genes with similarity to the Ail/PagC/Rck family of outer membrane proteins involved in adherence, invasion and serum resistance. Quantitative polymerase chain reaction analysis shows that RNA levels of one of these opacity genes, opaB, are higher in opaque relative to translucent colonies. We propose that in X. nematophila opaB may be one of several factors involved in immune suppression during infection, and expression of these factors can be co‐ordinately eliminated in a subpopulation, possibly through a phase variation mechanism.

CpxRA Regulates Mutualism and Pathogenesis in Xenorhabdus nematophila

ABSTRACT The CpxRA signal transduction system, which in Escherichia coli regulates surface structure assembly and envelope maintenance, is involved in the pathogenic and mutualistic interactions of the entomopathogenic bacterium Xenorhabdus nematophila. When ΔcpxR1 cells were injected into Manduca sexta insects, the time required to kill 50% of the insects was twofold longer than the time observed for wild-type cells and the ΔcpxR1 cells ultimately killed 16% fewer insects than wild-type cells killed. During mutualistic colonization of Steinernema carpocapsae nematodes, the ΔcpxR1 mutant achieved colonization levels that were only 38% of the wild-type levels. ΔcpxR1 cells exhibited an extended lag phase when they were grown in liquid LB or hemolymph, formed irregular colonies on solid medium, and had a filamentous cell morphology. A mutant with a cpxRp-lacZ fusion had peaks of expression in the log and stationary phases that were conversely influenced by CpxR; the ΔcpxR1 mutant produced 130 and 17% of the wild-type β-galactosidase activity in the log and stationary phases, respectively. CpxR positively influences motility and secreted lipase activity, as well as transcription of genes necessary for mutualistic colonization of nematodes. CpxR negatively influences the production of secreted hemolysin, protease, and antibiotic activities, as well as the expression of mrxA, encoding the pilin subunit. Thus, X. nematophila CpxRA controls expression of envelope-localized and secreted products, and its activity is necessary for both mutualistic and pathogenic functions.

Rhabdopeptides as insect-specific virulence factors from entomopathogenic bacteria.

Six novel linear peptides, named “rhabdopeptides”, have been identified in the entomopathogenic bacterium Xenorhabdus nematophila after the discovery of the corresponding rdp gene cluster by using a promoter trap strategy for the detection of insect‐inducible genes. The structures of these rhabdopeptides were deduced from labeling experiments combined with detailed MS analysis. Detailed analysis of an rdp mutant revealed that these compounds participate in virulence towards insects and are produced upon bacterial infection of a suitable insect host. Furthermore, two additional rhabdopeptide derivatives produced by Xenorhabdus cabanillasii were isolated, these showed activity against insect hemocytes thereby confirming the virulence of this novel class of compounds.

Xanthomonas perforans Colonization Influences Salmonella enterica in the Tomato Phyllosphere

ABSTRACT Salmonella enterica rarely grows on healthy, undamaged plants, but its persistence is influenced by bacterial plant pathogens. The interactions between S. enterica, Xanthomonas perforans (a tomato bacterial spot pathogen), and tomato were characterized. We observed that virulent X. perforans, which establishes disease by suppressing pathogen-associated molecular pattern (PAMP)-triggered immunity that leads to effector-triggered susceptibility, created a conducive environment for persistence of S. enterica in the tomato phyllosphere, while activation of effector-triggered immunity by avirulent X. perforans resulted in a dramatic reduction in S. enterica populations. S. enterica populations persisted at ∼10 times higher levels in leaves coinoculated with virulent X. perforans than in those where S. enterica was applied alone. In contrast, S. enterica populations were ∼5 times smaller in leaves coinoculated with avirulent X. perforans than in leaves inoculated with S. enterica alone. Coinoculation with virulent X. perforans increased S. enterica aggregate formation; however, S. enterica was not found in mixed aggregates with X. perforans. Increased aggregate formation by S. enterica may serve as the mechanism of persistence on leaves cocolonized by virulent X. perforans. S. enterica association with stomata was altered by X. perforans; however, it did not result in appreciable populations of S. enterica in the apoplast even in the presence of large virulent X. perforans populations. Gene-for-gene resistance against X. perforans successively restricted S. enterica populations. Given the effect of this interaction, breeding for disease-resistant cultivars may be an effective strategy to limit both plant disease and S. enterica populations and, consequently, human illness.

Diguanylate Cyclases AdrA and STM1987 Regulate Salmonella enterica Exopolysaccharide Production during Plant Colonization in an Environment-Dependent Manner

ABSTRACT Increasing evidence indicates that despite exposure to harsh environmental stresses, Salmonella enterica successfully persists on plants, utilizing fresh produce as a vector to animal hosts. Among the important S. enterica plant colonization factors are those involved in biofilm formation. S. enterica biofilm formation is controlled by the signaling molecule cyclic di-GMP and represents a sessile lifestyle on surfaces that protects the bacterium from environmental factors. Thus, the transition from a motile, planktonic lifestyle to a sessile lifestyle may represent a vital step in bacterial success. This study examined the mechanisms of S. enterica plant colonization, including the role of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), the enzymes involved in cyclic di-GMP metabolism. We found that two biofilm components, cellulose and curli, are differentially required at distinct stages in root colonization and that the DGC STM1987 regulates cellulose production in this environment independent of AdrA, the DGC that controls the majority of in vitro cellulose production. In addition, we identified a new function for AdrA in the transcriptional regulation of colanic acid and demonstrated that adrA and colanic acid biosynthesis are associated with S. enterica desiccation tolerance on the leaf surface. Finally, two PDEs with known roles in motility, STM1344 and STM1697, had competitive defects in the phyllosphere, suggesting that regulation of motility is crucial for S. enterica survival in this niche. Our results indicate that specific conditions influence the contribution of individual DGCs and PDEs to bacterial success, perhaps reflective of differential responses to environmental stimuli.

Few Differences in Metabolic Network Use Found Between Salmonella enterica Colonization of Plants and Typhoidal Mice

The human enteric pathogen Salmonella enterica leads a cross-kingdom lifestyle, actively colonizing and persisting on plants in between animal hosts. One of the questions that arises from this dual lifestyle is how S. enterica is able to adapt to such divergent hosts. Metabolic pathways required for S. enterica animal colonization and virulence have been previously identified, but the metabolism of this bacterium on plants is poorly understood. To determine the requirements for plant colonization by S. enterica, we first screened a library of metabolic mutants, previously examined in a systemic mouse typhoidal model, for competitive plant colonization fitness on alfalfa seedlings. By comparing our results to those reported in S. enterica-infected murine spleens, we found that the presence of individual nutrients differed between the two host niches. Yet, similar metabolic pathways contributed to S. enterica colonization of both plants and animals, such as the biosynthesis of amino acids, purines, and vitamins and the catabolism of glycerol and glucose. However, utilization of at least three metabolic networks differed during the bacteriums plant- and animal-associated lifestyles. Whereas both fatty acid biosynthesis and degradation contributed to S. enterica animal colonization, only fatty acid biosynthesis was required during plant colonization. Though serine biosynthesis was required in both hosts, S. enterica used different pathways within the serine metabolic network to achieve this outcome. Lastly, the metabolic network surrounding manA played different roles during colonization of each host. In animal models of infection, O-antigen production downstream of manA facilitates immune evasion. We discovered that manA contributed to S. enterica attachment, to seeds and germinated seedlings, and was essential for growth in early seedling exudates, when mannose is limited. However, only seedling attachment was linked to O-antigen production, indicating that manA played additional roles critical for plant colonization that were independent of surface polysaccharide production. The integrated view of S. enterica metabolism throughout its life cycle presented here provides insight on how metabolic versatility and adaption of known physiological pathways for alternate functions enable a zoonotic pathogen to thrive in niches spanning across multiple kingdoms of life.

Leafhopper-Induced Activation of the Jasmonic Acid Response Benefits Salmonella enterica in a Flagellum-Dependent Manner

Enteric human pathogens such as Salmonella enterica are typically studied in the context of their animal hosts, but it has become apparent that these bacteria spend a significant portion of their life cycle on plants. S. enterica survives the numerous stresses common to a plant niche, including defense responses, water and nutrient limitation, and exposure to UV irradiation leading to an increased potential for human disease. In fact, S. enterica is estimated to cause over one million cases of foodborne illness each year in the United States with 20% of those cases resulting from consumption of contaminated produce. Although S. enterica successfully persists in the plant environment, phytobacterial infection by Pectobacterium carotovorum or Xanthomonas spp. increases S. enterica survival and infrequently leads to growth on infected plants. The co-association of phytophagous insects, such as the Aster leafhopper, Macrosteles quadrilineatus, results in S. enterica populations that persist at higher levels for longer periods of time when compared to plants treated with S. enterica alone. We hypothesized that leafhoppers increase S. enterica persistence by altering the plant defense response to the benefit of the bacteria. Leafhopper infestation activated the jasmonic acid (JA) defense response while S. enterica colonization triggered the salicylic acid (SA) response. In tomato plants co-treated with S. enterica and leafhoppers, both JA- and SA-inducible genes were activated, suggesting that the presence of leafhoppers may affect the crosstalk that occurs between the two immune response pathways. To rule out the possibility that leafhoppers provide additional benefits to S. enterica, plants were treated with a chemical JA analog to activate the immune response in the absence of leafhoppers. Although bacterial populations continue to decline over time, analog treatment significantly increased bacterial persistence on the leaf surface. Bacterial mutant analysis determined that the bacterial flagellum, whether functional or not, was required for increased S. enterica survival after analog treatment. By investigating the interaction between this human pathogen, a common phytophagous insect, and their plant host, we hope to elucidate the mechanisms promoting S. enterica survival on plants and provide information to be used in the development of new food safety intervention strategies.