Virulence Factors of Erwinia amylovora: A Review Piqué, Núria; Miñana-Galbis, David; Merino, Susana ...
International Journal of Molecular Sciences,
06/2015, Letnik:
16, Številka:
6
Journal Article, Book Review
Recenzirano
Odprti dostop
Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae ...and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3'-5')-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.
Fire blight, caused by the pathogen Erwinia amylovora, is the most devastating bacterial disease of pome fruit in North America and worldwide. The primary method of dispersal for E. amylovora is ...through ooze, a mass of exopolysaccharides and bacterial cells that is exuded as droplets from infected host tissue. During the 2013 and 2014 field seasons, 317 ooze droplets were collected from field-inoculated apple trees. Populations of E. amylovora in ooze droplets were 10
CFU/μl on average. Ooze droplets harboring larger (>10
CFU/μl) cell populations were typically smaller in total volume and had darker coloring, such as orange, red, or dark red hues. Examination of apple host tissue at the site of emergence of ooze droplets using scanning electron microscopy revealed that ooze was not exuding through natural openings; instead, it was found on erumpent mounds and small (10-μm) tears in tissue. These observations suggested that E. amylovora-induced wounds in tissue provided the exit holes for ooze extrusion from the host. Analyses of E. amylovora populations in ooze droplets and within the stems from which ooze droplets emerged indicated that approximately 9% of the total bacterial population from infected stems is diverted to ooze. Gene expression analyses indicated that E. amylovora cells in stem sections located above ooze droplets and in ooze droplets were actively expressing critical pathogenicity genes such as hrpL, dspE, and amsK. Thus, our study identified ooze as a source of large, concentrated populations of E. amylovora that emerged from the host by rupturing host tissue. Because the cells in ooze droplets are expressing genes required for pathogenesis, they are already primed for infection should they be dispersed from ooze to new infection courts.
Summary
Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture ...of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c‐di‐GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three‐dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c‐di‐GMP‐dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c‐di‐GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.
Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a ...virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora.
Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger molecule that is an important virulence regulator in the plant pathogen
Intracellular levels of c-di-GMP are modulated by ...diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP and by phosphodiesterase (PDE) enzymes that degrade c-di-GMP. The regulatory role of the PDE enzymes in
has not been determined. Using a combination of single, double, and triple deletion mutants, we determined the effects of each of the four putative PDE-encoding genes (
,
,
, and
) in
on cellular processes related to virulence. Our results indicate that
and
are the two phosphodiesterases most active in virulence regulation in
Ea1189. The deletion of
resulted in a measurably significant increase in the intracellular pool of c-di-GMP, and the highest intracellular concentrations of c-di-GMP were observed in the Ea1189 Δ
and Ea1189 Δ
mutants. The regulation of virulence traits due to the deletion of the
genes showed two patterns. A stronger regulatory effect was observed on amylovoran production and biofilm formation, where both Ea1189 Δ
and Ea1189 Δ
mutants exhibited significant increases in these two phenotypes
In contrast, the deletion of two or more
genes was required to affect motility and virulence phenotypes. Our results indicate a functional redundancy among the
genes in
for certain traits and indicate that the intracellular degradation of c-di-GMP is mainly regulated by
and
, but they also suggest a role for
in regulating motility and virulence.
Precise control of the expression of virulence genes is essential for successful infection of apple hosts by the fire blight pathogen,
The presence and buildup of a signaling molecule called cyclic di-GMP enables the expression and function of some virulence determinants in
, such as amylovoran production and biofilm formation. However, other determinants, such as those for motility and the type III secretion system, are expressed and functional when cyclic di-GMP is absent. Here, we report studies of enzymes called phosphodiesterases, which function in the degradation of cyclic di-GMP. We show the importance of these enzymes in virulence gene regulation and the ability of
to cause plant disease.
The Gram-negative enterobacterium
causes fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essential
virulence factors. We found that mutations ...in
disrupted amylovoran production and virulence in apple fruits and tree shoots and that the deletion of
suppressed the
mutant phenotype. The level of expression of
was about 10-fold higher in the Δ
mutant than the wild type. A forward genetic suppressor screen in the Δ
mutant uncovered multiple mutations in
and supported the conclusion that the virulence defect of
mutants is due to reduced amylovoran production. The
and
genes are expressed as a two-gene operon,
The
gene encodes a previously uncharacterized putative polysaccharide subunit transporter, while
encodes a predicted glycosyltransferase. Mutation of
did not have a detectable effect on lipopolysaccharide patterns; however, the overexpression of
in both the wild-type and Δ
Δ
genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, the overexpression of
in the Δ
Δ
mutant inhibited bacterial growth in amylovoran-inducing medium. This growth inhibition phenotype was used in a forward genetic suppressor screen and reverse-genetics tests to identify several genes involved in lipopolysaccharide production, which, when mutated, restored the ability of the Δ
Δ
mutant overexpressing
to grow in amylovoran-inducing medium. Remarkably, all the lipopolysaccharide gene mutants tested were defective in lipopolysaccharide and amylovoran production. These results reveal a genetic connection between amylovoran and lipopolysaccharide production in
This study discovered previously unknown genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogen
This represents a step forward in our understanding of the biology underlying the production of these two macromolecules. Fire blight is an economically important disease that impacts the production of apples and pears worldwide. Few fire blight control measures are available, and growers rely heavily on antibiotic applications at bloom time. Both exopolysaccharide and lipopolysaccharide are
virulence factors. Our results indicate that the overexpression of the
gene in
disrupts both lipopolysaccharide production and exopolysaccharide production. This effect could potentially be used as the basis for the development of an antivirulence treatment for the prevention of fire blight disease.
The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora . Previous studies have demonstrated that the T3SS in E. ...amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp ⁰) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora . Expression of T3SS genes was greatly downregulated in the ppGpp ⁰ and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp ⁰ and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora . Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp -inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.
Fire blight, a disease of pome fruits caused by the bacterium
, has become increasingly difficult to manage after the emergence of streptomycin-resistant strains. Alternative antibiotics and copper ...are available; however, these chemicals have use restrictions in some countries and also can carry risks of phytotoxicity. Therefore, there is growing interest in biological-based management options, with bacteriophage (phages) showing promise, as these naturally occurring pathogens of bacteria are easy to isolate and grow. However, there are several technical challenges regarding the implementation of phage biocontrol in the field, as the viral molecules suffer from ultraviolet radiation (UVR) degradation and can die off rapidly in the absence of the host bacterium. In this work, we assessed the efficacy of
phages and a commercial phage product for blossom blight control in the field across multiple locations in the eastern United States. In these tests, disease control ranged from 0.0 to 82.7%, and addition of a UVR protectant only resulted in significantly increased disease control in 2 of 12 tests. We also analyzed microbial community population changes in response to phage application. Changes in bacterial community diversity metrics over time were not detected; however, relative abundances of target taxa were temporarily reduced after phage applications, indicating that these phage applications did not have deleterious effects on the flower microbiome. We have demonstrated that biological control of fire blight with phages is achievable, but a better understanding of phage-pathogen dynamics is required to optimize disease control efficacy.
The enterobacterial phytopathogen Erwinia amylovora causes fire blight, an invasive disease that threatens a wide range of commercial and ornamental Rosaceae host plants. The response elicited by E. ...amylovora in its host during disease development is similar to the hypersensitive reaction that typically leads to resistance in an incompatible host-pathogen interaction, yet no gene-for-gene resistance has been described for this host-pathogen system. Comparative genomic analysis has found an unprecedented degree of genetic uniformity among strains of E. amylovora, suggesting that the pathogen has undergone a recent genetic bottleneck. The genome of apple, an important host of E. amylovora, has been sequenced, creating new opportunities for the study of interactions between host and pathogen during fire blight development and for the identification of resistance genes. This review includes recent advances in the genomics of both host and pathogen.
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