Comparing Protein Expression in Erwinia amylovora Strain TS3128 Cultured under Three Sets of Environmental Conditions

Erwinia amylovora, the causal agent of fire-blight disease in apple and pear trees, was first isolated in South Korea in 2015. Although numerous studies, including omics analyses, have been conducted on other strains of E. amylovora, studies on South Korean isolates remain limited. In this study, we conducted a comparative proteomic analysis of the strain TS3128, cultured in three media representing different growth conditions. Proteins related to virulence, type III secretion system, and amylovoran production, were more abundant under minimal conditions than in rich conditions. Additionally, various proteins associated with energy production, carbohydrate metabolism, cell wall/membrane/envelope biogenesis, and ion uptake were identified under minimal conditions. The strain TS3128 expresses these proteins to survive in harsh environments. These findings contribute to understanding the cellular mechanisms driving its adaptations to different environmental conditions and provide proteome profiles as reference for future studies on the virulence and adaptation mechanisms of South Korean strains.     

Isolation and protein composition of the outer membrane ofErwinia amylovora

The outer membrane (OM) ofErwinia amylovora was separated from the cytoplasmic membrane either by isopycnic sucrose density gradient centrifugation or by treating the envelope preparation with sodium lauroylsarcosine. Outer membranes, prepared by using either method, were similar in the content of the major proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The wild-type strains ofE. amylovora (E9, E8, EA178, EA198, EA225, EA273) had—in common in the OM—two major protein bands of apparent molecular weights of 15,800 (protein z) and 38,000 (protein y). The OM protein profiles of the virulent wild-type (E9) and the avirulent mutant (E8) were identical but differed from otherE. amylovora strains by the presence of an additional major protein band of approximately 40,000 (protein x). In strain E9, protein x appeared to be associated with the peptidoglycan, whereas proteins y and z were apparently not peptidoglycan-bound.     

鸭疫里默氏杆菌强、弱菌株外膜蛋白的比较蛋白质组学研究

应用双向电泳及质谱技术对血清2型鸭疫里默氏杆菌强毒株及其体外传代200代(RA200)的弱毒菌株的外膜蛋白进行比较蛋白质组学研究,借此分析鸭疫里默氏杆菌的外膜蛋白表达特点,研究差异表达蛋白与细菌毒力的关系.在实验中检测到血清2型鸭疫里默氏杆菌原代及其体外传代获得的弱毒菌株的外膜蛋白约表达60个蛋白质点(n=3),其中相差5倍以上3个.胶内酶解和肽质量指纹图谱分析后鉴定,W1为热休克蛋白Hsp20家族成员,W2、W3为转座酶,推测它们可能与里默氏杆菌的毒力密切相关.     

2‐DE proteomic approach to the Botrytis cinerea secretome induced with different carbon sources and plant‐based elicitors

Botrytis cinerea is a phytopathogenic fungus infecting a number of crops (tomatoes, grapes and strawberries), which has been adopted as a model system in molecular phytopathology. B. cinerea uses a wide variety of infection strategies, which are mediated by a set of genes/proteins called pathogenicity/virulence factors. Many of these factors have been described as secreted proteins, and thus the study of this sub‐proteome, the secretome, under changing circumstances can help us to understand the roles of these factors, possibly revealing new loci for the fight against the pathogen. A 2‐DE, MALDI TOF/TOF‐based approach has been developed to establish the proteins secreted to culture media supplemented with different carbon sources and plant‐based elicitors (in this study: glucose, cellulose, starch, pectin and tomato cell walls). Secreted proteins were obtained from the culture media by deoxycholate‐trichloroacetic acid/phenol extraction, and 76 spots were identified, yielding 95 positive hits that correspond to 56 unique proteins, including several known virulence factors (i.e. pectin methyl esterases, xylanases and proteases). The observed increases in secretion of proteins with established virulence‐related functions indicate that this in vitro‐induction/proteome‐mining approach is a promising strategy for discovering new pathogenicity factors and dissecting infection mechanisms in a discrete fashion.     

Proteome variation among Filifactor alocis strains

Filifactor alocis, a Gram‐positive anaerobic rod, is now considered one of the marker organisms associated with periodontal disease. Although there was heterogeneity in its virulence potential, this bacterium was shown to have virulence properties that may enhance its ability to survive and persist in the periodontal pocket. To gain further insight into a possible mechanism(s) of pathogenesis, the proteome of F. alocis strains was evaluated. Proteins including several proteases, neutrophil‐activating protein A and calcium‐binding acid repeat protein, were identified in F. alocis. During the invasion of HeLa cells, there was increased expression of several of the genes encoding these proteins in the potentially more virulent F. alocis D‐62D compared to F. alocis ATCC 35896, the type strain. A comparative protein in silico analysis of the proteome revealed more cell wall anchoring proteins in the F. alocis D‐62D compared to F. alocis ATCC 35896. Their expression was enhanced by coinfection with Porphyromonas gingivalis. Taken together, the variation in the pathogenic potential of the F. alocis strains may be related to the differential expression of several putative virulence factors.     

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

The Type VII protein secretion system, found in Gram‐positive bacteria, secretes small proteins, containing a conserved W‐x‐G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.     

Response of life‐history traits to artificial and natural selection for virulence and nonvirulence in a Drosophila parastitoid,Asobara tabida

Co‐evolution of host–parasitoid interactions is determined by the costs of host resistance, which received empirical evidence, and the costs of parasitoid virulence, which have been mostly hypothesized. Asobara tabida is a parasitoid, which mainly parasitizes Drosophila melanogaster and D. subobscura, the first species being able to resist to the parasitoid development while the second species is not. To parasitize resistant hosts, including D. melanogaster, A. tabida develops sticky eggs, which prevent encapsulation, but this virulence mechanism may be costly. Interindividual and interpopulation variation in the proportion of sticky eggs respectively allowed us to (i) artificially select and compare life‐history traits of a virulent and a nonvirulent laboratory strain, and (ii) compare a virulent and a nonvirulent field strain, to investigate the hypothetical costs of virulence. We observed strong differences between the 2 laboratory strains. The nonvirulent strain invested fewer resources in reproduction and walked less than the virulent one but lived longer. Concerning the field strains, we observed that the nonvirulent strain had larger wings while the virulent one walked more and faster. All together, our results suggest that virulence may not always be costly, but rather that different life histories associated with different levels of virulence may coexist at both intra‐ and interpopulation levels.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

Plant nitrate supply regulates Erwinia amylovora virulence gene expression in Arabidopsis

We showed previously that nitrogen (N) limitation decreases Arabidopsis resistance to Erwinia amylovora (Ea). We show that decreased resistance to bacteria in low N is correlated with lower apoplastic reactive oxygen species (ROS) accumulation and lower jasmonic acid (JA) pathway expression. Consistently, pretreatment with methyl jasmonate (Me-JA) increased the resistance of plants grown under low N. In parallel, we show that in planta titres of a nonvirulent type III secretion system (T3SS)-deficient Ea mutant were lower than those of wildtype Ea in low N, as expected, but surprisingly not in high N. This lack of difference in high N was consistent with the low expression of the T3SS-encoding hrp virulence genes by wildtype Ea in plants grown in high N compared to plants grown in low N. This suggests that expressing its virulence factors in planta could be a major limiting factor for Ea in the nonhost Arabidopsis. To test this hypothesis, we preincubated Ea in an inducing medium that triggers expression of hrp genes in vitro, prior to inoculation. This preincubation strongly enhanced Ea titres in planta, independently of the plant N status, and was correlated to a significant repression of JA-dependent genes. Finally, we identify two clusters of metabolites associated with resistance or with susceptibility to Ea. Altogether, our data showed that high susceptibility of Arabidopsis to Ea, under low N or following preincubation in hrp-inducing medium, is correlated with high expression of the Ea hrp genes in planta and low expression of the JA signalling pathway, and is correlated with the accumulation of specific metabolites.     

Comparative genomics of Spiraeoideae‐infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low‐virulence strain

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae‐infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low‐virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.     

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.     

Effects of Pseudomonas syringae pv. morsprunorum Strain C28, Outer Membrane and LPS Preparations on Cherry Callus

Callus cultures were established from cherry (Prunus avium) cvs. Napoleon and Colt, respectively susceptible and resistant to race‐1 strains of Pseudomonas syringae pv. morsprunorum, by growth on Schenk–Hildebrandt medium. On Napoleon callus, necrosis began earlier and proceeded more rapidly when inoculated with the virulent race‐1 cherry isolate strain C28, than with mutants of diminished virulence derived from it, or with the virulent plum isolates D10 and D17. Colt tissue displayed poorer viability and showed susceptibility to strain C28 and the plum isolates. Callus from both sources was somewhat susceptible to the saprophytes P. aeruginosa NCIMB 8295 and P. fluorescens NCIMB 3756. Strain C28 grew on suspended Napoleon callus cells over a period of 3–4 days, causing leakage of UV‐absorbing compounds and K⁺, with a concomitant rise in extracellular pH. P. fluorescens NCIMB 3756 showed no growth on suspended callus for 6 days. EDTA‐extracted outer membrane (OM) from strain C28 caused leakage of UV‐absorbing material and K⁺, which was later reabsorbed, with little change in pH. The presence of OM suppressed the growth of a subsequent inoculum of strain C28, possibly due to complexation of the available Ca²⁺ and/or Mg²⁺ in the surrounding medium, by the component lipopolysaccharide (LPS). OM from the rough avirulent mutant strain C28‐2 induced leakage of K⁺, but not of UV‐absorbing material, and did not prevent the growth of subsequently inoculated strain C28. Smooth LPS from strain C28 did not cause leakage of K⁺ or of UV‐absorbing material and did not prevent growth of C28. The relevance of these findings is discussed in relation to disease.     

Comparative proteome analysis of fractions enriched for membrane-associated proteins from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica strains

The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.     

Genetic variability of plant-associated proteobacteria isolated from Slovak (Central European) territory

Genetic variability of 17 wild strains of γ-proteobacteria isolated from different host plants, locations and seasons, identified via routine diagnostics as Erwinia amylovora, was analysed. The ERIC-PCR confirmed the genetic homogeneity among 15 virulent strains while 2 avirulent strains were genetically distinct. These two avirulent strains differed in their antibiotic susceptibility from all virulent strains. The only avirulent Ra1051/98 strain showed significant ampicillin resistance. All 15 virulent strains were confirmed via the ERIC-PCR and MALDI-TOF MS, as closely related to the reference strain NCPPB 683 of E. amylovora. Avirulent strains were identified as Rahnella aquatilis and L. quercina subsp. britannica (formerly Brenneria quercina), by MALDI-TOF MS and the identification was confirmed by 16S rRNA gene sequence analysis. For the first time, L. quercina subsp britannica was identified in the fire blight-symptomatic samples in Central Europe, and for the first time this bacterium was isolated from the host different than oak.     

A chromosomally encoded type III secretion pathway in Yersinia enterocolitica is important in virulence

Numerous Gram-negative bacteria use a type III, or contact dependent, secretion system to deliver proteins into the cytosol of host cells. All of these systems identified to date have been shown to have a role in pathogenesis. We have identified 13 genes on the Yersinia enterocolitica chromosome that encode a type III secretion apparatus plus two associated putative regulatory genes. In order to determine the function of this chromosomally-encoded secretion apparatus, we created an in frame deletion of a gene that has homology to the hypothesized inner membrane pore, ysaV. The ysaV mutant strain failed to secrete eight proteins, called Ysps, normally secreted by the parental strain when grown at 28 degrees C in Luria-Bertani (LB) broth supplemented with 0.4 M NaCl. Disruption of the ysaV gene had no effect on motility or phospholipase activity, suggesting this chromosomally encoded type III secretion pathway is distinct from the flagella secretion pathway of Y. enterocolitica. Deletion of the ysaV gene in a virulence plasmid positive strain had no effect on in vitro secretion of Yops by the plasmid-encoded type III secretion apparatus. Secretion of the Ysps was unaffected by the presence or absence of the virulence plasmid, suggesting the chromosomally encoded and plasmid-encoded type III secretion pathways act independently. Y. enterocolitica thus has three type III secretion pathways that appear to act independently. The ysaV mutant strain was somewhat attenuated in virulence compared with the wild type in the mouse oral model of infection (an approximately 0.9 log difference in LD50). The ysaV mutant strain was nearly as virulent as the wild type when inoculated intraperitoneally in the mouse model. A ysaV probe hybridized to sequences in other Yersinia spp. and homologues were found in the incomplete Y. pestis genome sequence, indicating a possible role for this system throughout the genus.     

Characterization of the role of the Escherichia coli periplasmic chaperone SurA using differential proteomics

Little is known on how β‐barrel proteins are assembled in the outer membrane (OM) of Gram‐negative bacteria. SurA has been proposed to be the primary chaperone escorting the bulk mass of OM proteins across the periplasm. However, the impact of SurA deletion on the global OM proteome has not been determined, limiting therefore our understanding of the function of SurA. By using a differential proteomics approach based on 2‐D LC‐MSⁿ, we compared the relative abundance of 64 OM proteins, including 23 β‐barrel proteins, in wild‐type and surA strains. Unexpectedly, we found that the loss of SurA affects the abundance of eight β‐barrel proteins. Of all the decreased proteins, FhuA and LptD are the only two for which the decreased protein abundance cannot be attributed, at least in part, to decreased mRNA levels in the surA strain. In the case of LptD, an essential protein involved in OM biogenesis, our data support a role for SurA in the assembly of this protein and suggest that LptD is a true SurA substrate. Based on our results, we propose a revised model in which only a subset of OM proteins depends on SurA for proper folding and insertion in the OM.     

A proteomic evaluation of Pyrenophora tritici‐repentis,causal agent of tan spot of wheat,reveals major differences between virulent and avirulent isolates

Pyrenophora tritici‐repentis causes tan spot, an important foliar disease of wheat. The fungus produces multiple host‐specific toxins, including Ptr ToxB, a chlorosis‐inducing protein encoded by the ToxB gene. A homolog of ToxB is also found in avirulent isolates of the fungus. In order to improve understanding of the role of this homolog and evaluate the general pathogenic ability of P. tritici‐repentis, we compared the proteomes of avirulent race 4 and virulent race 5 isolates of the pathogen. Western blotting analysis revealed the presence of Ptr ToxB in spore germination and culture fluids of race 5 but not race 4. A comprehensive proteome‐level comparison by 2‐DE indicated 133 differentially abundant proteins in the secretome (29 proteins) and mycelium (104 proteins) of races 4 and 5, of which 63 were identified by MS/MS. A number of the proteins found to be up‐regulated in race 5 have been implicated in microbial virulence in other pathosystems, and included the secreted enzymes α‐mannosidase and exo‐β‐1,3‐glucanase, heat‐shock and BiP proteins, and various metabolic enzymes. These proteome‐level differences suggest a reduced general pathogenic ability in race 4 of P. tritici‐repentis, irrespective of toxin production. Such differences may reflect an adaptation to a saprophytic habit.