Riding the wave of genomics to investigate aquatic coliphage diversity and activity

Bacteriophages infecting Escherichia coli (coliphages) have been used as a proxy for faecal matter and water quality from a variety of environments. However, the diversity of coliphages that is present in seawater remains largely unknown, with previous studies largely focusing on morphological diversity. Here, we isolated and characterized coliphages from three coastal locations in the United Kingdom and Poland. Comparative genomics and phylogenetic analysis of phage isolates facilitated the identification of putative new species within the genera Rb69virus and T5virus and a putative new genus within the subfamily Tunavirinae. Furthermore, genomic and proteomic analysis combined with host range analysis allowed the identification of a putative tail fibre that is likely responsible for the observed differences in host range of phages vB_Eco_mar003J3 and vB_Eco_mar004NP2.     

Genetic diversity and biocontrol potential of fluorescent pseudomonads producing phloroglucinols and hydrogen cyanide from Swiss soils naturally suppressive or conducive to Thielaviopsis basicola-mediated black root rot of tobacco

Pseudomonas populations producing the biocontrol compounds 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) were found in the rhizosphere of tobacco both in Swiss soils suppressive to Thielaviopsis basicola and in their conducive counterparts. In this study, a collection of Phl+ HCN+Pseudomonas isolates from two suppressive and two conducive soils were used to assess whether suppressiveness could be linked to soil-specific properties of individual pseudomonads. The isolates were compared based on restriction analysis of the biocontrol genes phlD and hcnBC, enterobacterial repetitive intergenic consensus (ERIC)-PCR profiling and their biocontrol ability. Restriction analyses of phlD and hcnBC yielded very concordant relationships between the strains, and suggested significant population differentiation occurring at the soil level, regardless of soil suppressiveness status. This was corroborated by high strain diversity (ERIC-PCR) within each of the four soils and among isolates harboring the same phlD or hcnBC alleles. No correlation was found between the origin of the isolates and their biocontrol activity in vitro and in planta. Significant differences in T. basicola inhibition were however evidenced between the isolates when they were grouped according to their biocontrol alleles. Moreover, two main Pseudomonas lineages differing by the capacity to produce pyoluteorin were evidenced in the collection. Thus, Phl+ HCN+ pseudomonads from suppressive soils were not markedly different from those from nearby conducive soils. Therefore, as far as biocontrol pseudomonads are concerned, this work yields the hypothesis that the suppressiveness of Swiss soils may rely on the differential effects of environmental factors on the expression of key biocontrol genes in pseudomonads rather than differences in population structure of biocontrol Pseudomonas subcommunities or the biocontrol potential of individual Phl+ HCN+ pseudomonad strains.     

Mycobacterial proteomics: analysis of expressed proteomes and post-translational modifications to identify candidate virulence factors

The Mycobacterium tuberculosis bacillus has a number of unique features that make it a particularly effective human pathogen. Although genomic analysis has added to our current understanding of the molecular basis by which M. tuberculosis damages its host, proteomics may be better suited to describe the dynamic interactions between mycobacterial and host systems that underpin this disease. The M. tuberculosis proteome has been investigated using proteomics for over a decade, with increasingly sophisticated mass spectrometry technology and sensitive methods for comparative proteomic profiling. Deeper coverage of the M. tuberculosis proteome has led to the identification of hundreds of putative virulence determinants, as well as an unsurpassed coverage of post-translational modifications. Proteomics is therefore uniquely poised to contribute to our understanding of this pathogen, which may ultimately lead to better management of the disease.     

Abundance of the Quorum-Sensing Factor Ax21 in Four Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor.     

Global proteomic pattern of Tropheryma whipplei: A Whipple's disease bacterium

The proteome of Tropheryma whipplei, the intracellular bacterium responsible for Whipple's disease (WD), was analyzed using two complementary approaches: 2‐DE coupled with MALDI‐TOF and SDS‐PAGE with nanoLC‐MS/MS. This strategy led to the identification of 206 proteins of 808 predicted ORFs, resolving some questions raised by the genomic sequence of this bacterium. We successfully identified antibiotic targets and proteins with predicted N‐terminal signal sequences. Additionally, we identified a family of surface proteins (known as T. whipplei surface proteins (WiSPs)), which are encoded by a unique group of species‐specific genes and serve as both coding regions and DNA repeats that promote genomic recombination. Comparison of the protein expression profiles of the intracellular facultative host‐associated WD bacterium with other host‐associated, intracellular obligate, and environmental bacteria revealed that T. whipplei shares a proteomic expression profile with other host‐associated facultative intracellular bacteria. In summary, this study describes the global protein expression pattern of T. whipplei and reveals some specific features of the T. whipplei proteome.     

Comparative proteomic analysis reveals that T3SS,Tfp, and xanthan gum are key factors in initial stages of Citrus sinensis infection by Xanthomonas citri subsp. citri

The bacteria Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. The disease symptoms are characterized by localized host cell hyperplasia followed by tissue necrosis at the infected area. An arsenal of bacterial pathogenicity- and virulence-related proteins is expressed to ensure a successful infection process. At the post-genomic stage of Xac, we used a proteomic approach to analyze the proteins that are displayed differentially over time when the pathogen attacks the host plant. Protein extracts were prepared from infectious Xac grown in inducing medium (XAM1) for 24 h or from host citrus plants for 3 or 5 days after infection, detached times to evaluate the adaptation and virulence of the pathogen. The protein extracts were proteolyzed, and the peptides derived from tryptic digestion were investigated using liquid chromatography and tandem mass spectrometry. Changes in the protein expression profile were compared with the Xac genome and the proteome recently described under non-infectious conditions. An analysis of the proteome of Xac under infectious conditions revealed proteins directly involved in virulence such as the type III secretion system (T3SS) and effector proteins (T3SS-e), the type IV pilus (Tfp), and xanthan gum biosynthesis. Moreover, four new mutants related to proteins detected in the proteome and with different functions exhibited reduced virulence relative to the wild-type proteins. The results of the proteome analysis of infectious Xac define the processes of adaptation to the host and demonstrate the induction of the virulence factors of Xac involved in plant–pathogen interactions.     

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.     

Investigating pathogen biology at the level of the proteome

Bacterial infections are a major cause of morbidity and mortality throughout the world. By extending our understanding of the process of bacterial pathogenesis at the molecular level new strategies for their treatment and prevention can be developed. Proteomic technologies, along with other methods for global gene expression analysis, play an important role in understanding the mechanism(s) of bacterial pathogenesis. This review highlights the use of proteomics to identify protein biomarkers for virulent bacterial isolates and how these biomarkers can be correlated with the outcome of bacterial infection. Biomarker identification typically looks at the proteomes of bacteria grown under laboratory conditions. It is, however, the characterisation of the bacterial proteome during in vivo infection of its host that will eventually provide the most significant insights into bacterial pathogenesis. Although this area of research has significant technical challenges, a number of complementary proteome analytical approaches are being developed to identify and characterise the bacterial genes specifically expressed in vivo. Ultimately, the development of newly targeted therapies and vaccines using specific protein targets identified through proteomic analyses will be one of the major practical benefits arising from the proteomic analysis of bacterial pathogens.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.     

Comparative Analysis of Extracellular and Intracellular Proteomes of Listeria monocytogenes Strains Reveals a Correlation between Protein Expression and Serovar

Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern, with its frequent occurrence in food environments coupled with a high mortality rate. Among the 13 serovars, human listeriosis is mostly associated with the serovar 4b, 1/2b, and 1/2a strains. To investigate the diversity of L. monocytogenes, the intracellular and extracellular proteins of 12 strains were analyzed by two-dimensional gel electrophoresis. These strains had different origins, belonged to different serovars (4b, 1/2a, and 1/2b), and presented with different levels of virulence in chicken embryos. The clustering of the strains in two groups based on proteomic patterns is in agreement with the L. monocytogenes phylogenetic lineages. Statistical analysis did not allow for identification of proteins specific to the isolate origin or the virulence level of the strains, but 26 and 21 protein spots were shown to be significantly overexpressed and underexpressed, respectively, in the six strains of serovar 1/2a (lineage II) compared to strains of serovar 1/2b or 4b. Moreover, a penicillin-binding protein was specific for serovar 1/2b and two protein spots identified as a serine protease were specific to serovar 4b. These protein spots, identified through peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry, were essentially found in the extracellular proteome and may have uses as potential markers for serotyping and risk analysis.     

Phenotypic variation and genomic variation in insect virulence traits reveal patterns of intraspecific diversity in a locust-specific fungal pathogen

Intraspecific pathogen diversity is crucial for understanding the evolution and maintenance of adaptation in host–pathogen interactions. Traits associated with virulence are often a significant source of variation directly impacted by local selection pressures. The specialist fungal entomopathogen, Metarhizium acridum, has been widely implemented as a biological control agent of locust pests in tropical regions of the world. However, few studies have accounted for natural intraspecific phenotypic and genetic variation. Here, we examine the diversity of nine isolates of M. acridum spanning the known geographic distribution, in terms of (1) virulence towards two locust species, (2) growth rates on three diverse nutrient sources, and (3) comparative genomics to uncover genomic variability. Significant variability in patterns of virulence and growth was shown among the isolates, suggesting intraspecific ecological specialization. Different patterns of virulence were shown between the two locust species, indicative of potential host preference. Additionally, a high level of diversity among M. acridum isolates was observed, revealing increased variation in subtilisin-like proteases from the Pr1 family. These results culminate in the first in-depth analysis regarding multiple facets of natural variation in M. acridum, offering opportunities to understand critical evolutionary drivers of intraspecific diversity in pathogens.     

Comparison of cultural methods for the identification and molecular investigation of yeasts from sourdoughs for Italian sweet baked products

Twenty-five yeast strains isolated from sourdough samples for Panettone, Pandoro and Cornetto brioche manufactured by eight different bakeries in northern Italy were characterised. Classification was performed by the simplified identification method (SIM), Kurtzman and Fell's identification protocol, the API system from bioMérieux (France) and the MicroLog system from Biolog (USA). Genetic diversity was investigated by randomly amplified polymorphic DNA fingerprinting and mitochondrial-DNA restriction enzyme analysis. Sequences of the internal transcribed spacers between 18S and 26S rDNA genes were analysed. Candida humilis was the predominant species (56% of isolates), whereas the remaining strains (44%) were related to the Saccharomyces cerevisiae sensu stricto group. Identification systems based on phenotypic analysis proved to be unreliable to identify yeasts from sourdough. Either RAPD-PCR or mtDNA restriction analysis showed to be suitable for the identification of species, but could not be used to differentiate among the isolates at the strain level. Sequencing of the ITS region permitted a consistent classification of the sourdough yeasts.     

中国马铃薯晚疫病菌AFLP遗传多样性分析

应用AFLP分子标记检测了我国部分马铃薯主要产区马铃薯晚疫病菌的遗传多样性及不同地区菌株间的亲缘关系。在200对引物组合中,利用6个菌株筛选出12对多态性好、带型清晰的引物组合。利用这12对引物组合对1997-2002年间采自我国黑龙江、河北、四川和云南4省的50株菌株进行了PCR扩增,共扩增出922条谱带,其中多态性标记530条,占57.5%。利用NTSYSpc软件中UPGMA算法构建了我国马铃薯晚疫病菌的亲缘关系树状图,聚类分析结果表明我国马铃薯晚疫病菌的遗传多样性与病原菌的地理来源有一定的相关性,而与交配型、生理小种和对甲霜灵的抗性无明显的相关性。用POPGENE软件计算了各群体间的遗传多样性参数,结果表明我国马铃薯晚疫病菌的遗传多样性程度不高,不同地区种群间分化不明显。     

Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB

A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2-D gel analysis of the secreted M1T1 proteome, coupled with MALDI-TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA- M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase-shift to a SpeB-/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte-stimulating capacity. The proteome of the SpeB-in vivo phase-shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage-encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB- phase-shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.     

Host–pathogen interactions: a proteomic view

Host–pathogen interactions reflect the balance of host defenses and pathogen virulence mechanisms. Advances in proteomic technologies now afford opportunities to compare protein content between complex biologic systems ranging from cells to animals and clinical samples. Thus, it is now possible to characterize host–pathogen interactions from a global proteomic view. Most reports to date focus on cataloging protein content of pathogens and identifying virulence-associated proteins or proteomic alterations in host response. A more in-depth understanding of host–pathogen interactions has the potential to improve our mechanistic understanding of pathogenicity and virulence, thereby defining novel therapeutic and vaccine targets. In addition, proteomic characterization of the host response can provide pathogen-specific host biomarkers for rapid pathogen detection and characterization, as well as for early and specific detection of infectious diseases. A review of host–pathogen interactions focusing on proteomic analyses of both pathogen and host will be presented. Relevant genomic studies and host model systems will be also be discussed.     

Characterization of a Chromobacterium haemolyticum population from a natural tropical lake

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong β‐haemolytic activity, were nonviolacein producers and utilized i‐inositol, d ‐mannitol and d ‐sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S‐23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)‐PCR genomic fingerprinting using the BOX‐AR1 primer. tDNA‐ and ITS‐PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to β‐lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX‐PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.