Frontiers for research on the ecology of plant‐pathogenic bacteria: fundamentals for sustainability

Methods to ensure the health of crops owe their efficacy to the extent to which we understand the ecology and biology of environmental microorganisms and the conditions under which their interactions with plants lead to losses in crop quality or yield. However, in the pursuit of this knowledge, notions of the ecology of plant‐pathogenic microorganisms have been reduced to a plant‐centric and agro‐centric focus. With increasing global change, i.e. changes that encompass not only climate, but also biodiversity, the geographical distribution of biomes, human demographic and socio‐economic adaptations and land use, new plant health problems will emerge via a range of processes influenced by these changes. Hence, knowledge of the ecology of plant pathogens will play an increasingly important role in the anticipation and response to disease emergence. Here, we present our opinion on the major challenges facing the study of the ecology of plant‐pathogenic bacteria. We argue that the discovery of markedly novel insights into the ecology of plant‐pathogenic bacteria is most likely to happen within a framework of more extensive scales of space, time and biotic interactions than those that currently guide much of the research on these bacteria. This will set a context that is more propitious for the discovery of unsuspected drivers of the survival and diversification of plant‐pathogenic bacteria and of the factors most critical for disease emergence, and will set the foundation for new approaches to the sustainable management of plant health. We describe the contextual background of, justification for and specific research questions with regard to the following challenges:

• Development of terminology to describe plant–bacterial relationships in terms of bacterial fitness.
• Definition of the full scope of the environments in which plant‐pathogenic bacteria reside or survive.
• Delineation of pertinent phylogenetic contours of plant‐pathogenic bacteria and naming of strains independent of their presumed life style.
• Assessment of how traits of plant‐pathogenic bacteria evolve within the overall framework of their life history.
• Exploration of possible beneficial ecosystem services contributed to by plant‐pathogenic bacteria.

     

Targeted Proteomics for Studying Pathogenic Bacteria

Mass spectrometry‐based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria–host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS‐based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.     

Diffusible signal factor‐dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell‐to‐cell signals of the diffusible signal factor (DSF) family are cis‐2‐unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signalling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.     

Bacterial adaptation to life in association with plants - A proteomic perspective from culture to in situ conditions

Diverse bacterial taxa that live in association with plants affect plant health and development. This is most evident for those bacteria that undergo a symbiotic association with plants or infect the plants as pathogens. Proteome analyses have contributed significantly toward a deeper understanding of the molecular mechanisms underlying the development of these associations. They were applied to obtain a general overview of the protein composition of these bacteria, but more so to study effects of plant signaling molecules on the cytosolic proteome composition or metabolic adaptations upon plant colonization. Proteomic analyses are particularly useful for the identification of secreted proteins, which are indispensable to manipulate a host plant. Recent advances in the field of proteome analyses have initiated a new research area, the analysis of more complex microbial communities. Such studies are just at their beginning but hold great potential for the future to elucidate not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa when living in association with plants. These include not only the symbiotic and pathogenic bacteria, but also the commensal bacteria that are consistently found in association with plants and whose functions remain currently largely uncovered.     

Plant–rhizobacteria interactions alleviate abiotic stress conditions

Root-colonizing non-pathogenic bacteria can increase plant resistance to biotic and abiotic stress factors. Bacterial inoculates have been applied as biofertilizers and can increase the effectiveness of phytoremediation. Inoculating plants with non-pathogenic bacteria can provide 'bioprotection' against biotic stresses, and some root-colonizing bacteria increase tolerance against abiotic stresses such as drought, salinity and metal toxicity. Systematic identification of bacterial strains providing cross-protection against multiple stressors would be highly valuable for agricultural production in changing environmental conditions. For bacterial cross-protection to be an effective tool, a better understanding of the underlying morphological, physiological and molecular mechanisms of bacterially mediated stress tolerance, and the phenomenon of cross-protection is critical. Beneficial bacteria-mediated plant gene expression studies under non-stress conditions or during pathogenic rhizobacteria–plant interactions are plentiful, but only few molecular studies on beneficial interactions under abiotic stress situations have been reported. Thus, here we attempt an overview of current knowledge on physiological impacts and modes of action of bacterial mitigation of abiotic stress symptoms in plants. Where available, molecular data will be provided to support physiological or morphological observations. We indicate further research avenues to enable better use of cross-protection capacities of root-colonizing non-pathogenic bacteria in agricultural production systems affected by a changing climate.     

Crossing the kingdom border: Human diseases caused by plant pathogens

Interactions between pathogenic microorganisms and their hosts are varied and complex, encompassing open-field scale interactions to interactions at the molecular level. The capacity of plant pathogenic bacteria and fungi to cause diseases in human and animal systems was, until recently, considered of minor importance. However, recent evidence suggests that animal and human infections caused by plant pathogenic fungi, bacteria and viruses may have critical impacts on human and animal health and safety. This review analyses previous research on plant pathogens as causal factors of animal illness. In addition, a case study involving disruption of type III effector-mediated phagocytosis in a human cell line upon infection with an opportunistic phytopathogen, Pseudomonas syringae pv. tomato, is discussed. Further knowledge regarding the molecular interactions between plant pathogens and human and animal hosts is needed to understand the extent of disease incidence and determine mechanisms for disease prevention.     

Genetic control of processes of bacterial interactions with plants in associations

This review is devoted to the mechanisms and genetic control of processes underlying the formation and efficiency of associative relationships between bacteria and plants. The role of different polysaccharides and cellular fibrils in the appearance of associative relations and the biosynthetic pathway of these compounds and structures is considered. The molecular mechanisms of bacterial systems responsible for stimulating plant growth and development--nitrogen fixation and synthesis of a plant hormone, indoleacetic acid--are presented. The properties of associative bacteria are discussed in comparison with the relevant characteristics of the most studied free-living or symbiotic model species of bacteria.     

Evidences of lateral gene transfer between archaea and pathogenic bacteria

Acquisition of new genetic material through horizontal gene transfer has been shown to be an important feature in the evolution of many pathogenic bacteria. Changes in the genetic repertoire, occurring through gene acquisition and deletion, are the major events underlying the emergence and evolution of bacterial pathogens. However, horizontal gene transfer across the domains i.e. archaea and bacteria is not so common. In this context, we explore events of horizontal gene transfer between archaea and bacteria. In order to determine whether the acquisition of archaeal genes by lateral gene transfer is an important feature in the evolutionary history of the pathogenic bacteria, we have developed a scheme of stepwise eliminations that identifies archaeal-like genes in various bacterial genomes. We report the presence of 9 genes of archaeal origin in the genomes of various bacteria, a subset of which is also unique to the pathogenic members and are not found in respective non-pathogenic counterparts. We believe that these genes, having been retained in the respective genomes through selective advantage, have key functions in the organism’s biology and may play a role in pathogenesis.     

Plant models for animal pathogenesis

Several bacteria that are pathogenic to animals also infect plants. Mechanistic studies have proven that some human/animal pathogenic bacteria employ a similar subset of virulence determinants to elicit disease in animals, invertebrates and plants. Therefore, the results of plant infection studies are relevant to animal pathogenesis. This discovery has resulted in the development of convenient, cost-effective, and reliable plant infection models to study the molecular basis of infection by animal pathogens. Plant infection models provide a number of advantages in the study of animal pathogenesis. Using a plant model, mutations in animal pathogenic bacteria can easily be screened for putative virulence factors, a process which if done using existing animal infection models would be time-consuming and tedious. High-throughput screening of plants also provides the potential for unravelling the mechanisms by which plants resist animal pathogenic bacteria, and provides a means to discover novel therapeutic agents such as antibiotics and anti-infective compounds. In this review, we describe the developing technique of using plants as a model system to study Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus pathogenesis, and discuss ways to use this new technology against disease warfare and other types of bioterrorism.     

Mimicking small G-proteins: an emerging theme from the bacterial virulence arsenal

The identification of the Ras superfamily of small molecular weight GTPases (G-proteins) has opened up new fields in cancer biology, immunity and infectious disease research. Because of their ubiquitous role in cellular homeostasis, small G-proteins are common targets for several pathogens, including bacteria. It is well known that pathogenic bacteria have evolved virulence factors that chemically modify GTPases or directly mimic the activities of key regulatory proteins. However, recent studies now suggest that bacterial 'effector' proteins can also mimic the activities of Ras small G-proteins despite their lack of guanine nucleotide binding or GTPase enzymatic activity. The study of these unique pathogenic strategies continues to reveal novel mechanistic insights into host cellular communication networks and the role of small G-protein signalling during human infectious disease.     

Phytotoxins as tools in studying plant disease resistance

Many plant pathogenic fungi and bacteria produce phytotoxins which are a vital part of their arsenal in causing plant disease. Some host-specific toxins have proved useful in enhancing our understanding of the biochemical mechanisms underlying plant disease resistance.     

The interaction between bacteria and bile

Commensal and pathogenic microorganisms must resist the deleterious actions of bile in order to survive in the human gastrointestinal tract. Herein we review the current knowledge on the mechanisms by which Gram-positive and Gram-negative bacteria contend with bile stress. We describe the antimicrobial actions of bile, assess the variations in bile tolerance between bacterial genera and examine the interplay between bile stress and other stresses. The molecular mechanisms underlying bile tolerance are investigated and the relationship between bile and virulence is examined. Finally, the potential benefits of bile research are briefly discussed.     

Role of antigens and virulence factors of Salmonella enterica serovar Typhi in its pathogenesis

Salmonella enterica serovar Typhi (S. Typhi), the aetiologic agent of typhoid fever, is a human restricted pathogen. The molecular mechanism of Salmonella pathogenicity is complex. The investigations of the molecular mechanisms of Salmonella virulence factors have shown that pathogenic Salmonella spp. are distinguished from their non-pathogenic relatives by the presence of specific pathogenicity genes, often organized in so-called pathogenicity islands (PIs). The type III secretion system (T3SS) proteins encoded by two Salmonella PIs (SPIs) are associated with the pathogenicity at molecular level. The identification of T3SS has provided new insight into the molecular factors and mechanisms underlying bacterial pathogenesis. The T3SS encoded by SPI-1 contains invasion genes; while SPI-2 is responsible for intracellular pathogenesis and has a crucial role for systemic S. enterica infections. These studies reveal a complex set of pathogenic interferences between intracellular Salmonella and its host cells. The understanding of the mechanisms by which Salmonella evade the host defense system and establish pathogenesis will be important for proper disease management.     

A New Zebrafish Model of Oro-Intestinal Pathogen Colonization Reveals a Key Role for Adhesion in Protection by Probiotic Bacteria

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.     

Current concepts on the pathogenicity of phytopathogenic bacteria

What are the molecular determinants that make a bacterium a plant pathogen? In the last 10-20 years, important progress has been made in answering this question. In the early 20th century soon after the discovery of infectious diseases, the first studies of pathogenicity were undertaken. These early studies relied mostly on biochemistry and led to the discovery of several major pathogenicity determinants, such as toxins and hydrolytic enzymes which govern the production of major disease symptoms. From these pioneering studies, a simplistic view of pathogenicity arose. It was thought that only a few functions were sufficient to transform a bacterium into a pathogen. This view rapidly changed when modern techniques of molecular genetics were applied to analyse pathogenicity. Modern analyses of pathogenicity determinants took advantage of the relatively simple organization of the haploid genome of pathogenic bacteria. By creating non-pathogenic mutants, a large number of genes governing bacterium-host interactions were identified. These genes are required either for host colonization or for the production of symptoms. Even though the role of motility and chemotaxis in these processes is still unclear, it is clear that a strong attachment of Agrobacterium to plant cells is a prerequisite for efficient plant transformation and disease. Other important pathogenicity factors identified with a molecular genetic approach include hydrolytic enzymes such as pectinases and cellulases which not only provide nutrients to the bacteria but also facilitate pathogen invasion into host tissues. The precise role of exopolysaccharide in pathogenicity is still under discussion, however it is has been established that it is crucial for the induction of wilt symptoms caused by Ralstonia solanacearum. Trafficking of effector proteins from the invading bacterium into the host cell emerged recently as a new central concept. In plant pathogenic bacteria, protein translocation takes place through the so-called 'type II secretion machinery' encoded by hrp genes in the bacterium. These genes are present in representatives of all the major groups of Gram negative plant pathogenic bacteria except Agrobacterium. Most of these genes have counterparts in pathogens of mammals (including those of human) and they also play a central role in pathogenicity. Additionally, recent evidence suggests that a 'type IV secretion machinery' injects bacterial proteins into host cells. This machinery, originally found to be involved in the transfer of t-DNA from Agrobacterium into plant cells, was recently shown to translocate pathogenicity proteins in pathogens of mammals such as Helicobacter pylori and Brucella. Discovery of the trafficking of proteins from the pathogen into host cells revolutionized our conception of pathogenicity. First, it rather unexpectedly established the conservation of basic pathogenicity strategies in plant and animal pathogens. Second, this discovery changes our ideas about the overall strategy (or mechanism) of pathogenicity, although we still think the end result is exploitation of host cell nutritive components. Rather than killing the host cell from outside, we envision a more subtle approach in which pathogens inject effector proteins into the host cell to effect a change in host cell biology advantageous to the pathogen. Identification of the effector proteins, of their function and of the corresponding molecular targets in the host is a new challenge which will contribute to the conception of new strategies to control diseases.     

Novel tool in rheumatoid arthritis diagnosis—The usage of urease flap region peptidomimetics

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and non‐RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms. Thus, to better understand the role of bacteria in RA, we synthetized 6 peptidomimetics of bacterial ureases' flap region. These peptides were then used to distinguish RA patients from healthy people sera by immunoblotting. Most patients' sera were bound to peptidomimetic characteristic for Enterobacter sp. and Klebsiella sp. flap urease. We also found similarities between peptidomimetic sequence and human proteins connected with RA. This pilot study suggests that bacteria may trigger RA via mechanism of molecular mimicry of urease to host proteins and ureases flap peptidomimetics may be potential candidate as a new additional diagnostic test.     

Genomic clues for defining bacterial pathogenicity

Genomic sequences are becoming available from both pathogenic and nonpathogenic bacteria. Here we analyze an increasing body of information available on the molecular mechanisms Salmonella typhimurium uses to cause disease, in order to divine clues for identifying sequences that play a role in pathogenesis in other bacterial pathogens.     

Profiling Differentially Abundant Proteins by Overexpression of Three Putative Methyltransferases in Xanthomonas axonopodis pv. glycines

Methyltransferases (MTases) are enzymes that modify specific substrates by adding a methyl group using S‐adenosyl‐l ‐methionine. Functions of MTases have been extensively studied in eukaryotic organisms and animal pathogenic bacteria. Despite their importance, mechanisms underlying MTase function in plant pathogenic bacteria have not been studied in depth, as is the case of Xanthomonas axonopodis pv. glycines (Xag) that causes bacterial pustule disease in soybean crops worldwide. Here, the association between Xag proteome alterations and three MTase‐overexpressing strains, Xag(XgMT1), Xag(XgMT2), and Xag(XgMT3), compared to Xag carrying an empty vector, Xag(EV) is reported. Using label‐free shotgun comparative proteomic analysis, proteins are identified in all three biological replicates of the four strains and ranged from 1004 to 1082. In comparative analyses, 124, 135, and 134 proteins are differentially changed (over twofold) by overexpression of XgMT1, XgMT2, and XgMT3, respectively. These proteins are also categorized using cluster of orthologous group (COG) analyses, allowing postulation of biological mechanisms associated with three MTases in Xag. COGs reveal that the three MTases may play distinct roles, although some functions may overlap. These results are expected to allow new insight into understanding and predicting the biological functions of MTases in plant pathogenic bacteria. Data are available via ProteomeXchange (Identifier PXD012590).