Adam Schikora

Quorum Sensing of Bacteria and Trans-Kingdom Interactions of N -Acyl Homoserine Lactones with Eukaryotes

Many environmental and interactive important traits of bacteria, such as antibiotic, siderophore or exoenzyme (like cellulose, pectinase) production, virulence factors of pathogens, as well as symbiotic interactions, are regulated in a population density-dependent manner by using small signaling molecules. This phenomenon, called quorum sensing (QS), is widespread among bacteria. Many different bacterial species are communicating or “speaking” through diffusible small molecules. The production often is sophisticatedly regulated via an autoinducing mechanism. A good example is the production of N-acyl homoserine lactones (AHL), which occur in many variations of molecular structure in a wide variety of Gram-negative bacteria. In Gram-positive bacteria, other compounds, such as peptides, regulate cellular activity and behavior by sensing the cell density. The degradation of the signaling molecule—called quorum quenching—is probably another important integral part in the complex quorum sensing circuit. Most interestingly, bacterial quorum sensing molecules also are recognized by eukaryotes that are colonized by QS-active bacteria. In this case, the cross-kingdom interaction can lead to specific adjustment and physiological adaptations in the colonized eukaryote. The responses are manifold, such as modifications of the defense system, modulation of the immune response, or changes in the hormonal status and growth responses. Thus, the interaction with the quorum sensing signaling molecules of bacteria can profoundly change the physiology of higher organisms too. Higher organisms are obligatorily associated with microbial communities, and these truly multi-organismic consortia, which are also called holobionts, can actually be steered via multiple interlinked signaling substances that originate not only from the host but also from the associated bacteria.

N-acyl-homoserine lactone primes plants for cell wall reinforcement and induces resistance to bacterial pathogens via the salicylic acid/oxylipin pathway

The bacterial quorum-sensing molecule N-3-oxo-tetradecanoyl-l-homoserine lactone primes the plant for enhanced resistance to bacterial pathogens. The proposed mechanism is based on modifications of the cell wall composition and the responsiveness of stomatal guard cells to pathogen attack. The ability of plants to monitor their surroundings, for instance the perception of bacteria, is of crucial importance. The perception of microorganism-derived molecules and their effector proteins is the best understood of these monitoring processes. In addition, plants perceive bacterial quorum sensing (QS) molecules used for cell-to-cell communication between bacteria. Here, we propose a mechanism for how N-acyl-homoserine lactones (AHLs), a group of QS molecules, influence host defense and fortify resistance in Arabidopsis thaliana against bacterial pathogens. N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) primed plants for enhanced callose deposition, accumulation of phenolic compounds, and lignification of cell walls. Moreover, increased levels of oxylipins and salicylic acid favored closure of stomata in response to Pseudomonas syringae infection. The AHL-induced resistance seems to differ from the systemic acquired and the induced systemic resistances, providing new insight into inter-kingdom communication. Consistent with the observation that short-chain AHLs, unlike oxo-C14-HSL, promote plant growth, treatments with C6-HSL, oxo-C10-HSL, or oxo-C14-HSL resulted in different transcriptional profiles in Arabidopsis. Understanding the priming induced by bacterial QS molecules augments our knowledge of plant reactions to bacteria and suggests strategies for using beneficial bacteria in plant protection.

Interactions of Salmonella with animals and plants

Salmonella enterica species are Gram-negative bacteria, which are responsible for a wide range of food- and water-borne diseases in both humans and animals, thereby posing a major threat to public health. Recently, there has been an increasing number of reports, linking Salmonella contaminated raw vegetables and fruits with food poisoning. Many studies have shown that an essential feature of the pathogenicity of Salmonella is its capacity to cross a number of barriers requiring invasion of a large variety of cells and that the extent of internalization may be influenced by numerous factors. However, it is poorly understood how Salmonella successfully infects hosts as diversified as animals or plants. The aim of this review is to describe the different stages required for Salmonella interaction with its hosts: (i) attachment to host surfaces; (ii) entry processes; (iii) multiplication; (iv) suppression of host defense mechanisms; and to point out similarities and differences between animal and plant infections.

Homoserine Lactones Influence the Reaction of Plants to Rhizobia

Bacterial quorum sensing molecules not only grant the communication within bacterial communities, but also influence eukaryotic hosts. N-acyl-homoserine lactones (AHLs) produced by pathogenic or beneficial bacteria were shown to induce diverse reactions in animals and plants. In plants, the reaction to AHLs depends on the length of the lipid side chain. Here we investigated the impact of two bacteria on Arabidopsis thaliana, which usually enter a close symbiosis with plants from the Fabaceae (legumes) family and produce a long-chain AHL (Sinorhizobium meliloti) or a short-chain AHL (Rhizobium etli). We demonstrate that, similarly to the reaction to pure AHL molecules, the impact, which the inoculation with rhizosphere bacteria has on plants, depends on the type of the produced AHL. The inoculation with oxo-C14-HSL-producing S. meliloti strains enhanced plant resistance towards pathogenic bacteria, whereas the inoculation with an AttM lactonase-expressing S. meliloti strain did not. Inoculation with the oxo-C8-HSL-producing R. etli had no impact on the resistance, which is in agreement with our previous hypothesis. In addition, plants seem to influence the availability of AHLs in the rhizosphere. Taken together, this report provides new insights in the role of N-acyl-homoserine lactones in the inter-kingdom communication at the root surface.

AHL-priming functions via oxylipin and salicylic acid.

Collaborative action between the host plant and associated bacteria is crucial for the establishment of an efficient interaction. In bacteria, the synchronized behavior of a population is often achieved by a density-dependent communication called quorum sensing. This behavior is based on signaling molecules, which influence bacterial gene expression. N-acyl homoserine lactones (AHLs) are such molecules in many Gram-negative bacteria. Moreover, some AHLs are responsible for the beneficial effect of bacteria on plants, for example the long chain N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) can prime Arabidopsis and barley plants for an enhanced defense. This AHL-induced resistance phenomenon, named AHL-priming, was observed in several independent laboratories during the last two decades. Very recently, the mechanism of priming with oxo-C14-HSL was shown to depend on an oxylipin and salicylic acid (SA). SA is a key element in plant defense, it accumulates during different plant resistance responses and is the base of systemic acquired resistance. In addition, SA itself can prime plants for an enhanced resistance against pathogen attack. On the other side, oxylipins, including jasmonic acid (JA) and related metabolites, are lipid-derived signaling compounds. Especially the oxidized fatty acid derivative cis-OPDA, which is the precursor of JA, is a newly described player in plant defense. Unlike the antagonistic effect of SA and JA in plant–microbe interactions, the recently described pathway functions through a synergistic effect of oxylipins and SA, and is independent of the JA signaling cascade. Interestingly, the oxo-C14-HSL-induced oxylipin/SA signaling pathway induces stomata defense responses and cell wall strengthening thus prevents pathogen invasion. In this review, we summarize the findings on AHL-priming and the related signaling cascade. In addition, we discuss the potential of AHL-induced resistance in new strategies of plant protection.

Salmonella, a cross-kingdom pathogen infecting humans and plants

Infections with non-typhoidal Salmonella strains are constant and are a non-negligible threat to the human population. In the last two decades, salmonellosis outbreaks have increasingly been associated with infected fruits and vegetables. For a long time, Salmonellae were assumed to survive on plants after a more or less accidental infection. However, this notion has recently been challenged. Studies on the infection mechanism in vegetal hosts, as well as on plant immune systems, revealed an active infection process resembling in certain features the infection in animals. On one hand, Salmonella requires the type III secretion systems to effectively infect plants and to suppress their resistance mechanisms. On the other hand, plants recognize these bacteria and react to the infection with an induced defense mechanism similar to the reaction to other plant pathogens. In this review, we present the newest reports on the interaction between Salmonellae and plants. We discuss the possible ways used by these bacteria to infect plants as well as the plant responses to the infection. The recent findings indicate that plants play a central role in the dissemination of Salmonella within the ecosystem.

N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens.

The implementation of beneficial microorganisms for plant protection has a long history. Many rhizobia bacteria are able to influence the immune system of host plants by inducing resistance towards pathogenic microorganisms. In this report, we present a translational approach in which we demonstrate the resistance‐inducing effect of Ensifer meliloti (Sinorhizobium meliloti) on crop plants that have a significant impact on the worldwide economy and on human nutrition. Ensifer meliloti is usually associated with root nodulation in legumes and nitrogen fixation. Here, we suggest that the ability of S.u2009meliloti to induce resistance depends on the production of the quorum‐sensing molecule, oxo‐C14‐HSL. The capacity to enhanced resistance provides a possibility to the use these beneficial bacteria in agriculture. Using the Arabidopsis‐Salmonella model, we also demonstrate that the application of N‐acyl‐homoserine lactones‐producing bacteria could be a successful strategy to prevent plant‐originated infections with human pathogens.

The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

Image-based Analysis to Study Plant Infection with Human Pathogens

Our growing awareness that contaminated plants, fresh fruits and vegetables are responsible for a significant proportion of food poisoning with pathogenic microorganisms indorses the demand to understand the interactions between plants and human pathogens. Today we understand that those pathogens do not merely survive on or within plants, they actively infect plant organisms by suppressing their immune system. Studies on the infection process and disease development used mainly physiological, genetic, and molecular approaches, and image-based analysis provides yet another method for this toolbox. Employed as an observational tool, it bears the potential for objective and high throughput approaches, and together with other methods it will be very likely a part of data fusion approaches in the near future.

Editorial on plants as alternative hosts for human and animal pathogens

Fresh produce, in the form of raw or minimally processed fruits and vegetables, is recognized to be an important source of food-borne disease. Sporadic cases or outbreaks can arise from bacterial, viral, or parasitic contamination. Importantly, while the latter both require animal hosts for proliferation and cannot grow on or within crop plants, bacteria can do so and are able to use plants as secondary hosts. This type of interaction means that human pathogenic bacteria need to undergo adaptation to the plant host, which presents an environment quite distinct to animals or humans. Important differences lie in both physiochemical and biological properties, e.g., physiology, immunity, native microflora, physical barriers, mobility, and temperature. There is now good published evidence to describe different aspects of the interactions between plants and human pathogenic bacteria, and the subsequent effects on the outcome of colonization. However, in comparison to the interactions between the pathogens and their animal hosts, for which we know a great deal about both host and microbial factors, this area is relatively new with many important knowledge gaps. Research in this area is of most relevance to food safety, since the results can be and indeed, are already applied by public health agencies and food producers. It also reveals fascinating aspects of the bacterial life-cycle that were hitherto under appreciated. n nOne of the important features of plant host colonization is the adaptation of pathogens to the host defense response. Innate immunity is the first line of defense with both physical barriers and biological recognition of human pathogens, and the different layers of the response are reviewed in Melotto et al. (2014). An intriguing aspect is the finding that some human pathogens are able to subvert the plant defense, shown using Salmonella enterica and Arabidopsis as an example (Garcia and Hirt, 2014). Perception of human pathogens is based on recognition of PAMPs (e.g., flagellin) similar to those in phytopathogens and other plant-associated bacteria; perhaps unsurprising given that the mechanisms of recognition tend to be based on evolutionary conserved proteins. What was unexpected was the fact that some Salmonella serovars encode flagellin variants that are no longer recognized by the plant, presumably as a result of evolutionary selection (Garcia and Hirt, 2014). n nThere are good parallels in microbial suppression of the innate response of animal and plant hosts, for example via effectors secreted through the type 3 secretion (T3S) apparatus. Despite structural differences in the apparatus between human- and phyto-pathogens, the general function of host subversion is shared. Commonalities (and differences) in which pathways in animal and plant hosts are targeted by human pathogens are highlighted in Brunner and Fraiture (2014), showing where there is evolutionary conservation in the cellular hubs. Detailed examination of the function for one of the T3S effectors from S. enterica, SpvC, shows that it targets a signaling pathway that is shared in plant and animal hosts (Neumann et al., 2014). This effector is a phosphothreonine lyase able to de-phosphorylate activated MAP kinases, therefore attenuating the immune response in Arabidopsis. n nExamination of mechanisms of colonization shows that although some aspects are broadly the same, there are important differences in the interaction between S. enterica and either plant or animal hosts (Wiedemann et al., 2015). To better understand the molecular basis of the interaction, it is necessary to determine the protein-protein interactions (PPIs) between the pathogen and the plant host. An important comparison of PPIs between Salmonella and Arabidopsis proteins to known interactions between bacterial and animal proteins was presented in Schleker et al. (2015). In order to characterize the interactions on a global scale, an approach was used that combines several new as well as previously described algorithms predicting new PPIs (Kshirsagar et al., 2015). However, while this approach yields candidates worthy of further investigation, it also serves to highlight the paucity of reported functional data. To further explore the molecular interactions, methods must be developed that are appropriate to the bacteria-plant system, for example for the analysis of bacterial gene expression. An improved method for the extraction of RNA from mixed samples was presented in Holmes et al. (2014), which allows the analysis of expression patterns in bacteria and the plant host at the same time. n nColonization of plant hosts by human pathogens rarely, if ever, occurs in isolation and successful colonization is dependent on the ability of the bacteria to compete with the native microflora. Microbiome approaches show that crops not obviously associated with human pathogens may harbor potential pathogens. As shown with an example of grapevines, crop plants can support the endophytic growth of human pathogen, in this case Propionibacterium acnes, which probably arose from human contamination in the first place (Yousaf et al., 2014). Application of bulky organic fertilizers, such as manure, to crops may increase the chances of transmission of bacteria into the food chain. However, the potential for transmission clearly shows some specificity, not least from differences between species and sub-species of bacteria and the host plants (Hofmann et al., 2014). A combination of axenic system with susceptible plants (e.g., spinach) revealed that 400 bacteria in one ml are sufficient to successfully colonize the crop plant. n nThis collection of articles helps to highlight the underpinning mechanisms, but also shows the complexity of interactions between human pathogenic bacteria and plant hosts. It is becoming clear that it is not possible to apply a broad set of rules to these interactions: there is a great deal of specificity that depends on multiple factors. This is particularly important in the consideration of how to address issues related to food safety, for practices applied at the pre-harvest stage or for prevention of transmission and contamination during post-harvest processing.