Tyrosine-phosphorylated bacterial effector proteins: the enemies within

The tyrosine phosphorylation of proteins has a central role during signal transduction in eukaryotes. Recent progress shows that tyrosine phosphorylation is also a common feature of several effector proteins translocated by bacterial type III and type IV secretion systems. The involvement of these secretion systems in disease development is exemplified by a variety of pathogenic processes: pedestal formation (Tir of EPEC and Citrobacter), cell scattering (CagA of Helicobacter), invasion (Tarp of Chlamydia) and possibly proinflammatory responses and cell proliferation (BepD-F of Bartonella). The discovery that different bacterial pathogens use this common strategy to subvert host-cell function suggests that more examples will soon emerge.     

Type III effector proteins: doppelgangers of bacterial virulence

Bacterial pathogens have co-evolved with their hosts in their ongoing quest for advantage in the resulting interaction. These intimate associations have resulted in remarkable adaptations of prokaryotic virulence proteins and their eukaryotic molecular targets. An important strategy used by microbial pathogens of animals to manipulate host cellular functions is structural mimicry of eukaryotic proteins. Recent evidence demonstrates that plant pathogens also use structural mimicry of host factors as a virulence strategy. Nearly all virulence proteins from phytopathogenic bacteria have eluded functional annotation on the basis of primary amino-acid sequence. Recent efforts to determine their three-dimensional structures are, however, revealing important clues about the mechanisms of bacterial virulence in plants.     

Biologically plausible learning in neural networks: a lesson from bacterial chemotaxis

Learning processes in the brain are usually associated with plastic changes made to optimize the strength of connections between neurons. Although many details related to biophysical mechanisms of synaptic plasticity have been discovered, it is unclear how the concurrent performance of adaptive modifications in a huge number of spatial locations is organized to minimize a given objective function. Since direct experimental observation of even a relatively small subset of such changes is not feasible, computational modeling is an indispensable investigation tool for solving this problem. However, the conventional method of error back-propagation (EBP) employed for optimizing synaptic weights in artificial neural networks is not biologically plausible. This study based on computational experiments demonstrated that such optimization can be performed rather efficiently using the same general method that bacteria employ for moving closer to an attractant or away from a repellent. With regard to neural network optimization, this method consists of regulating the probability of an abrupt change in the direction of synaptic weight modification according to the temporal gradient of the objective function. Neural networks utilizing this method (regulation of modification probability, RMP) can be viewed as analogous to swimming in the multidimensional space of their parameters in the flow of biochemical agents carrying information about the optimality criterion. The efficiency of RMP is comparable to that of EBP, while RMP has several important advantages. Since the biological plausibility of RMP is beyond a reasonable doubt, the RMP concept provides a constructive framework for the experimental analysis of learning in natural neural networks.     

Plant LysM proteins: modules mediating symbiosis and immunity

Microbial glycans, such as bacterial peptidoglycans, fungal chitin or rhizobacterial Nod factors (NFs), are important signatures for plant immune activation or for the establishment of beneficial symbioses. Plant lysin motif (LysM) domain proteins serve as modules mediating recognition of these different N-acetylglucosamine (GlcNAc)-containing ligands, suggesting that this class of proteins evolved from an ancient sensor for GlcNAc. During early plant evolution, these glycans probably served as immunogenic patterns activating LysM protein receptor-mediated plant immunity and stopping microbial infection. The biochemical potential of plant LysM proteins for sensing microbial GlcNAc-containing glycans has probably since favored the evolution of receptors facilitating microbial infection and symbiosis.     

Ubiquitin-regulating effector proteins from Legionella

Ubiquitin is relatively modest in size but involves almost entire cellular signaling pathways. The primary role of ubiquitin is maintaining cellular protein homeostasis. Ubiquitination regulates the fate of target proteins using the proteasome- or autophagy-mediated degradation of ubiquitinated substrates, which can be either intracellular or foreign proteins from invading pathogens. Legionella, a gram-negative intracellular pathogen, hinders the host-ubiquitin system by translocating hundreds of effector proteins into the host cell’s cytoplasm. In this review, we describe the current understanding of ubiquitin machinery from Legionella. We summarize structural and biochemical differences between the host-ubiquitin system and ubiquitin-related effectors of Legionella. Some of these effectors act much like canonical host-ubiquitin machinery, whereas others have distinctive structures and accomplish non-canonical ubiquitination via novel biochemical mechanisms.     

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram‐negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.     

Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins

Plant disease resistance (R) genes mediate specific recognition of pathogens via perception of cognate avirulence (avr) gene products. The numerous highly similar AvrBs3-like proteins from the bacterial genus Xanthomonas provide together with their corresponding R proteins a unique biological resource to dissect the molecular basis of recognition specificity. A central question in this context is if R proteins that mediate recognition of structurally similar Avr proteins are themselves functionally similar or rather dissimilar. The recent isolation of rice xa5, rice Xa27 and tomato Bs4, R genes that collectively mediate recognition of avrBs3-like genes, provides a first clue to the molecular mechanisms that plants employ to detect AvrBs3-like proteins. Their initial characterization suggests that these R proteins are structurally and functionally surprisingly diverge. This review summarizes the current knowledge on R-protein-mediated recognition of AvrBs3-like proteins and provides working models on how recognition is achieved at the molecular level.     

A bacterial type III effector targets plant vesicle-associated membrane proteins

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.     

Plant pathogenic bacterial type III effectors subdue host responses

Like animals, plants sense bacterial pathogens through surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NB-LRR) and trigger defense responses. Many plant-pathogenic bacteria secrete a large repertoire of effector proteins into host cells to modulate host responses, enabling successful infection and multiplication in plants. A number of these effector proteins target plant innate immunity signaling pathways, while others induce specific host genes to enhance plant susceptibility. Substantial progress has been made in the past two years concerning biochemical function of effectors and their host targets. These advances provide new insights into regulatory mechanisms of plant immunity and host-pathogen co-evolution.     

Plant stomata function in innate immunity against bacterial invasion

Microbial entry into host tissue is a critical first step in causing infection in animals and plants. In plants, it has been assumed that microscopic surface openings, such as stomata, serve as passive ports of bacterial entry during infection. Surprisingly, we found that stomatal closure is part of a plant innate immune response to restrict bacterial invasion. Stomatal guard cells of Arabidopsis perceive bacterial surface molecules, which requires the FLS2 receptor, production of nitric oxide, and the guard-cell-specific OST1 kinase. To circumvent this innate immune response, plant pathogenic bacteria have evolved specific virulence factors to effectively cause stomatal reopening as an important pathogenesis strategy. We provide evidence that supports a model in which stomata, as part of an integral innate immune system, act as a barrier against bacterial infection.     

Bartonella henselae: Subversion of vascular endothelial cell functions by translocated bacterial effector proteins

Bartonella henselae (Bh) is a worldwide distributed zoonotic pathogen. Depending on the immune status of the infected individual this bacterium can cause a wide spectrum of clinical manifestations, ranging from cat scratch disease (CSD) to bacillary angiomatosis (BA) and bacillary peliosis (BP). BA and BP are characterized by tumor-like lesions at the skin or in the inner organs, respectively. These structures display pathological sprouting of capillaries with enlarged and hyperproliferated vascular endothelial cells (ECs) that are frequently found in close association with bacteria. Here we review the cellular changes observed upon Bh infection of ECs in vitro and outline the role of the VirB type IV secretion system (T4SS) and its translocated effector proteins in the modulation of EC signalling cascades. The current model how this virulence system could contribute to the vasoproliferative activity of Bh is described.     

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.     

A functional genomic yeast screen to identify pathogenic bacterial proteins

Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor approximately 1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.     

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer‐membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co‐occur and are from neighboring gene in Salmonella Typhi‐occurrence of homologs of both STY3178 and STY3179 are identified in many Gram‐negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra‐cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra‐cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin.     

WASp identity theft by a bacterial effector

EspF(U), a protein secreted by pathogenic enterohaemorrhagic E. coli (EHEC), activates N-WASp/WASp to induce actin pedestal formation in host cells. Two recent papers in Nature show that EspF(U) exploits a WASp activation strategy so extreme that it may effectively sequester WASp, blinding it to both autoinhibition and cellular regulation.     

Probing the cellular effects of bacterial effector proteins with the Yersinia toolbox

The type 3 secretion system (T3SS) is a powerful bacterial nanomachine that is able to modify the host cellular immune defense in favor of the pathogen by injection of effector proteins. In this regard, cellular Rho GTPases such as Rac1, RhoA or Cdc42 are targeted by a large group of T3SS effectors by mimicking cellular guanine exchange factors or GTPase-activating proteins. However, functional analysis of one type of T3SS effector that is translocated by bacterial pathogens is challenging because the T3SS effector repertoire can comprise a large number of proteins with redundant or interfering functions. Therefore, we developed the Yersinia toolbox to either analyze singular effector proteins of Yersinia spp. or different bacterial species in the context of bacterial T3SS injection into cells. Here, we focus on the WxxxE guanine exchange factor mimetic proteins IpgB1, IpgB2 and Map, which activate Rac1, RhoA or Cdc42, respectively, as well as the Rho GTPase inactivators YopE (a GTPase-activating mimetic protein) and YopT (cysteine protease), to generate a toolbox module for Rho GTPase manipulation.     

NACHT-LRR proteins (NLRs) in bacterial infection and immunity

Eukaryotes have evolved systems to detect bacterial intrusion. Recognition of bacteria relies on the sensing of pathogen associated molecular patterns (PAMPs) by host pattern recognition molecules (PRMs), which include various families of leucine-rich repeat (LRR) bearing proteins in plants and animals. Detection of microbes often occurs outside the cell. Recent findings now indicate that mammals have also evolved strategies to recognize bacteria inside the cell via members of the NACHT-LRR protein family (NLRs). Here, we review the biology of these mammalian NLRs and the emerging view of their important, role not solely as PRMs but as signalling platforms and regulators of immunity.