Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

Penicillium expansum is a necrotrophic wound fungal pathogen that secrets virulence factors to kill host cells including cell wall degrading enzymes (CWDEs), proteases, and mycotoxins such as patulin. During the interaction between P. expansum and its fruit host, these virulence factors are strictly modulated by intrinsic regulators and extrinsic environmental factors. In recent years, there has been a rapid increase in research on the molecular mechanisms of pathogenicity in P. expansum; however, less is known regarding the bacteria–fungal communication in the fruit environment that may affect pathogenicity. Many bacterial species use quorum-sensing (QS), a population density-dependent regulatory mechanism, to modulate the secretion of quorum-sensing signaling molecules (QSMs) as a method to control pathogenicity. N-acyl homoserine lactones (AHLs) are Gram-negative QSMs. Therefore, QS is considered an antivirulence target, and enzymes degrading these QSMs, named quorum-quenching enzymes, have potential antimicrobial properties. Here, we demonstrate that a bacterial AHL lactonase can also efficiently degrade a fungal mycotoxin. The mycotoxin is a lactone, patulin secreted by fungi such as P. expansum. The bacterial lactonase hydrolyzed patulin at high catalytic efficiency, with a kcat value of 0.724 ± 0.077 s−1 and KM value of 116 ± 33.98 μM. The calculated specific activity (kcat/KM) showed a value of 6.21 × 103 s−1M−1. While the incubation of P. expansum spores with the purified lactonase did not inhibit spore germination, it inhibited colonization by the pathogen in apples. Furthermore, adding the purified enzyme to P. expansum culture before infecting apples resulted in reduced expression of genes involved in patulin biosynthesis and fungal cell wall biosynthesis. Some AHL-secreting bacteria also express AHL lactonase. Here, phylogenetic and structural analysis was used to identify putative lactonase in P. expansum. Furthermore, following recombinant expression and purification of the newly identified fungal enzyme, its activity with patulin was verified. These results indicate a possible role for patulin and lactonases in inter-kingdom communication between fungi and bacteria involved in fungal colonization and antagonism and suggest that QQ lactonases can be used as potential antifungal post-harvest treatment.

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Multiple Xanthomonas euvesicatoria Type III Effectors Inhibit flg22-Triggered Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-16-0137-r ◽

2016 ◽

Vol 29 (8) ◽

pp. 651-660 ◽

Cited By ~ 17

Author(s):

Georgy Popov ◽

Malou Fraiture ◽

Frederic Brunner ◽

Guido Sessa

Keyword(s):

Pseudomonas Syringae ◽

Map Kinases ◽

Molecular Mechanisms ◽

Inducible Promoter ◽

Effector Proteins ◽

Host Cells ◽

In Planta ◽

Callose Deposition ◽

Type Iii ◽

Xanthomonas Euvesicatoria

Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast–based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid–inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.

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Proteoglycans in host–pathogen interactions: molecular mechanisms and therapeutic implications

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399409001367 ◽

2010 ◽

Vol 12 ◽

Cited By ~ 62

Author(s):

Allison H. Bartlett ◽

Pyong Woo Park

Keyword(s):

Cell Surface ◽

Virulence Factors ◽

Molecular Mechanisms ◽

Biological Significance ◽

Host Cells ◽

Microbial Pathogens ◽

Therapeutic Implications ◽

Core Proteins

Many microbial pathogens subvert proteoglycans for their adhesion to host tissues, invasion of host cells, infection of neighbouring cells, dissemination into the systemic circulation, and evasion of host defence mechanisms. Where studied, specific virulence factors mediate these proteoglycan–pathogen interactions, which are thus thought to affect the onset, progression and outcome of infection. Proteoglycans are composites of glycosaminoglycan (GAG) chains attached covalently to specific core proteins. Proteoglycans are expressed ubiquitously on the cell surface, in intracellular compartments, and in the extracellular matrix. GAGs mediate the majority of ligand-binding activities of proteoglycans, and many microbial pathogens elaborate cell-surface and secreted factors that interact with GAGs. Some pathogens also modulate the expression and function of proteoglycans through known virulence factors. Several GAG-binding pathogens can no longer attach to and invade host cells whose GAG expression has been reduced by mutagenesis or enzymatic treatment. Furthermore, GAG antagonists have been shown to inhibit microbial attachment and host cell entry in vitro and reduce virulence in vivo. Together, these observations underscore the biological significance of proteoglycan–pathogen interactions in infectious diseases.

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Characterisation of the Bartonella quintana YopJ Homologue

10.26686/wgtn.16830742 ◽

2021 ◽

Author(s):

◽

Alvey Little

Keyword(s):

Immune Modulation ◽

Effector Proteins ◽

Host Protein ◽

Host Cells ◽

Host Immune System ◽

Bartonella Quintana ◽

Type Iv ◽

Yersinia Species ◽

System A ◽

The Many

Bartonella is a genus of re-emerging bacterial pathogens that typically cause asymptomatic, intra-erythrocytic bacteraemia in their reservoir hosts and are highly specialised to evade host immunity. One of the many mechanisms by which Bartonella spp. modulate the host immune system is the type-IV-secretion system, a protein complex that delivers effector proteins directly into host cells to modulate their function. Some Bartonella species, including B. quintana, the causative agent of trench fever, possess an effector protein that is homologous to the effector YopJ of Yersinia species. Yersinia YopJ inhibits the MAPK and NF-kB pathways, and YopJ homologues in other species have similar effects, though through different targets. Very little is known about the function of the B. quintana YopJ homologue, but it may play a role in immune modulation by the bacteria. My aim was to characterise the function of the B. quintana YopJ homologue. I had evidence that it inhibits the NF-kB pathway, so I investigated which step of signalling activation is the target of this inhibition. I also sought to determine whether it impacts signalling pathways other than NF-kB, and to identify the specific host protein that it targets. I performed these investigations using ELISA, high-throughput fluorescence microscopy, Western blotting, LC/MS proteomic screening, and a yeast two-hybrid screen. I found that the B. quintana YopJ homologue inhibits the NF-kB pathway at or upstream of IKK activity, may also impact JNK signalling, the cell cycle, and the mTOR complex, and interacts with the host protein DCNL1, a component of neddylation machinery. Additionally, I determined that this interaction does not impact the neddylation of the protein Cullin-1.

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Characterisation of the Bartonella quintana YopJ Homologue

10.26686/wgtn.16830742.v1 ◽

2021 ◽

Author(s):

◽

Alvey Little

Keyword(s):

Immune Modulation ◽

Effector Proteins ◽

Host Protein ◽

Host Cells ◽

Host Immune System ◽

Bartonella Quintana ◽

Type Iv ◽

Yersinia Species ◽

System A ◽

The Many

Bartonella is a genus of re-emerging bacterial pathogens that typically cause asymptomatic, intra-erythrocytic bacteraemia in their reservoir hosts and are highly specialised to evade host immunity. One of the many mechanisms by which Bartonella spp. modulate the host immune system is the type-IV-secretion system, a protein complex that delivers effector proteins directly into host cells to modulate their function. Some Bartonella species, including B. quintana, the causative agent of trench fever, possess an effector protein that is homologous to the effector YopJ of Yersinia species. Yersinia YopJ inhibits the MAPK and NF-kB pathways, and YopJ homologues in other species have similar effects, though through different targets. Very little is known about the function of the B. quintana YopJ homologue, but it may play a role in immune modulation by the bacteria. My aim was to characterise the function of the B. quintana YopJ homologue. I had evidence that it inhibits the NF-kB pathway, so I investigated which step of signalling activation is the target of this inhibition. I also sought to determine whether it impacts signalling pathways other than NF-kB, and to identify the specific host protein that it targets. I performed these investigations using ELISA, high-throughput fluorescence microscopy, Western blotting, LC/MS proteomic screening, and a yeast two-hybrid screen. I found that the B. quintana YopJ homologue inhibits the NF-kB pathway at or upstream of IKK activity, may also impact JNK signalling, the cell cycle, and the mTOR complex, and interacts with the host protein DCNL1, a component of neddylation machinery. Additionally, I determined that this interaction does not impact the neddylation of the protein Cullin-1.

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YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00032-16 ◽

2016 ◽

Vol 80 (4) ◽

pp. 1011-1027 ◽

Cited By ~ 34

Author(s):

Ka-Wai Ma ◽

Wenbo Ma

Keyword(s):

Plant Pathogens ◽

Molecular Mechanisms ◽

Bacterial Species ◽

Cysteine Proteases ◽

Catalytic Triad ◽

Effector Proteins ◽

Host Cells ◽

Target Proteins ◽

Acetyltransferase Activity ◽

Type Iii

SUMMARYGram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, theYersiniaouter protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed.

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Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02566-16 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 17

Author(s):

Ahalieyah Anantharajah ◽

Julien M. Buyck ◽

Charlotta Sundin ◽

Paul M. Tulkens ◽

Marie-Paule Mingeot-Leclercq ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factors ◽

Transcriptional Activation ◽

Yersinia Pseudotuberculosis ◽

Effector Proteins ◽

Inflammasome Activation ◽

Protective Effects ◽

Secretion Systems ◽

Content Type

ABSTRACT Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosa in vitro. Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa. Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa. Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

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Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module

Microorganisms ◽

10.3390/microorganisms9081645 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1645

Author(s):

Tilman Schirmer ◽

Tjaart A. P. de Beer ◽

Stefanie Tamegger ◽

Alexander Harms ◽

Nikolaus Dietz ◽

...

Keyword(s):

Structural Changes ◽

Molecular Mechanisms ◽

Phylogenetic Analyses ◽

Substrate Binding ◽

Binding Pocket ◽

Effector Proteins ◽

Host Cells ◽

Cellular Functions ◽

X Ray Crystallography ◽

Evolutionary Diversification

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

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The TIR-domain containing effectors BtpA and BtpB from Brucella abortus block energy metabolism

10.1101/703330 ◽

2019 ◽

Cited By ~ 1

Author(s):

Julia María Coronas-Serna ◽

Arthur Louche ◽

María Rodríguez-Escudero ◽

Morgane Roussin ◽

Paul R.C. Imbert ◽

...

Keyword(s):

Energy Metabolism ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Cell Model ◽

Interleukin 1 ◽

Effector Proteins ◽

Host Cells ◽

Yeast Cells ◽

Tir Domain ◽

Tir Domains

ABSTRACTBrucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

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Role of the Salmonella Pathogenicity Island 1 Effector Proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica Subspecies 1 Serovar Typhimurium Colitis in Streptomycin-Pretreated Mice

Infection and Immunity ◽

10.1128/iai.72.2.795-809.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 795-809 ◽

Cited By ~ 164

Author(s):

Siegfried Hapfelmeier ◽

Kristin Ehrbar ◽

Bärbel Stecher ◽

Manja Barthel ◽

Marcus Kremer ◽

...

Keyword(s):

Salmonella Enterica ◽

Molecular Mechanisms ◽

Intestinal Inflammation ◽

Pathogenicity Island ◽

Effector Proteins ◽

Host Cells ◽

Starting Point ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.

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