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The aim of this dissertation is to enhance our fundamental understanding of host-pathogens interactions. I investigated these interactions by studying the relationship between the innate immune response and Yersinia pseudotuberculosis. Yersinia is a genus of Gram-negative bacteria that employ a type III secretion system (T3SS), an important virulence factor for Yersinia pathogenesis. The T3SS forms a pore in the membrane of host cells and injects Yersinia effector proteins into the host cell cytoplasm to impair the host immune response. YopD is a T3SS translocator protein involved in the post-transcriptional regulation of yop synthesis and pore formation in host cell membranes, both of which are directly linked to the effective translocation of effector proteins into the host cell cytosol. YopD is also engaged in interactions with several proteins including its chaperone LcrH/SycD, its fellow translocator proteins YopB and LcrV, the regulatory protein TyeA, the effector protein YopE, and it can even self-oligomerize. As YopD is a central player in key T3SS functions and associated protein interactions, there are many important questions that can be addressed through careful study of the YopD protein. The first part of this dissertation utilizes YopD as a tool to understand how host cells detect T3SSs during Yersinia infection. Different models have been proposed to address how the host cell senses the presence of T3SSs. Some research indicates that the formation of a pore and subsequent K+ efflux itself leads to the induction of an immune response. A competing hypothesis is that pore formation alone is insufficient and that the translocation of T3SS cargo is required for the induction of this immune response. We constructed a Y. pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopDdeltaTM) that formed pores in host cells but did not translocate T3SS cargo. Interestingly, this YopDdeltaTM mutant failed to induce significant production of pro-inflammatory cytokines, suggesting that host T3SS sensing requires either a translocation mediated event or the presence of a WT translocon.The second part of my dissertation focuses on how YopD is required for the optimal performance of the T3SS weaponry. While the YopD amino and carboxy termini participate in pore formation and T3SS regulation, the role of the central region between amino acids 150-227 remains unknown. I assessed the contribution of the YopD central region by generating Y. pseudotuberculosis YopDdelta150-170 and YopDdelta207-227 mutants and analyzing their T3SS functions. These mutants formed robust pores in macrophages and displayed wildtype levels of Yop secretion in vitro. However, both mutants exhibited defects when translocating into neutrophils and macrophages, could not prevent phagocyte ROS production, and displayed a virulence defect in disseminated Yersinia infection in vivo. These findings suggest that the YopD central region facilitates optimal T3SS effector protein delivery into target host cells to effectively disarm innate immune defenses such as ROS production. Together these findings enhance our understanding of host-pathogen interactions, both in terms of how T3SSs are detected by the host and how specific components contribute to the optimal performance of T3SS function.

Bacterial canker disease has become the largest threat to kiwifruit cultivation and production. A monomorphic subpopulation of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) is responsible for the pandemic worldwide. Diversity in pathogenicity has been found in the pandemic subpopulation and in other Psa3 subpopulations causing epidemics in China. However, the genetic bases have not yet been elucidated. In this study, 117 Psa3 isolates were identified by Psa- and Psa3-specific primers, and evaluated for pathogenicity. Three isolates G4, G40, and S2 are not pathogenic to kiwifruit and do not elicit hypersensitivity responses (HRs) in non-host Nicotiana benthamiana leaves. Two isolates, G25 and G35, exhibited attenuated HR-eliciting activity in non-host N. benthamiana, but they exhibited greatly and slightly reduced pathogenicity in host plants, respectively. The genomes of the five isolates were sequenced and compared with closely related isolates revealed by MLVA and whole-genome typing methods. The candidate genetic loci responsible for the changes in pathogenicity and HR elicitation, were further evaluated by allele replacement experiments. We found that the three non-pathogenic isolates were formed due to the independent, identical insertion events of ISPsy36 transposon in the hrpR gene, encoding a key regulator of type III secretion system (T3SS) and type III effectors (T3Es). In the symptomatic sample from which G4 was isolated, 27% HR negative isolates were detected. In isolate G25, transposon insertion of ISPsy32 at the non-coding sequence upstream of the hrpR gene was detected, similar to a previously reported low-virulent Psa3 strain M227. In isolate G35, we detected disruptions of T3Es hopBB1-1 and hopBB1-2, which induce HR in N. benthamiana leaves revealed by Agrobacterium tumefaciens infiltration. These phenotype-changed isolates were formed at low frequencies during the course of pathogen infection in host plants, supported by the binding assay of ISPsy32 and the non-coding DNA sequences upstream of the hrpR gene, the co-isolation of the virulent isolates belonging to the same MLVA clade, and the low levels of transcription of the transposon genes. Taken together, in terms of short-term field evolution, transposon insertions in the T3SS-related genes resulted in the formation of non-pathogenic and low-virulent Psa3 isolates.

The post-transcriptional regulator RsmA globally controls gene expression in bacteria. Previous studies showed that RsmA2 and RsmA3 played critical roles in regulating type III secretion system (T3SS), motility, syringafactin, and alginate productions in Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000). In this study, we investigated global gene expression profiles of the wild-type PstDC3000, the rsmA3 mutant, and the rsmA2/A3 double mutant in the hrp-inducing minimum medium (HMM) and King’s B (KB) medium. By comparing the rsmA2/A3 and rsmA3 mutants to PstDC3000, a total of 1358 and 1074 differentially expressed genes (DEGs) in HMM, and 870 and 1463 DEGs in KB were uncovered, respectively. When comparing the rsmA2/A3 mutant with the rsmA3 mutant, 277 and 741 DEGs in HMM and KB, respectively, were revealed. Transcriptomic analysis revealed that the rsmY, rsmZ, and rsmX1-5 non-coding small RNAs (ncsRNAs) were positively affected by RsmA2 and RsmA3, while RsmA3 positively regulates the expression of the rsmA2 gene and negatively regulates both rsmA1 and rsmA5 gene expression. Comparative transcriptomic analysis showed that RsmA2 and RsmA3 synergistically influenced the expression of genes involved in T3SS and alginate biosynthesis in HMM and chemotaxis in KB. RsmA2 and RsmA3 inversely affected genes involved in syringafactin production in HMM and ribosomal protein biosynthesis in KB. In addition, RsmA2 played a major role in influencing genes involved in sarcosine and thiamine biosynthesis in HMM and in mannitol and phosphate metabolism in KB. On the other hand, genes involved in fatty acid metabolism, cellulose biosynthesis, signal transduction, and stress responses were mainly impacted by RsmA3 in both HMM and KB; whereas RsmA3 played a major role in controlling genes involved in c-di-GMP, phosphate metabolism, chemotaxis, and capsular polysaccharide in HMM. Furthermore, regulation of syringafactin production and oxidative stress by RsmA2 and RsmA3 was experimentally verified. Our results suggested the potential interplay among the RsmA proteins, which exhibit distinct and overlapping roles in modulating virulence and survival in P. syringae under different nutritional conditions.

Background Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer. Results We dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements. Conclusions We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

Vibrio parahaemolyticus is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of V. parahaemolyticus to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans., Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion system 1 of the marine pathogen Vibrio parahaemolyticus utilizes the sequential action of four effectors to induce a rapid, proinflammatory cell death uniquely characterized by a prosurvival host transcriptional response (D. L. Burdette, M. L. Yarbrough, A Orvedahl, C. J. Gilpin, and K. Orth, Proc Natl Acad Sci USA 105:12497–12502, 2008, https://doi.org/10.1073/pnas.0802773105; N. J. De Nisco, M. Kanchwala, P. Li, J. Fernandez, C. Xing, and K. Orth, Sci Signal 10:eaa14501, 2017, https://doi.org/10.1126/scisignal.aal4501). Herein, we show that this prosurvival response is caused by the action of the channel-forming effector VopQ that targets the host V-ATPase, resulting in lysosomal deacidification and inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can interact with VopQ, causing a disruption in membrane integrity. Additionally, we observed that VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein response (UPR), resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and reveals new connections between V-ATPase function, UPR, and MAPK signaling. IMPORTANCE Vibrio parahaemolyticus is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of V. parahaemolyticus to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans. Here, we found that first system targets evolutionarily conserved signaling systems to manipulate host cells, eventually causing a rapid, orchestrated cells death within 3 h. We have found that the T3SS1 injects virulence factors that temporally manipulate host signaling. Within the first hour of infection, the effector VopQ acts first by activating host survival signals while diminishing the host cell apoptotic machinery. Less than an hour later, another effector, VopS, reverses activation and inhibition of these signaling systems, ultimately leading to death of the host cell. This work provides example of how pathogens have evolved to manipulate the interplay between T3SS effectors to regulate host signaling pathways.

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.

Shigella comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention.

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

The species Pseudomonas savastanoi, a member of the Pseudomonas syringae complex, includes five pathovars causing knots or excrescences in woody hosts: P. savastanoi pv. savastanoi, pv. fraxini, pv. nerii, pv. retacarpa (Psr) and pv. mandevillae, comprising isolates from olive, ash, oleander, broom and dipladenia plants, respectively. Comparative genomic analysis of all available P. savastanoi genomes included in these five pathovars defined a total of 45 type III secretion system effector (T3E) genes, including 24 core genes, four genes exclusive of Psr and several genes encoding pathovar-specific truncations. Noticeably, hierarchical clustering based on the presence, absence and truncations of T3E genes, correlated with the core genome phylogeny of these strains, indicating that the five pathovars contain characteristic sets of T3Es, which are thus likely contributing to define their pathogenicity profile. To unravel de role of T3Es in the host specificity of P. savastanoi pathovars we followed two different approaches: i) construction of knock out mutants affected in T3E genes exclusively encoded by specific pathovars and, ii) heterologous expression of T3E genes in strains belonging to pathovars lacking these T3Es or encoding specific truncations. Virulence assays of these strains in their natural hosts and in hosts infected by other P. savastanoi pathovars, allowed us to identify several T3Es with a role in the host specificity of this bacterial pathogen of woody hosts. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

The ability ofSalmonellato survive and replicate within mammalian host cells involves the generation of a membranous compartment known as theSalmonella-containing vacuole (SCV).Salmonellaemploys a number of effector proteins that are injected into host cells for SCV formation using its type-three secretion systems encoded in SPI-1 and SPI-2 (T3SSSPI-1and T3SSSPI-2, respectively). Recently, we reported thatS. Typhimurium requires T3SSSPI-1and T3SSSPI-2to survive in the model amoebaDictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two majorS. Typhimurium effectors SopB and SifA duringD. discoideumintracellular niche formation. First, we established thatS. Typhimurium resides in a vacuolar compartment withinD. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopBand ΔsifAmutant strains ofS. Typhimurium, and we characterized the composition of this compartment by quantitative proteomics. This comparative analysis suggests thatS. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche inD. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival ofS. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed bySalmonellato survive intracellularly in phagocytic amoebae.ImportanceThe molecular mechanisms involved inSalmonellasurvival to predation by phagocytic amoebae, such asD. discoideum, remains poorly understood. Although we established thatS. Typhimurium requires two specialized type-three secretion systems to survive inD. discoideum, no effector protein has been implicated in this process so far. Here, we confirmed the presence of a membrane-bound compartment containingS. Typhimurium inD. discoideum, and purified theD. discoideumSCV to characterize the associated proteome. In doing so, we established a key role for effector proteins SopB and SifA in remodeling the protein content of the SCV that ultimately allow the intracellular survival ofS. Typhimurium inD. discoideum. We also discuss similarities and differences with the proteomes of the human SCV. These findings contribute to unravel the mechanisms used bySalmonellato survive in the environment exploiting phagocytic amoebae as a reservoir.

[Background]: Ralstonia solanacearum is the causal agent of bacterial wilt, a devastating plant disease responsible for serious economic losses especially on potato, tomato, and other solanaceous plant species in temperate countries. In R. solanacearum, gene expression analysis has been key to unravel many virulence determinants as well as their regulatory networks. However, most of these assays have been performed using either bacteria grown in minimal medium or in planta, after symptom onset, which occurs at late stages of colonization. Thus, little is known about the genetic program that coordinates virulence gene expression and metabolic adaptation along the different stages of plant infection by R. solanacearum., [Results]: We performed an RNA-sequencing analysis of the transcriptome of bacteria recovered from potato apoplast and from the xylem of asymptomatic or wilted potato plants, which correspond to three different conditions (Apoplast, Early and Late xylem). Our results show dynamic expression of metabolism-controlling genes and virulence factors during parasitic growth inside the plant. Flagellar motility genes were especially up-regulated in the apoplast and twitching motility genes showed a more sustained expression in planta regardless of the condition. Xylem-induced genes included virulence genes, such as the type III secretion system (T3SS) and most of its related effectors and nitrogen utilisation genes. The upstream regulators of the T3SS were exclusively up-regulated in the apoplast, preceding the induction of their downstream targets. Finally, a large subset of genes involved in central metabolism was exclusively down-regulated in the xylem at late infection stages., [Conclusions]: This is the first report describing R. solanacearum dynamic transcriptional changes within the plant during infection. Our data define four main genetic programmes that define gene pathogen physiology during plant colonisation. The described expression of virulence genes, which might reflect bacterial states in different infection stages, provides key information on the R. solanacearum potato infection process., This work was funded by project PID2019-108595RB-I00 from the Spanish Ministry of Economy and Competitiveness. We also acknowledge financial support from the “Severo Ochoa Program for Centres of Excellence in R&D” 2016–2019 (SEV-2015-0533) and the CERCA Program of the Catalan Government (Generalitat de Catalunya). R. de Pedro-Jové received FI and FPU fellowships from Generalitat de Catalunya and the Spanish Ministry of Economy and Competitiveness. P. Sebastià received the support of a fellowship (code is LCF/BQ/IN17/11620004) from la Caixa Foundation (identifier [ID] 100010434). This project has received funding from the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 713673. M. Puigvert received an APIF doctoral fellowship from Universitat de Barcelona and travel fellowship funded by Fundació Montcelimar and Universitat de Barcelona to carry out a short stay in J.C. Setubal’s lab. J.C. Setubal holds a CNPq senior researcher fellowship. J.S. Monteiro is funded by a FAPESP doctoral fellowship under process number 2019/05287–2. A.P. Macho is funded by the Chinese Academy of Sciences and the Chinese 1000 Talents Program.

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells by translocating molecular toxins, called effector proteins. It resembles a molecular syringe, which establishes a cytosolic connection between bacteria and host cells. While the structure of the injectisome is well-defined, little is known about its molecular function and regulation. To investigate those, we focused on the adaptation of the injectisome to local pH as well as on the dynamics interactions of the cytosolic components with the effector proteins. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. We found that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. In Y. enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic injectisome components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the injectisome at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates injectisome activity in response to environmental conditions. In a next step, we study the binding of effector proteins to those dynamics of the cytosolic components, which also exchange between a free diffusing cytoplasmic and a injectisome bound state. An attempt to identify novel interaction partners of SctQ by co-IP did not yield in significantly new candidates and recent publications showed that protein dynamics are linked to the function of the injectisome. We analyzed the diffusion behavior of the cytosolic protein SctQ with single particle tracking photoactivated localization microscopy (sptPALM) in different strain backgrounds. Our data shows that SctQ diffuses in two populations and that the presence of effector proteins slows down that speed. This suggests that the cytosolic components are not only essential for secretion but participate actively in effector shuttling and recruitment to the injectisome., Viele bakterielle Pathogene nutzen ein Typ-III-Sekretionssystem (T3SS), um ihre Wirtszellen zu manipulieren, indem sie molekulare Toxine, sogenannte Effektor Proteine, translozieren. Es ähnelt einer molekularen Spritze, die eine direkte zytosolische Verbindung zwischen Bakterien und Wirtszellen herstellt. Während die Struktur des Injektisoms gut definiert ist, ist über seine molekulare Funktion und Regulation wenig bekannt. Um diese zu untersuchen, konzentrierten wir uns auf die Anpassung des Injektisoms an den lokalen pH-Wert sowie auf die dynamischen Interaktionen der zytosolischen Komponenten mit den Effektor Proteinen. Die Protein Sekretion durch das T3SS-Injektisom wird bei Kontakt mit einer beliebigen Wirtszelle aktiviert, und es war bisher unklar, wie eine vorzeitige Sekretion von Effektoren während einer Infektion verhindert wird. Wir fanden heraus, dass in den gastrointestinalen Pathogenen Y. enterocolitica und S. flexneri zytosolische Injektisome Komponenten bei niedrigem externem pH-Wert vorübergehend von der proximalen Schnittstelle des Injektisome dissoziieren, wodurch die Proteinsekretion in sauren Umgebungen, wie dem Magen, verhindert wird. In Yersinia enterocolitica führt ein niedriger externer pH-Wert im Periplasma zu einer teilweisen Dissoziation des inneren Membrane Ring des Injectisomes SctD, die wiederum hat die Dissoziation der zytosolischen Injektisom-Komponenten zufolge. Dieser Effekt wird bei der Wiederherstellung des neutralen pH-Wertes rückgängig gemacht, was eine schnelle Aktivierung des Injectisomes an den nativen Zielregionen im Wirt ermöglicht. Diese Ergebnisse deuten darauf hin, dass die zytosolischen Komponenten eine adaptive regulatorische Schnittstelle bilden, welche die Injectisome Aktivität in Abhängigkeit von den Umweltbedingungen reguliert. In einem nächsten Schritt untersuchen wir die Bindung von Effektorproteinen an die Dynamik der zytosolischen Komponenten. Diese wechseln zwischen einem frei diffundierenden zytoplasmatischen und einem an das Injektisom gebundenen Zustand. Der Versuch, neue Interaktionspartner von SctQ durch Co-IP zu identifizieren, ergab keine signifikant neuen Kandidaten, und neuere Publikationen zeigten, dass die Dynamik des Proteins mit der Funktion des Injektisoms verknüpft ist. Wir analysierten das Diffusionsverhalten des zytosolischen Proteins SctQ haben könnten, haben wir das Diffusionsverhalten des zytosolischen Proteins SctQ mit Single Particle Tracking Photoactivated Localization Microscopy (sptPALM) in verschiedenen Stammhintergründen analysiert. Unsere Daten zeigen, dass SctQ in zwei Populationen diffundiert und dass die Anwesenheit von Effektorproteinen diese Geschwindigkeit verlangsamt. Dies deutet darauf hin, dass die zytosolischen Komponenten nicht nur für die Sekretion essentiell sind, sondern auch aktiv am Effektor Transport und der Rekrutierung zum Injectisome teilnehmen. Insgesamt tragen unsere gesammelten Daten zum Wissen darüber bei, wie der Effektor Transport zum Injectisome und in die Wirtszelle reguliert wird und wie dieser überhaupt erst ermöglicht werden kann.

The two subspecies of Photobacterium damselae, namely subsp. damselae and subsp. piscicida, are pathogenic for diverse fish species of financial importance in marine aquaculture. In the present thesis we have characterized the variable genome in these two subspecies, which proved to be highly diverse. We have demonstrated that horizontal gene transfer has played a major role in the acquisition of the ability to metabolize sucrose, and in antimicrobial resistance in the subspecies damselae. Notably, this study has unveiled the existence of a novel, highly unstable virulence plasmid, encoding a Type III secretion system (T3SS) that is highly prevalent in the subspecies piscicida, and which constitutes a new virulence factor in this pathogen.

The marine bacterium Photobacterium damselae subsp. piscicida (Pdp) causes photobacteriosis in fish and important financial losses in aquaculture, but knowledge of its virulence factors is still scarce. We here demonstrate that an unstable plasmid (pPHDPT3) that encodes a type III secretion system (T3SS) is highly prevalent in Pdp strains from different geographical origins and fish host species. We found that pPHDPT3 undergoes curing upon in vitro cultivation, and this instability constitutes a generalized feature of pPHDPT3-like plasmids in Pdp strains. pPHDPT3 markers were detected in tissues of naturally-infected moribund fish and in the Pdp colonies grown directly from the fish tissues but were undetectable in a fraction of the colonies produced upon the first passage of the primeval colonies on agar plates. Notably, cured strains exhibited a marked reduction in virulence for fish, demonstrating that pPHDPT3 is a major virulence factor of Pdp. The attempts to stabilize pPHDPT3 by insertion of antibiotic resistance markers by allelic exchange caused an even greater reduction in virulence. We hypothesize that the existence of a high pressure to shed pPHDPT3 plasmid in vitro caused the selection of clones with off-target mutations and gene rearrangements during the process of genetic modification. Collectively, these results show that pPHDPT3 constitutes a novel, hitherto unreported virulence factor of Pdp that shows a high instability in vitro and warn that the picture of Pdp virulence genes has been historically underestimated, since the loss of the T3SS and other plasmid-borne genes may have occurred systematically in laboratories for decades.

The post-transcriptional regulator RsmA globally controls gene expression in bacteria. Previous studies showed that RsmA2 and RsmA3 played critical roles in regulating type III secretion system (T3SS), motility, syringafactin, and alginate productions in Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000). In this study, we investigated global gene expression profiles of the wild-type PstDC3000, the rsmA3 mutant, and the rsmA2/A3 double mutant in the hrp-inducing minimum medium (HMM) and King’s B (KB) medium. By comparing the rsmA2/A3 and rsmA3 mutants to PstDC3000, a total of 1358 and 1074 differentially expressed genes (DEGs) in HMM, and 870 and 1463 DEGs in KB were uncovered, respectively. When comparing the rsmA2/A3 mutant with the rsmA3 mutant, 277 and 741 DEGs in HMM and KB, respectively, were revealed. Transcriptomic analysis revealed that the rsmY, rsmZ, and rsmX1-5 non-coding small RNAs (ncsRNAs) were positively affected by RsmA2 and RsmA3, while RsmA3 positively regulates the expression of the rsmA2 gene and negatively regulates both rsmA1 and rsmA5 gene expression. Comparative transcriptomic analysis showed that RsmA2 and RsmA3 synergistically influenced the expression of genes involved in T3SS and alginate biosynthesis in HMM and chemotaxis in KB. RsmA2 and RsmA3 inversely affected genes involved in syringafactin production in HMM and ribosomal protein biosynthesis in KB. In addition, RsmA2 played a major role in influencing genes involved in sarcosine and thiamine biosynthesis in HMM and in mannitol and phosphate metabolism in KB. On the other hand, genes involved in fatty acid metabolism, cellulose biosynthesis, signal transduction, and stress responses were mainly impacted by RsmA3 in both HMM and KB; whereas RsmA3 played a major role in controlling genes involved in c-di-GMP, phosphate metabolism, chemotaxis, and capsular polysaccharide in HMM. Furthermore, regulation of syringafactin production and oxidative stress by RsmA2 and RsmA3 was experimentally verified. Our results suggested the potential interplay among the RsmA proteins, which exhibit distinct and overlapping roles in modulating virulence and survival in P. syringae under different nutritional conditions.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC50 of 1.8 ± 0.4 μM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC50 of 0.65 ± 0.09 μM.

Activation of the NLRP3 inflammasome requires the expression of NLRP3, which is strictly regulated by its capacity to directly recognize microbial-derived substances. Even though the involvement of caspase-1 activation in macrophages via NLRP3 and NLRC4 has been discovered, the accurate mechanisms by which Shigella infection triggers NLRP3 activation remain inadequately understood. Here, we demonstrate that IpaH4.5, a Shigella T3SS effector, triggers inflammasome activation by regulating NLRP3 expression through the E3 ubiquitin ligase activity of IpaH4.5. First, we found that IpaH4.5 interacted with NLRP3. As a result, IpaH4.5 modulated NLRP3 protein stability and inflammasome activation. Bacteria lacking IpaH4.5 had dramatically reduced ability to induce pyroptosis. Our results identify a previously unrecognized target of IpaH4.5 in the regulation of inflammasome signaling and clarify the molecular basis for the cytosolic response to the T3SS effector.

The type III secretion system (T3SS) consists of a syringe-like export machine injecting effectors from the bacterial cytosol directly into host cells to establish infection. This mechanism is widely distributed in gram-negative bacteria and can be targeted as an innovative strategy for the developing of anti-virulence drugs. In this study, we present an effective T3SS inhibitor, myricanol, inspired by the use of folk medicinal plants traditionally used against infections. Myricanol is a cyclic diarylheptanoid isolated from the medicinal plant Myrica nagi, which is found in South and East Asia. Bioassay-guided fractionation revealed that myricanol inhibited not only the secretion of type III effector proteins of Salmonella enterica serovar Typhimurium UK-1 χ8956 (S. Typhimurium) but also the invasion of S. Typhimurium into mammalian cells, but showed no toxicity to bacterial growth or the host cells. RNA-Seq data analysis showed that the transcription of the pathogenesis-related SPI-1 gene was significantly inhibited by myricanol. Further study demonstrated that myricanol binds physically to HilD and interferes with its DNA-binding activity to the promoters of the hilA and invF genes. In conclusion, we propose that myricanol is responsible for the anti-infectious properties of M. nagi and is a novel T3SS inhibitor of S. Typhimurium through a previously unappreciated mechanism of action.

Background For successful colonization, enterohaemorrhagic Escherichia coli (EHEC) injects virulence factors, called effectors, into target cells through the type three secretion system (T3SS), which is composed of a needle and basal body. Under anaerobic conditions, the T3SS machinery remains immature and does not have a needle structure. However, activation of nitrate respiration enhances the completion of the T3SS machinery. Because nitric oxide released by the host inflammatory response increases nitrate concentration, we sought to determine the effect of the inflammatory response on initiation of EHEC microcolony-formation. Results The colony-forming capacity was increased in accordance with the increase of nitrate in the medium. The addition of the nitric oxide-producing agent NOR-4 also enhanced the adherence capacity, which was dependent on nitrate reductase encoded by the narGHJI genes. Culture supernatant of epithelial cells, which was stimulated by a cytokine mixture, enhanced the colony-forming capacity of wild-type EHEC but not of the narGHJI mutant. Finally, colony formation by wild-type EHEC on epithelial cells, which were preincubated with heat-killed bacteria, was higher than the narGHJI mutant, and this effect was abolished by aminoguanidine hydrochloride, which is an iNOS (inducible nitric oxide synthase) inhibitor. Conclusions These results indicate that the inflammatory response enhances EHEC adherence by increasing nitrate concentration.

Diarrheal disease is caused by a diverse group of enteric pathogens and is one of the leading causes of preventable deaths worldwide. Some of the most common causes of diarrheal disease are bacterial pathogens such as Salmonella, E. coli, Cholera, and Shigella. Many bacterial enteric pathogens use a type three secretion system (T3SS) to cause infection. The T3SS uses a needle and syringe like apparatus that allows the bacteria to inject proteins into the host cell to cause infection. Striking similarities in T3SSs from all pathogens that utilize them makes the T3SS an attractive target for the development of non-antibiotic drugs to combat them. With its high global burden, emergence of multi drug resistant strains, and lack of vaccine, Shigella is a prime target for study of the T3SS and is the focus of this dissertation. In Shigella, the ATPase Spa47 resides at the base of the T3SS and is responsible for powering the system. Here, we explore how Shigella regulates Spa47 activity finding two proteins that can both increase and decrease Spa47 activity depending on the oligomeric state of Spa47. Additionally, we explore chemical inhibitors and identify several that efficiently inhibit Spa47 activity and Shigella’s ability to secrete proteins necessary to cause infection. Testing the Shigella ATPase inhibitors on T3SS ATPases from E. Coli and Salmonella showed similar levels of inhibition, demonstrating the potential for developing cross pathogen therapeutics against pathogens that use one or more T3SSs. Together, these findings advance the understanding of how Shigella regulate its T3SS and move us one step closer to developing broadly active therapeutics against an important class of human pathogens.

Type III secreted effector (T3SE) prediction remains a big computational challenge. In practical applications, current software tools often suffer problems of high false-positive rates. One of the causal factors could be the relatively unitary type of biological features used for the design and training of the models. In this research, we made a comprehensive survey on the sequence-based features of T3SEs, including signal sequences, chaperone-binding domains, effector domains, and transcription factor binding promoter sites, and assembled a unified prediction pipeline integrating multi-aspect biological features within homology-based and multiple machine learning models. To our knowledge, we have compiled the most comprehensive biological sequence feature analysis for T3SEs in this research. The T3SEpp pipeline integrating the variety of features and assembling different models showed high accuracy, which should facilitate more accurate identification of T3SEs in new and existing bacterial whole-genome sequences., Many Gram-negative bacteria infect hosts and cause diseases by translocating a variety of type III secreted effectors (T3SEs) into the host cell cytoplasm. However, despite a dramatic increase in the number of available whole-genome sequences, it remains challenging for accurate prediction of T3SEs. Traditional prediction models have focused on atypical sequence features buried in the N-terminal peptides of T3SEs, but unfortunately, these models have had high false-positive rates. In this research, we integrated promoter information along with characteristic protein features for signal regions, chaperone-binding domains, and effector domains for T3SE prediction. Machine learning algorithms, including deep learning, were adopted to predict the atypical features mainly buried in signal sequences of T3SEs, followed by development of a voting-based ensemble model integrating the individual prediction results. We assembled this into a unified T3SE prediction pipeline, T3SEpp, which integrated the results of individual modules, resulting in high accuracy (i.e., ∼0.94) and >1-fold reduction in the false-positive rate compared to that of state-of-the-art software tools. The T3SEpp pipeline and sequence features observed here will facilitate the accurate identification of new T3SEs, with numerous benefits for future studies on host-pathogen interactions. IMPORTANCE Type III secreted effector (T3SE) prediction remains a big computational challenge. In practical applications, current software tools often suffer problems of high false-positive rates. One of the causal factors could be the relatively unitary type of biological features used for the design and training of the models. In this research, we made a comprehensive survey on the sequence-based features of T3SEs, including signal sequences, chaperone-binding domains, effector domains, and transcription factor binding promoter sites, and assembled a unified prediction pipeline integrating multi-aspect biological features within homology-based and multiple machine learning models. To our knowledge, we have compiled the most comprehensive biological sequence feature analysis for T3SEs in this research. The T3SEpp pipeline integrating the variety of features and assembling different models showed high accuracy, which should facilitate more accurate identification of T3SEs in new and existing bacterial whole-genome sequences.

The type III secretion system (T3SS) consists of a syringe-like export machine injecting effectors from the bacterial cytosol directly into host cells to establish infection. This mechanism is widely distributed in gram-negative bacteria and can be targeted as an innovative strategy for the developing of anti-virulence drugs. In this study, we present an effective T3SS inhibitor, myricanol, inspired by the use of folk medicinal plants traditionally used against infections. Myricanol is a cyclic diarylheptanoid isolated from the medicinal plant Myrica nagi, which is found in South and East Asia. Bioassay-guided fractionation revealed that myricanol inhibited not only the secretion of type III effector proteins of Salmonella enterica serovar Typhimurium UK-1 χ8956 (S. Typhimurium) but also the invasion of S. Typhimurium into mammalian cells, but showed no toxicity to bacterial growth or the host cells. RNA-Seq data analysis showed that the transcription of the pathogenesis-related SPI-1 gene was significantly inhibited by myricanol. Further study demonstrated that myricanol binds physically to HilD and interferes with its DNA-binding activity to the promoters of the hilA and invF genes. In conclusion, we propose that myricanol is responsible for the anti-infectious properties of M. nagi and is a novel T3SS inhibitor of S. Typhimurium through a previously unappreciated mechanism of action.

Background The nucleotide second messengers, i.e., guanosine tetraphosphate and pentaphosphate [collectively referred to as (p) ppGpp], trigger the stringent response under nutrient starvation conditions and play an essential role in virulence in the fire blight pathogen Erwinia amylovora. Here, we present transcriptomic analyses to uncover the overall effect of (p) ppGpp-mediated stringent response in E. amylovora in the hrp-inducing minimal medium (HMM). Results In this study, we investigated the transcriptomic changes of the (p) ppGpp0 mutant under the type III secretion system (T3SS)-inducing condition using RNA-seq. A total of 1314 differentially expressed genes (DEGs) was uncovered, representing more than one third (36.8%) of all genes in the E. amylovora genome. Compared to the wild-type, the (p) ppGpp0 mutant showed down-regulation of genes involved in peptide ATP-binding cassette (ABC) transporters and virulence-related processes, including type III secretion system (T3SS), biofilm, and motility. Interestingly, in contrast to previous reports, the (p) ppGpp0 mutant showed up-regulation of amino acid biosynthesis genes, suggesting that it might be due to that these amino acid biosynthesis genes are indirectly regulated by (p) ppGpp in E. amylovora or represent specific culturing condition used. Furthermore, the (p) ppGpp0 mutant exhibited up-regulation of genes involved in translation, SOS response, DNA replication, chromosome segregation, as well as biosynthesis of nucleotide, fatty acid and lipid. Conclusion These findings suggested that in HMM environment, E. amylovora might use (p) ppGpp as a signal to activate virulence gene expression, and simultaneously mediate the balance between virulence and survival by negatively regulating DNA replication, translation, cell division, as well as biosynthesis of nucleotide, amino acid, fatty acid, and lipid. Therefore, (p) ppGpp could be a promising target for developing novel control measures to fight against this devastating disease of apples and pears.

Bradyrhizobium elkanii BLY3-8 does not form nodules on the roots of Rj3-genotype soybean (cultivar D-51). This is a cultivar-specific nodulation restriction. The genes A6X20_40975 and A6X20_41030 in strain BLY3-8 were predicted to encode the transcriptional activator and apparatus of the type III secretion system (T3SS) (the proteins TtsI and RhcJ), respectively. Mutants disrupted in these genes overcame the nodulation restriction. These results suggest that an effector injected via T3SS into Rj3-genotype soybean is involved in nodulation restriction by Rj3-genotype soybean.

Relative to peritrichous bacteria, polar flagellates possess regulatory systems that order flagellar gene transcription differently and produce flagella in specific numbers only at poles. How transcriptional and flagellar biogenesis regulatory systems are interlinked to promote the correct synthesis of polar flagella in diverse species has largely been unexplored. We found evidence for many Gram-negative polar flagellates encoding two-component signal transduction systems with activity linked to the formation of flagellar type III secretion systems to enable production of flagellar rod and hook proteins at a discrete, subsequent stage during flagellar assembly. This polar flagellar transcriptional program assists, in some manner, the FlhF/FlhG flagellar biogenesis regulatory system, which forms specific flagellation patterns in polar flagellates in maintaining flagellation and motility when activity of FlhF or FlhG might be altered. Our work provides insight into the multiple regulatory processes required for polar flagellation., Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, C ring, and flagellar type III secretion system (fT3SS) core proteins that form a competent fT3SS. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program have not been thoroughly assimilated. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation to possess flagellum-associated two-component signal transduction systems (TCSs). We present data supporting a general theme in polar flagellates whereby MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. We demonstrate that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCSs to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, we discovered a link between the polar flagellar transcription program and the activity of FlhF/FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. Our findings integrate flagellar transcriptional and biogenesis regulatory processes involved in polar flagellation in many species.

La especie Pseudomonas savastanoi está constituida por cuatro patovares que causan tumores o excrecencias en huéspedes leñosos: pv. savastanoi (Psv), pv. nerii, pv. fraxini y pv. retacarpa, constituidos por cepas aisladas de olivo, adelfa, fresno y retama, respectivamente. Además, recientemente se ha identificado a P. savastanoi como el agente causal de la necrosis bacteriana de la dipladenia (Mandevilla spp.), el cual se ha propuesto como un nuevo patovar, pv. mandevillae. La patogenicidad de P. savastanoi depende, entre otros factores, del sistema de secreción tipo III (T3SS) y de su repertorio de efectores (T3E), los cuales se han identificado como uno de los factores principales en la especificidad de huésped. La reciente disponibilidad de nuevas secuencias genómicas nos ha permitido realizar un estudio genómico comparativo de los genes que codifican el T3SS y sus T3E en 21 cepas de P. savastanoi dirigido a la identificación de posibles genes implicados en el rango de huésped. Estos análisis se han acompañado con pruebas de patogenicidad cruzada que nos han permitido correlacionar los resultados de los análisis genómicos con la especificidad de huésped de algunas de estas cepas. De los T3E identificados en este análisis, se ha realizado un estudio en mayor profundidad del papel en el rango de huésped de HopBL1, HopBL2, AvrPto1, AvrRps4, HopF1 y HopZ5. Además, hemos realizado un análisis RNAseq y una predicción bioinformática dirigida a la caracterización del regulón HrpL de la cepa modelo Psv NCCPB 3335, principal regulador del T3SS y sus T3E. En resumen, en esta Tesis Doctoral se ha demostrado que el T3SS es esencial para la patogénesis de todos los patovares de P. savastanoi aislados de huéspedes leñosos y que su repertorio de T3E se ha diversificado en el proceso de adaptación al huésped, siendo elementos determinantes en la definición de su rango de huésped.

Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...