T4SS Effector Protein Prediction with Deep Learning

Extensive research has been carried out on bacterial secretion systems, as they can pass effector proteins directly into the cytoplasm of host cells. The correct prediction of type IV protein effectors secreted by T4SS is important, since they are known to play a noteworthy role in various human pathogens. Studies on predicting T4SS effectors involve traditional machine learning algorithms. In this work we included a deep learning architecture, i.e., a Convolutional Neural Network (CNN), to predict IVA and IVB effectors. Three feature extraction methods were utilized to represent each protein as an image and these images fed the CNN as inputs in our proposed framework. Pseudo proteins were generated using ADASYN algorithm to overcome the imbalanced dataset problem. We demonstrated that our framework predicted all IVA effectors correctly. In addition, the sensitivity performance of 94.2% for IVB effector prediction exhibited our framework’s ability to discern the effectors in unidentified proteins.publishedVersio

Genetic rearrangements in <i>Pseudomonas amygdali</i> pathovar <i>aesculi </i>shape coronatine plasmids

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

Protein phosphorylation regulates a large variety of biological processes in all living cells. In pathogenic bacteria, the study of serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation has shed light on the course of infectious diseases, from adherence to host cells to pathogen virulence, replication, and persistence. Mass spectrometry (MS)-based phosphoproteomics has provided global maps of Ser/Thr/Tyr phosphosites in bacterial pathogens. Despite recent developments, a quantitative and dynamic view of phosphorylation events that occur during bacterial pathogenesis is currently lacking. Temporal, spatial, and subpopulation resolution of phosphorylation data is required to identify key regulatory nodes underlying bacterial pathogenesis. Herein, we discuss how technological improvements in sample handling, MS instrumentation, data processing, and machine learning should improve bacterial phosphoproteomic datasets and the information extracted from them. Such information is expected to significantly extend the current knowledge of Ser/Thr/Tyr phosphorylation in pathogenic bacteria and should ultimately contribute to the design of novel strategies to combat bacterial infections

Structural Analysis of Complex Bacterial Secretion Systems

From the moment Anton van Leeuwenhoek saw bacteria for the first time to the modern era of the ‘resolution revolution’ by the Nobel prize-winning method single particle cryo-electron microscopy (EM), advances in microscopy have led to visual observations that enhance our understanding of life at a wide range of scales, from millimeters to angstroms. The increasingly popular imaging technique cryo-electron tomography (cryo-ET) is uniquely suited for visualizing protein complexes and cellular features at high resolution in their native environment. This technology presents great potential for visualizing host-pathogen interactions. The work within this dissertation utilizes cutting-edge cryo-ET technologies to visualize different stages of bacterial infection and host defenses with an unprecedented level of detail. The first part of this dissertation focuses on determining the in situ structure of bacterial secretion machine. Legionella pneumophila (L. pneumophila) uses a sophisticated nanomachine called the type IV secretion system (T4SS) to inject bacterial substrates into target cells to achieve infection and survival inside hosts. Subtomogram averaging and 3D classification analysis of \u3e9000 nanomachines revealed structural intermediates, suggesting that the T4SS machine is built from the outer membrane towards the cytoplasm. We propose an ‘outside-inside’ model of T4SS machine assembly. Furthermore, focused refinement of the cytoplasmic ATPase complex, composed on DotO and DotB ATPases, revealed that binding of DotB to DotO creates conformational changes leading to opening of a secretion channel that crosses the inner membrane. The second part of this dissertation focuses on how a bacterial pathogen uses the secretion machine to mediate interactions with the host membrane and initiate direct secretion of substrates across the host membrane. Salmonella uses its type III secretion (T3SS) machine, the injectisome, to create intimate contact with the host membrane for substrate translocation to initiate infection. Subtomogram averaging of the contact site between the injectisome needle tip and membrane allowed the first direct visualization of the T3SS translocon embedded in the host membrane. This finding provides critical evidence supporting the long-postulated direct substrate secretion model. The final part of this dissertation focuses on the use of cryo-ET and cutting-edge auxiliary tools, such as cryo-correlative light and electron microscopy (CLEM) and cryo-focused ion beam (FIB) milling, to visualize bacterial pathogens inside host cells. Chapter 4 contains studies characterizing the developmental transitions of Coxiella burnetii (C. burnetii) inside host cells and describes how these transitions impact assembly of T4SS machines. Moreover, high-resolution tomograms of C. burnetii at different stages of developmental transitions revealed that rapid changes in cell size can be facilitated by preserving the inner membrane

Investigating the protein subcomplexes from a conjugative Type IV Secretion System

Type IV secretion system (T4SS) are versatile nanomachines that enable the efficient transport of substrates in bacteria. In general, they are formed from two major membrane embedded subassemblies: an outer membrane core complex (OMCC) and an inner membrane complex (IMC). The conjugative T4SS encoded by the F plasmid is of particular interest due to its clinical relevance as it facilitates the spread of antibiotic resistance amongst bacterial population. Despite its importance, atomic details of the F-T4SS structure and protein-protein interactions were rudimentary which in turn precludes thorough understanding of how conjugation is orchestrated. Therefore, this thesis aimed to improve knowledge on the F-T4SS by studying the structure of the F-OMCC and investigating other proteins the complex may interact with. After optimising the detergent solubilisation of the F-OMCC expressed from the pED208 F-like plasmid, and improving the purification of the complex, a cryo-EM dataset was collected. Using single particle analysis, the structure was solved with an overall resolution of 3.3 Å. The F-OMCC is formed from two concentric rings which have two distinct symmetries. The outer ring adopts 13-fold symmetry whereas the inner ring showed 17-fold symmetry, together they form a 2.1 MDa complex. The atomic models of TraB, TraK and TraV were built into the structure, and they revealed a unique stoichiometric arrangement. Interestingly, TraV and TraK proteins were found to adopt two different conformations within the outer ring. TraV and TraB were found to accommodate the symmetry mismatch by existing in both F-OMCC rings, and also appeared to confer flexibility. This makes the F-OMCC a dynamic complex which is likely to have important implications in the pilus and T4SS activity during conjugation. The interactions between the F-OMCC and other Tra/Trb proteins were also investigated to decipher how the concerted dynamics of the pilus may be connected to the complex. A potential interaction between F-OMCC and the proteins TraH and TraN was observed by pull-down assays. Furthermore, initial work on TraG found that it seems to assemble as a high order oligomer in solution. The results are reminiscent of a hexameric protein which may be functionally important. Together, the findings of this thesis reveal novel insights into the F-T4SS and its subassemblies. The approach used to purify the F-OMCC and study the interactions will act as the basis of future work on the F-T4SS and is directly applicable to the other protein complexes within the conjugative nanomachine.Open Acces

Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella species

Shining light on T6SS mode of action and function within single cells and bacterial communities

Bacteria are ubiquitously found in the environment and form the basis for all known ecosystems on our planet. Most bacterial cells reside in complex multi-species bacterial communities, which are often associated with a host, such as the human microbiota. These bacterial communities are shaped by cooperative and competitive interactions amongst their members. Like higher animals, bacteria also compete with their conspecifics for nutrients and space. This evolutionary arms race resulted in a diverse set of strategies for microbial competition. In particular, bacteria residing on solid surfaces can compete with their neighbors through the use of specialized nanomachines, called secretion systems, enabling the direct delivery of toxic effector molecules into by-standing target cells. The most commonly used weapon for contact-dependent antagonism is the bacterial Type VI secretion system (T6SS). The T6SS belongs to the family of contractile injection systems (CISs). All CISs are structurally and functionally related to contractile bacteriophages (e.g. phage T4) and translocate proteins into target cells by means of physical force, which is generated by rapid sheath contraction. This results in the ejection of the inner tube associated with a sharp tip and effector proteins at its end. Effector translocation leads ultimately to target cell death. Importantly, the T6SS is capable translocating effectors across broad ranges of biological membranes making it a powerful weapon in microbial warfare as well as potent virulence mechanism towards eukaryotic host cells. Our current understanding of T6SS mode of action is primarily based on the combination of structural biology and fluorescence live-cell microscopy studies. While in particular cryo-electron microscopy (cryo-EM) revealed the detailed architecture of the T6SS in situ and of isolated subassemblies, fluorescence live-cell microscopy uncovered the remarkable dynamics of T6SS biogenesis. However, a complete understanding of T6SS dynamics is hampered in standard fluorescent microscopy due to: (i) the spatial and temporal resolution limit, (ii) the inability to efficiently label secreted components of the machinery, (iii) the weak signals due to low protein abundance and rapid photobleaching, (iv) the difficulty to perform long-term co-incubation experiments as well as (v) the inability to precisely control spatial and chemical environment. My doctoral thesis aimed to overcome these limitations to allow novel insights into dynamics of the T6SSs of Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi. Specifically sheath assembly, initiation of sheath contraction, T6SS mediated protein translocation in to sister cells as well as strategies for prey cell inhibition were studied in this thesis. First, I studied sheath assembly in ampicillin induced V. cholerae spheroplasts. These enlarged cells assemble T6SS sheaths which are up to 10x longer as compared to rod shaped cells. This allowed us to photobleach an assembling sheath structure and demonstrate that new sheath subunits are added to the growing sheath polymer at the distal end opposite the baseplate. Importantly, this was the first direct observation made for any contractile machines studied to date. Moreover, I showed that unlike for all other CISs, T6SS sheath length is not regulated and correlates with cell size. In order to monitor protein translocation into target cells, we developed a T6SS dependent interbacterial protein complementation assay, enabling the indirect detection of translocated T6SS components into the cytosol of recipient cells. This allowed us to demonstrate that secreted T6SS components are exchanged among by-standing sister cells within minutes upon initial cell contact. Importantly, these results were the first experimental indication that T6SS is capable of translocating its components into the cytosol of Gram-negative target cells. Furthermore, we showed that the amount and the composition of the secreted tip influences the number of T6SS assemblies per cell, whereas different concentration of the tube protein influenced sheath length. We also provided evidence that precise aiming of T6SS assemblies through posttranslational regulation in P. aeruginosa increases the efficiency of substrate delivery. In addition, together with two Nanoscience master students we have also been implementing microfluidics in the Basler laboratory. This powerful technology enabled us to control the spatial arrangements of aggressor and prey populations and to follow these populations at single-cell level over time scales of several hours. In collaboration with Prof. Kevin Forster, University of Oxford, we demonstrated that the rate of target cell lysis heavily influences the outcome of contact-dependent T6SS killing and thus drives evolution of lytic effectors. Moreover, microfluidics allows for the dynamic change of the chemical microenvironment during imaging experiments. By following the T6SS dynamics in response to hyperosmotic shocks resulting in a rapid cell volume reduction, we found that physical pressure from the collapsing cell envelope could trigger sheath contraction. This led us to propose a model for sheath contraction under steady-state conditions where continued sheath polymerization against membrane contact site leads to a gradual increase in pressure applied to the assembled sheath. We propose that this could be potentially sensed by the baseplate, which in turn would trigger sheath contraction