IT’S THE LITTLE THINGS: AN EXPLORATION OF SMALL RNAS AND SELFISH GENETIC ELEMENTS OF THE HUMAN BACTERIAL PATHOGENS COXIELLA BURNETII AND BARTONELLA BACILLIFORMIS

Coxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever in humans. Previous work in our lab has demonstrated that C. burnetii codes for several small RNAs (sRNAs) that are differentially expressed between in vivo and in vitro growth conditions. sRNAs serve as post-transcriptional regulatory effectors involved in the control of nearly all biological processes. We demonstrated that several of the identified sRNAs, namely Coxiella burnetii small RNA 3 (CbsR3), Cbsr13, and CbsR16, represent members of two novel families of miniature inverted-repeat transposable elements (MITEs), termed QMITE1 and QMITE2. Furthermore, we have characterized a highly expressed, infection-specific sRNA, CbsR12, and have determined that it is necessary for expansion of the C. burnetii intracellular niche in a human monocyte-derived alveolar macrophage cell line. We have determined that CbsR12 may participate in broad gene regulation by acting as an RNA sponge for the global regulatory RNA-binding protein CsrA. Additionally, CbsR12 is a trans-acting sRNA that targets transcripts of the carA, metK, and cvpD genes in vitro and in vivo. Bartonella bacilliformis is a Gram-negative alphaproteobacterium and the etiological agent of Carrión\u27s disease in humans. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant environmental shifts during its life cycle, including changes in pH and temperature. Bacterial sRNAs can serve as a means of rapid regulation under shifting environmental conditions. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we verified the predicted molecular targeting of a sand fly-specific sRNA, Bartonella bacilliformis small RNA 9 (BbsR9), to transcripts of the ftsH, nuoF, and gcvT genes, in vitro

Exploring environmental adaptations and habitat preferences in three microbial lineages using comparative (meta)genomic approaches

The utilization of -omics based approaches (metagenomics, genomics, transcriptomics, proteomics, and metabolomics) in the field of microbiology has greatly advanced our understanding of the microbial world. The utilization of such approaches, either on pure cultures, or directly on environmental samples has provided novel insights into the role of microorganisms in earth biogeochemical cycles, microbial evolutionary dynamics, and their potential biotechnological applications. In the field of microbial pathogenesis, informatics-based methods have helped in uncovering several venues of pathogenesis including pathogens strain-specific characteristics, virulence genes, antimicrobial resistance, and understanding the landscape of various diseases.Here, I present my 3 research projects based on exploiting various -omics based approaches to understand the ecology, evolution, and pathogenic determinants of various groups of cultured, and yet- uncultured microorganisms. In chapter I, I implemented genome-resolved metagenomics to elucidate the ecological roles, metabolic capabilities, and physiological preferences of a novel yet-uncultured microbial phylum recovered from enrichments of tertiary oil reservoir. I showed that this lineage is a slow-growing member of rare biosphere and an aminolytic halothermophilic organism. We proposed creating a new candidate phylum "Mcinerneybacteriota" to accommodate this organism. This work has been published in the journal “Systematic and Applied Microbiology”. In chapter II, I analyzed multiple genome-resolved metagenomes of uncultured Group 18 Acidobacteria to understand their biogeochemical roles and elucidate the key evolutionary innovations that enable Acidobacteria to thrive in soil ecosystems. I demonstrated that soil-dwelling genera were characterized by larger genomes, higher CRISPR loci, expanded CAZyme machinery, possession of a C1 metabolism, and a sole dependence on aerobic respiration, whereas nonsoil genomes encoded a more versatile respiratory capacity and potential for utilizing the Wood-Ljungdahl (WL) pathway as an electron sink. This work is published in the journal “Applied and Environmental Microbiology”. Lastly, my third project (Chapter III) is about utilizing genomics and transcriptomics for an intracellular pathogen, Coxiella burnetii, to understand the changes in its genes crucial for intracellular success during long-term culturing in an axenic media. Here, I showed the expression changes and mutations in multiple genes that are known or most likely predicted to be crucial to their normal intracellular growth lifestyle or pathogenesis

Understanding the role of Coxiella outer membrane protein-1 in relation to the Type IVB Secretion system of Coxiella burnetii

Obligate intracellular bacterial pathogens, like Coxiella burnetii, the causative agent of Q Fever, rely upon the host for metabolites and carbon sources for energy and biosynthesis of nucleic acids, proteins, and energy rich molecules necessary for active vegetative growth in the host. Deficiencies in biosynthetic pathways were previously identified through genomic analyses of C. burnetii, but bacterial factors contributing to pathogenesis, with the exception of the O-lipopolysaccharide (O-LPS) and the Type IVb Secretion System (T4BSS), remain elusive. The poor efficacy of treatment and vaccine options necessitates understanding how bacterial factors contribute to disease severity, persistence of infections, and inconsistent treatment outcomes. Disulfide bond (Dsb) proteins are integral in the formation and isomerization of disulfide bonds in the T4bSS. Dsb proteins in other bacterial pathogens act upon known virulence factors that promote pathogenicity. The purpose of this study was to characterize the Coxiella outer membrane protein 1 (Com1), a putative Dsb protein, establish that it is a functional Dsb protein, and that it is linked to known virulence factors. This work will deepen the understanding in the Coxiella field of factors that might serve as alternative targets for therapeutics.Includes bibliographical references

Lysosomal evasion of legionella pneumophilia the effector.

Legionella pneumophila is a Gram-negative facultative intracellular bacterium found in freshwater environments that has co-evolved to survive and proliferate in various amoeba and protozoan species, which serve as the natural host for the bacterium. Humans are an accidental host of L. pneumophila, where infection occurs upon inhalation of aerosolized water droplets that contain the bacteria. Intracellular proliferation of L. pneumophila in alveolar macrophages is essential for manifestation of pneumonia, designated as Legionnaires’ Disease. Biogenesis of the legionella containing vacuole (LCV) occurs via interception of ER-Golgi vesicle trafficking and avoids the default endosomal/lysosomal degradation pathway. Intracellular proliferation of L. pneumophila within protozoa and macrophages is dependent on the Dot/Icm type IV secretion system (T4SS) apparatus, which is comprised of 27 proteins and is responsible for translocating over 350 different effector proteins into the host cell. Many of these effector proteins contain eukaryotic-like domains and motifs, which have been acquired through interkingdom horizontal gene transfer from various aquatic eukaryotic hosts. While L. pneumophila contains the largest repertoire of effector proteins, known for an intracellular pathogen, most of which are not required for survival and proliferation in mammalian macrophages. It is more likely that the large repertoire of effector proteins constitutes a toolbox utilized by L. pneumophila to survive and replicate within various protozoan species. The diversion of the L. pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for disease manifestation. Many of the ~350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes; but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophilia in human monocyte-derived macrophages (hMDMs), amoebae and for intrapulmonary proliferation in mice. This is shown by both single cell analysis during confocal microscopy and by quantifying colony forming units (CFUs). We have shown the mavE null mutant fails to remodel the LCV with ER-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. Importantly, during infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE (39-172) was resolved to 1.8 Å, revealing a eukaryotic NPxY motif that binds with phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. We show that point mutations within the NPxY motif results in attenuation of L. pneumophila in both hMDMs and amoeba, and the substitution defects of P78 and D64 results in fusion of the LCV to the lysosomes, with no remodeling by the ER, leading to bacterial degradation. Following ectopic expression of MavE, a proximity-dependent biotin identification (BioID) strategy was used to screen for MavE-interacting proteins in mammalian cells. These data show that MavE interacts with a host protein, acyl-CoA binding domain containing 3 (ACBD3), which co-localizes with the LCV. ACBD3 plays an essential role in the sorting and modification of proteins exported from the endoplasmic reticulum through its interaction with the integral membrane protein giantin. We have shown the mavE null mutant-containing LCV fails to colocalize with ACBD3, similar to the Dot/Icm translocation-defective mutant. There are areas of homology of ACBD3 with proteins found in Amoebozoa, indicative of a possible conserved binding motif. We conclude that the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion by interacting with the host protein ACBD3, which is involved in ER-Golgi vesicle trafficking and is likely conserved throughout evolution

Galleria mellonella-intracellular bacteria pathogen infection models: the ins and outs

Galleria mellonella (greater wax moth) larvae are used widely as surrogate infectious disease models, due to ease of use and the presence of an innate immune system functionally similar to that of vertebrates. Here, we review G. mellonella–human intracellular bacteria pathogen infection models from the genera Burkholderia, Coxiella, Francisella, Listeria, and Mycobacterium. For all genera, G. mellonella use has increased understanding of host–bacterial interactive biology, particularly through studies comparing the virulence of closely related species and/or wild-type versus mutant pairs. In many cases, virulence in G. mellonella mirrors that found in mammalian infection models, although it is unclear whether the pathogenic mechanisms are the same. The use of G. mellonella larvae has speeded up in vivo efficacy and toxicity testing of novel antimicrobials to treat infections caused by intracellular bacteria: an area that will expand since the FDA no longer requires animal testing for licensure. Further use of G. mellonella–intracellular bacteria infection models will be driven by advances in G. mellonella genetics, imaging, metabolomics, proteomics, and transcriptomic methodologies, alongside the development and accessibility of reagents to quantify immune markers, all of which will be underpinned by a fully annotated genome

Immunological correlates of illness severity and course in acute Q fever

Acute Q fever is the disease manifestation of Coxiella burnetii infection. This obligate intracellular bacterium is phagocytosed by innate immune cells, where it replicates within the usually bactericidal environment of the phagolysosome. As the immune response is activated, the resultant pro-inflammatory cytokines aid in pathogen clearance but also trigger an acute sickness response in the host. This thesis describes the natural history of acute Q fever in a prospective cohort – the Dubbo Infection Outcomes Study (DIOS). In these subjects, the acute febrile illness was characterised by severe headache, drenching sweats and fatigue. In approximately 10% of subjects, symptomatic illness marked by fatigue remained present for months, or occasionally years, after the acute illness. Subjects with more severe acute illness were more likely to develop this post Q fever fatigue syndrome (QFS). The aim of this thesis was to determine whether ongoing infection or aberrant immune activation drive the prolonged symptoms of QFS. Sensitive real time PCR detection of Coxiella DNA revealed a significant minority of subjects had very low copy numbers in circulating monocytes, with an increased prevalence in those with QFS. However, the detection was not consistently found within individual subjects and the copy number was at the threshold of reliable detection. C. burnetii was shown here to stimulate cytokine production in monocytic cells via interaction with Toll-like receptor (TLR)-2 and not TLR-4. Functional polymorphisms in these TLRs were identified in subjects with Q fever, but were not associated with Q fever susceptibility, severity or duration. Phase I-specific responses are believed to be critical in the generation of protective immunity to C. burnetii, yet the phase II-specific responses of innate and adaptive immune components were consistently of higher magnitude. Whole C. burnetii organisms induced antigen-non-specific T cell activation, presumably via the indirect activation of monocytes by C. burnetii LPS. No significant differences were found in the magnitude or kinetics of the host response to infection, or in the carriage of genetic polymorphisms, when comparing subjects who developed QFS with subjects who had promptly resolving illness. It remains unclear what factors mediate the progression of acute Q fever to QFS

APPROACHES TO IDENTIFY SURFACE PROTEINS OF ANAPLASMA PHAGOCYTOPHILUM DENSE-CORED ORGANISMS AS ADHESINS TO HUMAN P-SELECTIN GLYCOPROTEIN LIGAND-1

Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects neutrophils to cause human granulocytic anaplasmosis. Sialyl Lewis x (sLex)-modified P-selectin glycoprotein ligand-1 (PSGL-1) is the confirmed receptor utilized by A. phagocytophilum to bind and invade human neutrophils and myeloid cell lines. As an obligate intracellular pathogen, the binding of A. phagocytophilum to a host cell receptor is a prerequisite step for entry and replication, and thus its survival. However, the bacterial adhesins mediating this process have yet to be identified. In this study, we sought to identify surface proteins of A. phagocytophilum as putative adhesins. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between a smaller electron dense-cored cell (DC), which has a dense nucleoid, and a larger, pleomorphic electron lucent reticulate cell (RC), which has a dispersed nucleoid. We determined that the respective roles of the A. phagocytophilum DCs and RCs are adherence/infection and vacuolar replication, respectively, which is a finding that is consistent with the life cycles of other obligate intravacuolar pathogens that undergo biphasic development. Most importantly, we demonstrated the A. phagocytophilum DC is responsible for recognizing human PSGL-1. To identify surface proteins as putative adhesins we tested a variety of approaches. Three different computer prediction programs were compared, resulting in identification of 16 to 130 potential membrane proteins. As a more direct means to identify A. phagocytophilum surface proteins as PSGL-1 adhesins, several affinity capture approaches were tested. We used commercially available recombinant human PSGL-1 (rhPSGL-1) to try and capture adhesins by crosslinking and affinity purification. We were unsuccessful, but nevertheless gained insight into the binding properties of A. phagocytophilum. We next chose to take a broader approach to identify outer membrane proteins of the adherent DC by biotinylation. In the process we developed new density-gradient centrifugation approaches which successfully purified an RC-enriched population as well as a mixed population of RC and DC organisms. Results from this work demonstrate that A. phagocytophilum DC organisms are responsible for binding PSGL-1. Additionally, the results obtained thus far of gradient-purified bacteria will serve as a foundation for future experiments in identifying surface and developmental form specific proteins

Prospects from systems serology research

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142338/1/imm12861.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142338/2/imm12861_am.pd

Conjugative relaxases as drivers of protein and DNA translocation through Type IV Secretion Systems: biological and biotechnological implications

Este trabajo se ha centrado en el estudio de diferentes aspectos de la actividad integrasa de las relaxasas conjugativas. Hemos analizado la relación entre la capacidad de oligomerizar de las relaxasas en presencia de su ADN diana y de catalizar la integración del ADN, utilizando como modelo la relaxasa TrwC. También, hemos estudiado el posible papel biológico de esta reacción en la colonización de huéspedes no permisivos. Además, hemos analizado la habilidad de la relaxasa MobA del plásmido RSF1010 en mediar la transferencia de ADN a una célula eucariota a través del SST4 del patógeno B. henselae, así como de promover la integración de este ADN en el genoma eucariota. Finalmente, hemos estudiado el potencial uso biotecnológico de las relaxasas conjugativas como sistemas de envío de ADN y proteínas, fusionando la relaxasa TrwC a la proteína Cas12a. Hemos comprobado que la proteína de fusión es activa y funcional en la célula receptora procariota, tras ser translocada a través del SST4.This work has focused on the study of different aspects of the integrase activity of conjugative relaxases. We have analyzed the relationship between the ability of relaxases to oligomerize in the presence of their target DNA and to catalyze DNA integration, using the TrwC relaxase as a model. Also, we have studied the possible biological role of this reaction in the colonization of non-permissive hosts. Furthermore, we have analyzed the ability of the MobA relaxase from plasmid RSF1010 to mediate the transfer of DNA to a eukaryotic cell through the T4SS of the pathogen B. henselae, as well as to promote the integration of this DNA into the eukaryotic genome. Finally, we have studied the potential biotechnological use of conjugative relaxases as DNA and protein delivery systems, fusing the TrwC relaxase to the Cas12a protein. We have verified that the fusion protein is active and functional in the prokaryotic receptor cell, after being translocated through T4SS.Esta Investigación ha sido financiada por una ayuda para contratos predoctorales en el área de la Biomedicina, Biotecnología y Ciencias de la Salud de la Universidad de Cantabria: 7665391046 Y0SC001170. El trabajo en el laboratorio de Matxalen Llosa Blas ha sido financiado por el Ministerio de Economía, Industria y Competitividad de España: BIO2013-46414-P y BIO2017-87190-R. Las estancias de investigación en el Instituto de Productos Lácteos de Asturias (IPLA) y en el Instituto Pasteur se realizaron gracias a sendas ayudas de la Universidad de Cantabria

Characterization of Legionella pneumophila Effector Proteins, LneB and MavA

The crucial virulence factor of accidental human pathogen Legionella pneumophila during the course of Legionnaire disease is the over 300 effector proteins secreted from its Dot/Icm secretion system. Eukaryotic host cells usually elicit an arsenal of immune responses against invading L. pneumophila. Nonetheless, the bacteria unexpectedly subvert these defense mechanisms to survive and proliferate unhindered in the host. Although some effector proteins have been proposed to play a significant role in this host-pathogen interaction, many still need to be characterized. The LneB and MavA proteins are examples of those effectors that need characterization. Thus, this study aimed to investigate the structural and functional characteristics of LneB and MavA proteins using several bioinformatics predictive pipelines and transcriptomics data supplemented experimentally through cell-based and biochemical assays to support the prediction. The LneB protein was predicted to have histone acetylation activity (HAT) based on bioinformatics analysis. To investigate the HAT activity of LneB in vitro, the protein was ectopically expressed in the Escherichia coli BL21 strain and purified using nickel ion chromatography. The HAT activity assay was carried out on the purified LneB protein and on the nuclear extracts from LneB-GFP transfected 293T cells. Transcriptomics analysis shows that the LneB protein differentially induces upregulation of early growth factor and dehydrogenase (DHRS2) compared to the GFP control. There was no significant difference between the in vitro HAT activity of LneB protein and the elution buffer (p-value = 0.1137, t-value = 5.537). In vivo, HAT activity was significantly reduced in cells transfected with LneB protein compared to the GFP control (p-value = 0.0025, t-value = 20.08). The HAT activity is not significantly different at a MOI of 10 or 100 when infected cells (Dot/Icm mutant and wild-type L. pneumophila) are compared to uninfected U937 cells (p-value = 0.8969 and 0.5384, respectively). However, the HAT activity in cells infected with an L. pneumophila Dot/Icm mutant at MOI of 100 was significantly lower than in cells that were not infected (p-value = 0.0236). This result suggests that the effector protein from the wild type plays a significant role in acetylating histone protein in the host. Further investigation is required to understand the HAT activity of LneB and other roles the protein could play in the host. Our bioinformatics analysis suggested that the MavA protein possesses Ras-GEF domains and potentially binds to GTP. The protein is predicted to possess two coiled-coil domains and also interact with GTP, Ras and actin. The transcriptomic data from cells expressing MavA protein showed significant upregulation of sixteen genes, which are involve in steroid hormone metabolic processes, endocytic recycling, cilia movement among others. The sortilin receptor protein was the only repressed gene in the cell when compared to a GFP protein control. Connecting the bioinformatics finding and the review of literature, we suggested that the MavA protein could be involved in the biological process in the cell such as internalization of L. pneumophila, creation of Legionella-containing vacuoles in host cells through endosomal remodeling or cytoskeletal reorganization