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Le peptidoglycane (ou muréine) est un élément essentiel de la paroi chez la plupart des bactéries. Il est également la cible de nombreuses molécules antimicrobiennes : des défenses immunitaires de l’hôte, aux antibiotiques, en passant par différentes enzymes lytiques. Il a été mis en évidence que des modifications de la structure du peptidoglycane permettent aux bactéries d’acquérir des résistances face à ces différents agents. Les Corynebacteriales, un ordre de bactérie connu pour leur enveloppe atypique composée d’une membrane cytoplasmique classique et d’un complexe peptidoglycane – arabinogalactane - acides mycoliques (mAGP), montrent différents types de modifications du peptidoglycane. Au cours de ce travail, nous avons étudié deux types de modifications chez notre modèle Corynebacterium glutamicum,- une bactérie non-pathogène, souvent utilisée comme modèle de laboratoire - qui sont les amidations de deux résidus (meso-DAP et D-Glu) du domaine peptidique du peptidoglycane. Nous avons pu identifier les gènes responsables de ces modifications : ltsACg et murT/gatDCg. Une caractérisation biochimique de LtsACg a permis de montrer son rôle dans l’amidation du meso-DAP, d’identifier son substrat physiologique et l’étude du mutant inactivé pour ltsACg- a montré son rôle dans la résistance au lysozyme et aux β-lactamines. Dans un deuxième temps, nous avons débuté la caractérisation biochimique de MurT/GatDCg et l’étude du mutant conditionnel gatDCg-. Les résultats préliminaires montrent une essentialité de ce gène chez C. glutamicum, et une activité D-Glu amidase de l’opéron MurtT/GatDCg par l’expression hétérologue chez E. coli. Ces enzymes sont conservées au sein de l’ordre Corynebacteriales et les amidations du D-Glu et du meso-DAP sont retrouvées chez différentes espèces pathogènes. Cette étude ouvre donc de nouvelles perspectives pour l’étude de ces modifications chez d’autres espèces de Corynebacteriales pathogènes et tirer profit du rôle de ces amidations dans la résistance aux β-lactamines pour trouver de nouvelles cibles thérapeutiques. / Peptidoglycan is an essential and unique element in cell wall of most bacteria. It is also the target of many antimicrobial molecules as immune defense molecules, antibiotics or lytic enzymes. It has been shown that modifications of its structure can allow bacteria to become resistant for those molecules. Corynebacteriales, is an order of bacteria, known for their atypical envelope with a classical plasmic membrane surrounded by a huge complex made of peptidoglycan, arabinogalactan and mycolic acids (mAGP). Peptidoglycan of different species in this order have distinct types of modifications. During this work, we study two types of modifications in our model Corynebacterium glutamicum – a non-pathogenic bacteria usually used as a lab model – which are amidation of both residues (meso-DAP and D-Glu) of the peptide chain of peptidoglycan. We identified genes responsible for those modifications: ltsACg and murT/gatDCg. Biochemical characterization of LtsACg showed its role in amidation of meso-DAP and the study of the mutant ltsACg- show a high impact on lysozyme and β-lactam resistance. In a second time, we have initiated biochemical characterization of MurT/GatDCg and analysis of the conditional mutant gatDCg-. Preliminary results show that gatDcg is essential in C. glutamicum and an activity of D-Glu muropeptide amidation during heterologous expression in E. coli have been detected. Those enzymes are conserved through the order of Corynbebacteriales and both modifications of D-Glu and meso-DAP were found in different species including pathogenic. This work gives new perspectives for study of modifications in other Corynebacteriales species, in particular for pathogenic species because those enzyme constitute new therapeutic targets for β-lactam resistance.

In my dissertation, I have worked on two distinct projects related to the immune system. The abstracts for the two projects that make up my dissertation work are below. In the first project (presented in Chapter 2), we study regulatory T (Treg) cells in chronic liver disease. Current dogma holds that Treg cells preserve tissue function in settings of inflammation and damage. Consistent with this, herein we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis, the key prognostic factor for patients – through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance has hitherto been unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner, also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, in the setting of NASH, we find that Areg from Treg cells promotes hepatocyte gluconeogenesis – through hepatocyte detection of fibrosis development and soluble mediators, including IL-6, produced by activated hepatic stellate cells – offering new insight into the cellular interplay of how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH. In the second project (presented in Chapter 3), we use engineered bacteria to produce chemokines in the tumor to promote anti-tumor immunity. Tumors employ multiple mechanisms to actively exclude immune cells involved in anti-tumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models – an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells – using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive anti-tumor immune responses, offering a new cancer immunotherapy strategy.

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of monotherapies. A major challenge facing tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. In contrast, certain strains of bacteria are gaining recognition as a new class of antigen-agnostic cell therapy due to their selective growth within the immunosuppressive niche of the solid tumor microenvironment (TME). In response, this dissertation aims to pair the cytotoxicity of CAR-T cells with the antigen-independent specificity of tumor-colonizing bacteria to create a new strategy for solid tumor recognition. Here, we reprogram the probiotic strain of E. coli Nissle 1917 to release synthetic CAR targets and human chemokines directly within the solid tumor core. To enable universal targeting, we design synthetic targets to bind ubiquitous components of the TME and broadly tag tumor tissue for CAR-mediated lysis. We demonstrate that these targets robustly coat the surface of cancer cell lines and lead to effective killing by CAR-T cells across various cancer types. We additionally show that injected probiotics selectively grow within the tumor core and maintain target production ¬ in situ – leading to therapeutic efficacy across multiple genetically distinct tumor models. Within this dissertation, we also reveal that intratumoral bacteria provide natural adjuvant effects that serve to activate and increase the effector functions of CAR-T cells in vivo. However, we discover that this can lead to early T cell exhaustion and terminal effector differentiation. To mitigate the counterproductive effects of overstimulation, we generate a new probiotic strain with reduced inflammatory properties that significantly improves CAR-T cell phenotype – leading to enhanced therapeutic benefit in a human model of leukemia. We conclude by discussing the numerous avenues available to optimize cross-Kingdom signaling and to ultimately leverage the full therapeutic benefit of combined cell therapies for future translation. Altogether, this dissertation highlights the potential of the probiotic-guided CAR-T cell (ProCAR) platform to address the critical roadblock of identifying suitable CAR targets by providing an antigen in situ that is orthogonal to both healthy tissue and tumor genetics – and, in turn, aims to establish the foundation for engineered communities of living medicines.

Aquifers are an important storage location and source of fresh groundwater. They may become polluted by a number of contaminants including mobile divalent radionuclides such as strontium-90 which is a byproduct of uranium fission. A method for remediating such divalent radionuclides is sequestration through co-precipitation into calcium carbonate. Calcium carbonate precipitation occurs naturally but can be enhanced by the use of ureolytic microorganisms living within the aquifer. The microbial enzyme urease cleaves ammonia from urea (added as a stimulant to the aquifer) increasing the pH and subsequently pushing the bicarbonate equilibrium towards precipitation. Laboratory experimentation is necessary to better predict field scale outcomes of remediation that is driven by ureolytic calcium carbonate co-precipitation. To aid in such laboratory experiments, I constructed two ureolytic organisms which contain green fluorescent protein (GFP) so that the location of the microbes in relation to media flow paths and precipitation can be viewed by microscopy in a 2- dimensional porous medium flow cell reactor. The reactor was operated with a parallel flow regime where the two influent media would not promote microbially induced calcium carbonate precipitation until they were mixed in the flow cell. A demonstration study compared the results of parallel flow and mixing in the reactor operated with and without one of the GFP-containing ureolytic organisms. The growth and precipitation of calcium carbonate within the reactor pore space altered flow paths to promote a wider mixing zone and a more widely distributed overall calcium carbonate precipitation pattern. This study will allow optimization of remediation efforts of contaminants such as strontium-90 in aquifers. / Graduation date: 2012