Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria

During the bacterial cell cycle, chromosome replication and cell division must be coordinated with overall cell growth in order to maintain the correct ploidy and cell size. The spatial and temporal coordination of these processes in mycobacteria is not understood. Here we use microfluidics and time-lapse fluorescence microscopy to measure the dynamics of cell growth, division and chromosome replication in single cells of Mycobacterium smegmatis. We find that single-cell growth is size-dependent (large cells grow faster than small cells), which implicates a size-control mechanism in cell-size homoeostasis. Asymmetric division of mother cells gives rise to unequally sized sibling cells that grow at different velocities but show no differential sensitivity to antibiotics. Individual cells are restricted to one round of chromosome replication per cell division cycle, although replication usually initiates in the mother cell before cytokinesis and terminates in the daughter cells after cytokinesis. These studies reveal important differences between cell cycle organization in mycobacteria compared with better-studied model organisms

Noninvasive immuno-PET imaging of CD8 + T cell behavior in influenza A virus-infected mice

Immuno-positron emission tomography (immuno-PET) is a noninvasive imaging method that enables tracking of immune cells in living animals. We used a nanobody that recognizes mouse CD8α and labeled it wit

Commensal-specific immune responses at the intestinal mucosa

Thesis: Ph. D. to the Microbiology Graduate Program, Massachusetts Institute of Technology, Department of Biology, February, 2020Cataloged from student-submitted PDF of thesis.Includes bibliographical references.The intestinal mucosa harbors a dense community of microbes that breaks down polysaccharides indigestible by the host, synthesizes essential vitamins, stimulates maturation of the immune system, and outcompetes the growth of pathogenic species. In return, the host provides commensals with a habitat rich in energy derived from ingested food. The intestinal immune system faces the daunting task of maintaining homeostasis despite the enormous load and diversity of antigens present at this site. Failure to maintain this balance has dramatic consequences and can cause food allergies, inflammatory bowel disease or invasive infections. Peripherally-induced Foxp3⁺-regulatory T cells (pTregs) maintain immune homeostasis at the intestinal mucosa by regulating effector T cell responses against dietary antigens and microbes.Similarly to pTregs, a subset of small intestine intraepithelial lymphocytes CD4⁺CD8[alpha][alpha]⁺ (CD4[subscript IELs]) exhibit regulatory properties and promote tolerance against dietary antigens. In this thesis, I describe a new commensal-specific CD4+ T cell model obtained by somatic cell nuclear transfer using, as a donor, a single pTreg from the mesenteric lymph node. In chapter 1, I provide an overview of the interplay between the microbiota and the mucosal immune system. In chapter 2, we describe our newly developed model and use it to assess how the identity of the T cell receptor (TCR) affect the fate of a T cell. In chapter 3, I describe the antigen and epitope recognized by this transnuclear (TN) TCR and show that TN cells can protect against intestinal inflammation in a colitis model. In chapter 4, I describe how TN cells can also differentiate into T follicular helper and promote systemic responses.In chapter 5, we developed a strategy to target antigens to outer membrane vesicles (OMVs) of Bacteroides and thereby assess the antigen-specific responses to OMVs. In chapter 6, I provide concluding remarks and discuss future prospective for our findings. In the appendix, I describe a new mouse model to site-specifically label and track the B cell receptor of primary B cells.by Djenet Bousbaine.Ph. D. to the Microbiology Graduate ProgramPh.D.totheMicrobiologyGraduateProgram Massachusetts Institute of Technology, Department of Biolog

The studies of ParA and ParB dynamics reveal asymmetry of chromosome segregation in mycobacteria

Active segregation of bacterial chromosomes usually involves the action of ParB proteins, which bind in proximity of chromosomal origin (oriC) regions forming nucleoprotein complexes - segrosomes. Newly duplicated segrosomes are moved either uni- or bidirectionally by the action of ATPases - ParA proteins. In Mycobacterium smegmatis the oriC region is located in an off-centred position and newly replicated segrosomes are segregated towards cell poles. The elimination of M. smegmatis ParA and/or ParB leads to chromosome segregation defects. Here, we took advantage of microfluidic time-lapse fluorescent microscopy to address the question of ParA and ParB dynamics in M. smegmatis and M. tuberculosis cells. Our results reveal that ParB complexes are segregated in an asymmetrical manner. The rapid movement of segrosomes is dependent on ParA that is transiently associated with the new pole. Remarkably in M. tuberculosis, the movement of the ParB complex is much slower than in M. smegmatis, but segregation as in M. smegmatis lasts approximately 10% of the cell cycle, which suggests a correlation between segregation dynamics and the growth rate. On the basis of our results, we propose a model for the asymmetric action of segregation machinery that reflects unequal division and growth of mycobacterial cells

One-pot dual labeling of IgG 1 and preparation of C-to-C fusion proteins through a combination of sortase A and butelase 1

Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule

One-step generation of monoclonal B cell receptor mice capable of isotype switching and somatic hypermutation

We developed a method for rapid generation of B cell receptor (BCR) monoclonal mice expressing prerearranged Igh and Igk chains monoallelically from the Igh locus by CRISPR-Cas9 injection into fertilized oocytes. B cells from these mice undergo somatic hypermutation (SHM), class switch recombination (CSR), and affinity-based selection in germinal centers. This method combines the practicality of BCR transgenes with the ability to study Ig SHM, CSR, and affinity maturation

Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease

Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach diseaseassociated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4⁺ and CD8⁺ T cells) in an antigenspecific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.United States. Defense Advanced Research Projects Agency (Contract HR0011-12-2-0015