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The type III secretion system (T3SS) is a syringe-like virulence factor that delivers bacterial proteins directly into the cytoplasm of host cells. An essential component of the system is the translocon, which creates a pore in the host cell membrane through which proteins are injected. In Pseudomonas aeruginosa, the translocation pore is formed by proteins PopB and PopD and attaches to the T3SS needle via the needle tip protein PcrV. The structure and stoichiometry of the multimeric pore are unknown. We took a genetic approach to map contact points within the system by taking advantage of the fact that the translocator proteins of P. aeruginosa and the related Aeromonas hydrophila T3SS are incompatible and cannot be freely exchanged. We created chimeric versions of P. aeruginosa PopB and A. hydrophila AopB to intentionally disrupt and restore protein-protein interactions. We identified a chimeric B-translocator that specifically disrupts an interaction with the needle tip protein. This disruption did not affect membrane insertion of the B-translocator but did prevent formation of the translocation pore, arguing that the needle tip protein drives the formation of the translocation pore. IMPORTANCE Type III secretion systems are integral to the pathogenesis of many Gram-negative bacterial pathogens. A hallmark of these secretion systems is that they deliver effector proteins vectorially into the targeted host cell via a translocation pore. The translocon is crucial for T3SS function, but it has proven difficult to study biochemically and structurally. Here, we used a genetic approach to identify protein-protein contacts among translocator proteins that are important for function. This genetic approach allowed us to specifically break a contact between the translocator PopB and the T3SS needle tip protein PcrV. Breaking this contact allowed us to determine, for the first time, that the needle tip actively participates in the assembly of the translocation pore by the membrane-bound pore-forming translocator proteins. Our study therefore both expands our knowledge of the network of functionally important interactions among translocator proteins and illuminates a new step in the assembly of this critical host cell interface.

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Broadly HIV-1-neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02-derived heavy chains paired with light chains expressing five-amino acid-long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line-targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans.

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The process of neuroinflammation contributes to the pathogenic mechanism of many neurodegenerative diseases. The deleterious attributes of neuroinflammation involve aberrant and uncontrolled activation of glia, which can result in damage to proximal brain parenchyma. Failure to distinguish self from non-self, as well as leukocyte reaction to aggregation and accumulation of proteins in the CNS, are the primary mechanisms by which neuroinflammation is initiated. While processes local to the CNS may instigate neurodegenerative disease, the existence or dysregulation of systemic homeostasis can also serve to improve or worsen CNS pathologies, respectively. One fundamental component of systemic homeostasis is the gut microbiota, which communicates with the CNS via microbial metabolite production, the peripheral nervous system, and regulation of tryptophan metabolism. Over the past 10-15 years, research focused on the microbiota-gut-brain axis has culminated in the discovery that dysbiosis, or an imbalance between commensal and pathogenic gut bacteria, can promote CNS pathologies. Conversely, a properly regulated and well-balanced microbiome supports CNS homeostasis and reduces the incidence and extent of pathogenic neuroinflammation. This review will discuss the role of the gut microbiota in exacerbating or alleviating neuroinflammation in neurodegenerative diseases, and potential microbiota-based therapeutic approaches to reduce pathology in diseased states.

RESUMEN

Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2∗02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies.

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