Biology subjects

Hill, J. D.

Publications and source records attributed to Hill, J. D..

4 recordsLinked to original sources

bioRxiv2024

CCR2 limits inflammatory functions of CD8 TRM cells that impair recognition memory during recovery from WNV encephalitis

Central nervous system (CNS) resident memory CD8 T cells (TRM) that express IFN-{gamma} contribute to neurodegenerative processes, including synapse loss, leading to memory impairments. Here, we show that CCR2 signalling in CD8 TRM that persist within the hippocampus after recovery from CNS infection with West Nile virus (WNV) significantly prevents the development of memory impairments. Using CCR2-deficient mice, we determined that CCR2 expression is not essential for CNS T cell recruitment or virologic control during acute WNV infection. However, transcriptomic analyses of forebrain CCR2+ versus CCR2- CD8 TRM during WNV recovery reveal that CCR2 signalling significantly regulates hippocampal CD8 TRM phenotype and function via extrinsic and intrinsic effects, decreasing the expression of CD103 and granzyme A and IFN-{gamma}, respectively. Consistent with this, WNV-recovered Cd8acreCcr2fl/fl mice exhibit decreased recognition memory. Our findings highlight a neuroprotective role for CCR2 in limiting CD8 T cell-mediated neuroinflammation and cognitive deficits, providing insights into potential therapeutic targets for CNS infections.

immunology↗

bioRxiv2024

Butyrate as a growth factor of Clostridium acetobutylicum

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. Proteomic data suggest that butyrate may lead to lysine butyrylation, a lesser-known post-translational modification, which might affect enzyme catalysis and thus cellular metabolism. Although high levels of butyrate induce toxic stress responses, it is not known if butyrate at non-toxic levels influences cellular processes such as growth, health, metabolism, and sporulation. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome in order to eliminate the butyrate synthetic pathway and thus butyrate formation. For rapid genome engineering, a xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. The addition of non-toxic levels of butyrate revealed that butyrate has a profound effect on the growth, health, and sporulation of C. acetobutylicum. By further deleting spo0A, the gene of the master regulator of sporulation, and followed by butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of the precursor of butyrate biosynthesis crotonate to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and other Clostridium organisms, and may thus be as a modulator of microbial population dynamics. HighlightsO_LIDeployed chromosomally integrated spCas9 for markerless one-step Clostridium acetobutylicum genome engineering. C_LIO_LIDeleted 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from Clostridium acetobutylicum to elucidate the roles of butyrate in cellular processes. C_LIO_LIDemonstrated butyrate as a growth factor stimulating cellular processes in Clostridium acetobutylicum and potentially other Clostridium species. C_LIO_LISuggested butyrate as a potential modulator of microbial population based on different responses of microbes against butyrate. C_LI

bioengineering↗

bioRxiv2024

Species-Specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably, the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that rRNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantitate individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii and Clostridium kluyverii. In pursuing our goal to capture each organisms population dynamics, we demonstrate the dynamic rRNA, and thus ribosome, exchange between the three species leading to formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct dynamic localization patterns among the three organisms. Our data also support the use of rRNA-FISH to assess the cultures health and expansion potential, and here again our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCEThough dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in cocultures, we hypothesized that rRNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to probe, quantitatively, complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, "state-of-the-art" labelling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved key challenges and applied the technique to co-cultures of C. acetobutylicum, C. ljungdahlii, and C. kluyveri. We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, it can capture individual population dynamics in co-cultures.

microbiology↗

bioRxiv2023

Myeloid cell activation during Zika virus encephalitis predicts recovery of functional cortical connectivity

Neurologic complications of Zika virus (ZIKV) infection across the lifespan have been described during outbreaks in Southeast Asia, South America, and Central America since 2016. In the adult CNS ZIKV tropism for neurons is tightly linked to its effects, with neuronal loss within the hippocampus during acute infection and protracted synapse loss during recovery, which is associated with cognitive deficits. The effects of ZIKV on cortical networks have not been evaluated. Although animal behavior assays have been used previously to model cognitive impairment, in vivo brain imaging can provide orthogonal information regarding the health of brain networks in real time, providing a tool to translate findings in animal models to humans. In this study, we use widefield optical imaging to measure cortical functional connectivity (FC) in mice during acute infection with, and recovery from, intracranial infection with a mouse-adapted strain of ZIKV. Acute ZIKV infection leads to high levels of myeloid cell activation, with loss of neurons and presynaptic termini in the cerebral cortex and associated loss of FC primarily within the somatosensory cortex. During recovery, neuron numbers, synapses and FC recover to levels near those of healthy mice. However, hippocampal injury and impaired spatial cognition persist. The magnitude of activated myeloid cells during acute infection predicted both recovery of synapses and the degree of FC recovery after recovery from ZIKV infection. These findings suggest that a robust inflammatory response may contribute to the health of functional brain networks after recovery from infection. Significance StatementDetermining the long-term cognitive impact of infections is clinically challenging. We found that the degree of myeloid cell activation correlated with the degree of recovery of functional connectivity after recovery from ZIKV encephalitis. Using functional cortical connectivity, we demonstrate that interhemispheric cortical connectivity is decreased in individuals with acute ZIKV encephalitis. This correlates with decreased presynaptic terminals in the somatosensory cortex. During recovery from ZIKV infection, presynaptic terminals recover, which is associated with recovered interhemispheric connectivity. This suggests a role for activated myeloid cells in maintenance of cognition and further supports the contribution of synapses in the cortex to functional networks in the brain, which can be detected by widefield optical imaging. These findings also suggest neuroinflammation may play a neuroprotective role in addition to aiding in local virologic control.

biophysics↗