mBio最新文献

The bioRxiv and medRxiv preprint servers brought preprinting to the life sciences and played a critical role in disseminating COVID research during the pandemic. Here, I reflect on the birth of bioRxiv and medRxiv and the crucial role so many members of the community played, our experience during the pandemic, and the launch of the new non-profit organization set up to oversee the servers. The pandemic was a stress test for bioRxiv and medRxiv that demonstrated their value and robustness. Under the umbrella of openRxiv, they are now poised to become long-term infrastructure underpinning a new publishing ecosystem.

Toxoplasma gondii exploits host phagocytes to disseminate systemically and establish infection in the central nervous system (CNS). Yet, the mechanisms governing the interaction between parasitized phagocytes and the brain endothelium remain elusive. Here, we show that T. gondii infection, but not parasite lysates, robustly induces transcriptional and secretory upregulation of the chemokine C-C motif ligand 5 (CCL5/RANTES) in primary brain endothelial cells and dendritic cells (DCs). This response was triggered by the parasite effector GRA15 through NF-κB signaling, while the effector TEEGR counteracted CCL5 induction in an MYR-translocon-dependent manner. In response to recombinant CCL5, infected DCs displayed increased hypermotility, chemotaxis toward CCL5 gradients, and enhanced transmigration across polarized endothelial monolayers. Intraperitoneally infected mice rapidly upregulated CCL5 in the blood and Ccl5 expression in the cerebral microvasculature, thereby increasing the adhesion of parasitized DCs and cerebral parasite loads. Pretreatment of mice with recombinant CCL5 dramatically elevated the sequestration of infected DCs, while treatment with the selective chemokine receptor 5 (CCR5) antagonist Maraviroc reverted sequestration. Together, these findings reveal that T. gondii co-opts the host CCL5/CCR5 axis via GRA15-mediated signaling to promote leukocyte-dependent dissemination and early colonization of the CNS.

Importance: The intracellular parasite Toxoplasma gondii invades immune cells to spread through the circulatory system, eventually reaching the brains of humans and animals. It is not well understood how parasitized immune cells interact with blood vessel walls, a process that ultimately helps Toxoplasma colonize the brain tissue. We found that when Toxoplasma infects the cells lining the blood vessels (endothelium), these produce C-C motif chemokine ligand 5 (CCL5), a potent signaling and attractant molecule. CCL5 production was triggered by a parasite-derived secreted protein, GRA15. CCL5 activated and attracted infected immune cells. In mice, the levels of CCL5 increased quickly in the brain microvasculature after infection, helping the infected immune cells adhere to brain vessels. When the effect of CCL5 was pharmacologically blocked, fewer infected cells sequestered in the brain vessels, lowering the parasite loads. These findings reveal a mechanism through which Toxoplasma manipulates host cells to produce factors that facilitate its colonization of the brain.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. Inbred mouse models of TB do not reflect the pathological states and heterogeneity seen in human TB disease. Thus, we recently established a model of TB in diversity outbred (DO) mice, which displayed heterogeneity in inflammatory and protective responses following aerosol Mtb infection. In the current study, we show that DO mice vaccinated with M. bovis Bacille Calmette Guerin (BCG) are significantly protected upon Mtb HN878 infection, and protection is associated with the induction of transcriptional pathways involved in transforming growth factor B (TGF-β) and Toll-like receptor (TLR)-10 signaling. Targeting lung innate pathways in BCG-vaccinated DO mice using adjuvants also further improved protection upon Mtb infection by inducing genes associated with cellular responses to external stimuli, B-cell responses, as well as IL-17-producing CD4⁺ T-cell responses. Depletion of CD4⁺ T cells resulted in loss of vaccine-induced protection in DO BCG-vaccinated and adjuvant-treated Mtb-infected mice. Together, our new results show that innate targeting of the lung by activating TLR pathways could induce protective pathways in T cells that significantly improve upon the protection induced by BCG vaccination. Additionally, the DO mouse model of vaccination and Mtb infection can provide novel insights into immune pathways that are important for improving vaccine-induced protection against TB.

Importance: Bacille Calmette Guerin (BCG) vaccination in genetically diverse outbred (DO) mice provides significant protection against Mycobacterium tuberculosis (Mtb) challenge. This protection induced pathways associated with transforming growth factor B (TGF-β) receptor complex, genes associated with lung repair, and Toll-like receptor (TLR)-10 pathway. The enhanced protection observed in BCG-vaccinated mice correlated with improved formation of B-cell follicles and IL-17-producing CD4⁺ T-cell responses. CD4⁺ T-cell responses mediated the enhanced protection in the lungs of DO mice vaccinated with BCG + adjuvant, as depletion of CD4⁺ T-cell responses reversed the enhanced protection. The DO mouse model of tuberculosis vaccination is a highly relevant model to probe mechanisms of vaccine-induced protection and provide novel insights into lung pathways that mediate protection. The study also found that genes associated with lung repair, including TGF-β receptor complex pathways, were induced in BCG-vaccinated Mtb-infected DO mouse lungs. The study suggests that the activation of lung innate pathways in BCG vaccination through the use of mucosal Amph CpG delivery, CD40L activation, and IL-10 neutralization could significantly enhance protection upon Mtb challenge.

Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.

Importance: Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.

Aspects of transcriptional regulation of plant cell wall-degrading enzyme (PCWDE) genes have been characterized in the filamentous fungus Neurospora crassa. However, the upstream signaling pathways that regulate PCWDE expression are not well understood. We have previously reported roles for heterotrimeric G-proteins and adenylyl cyclase in the degradation of cellulose to glucose in N. crassa. Here, we performed mRNA-seq to identify patterns of gene expression after transfer from glucose to cellulose medium in wild type, the Gα mutants Δgna-1 and Δgna-3, and the adenylyl cyclase mutant Δcr-1. In wild type, 3719 genes were regulated at least twofold during growth on cellulose vs glucose. Analysis of transcriptomics data for the strains after transfer from glucose to cellulose demonstrated that the Δcr-1 mutant had the most misregulated genes, with 2,232, followed by Δgna-3 with 1,182 and Δgna-1 with 648 genes. Metabolic genes were the most prevalent differentially expressed genes in the mutants. Expression of PCWDEs, including most of the cellulases, was downregulated in the three mutants, with Δcr-1 displaying the greatest deficiency. Furthermore, several transcription factors essential for cellulase expression were misregulated in the mutants. The primary factors clr-1 and clr-2 were downregulated in Δgna-3 and Δcr-1 strains, and clr-2 was reduced in Δgna-1 mutants. Overexpression of clr-2 restored cellulase activity and increased the expression of two major cellulase genes in all three mutants. Taken together, our results demonstrate that heterotrimeric G-proteins and cAMP signaling strongly impact transcriptional control of cellulase activity, culminating in the expression of the transcription factor clr-2 in N. crassa.IMPORTANCEFilamentous fungi are important organisms for degradation of plant biomass. Both nonpathogens and plant pathogens secrete plant cell wall degrading enzymes to release simple sugars from the plant cell wall to use as carbon sources for growth. Much is known about the transcription factors that control production of plant cell wall-degrading enzymes by fungi. However, mechanistic details for how different lignocellulosic compounds are sensed by these organisms and the resultant cellular responses that operate upstream of cellulase-regulating transcription factors are lacking. Our research helps bridge this gap by identifying the role of G-protein subunits and cAMP in the regulation of gene expression during growth on cellulose. Understanding the environmental sensing and signal transduction pathways that regulate cellulase gene expression will have applications to agricultural losses due to plant pathogens, carbon recycling in the environment, and production of biofuels.

Adeno-associated virus (AAV) vectors have taken center stage for gene therapy and have shown clinical efficacy in 15 human diseases to date. The Food and Drug Administration has approved seven AAV "drugs" for one-time treatment respectively for Leber's congenital amaurosis, spinal muscular atrophy, hemophilia B, Duchenne muscular dystrophy, hemophilia A, and aromatic L-amino acid decarboxylase deficiency. Despite these remarkable developments, it has become increasingly clear that the first generation of AAV vectors is less than optimal since in most, if not all, cases, exceedingly high doses are needed to achieve clinical efficacy, and as a consequence, in some patients, serious adverse events have been observed, and to date, at least 21 patients have died. Thus, there is a need to reassess the limitations of the first generation of AAV vectors as well as an urgent need to develop the next generation of AAV vectors that are safe and effective.

Salmonella enterica serovar (S.) Typhi, the etiological agent of typhoid fever, is strictly human adapted, which presents a significant challenge for studying its pathogenesis in animal models. A common strategy to overcome this limitation is to infect mice with S. Typhimurium as a surrogate pathogen. Since S. Typhimurium is a non-typhoidal serovar that does not encode the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi, we explored whether the mouse virulent typhoidal Salmonella serovar Paratyphi C, which expresses the Vi antigen, would be better suited as a surrogate pathogen to study typhoid fever pathogenesis in the mouse. In contrast to the nontyphoidal serovar Typhimurium, which produced lethal morbidity in C57BL/6 mice within a few days after infection, S. Paratyphi C demonstrated prolonged colonization of systemic organs for up to 28 days after infection. Analysis of virulence factors revealed that the Vi antigen was important at very early stages after infection (up to 2 days), whereas the type III secretion system encoded by Salmonella pathogenicity island 2 became critical at later stages. Vaccination with purified Vi antigen suppressed S. Paratyphi C dissemination. Implantation of a biotelemetry device revealed that S. Paratyphi C triggered fever after an incubation period of 3 days, which was reminiscent of the prolonged incubation period of typhoid fever. In conclusion, our findings suggest that the use of S. Paratyphi C as a surrogate pathogen provides a mouse model for studying typhoid fever pathogenesis and vaccine development.IMPORTANCEThe emergence of extensively drug-resistant Salmonella enterica serovar (S.) Typhi poses a serious threat to public health, but its host restriction to humans poses a challenge for studying pathogenesis and vaccine development in animal models. Here, we used S. Paratyphi C, a mouse virulent typhoidal serovar that expresses the virulence-associated Vi capsular polysaccharide, as a surrogate pathogen for studying typhoid fever in a mouse model. Our model recapitulates key features of typhoid fever, including clinical symptoms such as a prolonged incubation period, fever, and splenomegaly. Notably, disseminated infection with S. Paratyphi C developed after inoculation by the natural oral route. We demonstrate the utility of this model for studying pathogenesis and vaccination. We conclude that our new mouse model for typhoid fever offers a promising platform for evaluating novel therapeutics and vaccine candidates to address the problem of drug resistance in S. Typhi and reduce the global burden of typhoid fever.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is a potent inducer of cell-mediated immunity, which has led to the development of attenuated, Listeria-based cancer vaccines. L. monocytogenes strains, such as live-attenuated double-deleted Listeria (LADD), lacking two key virulence factors, ΔactA and ΔinlB, have been used safely in clinical trials and showed promising anti-tumor activity. Despite early clinical success, improving potency and safety by preventing extracellular bacterial growth is paramount for the development of further clinical applications. We describe a quadruple attenuated intracellular Listeria (QUAIL) strain that, in addition to ΔactAΔinlB, lacks ribC and ribF, which encode enzymes required for generating the essential flavin cofactors flavin mononucleotide (FMN) and flavin adenine nucleotide (FAD). QUAIL imported FMN and FAD during intracellular growth but was unable to grow extracellularly in blood or on vascular catheters in mice, which reduced its lethality. Despite its lack of extracellular growth, QUAIL maintained its immunoprotective properties, which were comparable to LADD. Furthermore, we showed that QUAIL can be engineered to synthesize riboflavin, leading to expansion and activation of mucosal-associated invariant T cells. Together, our data support the use of QUAIL as a promising therapeutic platform with an improved safety profile that is amenable to further modifications to expand its immune-activating potential.IMPORTANCEListeria-based live-attenuated cancer vaccines represent a promising therapy in many different pre-clinical tumor models and in clinical trials. Enhancing its anti-cancer immunity and increasing its safety profile will advance the clinical applications of Listeria vaccines. By manipulating Listeria monocytogenes flavin metabolism, we engineered a quadruple attenuated intracellular Listeria (QUAIL) vaccine candidate strain that has limited toxicity associated with extracellular growth in major extracellular niches in vivo, including blood and implanted catheter ports. Furthermore, we showed that QUAIL can be effectively programmed to engage innate-like T cells known as mucosal-associated invariant T cells, which could be harnessed for future cancer immunotherapies. The results presented here lay the foundation for further analysis of QUAIL as a safer, yet immunopotent L. monocytogenes vaccine or therapeutic vector.