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Streptococcus agalactiae (Group B Streptococcus ) strain COH1 is a representative strain of serotype III, multi-locus sequence type 17, which is disproportionately associated with neonatal meningitis. Here we report the transcriptome of COH1 when interacting with human brain endothelial cells compared with COH1 alone., Competing Interests: The authors declare no conflict of interest.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae ) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY , encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text] codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators., Importance: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries., Competing Interests: The authors declare no conflict of interest.

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6 , MetSVORF5 , MetSVORF2 , and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study's results led to the first preliminary infection model of MetSV infecting M. mazei , summarizing the key infection steps as follows: replication, assembly, and host cell lysis., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus is a leading cause of difficult-to-treat infections. The capacity of S. aureus to survive and persist within phagocytic cells is an important factor contributing to therapy failures and infection recurrence. Therefore, interfering with S. aureus intracellular persistence is key to treatment success. In this study, we used a S. aureus strain carrying the reporter mKikumeGR that enables the monitoring of the metabolic status of intracellular bacteria to achieve a better understanding of the molecular mechanisms facilitating S. aureus survival and persistence within macrophages. We found that shortly after bacteria internalization, a large fraction of macrophages harbored mainly S. aureus with high metabolic activity. This population decreased gradually over time with the concomitant increase of a macrophage subpopulation harboring S. aureus with low metabolic activity, which prevailed at later times. A dual RNA-seq analysis performed in each macrophage subpopulation showed that the host transcriptional response was similar between both subpopulations. However, intracellular S. aureus exhibited disparate gene expression profiles depending on its metabolic state. Whereas S. aureus with high metabolic activity exhibited a greater expression of genes involved in protein synthesis and proliferation, bacteria with low metabolic activity displayed a higher expression of oxidative stress response-related genes, silenced genes involved in energy-consuming processes, and exhibited a dormant-like state. Consequently, we propose that reducing metabolic activity and entering into a dormant-like state constitute a survival strategy used by S. aureus to overcome the adverse environment encountered within macrophages and to persist in the intracellular niche. IMPORTANCE The capacity of Staphylococcus aureus to survive and persist within phagocytic cells has been associated with antibiotic treatment failure and recurrent infections. Here, we investigated the molecular mechanisms leading to S. aureus persistence within macrophages using a reporter system that enables to distinguish between intracellular bacteria with high and low metabolic activity in combinstion with a dual RNA-seq approach. We found that with the progression of infection, intracellular S. aureus transitions from a high metabolic state to a low metabolic dormant-like state by turning off major energy-consuming processes while remaining viable. This process seems to be driven by the level of stress encountered in the intracellular niche. Our study indicates that effective therapies by which to treat S. aureus infections should be able to target not only high metabolic bacteria but also intracellular dormant-like S. aureus.

Strategically located at mucosal sites, mast cells are instrumental in sensing invading pathogens and modulating the quality of the ensuing immune responses depending on the nature of the infecting microbe. It is believed that mast cells produce type I IFN (IFN-I) in response to viruses, but not to bacterial infections, because of the incapacity of bacterial pathogens to internalize within mast cells, where signaling cascades leading to IFN-I production are generated. However, we have previously reported that, in contrast with other bacterial pathogens, Staphylococcus aureus can internalize into mast cells and therefore could trigger a unique response. In this study, we have investigated the molecular cross-talk between internalized S. aureus and the human mast cells HMC-1 using a dual RNA sequencing approach. We found that a proportion of internalized S. aureus underwent profound transcriptional reprogramming within HMC-1 cells to adapt to the nutrients and stress encountered in the intracellular environment and remained viable. HMC-1 cells, in turn, recognized intracellular S. aureus via cGMP-AMP synthase-STING-TANK-binding kinase 1 signaling pathway, leading to the production of IFN-I. Bacterial internalization and viability were crucial for IFN-I induction because inhibition of S. aureus internalization or infection with heat-killed bacteria completely prevented the production of IFN-I by HMC-1 cells. Feeding back in an autocrine manner in S. aureus -harboring HMC-1 cells and in a paracrine manner in noninfected neighboring HMC-1 cells, IFN-I promoted a cell-autonomous antimicrobial state by inducing the transcription of IFN-I-stimulated genes. This study provides unprecedented evidence of the capacity of mast cells to produce IFN-I in response to a bacterial pathogen., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis ( Nm ) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6 , CXCL1 , CXCL2 , CXCL8 , and CCL20 , and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans- endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm -responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.

Objective: We hypothesized that diabetes-related distress would vary by type of diabetes and medication regimen [Type 1 diabetes (T1DM), Type 2 diabetes with insulin use (T2DM-i), Type 2 diabetes without insulin use (T2DM)]. Thus, the aim of this study was to identify groups with elevated diabetes-related distress., Methods: We administered the 17-item Diabetes-related Distress Scale (DDS-17) to 585 patients. We collected demographics, medications, and lab results from patient records., Results: Patients were categorized by type of diabetes and medication: T1DM (n = 149); T2DM-i (n = 333); and T2DM (n = 103). ANOVA revealed significant differences in sample characteristics. ANCOVA were conducted on all four DDS-17 domains [Emotional Burden (EB); Physician-related Distress (PD); Regimen-related Distress (RD); and Interpersonal Distress (ID)]; covariates included in the models were sex, age, duration of diabetes, BMI, and HbA1c. EB was significantly lower in T1DM than T2DM-i, p < 0.05. In addition, RD was significantly lower in T1DM than either T2DM-i, p < 0.05 and T2DM, p < 0.05., Conclusions: EB and RD are higher for those with type 2 diabetes. Thus, interventions to reduce EB and RD need to be considered for patients with type 2 diabetes., Implications: DDS-17 is useful in identifying diabetes-related distress in patients with diabetes. Efforts need to be made to reduce EB and RD., (Published by Elsevier B.V.)