Perforin-2 Breaches the Envelope of Phagocytosed Bacteria Allowing Antimicrobial Effectors Access to Intracellular Targets.

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study, we used Salmonella enterica serovar Typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2's bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of Ags contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface Ag was found to be independent of Perforin-2. Consistent with our in vitro results, a protease-sensitive, periplasmic superoxide dismutase (SodCII) contributed to the virulence of S. Typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate, our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria, facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial cell.

Association of Rideshare Use With Alcohol-Associated Motor Vehicle Crash Trauma.

Importance: Motor vehicle crashes (MVCs) are an important public health concern. Recent trends suggest that introducing rideshare services has decreased the incidence of MVCs. However, detailed analyses linking rideshare volume, convictions for impaired driving, and nonfatal MVC traumas remain inconclusive. Objective: To determine if there is an association between rideshare use and MVC traumas and convictions for impaired driving in Houston, Texas. Design, Setting, and Participants: This multicenter cohort study was conducted between January 2007 and November 2019 with hospital data from the Red Duke Trauma Institute within the Memorial Hermann Hospital-Texas Medical Center and Ben Taub General Hospital. Rideshare data from Uber and Google covered trips taken within Houston, Texas, from February 2014 (the date of deployment of Uber to Houston) to December 2018. Impaired driving convictions included all indictments made by the Harris County, Texas, District Attorney's office from January 2007 to December 2018. All adults with MVC traumas evaluated at both centers in the study population (individuals >16 years with a mechanism of injury classified under "motor vehicle collision") were included. Impaired driving incidents were included only if the final legal outcome was conviction. Main Outcomes and Measures: The primary study outcomes were the incident rate ratios for hourly MVC traumas and daily impaired driving convictions. Results: A total of 23 491 MVC traumas (involving patients with a mean [SD] age of 37.9 [17.8] years and 14 603 male individuals [62.1%]), 93 742 impaired driving convictions, and more than 24 million Uber rides were analyzed. Following the introduction of Uber in February 2014, MVC traumas decreased by 23.8% (from a mean [SD] of 0.26 [0.04] to 0.21 [0.06] trauma incidents per hour) during peak trauma periods (Friday and Saturday nights). The incident rate ratio of MVC traumas following Uber deployment was 0.33 (95% CI, 0.17-0.67) per 1000 indexed rides (P = .002). Furthermore, rideshare use was associated with a significant, geographically linked reduction in impaired driving convictions between January 2014 to December 2019 (incidence rate ratio, 0.76 [95% CI, 0.73-0.78]; P < .001). Conclusions and Relevance: In this study, introducing rideshare services in the Houston metropolitan area was associated with significant reductions in MVC traumas and impaired driving convictions. Increased use of rideshares may be an effective means of reducing impaired driving and decreasing rate of MVC traumas.

Role of Ethnicity and Geographic Location on Glioblastoma IDH1/IDH2 Mutations.

BACKGROUND: Previous studies have demonstrated possible differences in glioblastoma (GBM) survival attributable to ethnicity. The goal of this study was to quantify oncogenic differences and evaluate the overall survival (OS) and progression-free survival (PFS) differences in GBM patients across race/ethnicity using both population-based surveillance and institutional data sets from the United States (US) and Mexico. METHODS: Retrospective cohort study comprising the Texas Cancer Registry (TCR, n = 4134) and referral institutions located in US (n = 254) and Mexico (n = 47) were evaluated. Primary outcomes include OS and PFS. Oncogenic differences attributable to ethnicity were assessed. IDH1/IDH2 status was evaluated by sequencing in US and Mexico samples. Kaplan-Meier and Cox proportional hazards regression for survival analysis. RESULTS: A total of 4134 GBM patients were identified from the TCR data set, ethnicity comparison demonstrated that Hispanic patients were diagnosed at a significantly younger age compared to non-Hispanic white patients (NHW) (median: 58 vs. 62, P < 0.001) and had improved OS (hazard ratio: 0.82, P < 0.001). In the oncogenic analysis, we observed a significant enrichment of IDH1/IDH2 mutations in Mexican Hispanic patients compared to US Hispanic patients (29.8% vs. 7.9%, P = 0.012); IDH2 mutations drove this difference. Post-progression survival was significantly shorter in patients from Mexico than US (3.0 vs. 11.4 months; P < 0.001), while OS remained similar. CONCLUSIONS: IDH2 mutations are more prevalent in Mexican Hispanic individuals compared to US individuals and may be a crucial contributor to the previously reported survival benefit of Hispanic individuals in large population databases. These findings are critical for both screening of IDH2 mutations and targeted interventions in GBM.

CexE Is a Coat Protein and Virulence Factor of Diarrheagenic Pathogens.

CexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenic Escherichia coli (ETEC); a frequent cause of diarrheal illnesses worldwide. However, an examination of sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. In addition, homologs of CexE are present in enteroaggregative E. coli (EAEC), Yersinia enterocolitica, Providencia alcalifaciens, and Citrobacter rodentium. Although it has been hypothesized that CexE and its homologs are virulence factors, this has yet to be tested. Thus the primary aim of this study was to determine if these proteins contribute to pathogenicity. Our secondary aim was determine if they are secreted coat proteins. Here we report that all neonatal mice infected with a wild-type strain of C. rodentium perished. In contrast a cexE mutant was significantly attenuated with 45% neonate survival. In adult mice the wild-type strain reached significantly higher loads in the large intestines and were shed in greater numbers than cexE mutants. Secretion of the CexE homolog in EAEC is dependent upon an atypical Type I secretion system that accepts its client from the periplasm rather than the cytoplasm. Insertion mutants of cexC, the putative ATPase of the CexE secretion system, were attenuated in our murine model. In vitro we found that CexC is required for the secretion of CexE to the outer membranes of both ETEC and C. rodentium. Secretion is not constitutive because CexE accumulates in the periplasm when the two pathogens are cultured under noninducing conditions. Although secretion conditions differ between ETEC and C. rodentium, secreted CexE remains predominantly associated with the outer membranes of both species. In aggregate these findings demonstrate that CexE is a secreted coat protein and virulence factor that promotes colonization of host intestinal tissues by enteric pathogens.

Impaired B Cell Function in Mice Lacking Perforin-2.

Perforin-2 (P2) is a pore-forming protein with cytotoxic activity against intracellular bacterial pathogens. P2 knockout (P2KO) mice are unable to control infections and die from normally non-lethal bacterial infections. Here we show that P2KO mice as compared to WT mice show significantly higher levels of systemic inflammation, measured by inflammatory markers in serum, due to continuous microbial translocation from the gut which cannot be controlled as these mice lack P2. Systemic inflammation in young and old P2KO mice induces intrinsic B cell inflammation. Systemic and B cell intrinsic inflammation are negatively associated with in vivo and in vitro antibody responses. Chronic inflammation leads to class switch recombination defects, which are at least in part responsible for the reduced in vivo and in vitro antibody responses in young and old P2KO vs. WT mice. These defects include the reduced expression of activation-induced cytidine deaminase (AID), the enzyme for class switch recombination, somatic hypermutation and IgG production and of its transcriptional activators E47 and Pax5. Of note, the response of young P2KO mice is not different from the one observed in old WT mice, suggesting that the chronic inflammatory status of mice lacking P2 may accelerate, or be equivalent, to that seen in old mice. The inflammatory status of the splenic B cells is associated with increased frequencies and numbers of the pro-inflammatory B cell subset called Age-associated B Cells (ABCs) in the spleen and the visceral adipose tissue (VAT) of P2KO old mice. We show that B cells differentiate into ABCs in the VAT following interaction with the adipocytes and their products, and this occurs more in the VAT of P2KO mice as compared to WT controls. This is to our knowledge the first study on B cell function and antibody responses in mice lacking P2.

Glioma induced alterations in fecal short-chain fatty acids and neurotransmitters.

Aim: To explore fecal short-chain fatty acids and neurotransmitter alterations in a mouse-glioma model and glioma patients. Methods: Liquid chromatography-mass spectrometry and 16S rRNA-sequencing from fecal samples were performed to measure metabolite levels and taxa abundance in mice/humans. Mice underwent GL261 implantation with/without temozolomide. Glioma patients were compared with healthy controls. Results: Glioma altered several short-chain fatty acids and neurotransmitter levels. Reduced 5-hydroxyindoleaceic acid and norepinephrine levels were seen in mice and humans. Interestingly, temozolomide treatment abrogates the effects of glioma on fecal metabolites. Conclusion: Our findings demonstrate the interplay between glioma and the gut-brain axis. Further work is required to identify pathways within the gut-brain axis by which glioma influences and promotes the modulation of fecal metabolites and microbiome.

MPEG1/perforin-2 mutations in human pulmonary nontuberculous mycobacterial infections.

Perforin-2 is a highly conserved pore-forming protein encoded by macrophage expressed gene 1 (MPEG1). A number of studies have shown that Perforin-2-deficient mice are unable to survive following a bacterial challenge that is nonlethal in WT mice. There is also recent evidence that Mpeg1+/- heterozygous mice display an intermediate killing ability compared with Mpeg1 WT and Mpeg1-/- mice. Despite these in vivo findings, to date, no perforin-2 deficiencies have been associated with human disease. Here, we report four patients with persistent nontuberculous mycobacterial infection who had heterozygous MPEG1 mutations. In vitro, neutrophils, macrophages, and B cells from these patients were unable to kill Mycobacterium avium as efficiently as normal controls. CRISPR mutagenesis validated the deleterious antibacterial activity of these mutations. These data suggest that perforin-2 haploinsufficiency may contribute to human susceptibility to infections with intracellular bacteria.

Corrigendum: Distinct Transcriptomic Features Are Associated with Transitional and Mature B-Cell Populations in the Mouse Spleen.

[This corrects the article on p. 30 in vol. 6, PMID: 25717326.].

Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2.

Perforin-2 (MPEG1) is an effector of the innate immune system that limits the proliferation and spread of medically relevant Gram-negative, -positive, and acid fast bacteria. We show here that a cullin-RING E3 ubiquitin ligase (CRL) complex containing cullin-1 and ßTrCP monoubiquitylates Perforin-2 in response to pathogen associated molecular patterns such as LPS. Ubiquitylation triggers a rapid redistribution of Perforin-2 and is essential for its bactericidal activity. Enteric pathogens such as Yersinia pseudotuberculosis and enteropathogenic Escherichia coli disarm host cells by injecting cell cycle inhibiting factors (Cifs) into mammalian cells to deamidate the ubiquitin-like protein NEDD8. Because CRL activity is dependent upon NEDD8, Cif blocks ubiquitin dependent trafficking of Perforin-2 and thus, its bactericidal activity. Collectively, these studies further underscore the biological significance of Perforin-2 and elucidate critical molecular events that culminate in Perforin-2-dependent killing of both intracellular and extracellular, cell-adherent bacteria.

Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria.

Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system.