ABSTRACT

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

IMPORTANCE Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ~$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.

ABSTRACT

Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.

IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

ABSTRACT

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro. Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11b^lo/– pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.

IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro. Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

ABSTRACT

Microbes affect vertebrates on timescales from daily to evolutionary, and the cumulative effect of these interactions is immense. However, how microbiomes compare across (host) species is poorly understood, as most studies focus on relatively few species. A recent mBio article by S. J. Song, J. G. Sanders, F. Delsuc, J. Metcalf, et al. (mBio 11:e02901-19, 2019, https://doi.org/10.1128/mBio.02901-19) expands our collective understanding of the vertebrate microbiome by analyzing ~900 species. They demonstrate that patterns within mammals contrast with those within birds. Their results suggest many hypotheses about the role of host ecology and evolution on microbiome variation. Bats, the only volant mammals, appear to contradict many of the general mammal microbiome trends, in some ways resembling birds. What role has powered flight, and the evolution thereof, played in microbiome structure and function? Comparative methods, mechanistic hypotheses, and theory will elucidate this exciting question (and others) that we can ask using Song, Sanders et al.’s data and results.

ABSTRACT

Recent global advocacy efforts have highlighted the importance of development of a vaccine against group A Streptococcus (GAS). Combo5 is a non-M protein-based vaccine that provides protection against GAS skin infection in mice and reduces the severity of pharyngitis in nonhuman primates. However, Combo5 with the addition of aluminum hydroxide (alum) as an adjuvant failed to protect against invasive GAS infection of mice. Here, we show that formulation of Combo5 with adjuvants containing saponin QS21 significantly improves protective efficacy, even though all 7 adjuvants tested generated high antigen-specific IgG antibody titers, including alum. Detailed characterization of Combo5 formulated with SMQ adjuvant, a squalene-in-water emulsion containing a TLR4 agonist and QS21, showed significant differences from the results obtained with alum in IgG subclasses generated following immunization, with an absence of GAS opsonizing antibodies. SMQ, but not alum, generated strong interleukin-6 (IL-6), gamma interferon (IFN-), and tumor necrosis alpha (TNF-α) responses. This work highlights the importance of adjuvant selection for non-M protein-based GAS vaccines to optimize immune responses and protective efficacy.

IMPORTANCE Availability of a group A Streptococcus vaccine remains an unmet public health need. Here, we tested different adjuvant formulations to improve the protective efficacy of non-M protein vaccine Combo5 in an invasive disease model. We show that novel adjuvants can dramatically shape the type of immune response developed following immunization with Combo5 and significantly improve protection. In addition, protection afforded by Combo5 is not mediated by opsonizing antibodies, believed to be the main correlate of protection against GAS infections. Overall, this report highlights the importance of adjuvant selection in raising protective immune responses against GAS invasive infection. Adjuvants that can provide a more balanced Th1/Th2-type response may be required to optimize protection of GAS vaccines, particularly those based on non-M protein antigens.

ABSTRACT

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.

IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

ABSTRACT

Environmental exposure has a significant impact on human health. While some airborne fungi can cause life-threatening infections, the impact of environment on fungal spore dispersal and transmission is poorly understood. The democratization of shotgun metagenomics allows us to explore important questions about fungal propagation. We focus on Pneumocystis, a genus of host-specific fungi that infect mammals via airborne particles. In humans, Pneumocystis jirovecii causes lethal infections in immunocompromised patients if untreated, although its environmental reservoir and transmission route remain unclear. Here, we attempt to clarify, by analyzing human exposome metagenomic data sets, whether humans are exposed to different Pneumocystis species present in the air but only P. jirovecii cells are able to replicate or whether they are selectively exposed to P. jirovecii. Our analysis supports the latter hypothesis, which is consistent with a local transmission model. These data also suggest that healthy carriers are a major driver for the transmission.

ABSTRACT

Recognition modes of individual T cell receptors (TCRs) are well studied, but factors driving the selection of TCR repertoires from primary through persistent human virus infections are less well understood. Using deep sequencing, we demonstrate a high degree of diversity of Epstein-Barr virus (EBV)-specific clonotypes in acute infectious mononucleosis (AIM). Only 9% of unique clonotypes detected in AIM persisted into convalescence; the majority (91%) of unique clonotypes detected in AIM were not detected in convalescence and were seeming replaced by equally diverse "de novo" clonotypes. The persistent clonotypes had a greater probability of being generated than nonpersistent clonotypes due to convergence recombination of multiple nucleotide sequences to encode the same amino acid sequence, as well as the use of shorter complementarity-determining regions 3 (CDR3s) with fewer nucleotide additions (i.e., sequences closer to germ line). Moreover, the two most immunodominant HLA-A2-restricted EBV epitopes, BRLF1₁₀₉ and BMLF1₂₈₀, show highly distinct antigen-specific public (i.e., shared between individuals) features. In fact, TCRα CDR3 motifs played a dominant role, while TCRβ played a minimal role, in the selection of TCR repertoire to an immunodominant EBV epitope, BRLF1. This contrasts with the majority of previously reported repertoires, which appear to be selected either on TCRβ CDR3 interactions with peptide/major histocompatibility complex (MHC) or in combination with TCRα CDR3. Understanding of how TCR-peptide-MHC complex interactions drive repertoire selection can be used to develop optimal strategies for vaccine design or generation of appropriate adoptive immunotherapies for viral infections in transplant settings or for cancer.

IMPORTANCE Several lines of evidence suggest that TCRα and TCRβ repertoires play a role in disease outcomes and treatment strategies during viral infections in transplant patients and in cancer and autoimmune disease therapy. Our data suggest that it is essential that we understand the basic principles of how to drive optimum repertoires for both TCR chains, α and β. We address this important issue by characterizing the CD8 TCR repertoire to a common persistent human viral infection (EBV), which is controlled by appropriate CD8 T cell responses. The ultimate goal would be to determine if the individuals who are infected asymptomatically develop a different TCR repertoire than those that develop the immunopathology of AIM. Here, we begin by doing an in-depth characterization of both CD8 T cell TCRα and TCRβ repertoires to two immunodominant EBV epitopes over the course of AIM, identifying potential factors that may be driving their selection.

ABSTRACT

Two-component signaling systems (TCSs) function to detect environmental cues and transduce this information into a change in transcription. In its simplest form, TCS-dependent regulation of transcription entails phosphoryl-transfer from a sensory histidine kinase to its cognate DNA-binding receiver protein. However, in certain cases, auxiliary proteins may modulate TCSs in response to secondary environmental cues. Caulobacter crescentus FixT is one such auxiliary regulator. FixT is composed of a single receiver domain and functions as a feedback inhibitor of the FixL-FixJ (FixLJ) TCS, which regulates the transcription of genes involved in adaptation to microaerobiosis. We sought to define the impact of fixT on Caulobacter cell physiology and to understand the molecular mechanism by which FixT represses FixLJ signaling. fixT deletion results in excess production of porphyrins and premature entry into stationary phase, demonstrating the importance of feedback inhibition of the FixLJ signaling system. Although FixT is a receiver domain, it does not affect dephosphorylation of the oxygen sensor kinase FixL or phosphoryl-transfer from FixL to its cognate receiver FixJ. Rather, FixT represses FixLJ signaling by inhibiting the FixL autophosphorylation reaction. We have further identified a 4-cysteine motif in Caulobacter FixT that binds an Fe-S cluster and protects the protein from degradation by the Lon protease. Our data support a model in which the oxidation of this Fe-S cluster promotes the degradation of FixT in vivo. This proteolytic mechanism facilitates clearance of the FixT feedback inhibitor from the cell under normoxia and resets the FixLJ system for a future microaerobic signaling event.

IMPORTANCE Two-component signal transduction systems (TCSs) are broadly conserved in the bacterial kingdom and generally contain two molecular components, a sensor histidine kinase and a receiver protein. Sensor histidine kinases alter their phosphorylation state in direct response to a physical or chemical cue, whereas receiver proteins "receive" the phosphoryl group from the kinase to regulate a change in cell physiology. We have discovered that a single-domain receiver protein, FixT, binds an Fe-S cluster and controls Caulobacter heme homeostasis though its function as a negative-feedback regulator of the oxygen sensor kinase FixL. We provide evidence that the Fe-S cluster protects FixT from Lon-dependent proteolysis in the cell and endows FixT with the ability to function as a second, autonomous oxygen/redox sensor in the FixL-FixJ signaling pathway. This study introduces a novel mechanism of regulated TCS feedback control by an Fe-S-binding receiver domain.

ABSTRACT

Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.

IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.

ABSTRACT

The appressoria that are generated by the rice blast fungus Magnaporthe oryzae in response to surface cues are important for successful colonization. Previous work showed that regulators of G-protein signaling (RGS) and RGS-like proteins play critical roles in appressorium formation. However, the mechanisms by which these proteins orchestrate surface recognition for appressorium induction remain unclear. Here, we performed comparative transcriptomic studies of Morgs mutant and wild-type strains and found that M. oryzae Aa91 (MoAa91), a homolog of the auxiliary activity family 9 protein (Aa9), was required for surface recognition of M. oryzae. We found that MoAA91 was regulated by the MoMsn2 transcription factor and that its disruption resulted in defects in both appressorium formation on the artificial inductive surface and full virulence of the pathogen. We further showed that MoAa91 was secreted into the apoplast space and was capable of competing with the immune receptor chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immune responses. In summary, we have found that MoAa91 is a novel signaling molecule regulated by RGS and RGS-like proteins and that MoAa91 not only governs appressorium development and virulence but also functions as an effector to suppress host immunity.

IMPORTANCE The rice blast fungus Magnaporthe oryzae generates infection structure appressoria in response to surface cues largely due to functions of signaling molecules, including G-proteins, regulators of G-protein signaling (RGS), mitogen-activated protein (MAP) kinase pathways, cAMP signaling, and TOR signaling pathways. M. oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 were found to be particularly important in appressorium development. To explore the mechanisms by which these proteins regulate appressorium development, we have performed a comparative in planta transcriptomic study and identified an auxiliary activity family 9 protein (Aa9) homolog that we named MoAa91. We showed that MoAa91 was secreted from appressoria and that the recombinant MoAa91 could compete with a chitin elicitor-binding protein precursor (CEBiP) for chitin binding, thereby suppressing chitin-induced plant immunity. By identifying MoAa91 as a novel signaling molecule functioning in appressorium development and an effector in suppressing host immunity, our studies revealed a novel mechanism by which RGS and RGS-like proteins regulate pathogen-host interactions.

ABSTRACT

Host persistence of bacteria is facilitated by mutational and recombinatorial processes that counteract loss of genetic variation during transmission and selection from evolving host responses. Genetic variation was investigated during persistent asymptomatic carriage of Neisseria meningitidis. Interrogation of whole-genome sequences for paired isolates from 25 carriers showed that de novo mutations were infrequent, while horizontal gene transfer occurred in 16% of carriers. Examination of multiple isolates per time point enabled separation of sporadic and transient allelic variation from directional variation. A comprehensive comparative analysis of directional allelic variation with hypermutation of simple sequence repeats and hyperrecombination of class 1 type IV pilus genes detected an average of seven events per carrier and 2:1 bias for changes due to localized hypermutation. Directional genetic variation was focused on the outer membrane with 69% of events occurring in genes encoding enzymatic modifiers of surface structures or outer membrane proteins. Multiple carriers exhibited directional and opposed switching of allelic variants of the surface-located Opa proteins that enables continuous expression of these adhesins alongside antigenic variation. A trend for switching from PilC1 to PilC2 expression was detected, indicating selection for specific alterations in the activities of the type IV pilus, whereas phase variation of restriction modification (RM) systems, as well as associated phasevarions, was infrequent. We conclude that asymptomatic meningococcal carriage on mucosal surfaces is facilitated by frequent localized hypermutation and horizontal gene transfer affecting genes encoding surface modifiers such that optimization of adhesive functions occurs alongside escape of immune responses by antigenic variation.

IMPORTANCE Many bacterial pathogens coexist with host organisms, rarely causing disease while adapting to host responses. Neisseria meningitidis, a major cause of meningitis and septicemia, is a frequent persistent colonizer of asymptomatic teenagers/young adults. To assess how genetic variation contributes to host persistence, whole-genome sequencing and hypermutable sequence analyses were performed on multiple isolates obtained from students naturally colonized with meningococci. High frequencies of gene transfer were observed, occurring in 16% of carriers and affecting 51% of all nonhypermutable variable genes. Comparative analyses showed that hypermutable sequences were the major mechanism of variation, causing 2-fold more changes in gene function than other mechanisms. Genetic variation was focused on genes affecting the outer membrane, with directional changes in proteins responsible for bacterial adhesion to host surfaces. This comprehensive examination of genetic plasticity in individual hosts provides a significant new platform for rationale design of approaches to prevent the spread of this pathogen.

ABSTRACT

Mobile elements—plasmids and phages—are important components of microbial function and evolution via traits that they encode and their capacity to shuttle genetic material between species. We here report the unusually rich array of mobile elements within the genome of Arsenophonus nasoniae, the son-killer symbiont of the parasitic wasp Nasonia vitripennis. This microbe’s genome has the highest prophage complement reported to date, with over 50 genomic regions that represent either intact or degraded phage material. Moreover, the genome is predicted to include 17 extrachromosomal genetic elements, which carry many genes predicted to be important at the microbe-host interface, derived from a diverse assemblage of insect-associated gammaproteobacteria. In our system, this diversity was previously masked by repetitive mobile elements that broke the assembly derived from short reads. These findings suggest that other complex bacterial genomes will be revealed in the era of long-read sequencing.

IMPORTANCE The biology of many bacteria is critically dependent on genes carried on plasmid and phage mobile elements. These elements shuttle between microbial species, thus providing an important source of biological innovation across taxa. It has recently been recognized that mobile elements are also important in symbiotic bacteria, which form long-lasting interactions with their host. In this study, we report a bacterial symbiont genome that carries a highly complex array of these elements. Arsenophonus nasoniae is the son-killer microbe of the parasitic wasp Nasonia vitripennis and exists with the wasp throughout its life cycle. We completed its genome with the aid of recently developed long-read technology. This assembly contained over 50 chromosomal regions of phage origin and 17 extrachromosomal elements within the genome, encoding many important traits at the host-microbe interface. Thus, the biology of this symbiont is enabled by a complex array of mobile elements.

ABSTRACT

Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG. The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975. legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.

IMPORTANCE Legionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires’ disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

ABSTRACT

Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa. Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducens fabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction.

IMPORTANCE Geobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria.

ABSTRACT

To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance.

IMPORTANCE Abuse of antibiotics often leads to increase of multidrug-resistant (MDR) bacteria, which threatens the life of human beings. To overcome threat of antibiotic resistance, scientists are developing a novel class of antibiotics, antimicrobial peptides, that can eradicate MDR bacteria. Unfortunately, these antibiotics have mainly been developed to cure bacterial skin infections rather than others, such as life-threatening sepsis. Major pharmaceutical companies have tried to develop antiseptic drugs; however, they have not been successful. Here, we report that AMPR-11, the antimicrobial peptide (AMP) derived from mitochondrial nonselective channel Romo1, has antimicrobial activity against Gram-positive and Gram-negative bacteria comprising many clinically isolated MDR strains. Moreover, AMPR-11 increased the survival rate in a murine model of sepsis caused by MDR bacteria. We propose that AMPR-11 could be a novel antiseptic drug candidate with a broad antimicrobial spectrum to overcome MDR bacterial infection.

ABSTRACT

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

ABSTRACT

Symbiotic microorganisms can have a profound impact on the host physiology and behavior, and novel relationships between symbionts and their hosts are continually discovered. A colony of social ants consists of various castes that exhibit distinct lifestyles and is, thus, a unique model for investigating how symbionts may be involved in host eusociality. Yet our knowledge of social ant-symbiont dynamics has remained rudimentary. Through 16S rRNA gene deep sequencing of the carpenter ant Camponotus japonicus symbiont community across various castes, we here report caste-dependent diversity of commensal gut microbiota and lineage divergence of "Candidatus Blochmannia," an obligate endosymbiont. While most prevalent gut-associated bacterial populations are found across all castes (Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria), we also discovered uncultured populations that are found only in males (belonging to Corynebacteriales, Alkanindiges, and Burkholderia). Most of those populations are not detected in laboratory-maintained queens and workers, suggesting that they are facultative gut symbionts introduced via environmental acquisition. Further inspection of "Ca. Blochmannia" endosymbionts reveals that two populations are dominant in all individuals across all castes but that males preferentially contain two different sublineages that are diversified from others. Clearly, each caste has distinct symbiont communities, suggesting an overlooked biological aspect of host-symbiont interaction in social insects.

IMPORTANCE Social animals, such as primates and some insects, have been shown to exchange symbiotic microbes among individuals through sharing diet or habitats, resulting in increased consistency of microbiota among social partners. The ant is a representative of social insects exhibiting various castes within a colony; queens, males, and nonreproductive females (so-called workers) show distinct morphologies, physiologies, and behaviors but tightly interact with each other in the nest. However, how this social context affects their gut microbiota has remained unclear. In this study, we deeply sequenced the gut symbiont community across various castes of the carpenter ant Camponotus japonicus. We report caste-dependent diversity of commensal gut microbial community and lineage divergence of the mutualistic endosymbiont "Candidatus Blochmannia." This report sheds light on the hidden diversity in microbial populations and community structure associated with guts of males in social ants.

ABSTRACT

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C–DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia-induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia-mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation.

IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies.

ABSTRACT

The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ~75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest.

IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a "do-it-yourself" kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

Drivers with blood-alcohol levels below legal limits cause 15% of all crash deaths that involve alcohol, a study in the March issue of the American Journal of Preventive Medicine finds.

To the Editor—The North Carolina Medical Board and North Carolina Medical Society have concerns regarding the Correspondence To the Editor in the March/April 2019 issue of the North Carolina Medical Journal titled, "Aid in Dying in North Carolina" [1]. Although we recognize the beliefs shared by the individual authors were not intended to be conclusive guidance regarding the status of aid in dying (AID) in North Carolina, we feel compelled to respond with a few clarifying notes.

The authors of the correspondence opine: "In light of the legal analysis of North Carolina law, we feel confident that AID can be provided to patients who request it" and that "physicians can provide AID ... without risk of a viable criminal or disciplinary action" [1].

In all matters of medical practice, including end-of-life matters, physicians and physician assistants must meet the standards of acceptable and prevailing medical practice and the ethics of the medical profession. If the Medical Board receives a complaint related to AID, it will evaluate the complaint and determine, utilizing expert consultants, whether the physician engaged in unprofessional conduct as defined by the North Carolina Medical Practice Act.

Further, disagreement exists within the medical community regarding the role of clinicians in medical AID. In one national survey, there was no consensus about the acceptability of AID among physicians and other health care professionals caring for older adults [2]. Respondents also expressed concerns about AID applied to vulnerable populations, including those with low health literacy, low English proficiency, disability, dependency,...

BACKGROUND Pregnant patients from rural counties of Western North Carolina face additional barriers when accessing comprehensive perinatal substance use disorders care at Project CARA as compared to patients local to the program in Buncombe County. We hypothesized regional patients would be less engaged in care.

METHOD Using a retrospective cohort design, univariate analyses (², t-test; P < .05) compared patients' characteristics, engagement in care, and delivery outcomes. Engagement in care, the primary outcome, was operationalized as: attendance at expected, program-specific prenatal and postpartum visits, utilization of in-house counseling, community-based and/or inpatient substance use disorders treatment, and maternal urine drug screen at delivery negative for illicit substances.

RESULTS Regional patients (n = 324) were more likely than Buncombe County patients (n = 284) to have opioid [209 (64.5%) versus 162 (57.0%)] or amphetamine/methamphetamine use disorders (25 [7.7%] versus 13 [4.6%]), but less likely to have cannabis use (19 [5.9%] versus 38 [13.4%]; P = .009) and concurrent psychiatric disorders (214 [66.0%] versus 220 [77.5%]; P = .002). Engagement at postpartum visits was the significantly different outcome between patients (110/221 [49.8%] versus 146/226 [64.6%]; P = .002).

LIMITATIONS Outcomes were available for 66.8% of regional and 79.6% of Buncombe County patients of one program in one predominately white, non-Hispanic region of the state.

CONCLUSION Contrary to our hypothesis, regional and Buncombe County women engaged in prenatal care equally. However, a more formal transition into the postpartum period is needed, especially for regional women. A "hub-and-spokes" model that extends delivery of perinatal substance use disorders care into rural communities may be more effective for engagement retention.

BACKGROUND Trauma—emotional, physical, and psychological—is common and associated with increased risk behaviors, low rates of care engagement and viral suppression, and overall poor health outcomes for people living with HIV (PLWH). This article presents the results of 15 in-depth, semi-structured interviews with PLWH in the Southeastern United States in which participants identified a trauma and described its long-lasting impact on their lives. Participants' trauma narratives described a wide range of traumas, including childhood sexual abuse, the loss of a loved one, and their HIV diagnosis.

METHODS Systematic qualitative analysis was used to delineate beliefs about causes, symptoms, treatments, quality of life, and health implications of trauma.

RESULTS: Fifteen participants completed semi-structured interviews that lasted on average 32 minutes. Participants described a wide spectrum of personal trauma that occurred both prior and subsequent to their HIV diagnosis. The types of trauma identified included physical, sexual, and psychological abuse inflicted by intimate partners, family members, and/or strangers.

LIMITATIONS A chief limitation of this study is selection bias. Additionally, the participant selection and content of the trauma narratives might have been affected by the surrounding context of the parent study centered on HIV, aging, and psychosocial stress. It is also difficult to interpret the distinction between discrete trauma experiences and the diagnosis of HIV, leading to potential information bias.

CONCLUSION This study highlights the importance of social support in coping with trauma and the effect of trauma on health-related behaviors. It also illustrates the need for additional research on the topic of trauma and trauma-informed care for PLWH. Understanding how different types of trauma affect individuals' lives is necessary to inform recommendations to provide better care for PLWH.

A number of partial articulated specimens of Cheiracanthus peachi nov. sp. have been collected from the Mey Flagstone Formation and Rousay Flagstone Formation within the Orcadian Basin of northern Scotland. The new, robust-bodied species is mainly distinguished by the scale ornament of radiating grooves rather than ridges. Compared to other Cheiracanthus species in the Orcadian Basin, C. peachi nov. sp. has quite a short range making it a useful zone fossil. As well as describing the general morphology of the specimens, we have also described and figured SEM images of scales and histological sections of all elements, enabling identification of other, isolated remains. Of particular biological interest is the identification of relatively robust, tooth-like gill rakers. Finally, the species has also been identified from isolated scales in Belarus, where it appears earlier and has a longer stratigraphical range, implying the species evolved in the marine deposits of the east and migrated west into the Orcadian Basin via the river systems.

Plants have evolved effective strategies to defend themselves against pathogen invasion. Starting from the plasma membrane with the recognition of microbe-associated molecular patterns (MAMPs) via pattern recognition receptors, internal cellular signaling pathways are induced to ultimately fend off the attack. Phospholipase D (PLD) hydrolyzes membrane phospholipids to produce phosphatidic acid (PA), which has been proposed to play a second messenger role in immunity. The Arabidopsis (Arabidopsis thaliana) PLD family consists of 12 members, and for some of these, a specific function in resistance toward a subset of pathogens has been shown. We demonstrate here that Arabidopsis PLD1, but not its close homologs PLD2 and PLD3, is specifically involved in plant immunity. Genetic inactivation of PLD1 resulted in increased resistance toward the virulent bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Botrytis cinerea. As pld1 mutant plants responded with elevated levels of reactive oxygen species to MAMP treatment, a negative regulatory function for this PLD isoform is proposed. Importantly, PA levels in pld1 mutants were not affected compared to stressed wild-type plants, suggesting that alterations in PA levels are not likely the cause for the enhanced immunity in the pld1 line. Instead, the plasma-membrane-attached PLD1 protein colocalized and associated with the BAK1-INTERACTING RECEPTOR-LIKE KINASES BIR2 and BIR3, which are known negative regulators of pattern-triggered immunity. Moreover, complex formation of PLD1 and BIR2 was further promoted upon MAMP treatment. Hence, we propose that PLD1 acts as a negative regulator of plant immune responses in complex with immunity-related proteins BIR2 and BIR3.

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that (1) RIN is indispensable for full ripening but not for the induction of ripening; and (2) the rin mutation, which produces nonripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that, contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.

The current coronavirus 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, raises important questions as to whether pre-morbid use or continued administration of inhaled corticosteroids (ICS) affects the outcomes of acute respiratory infections due to coronavirus. Many physicians are concerned about whether individuals positive for SARS-CoV-2 and taking ICS should continue them or stop them, given that ICS are often regarded as immunosuppressive. A number of key questions arise. Are people with asthma or COPD at increased risk of developing COVID-19? Do ICS modify this risk, either increasing or decreasing it? Do ICS influence the clinical course of COVID-19? (figure 1). Whether ICS modify the risk of developing COVID-19 or the clinical course of COVID-19 in people who do not have lung disease should also be considered (figure 1).

The aim of this study was to identify factors associated with the death of patients with COVID-19 pneumonia caused by the novel coronavirus SARS-CoV-2.

All clinical and laboratory parameters were collected prospectively from a cohort of patients with COVID-19 pneumonia who were hospitalised to Wuhan Pulmonary Hospital (Wuhan City, Hubei Province, China) between 25 December 2019 and 7 February 2020. Univariate and multivariate logistic regression was performed to investigate the relationship between each variable and the risk of death of COVID-19 pneumonia patients.

In total, 179 patients with COVID-19 pneumonia (97 male and 82 female) were included in the present prospective study, of whom 21 died. Univariate and multivariate logistic regression analysis revealed that age ≥65 years (OR 3.765, 95% CI 1.146-17.394; p=0.023), pre-existing concurrent cardiovascular or cerebrovascular diseases (OR 2.464, 95% CI 0.755-8.044; p=0.007), CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 (OR 3.982, 95% CI 1.132-14.006; p<0.001) and cardiac troponin I ≥0.05 ng·mL^–1 (OR 4.077, 95% CI 1.166-14.253; p<0.001) were associated with an increase in risk of mortality from COVID-19 pneumonia. In a sex-, age- and comorbid illness-matched case–control study, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1 remained as predictors for high mortality from COVID-19 pneumonia.

We identified four risk factors: age ≥65 years, pre-existing concurrent cardiovascular or cerebrovascular diseases, CD3⁺CD8⁺ T-cells ≤75 cells·μL^–1 and cardiac troponin I ≥0.05 ng·mL^–1. The latter two factors, especially, were predictors for mortality of COVID-19 pneumonia patients.

The treatment for obstructive sleep apnoea (OSA) with continuous positive airway pressure (CPAP) or mandibular advancement devices (MADs) is associated with blood pressure (BP) reduction; however, the overall effect is modest. The aim of this systematic review and meta-analysis of randomised controlled trials (RCTs) comparing the effect of such treatments on BP was to identify subgroups of patients who respond best to treatment.

The article search was performed in three different databases with specific search terms and selection criteria. From 2289 articles, we included 68 RCTs that compared CPAP or MADs with either passive or active treatment. When all the studies were pooled together, CPAP and MADs were associated with a mean BP reduction of –2.09 (95% CI –2.78– –1.40) mmHg for systolic BP and –1.92 (95% CI –2.40– –1.43) mmHg for diastolic BP and –1.27 (95% CI –2.34– –0.20) mmHg for systolic BP and –1.11 (95% CI –1.82– –0.41) mmHg for diastolic BP, respectively. The subgroups of patients who showed a greater response were those aged <60 years (systolic BP –2.93 mmHg), with uncontrolled BP at baseline (systolic BP –4.14 mmHg) and with severe oxygen desaturations (minimum arterial oxygen saturation measured by pulse oximetry <77%) at baseline (24-h systolic BP –7.57 mmHg).

Although this meta-analysis shows that the expected reduction of BP by CPAP/MADs is modest, it identifies specific characteristics that may predict a pronounced benefit from CPAP in terms of BP control. These findings should be interpreted with caution; however, they are particularly important in identifying potential phenotypes associated with BP reduction in patients treated for OSA.

Consider consumption of annonacin-containing plant products, including pawpaw, as a possible environmental risk factor for atypical parkinsonism.

Pediatricians are encouraged to address male adolescent sexual and reproductive health on a regular basis, including taking a sexual history, discussing healthy sexuality, performing an appropriate physical examination, providing patient-centered and age-appropriate anticipatory guidance, and administering appropriate vaccinations. These services can be provided to male adolescent patients in a confidential and culturally appropriate manner, can promote healthy sexual relationships and responsibility, can and involve parents in age-appropriate discussions about sexual health.

BACKGROUND AND OBJECTIVES:

High-quality cardiopulmonary resuscitation (CPR) increases the likelihood of survival of pediatric out-of-hospital cardiac arrest (OHCA). Maintenance of high-quality CPR during transition of care between prehospital and pediatric emergency department (PED) providers is challenging. Our objective for this initiative was to minimize pauses in compressions, in alignment with American Heart Association recommendations, for patients with OHCA during the handoffs from prehospital to PED providers. We aimed to decrease interruptions in compressions during the first 2 minutes of PED care from 17 seconds (baseline data) to 10 seconds over 12 months. Our secondary aims were to decrease the length of the longest pause in compressions to <10 seconds and eliminate encounters in which time to defibrillator pad placement was >120 seconds.

Multiple births are increasing in frequency related to advanced maternal age and fertility treatments, and they have an increased risk for congenital anomalies compared to singleton births. However, twins have the same congenital anomalies <15% of the time. Thus, having multiple births with discordant anomalies is a growing challenge for neonatologists. Although external anomalies can often be spotted quickly at delivery or sex differences between multiples can rapidly identify those with internal anomalies described on prenatal ultrasound, we present a case of male multiples, who would optimally receive different initial resuscitation strategies on the basis of the presence or absence of an internal anomaly. The similar size of 4 extremely preterm quadruplets raises concern for whether accurate, immediate identification of 1 neonate with a congenital diaphragmatic hernia will be reliable in the delivery room. Clinicians discuss the ethical considerations of an "all for one" approach to this resuscitation.

Positive selection causes beneficial alleles to rise to high frequency, resulting in a selective sweep of the diversity surrounding the selected sites. Accordingly, the signature of a selective sweep in an ancestral population may still remain in its descendants. Identifying signatures of selection in the ancestor that are shared among its descendants is important to contextualize the timing of a sweep, but few methods exist for this purpose. We introduce the statistic SS-H12, which can identify genomic regions under shared positive selection across populations and is based on the theory of the expected haplotype homozygosity statistic H12, which detects recent hard and soft sweeps from the presence of high-frequency haplotypes. SS-H12 is distinct from comparable statistics because it requires a minimum of only two populations, and properly identifies and differentiates between independent convergent sweeps and true ancestral sweeps, with high power and robustness to a variety of demographic models. Furthermore, we can apply SS-H12 in conjunction with the ratio of statistics we term and to further classify identified shared sweeps as hard or soft. Finally, we identified both previously reported and novel shared sweep candidates from human whole-genome sequences. Previously reported candidates include the well-characterized ancestral sweeps at LCT and SLC24A5 in Indo-Europeans, as well as GPHN worldwide. Novel candidates include an ancestral sweep at RGS18 in sub-Saharan Africans involved in regulating the platelet response and implicated in sudden cardiac death, and a convergent sweep at C2CD5 between European and East Asian populations that may explain their different insulin responses.

The way genes contribute to behavior is complicated. Although there are some single genes with large contributions, most behavioral differences are due to small effects from many interacting genes. This makes it hard to identify the genes that cause behavioral differences. Mutagenesis screens in model organisms, selective breeding experiments in animals, comparisons between related populations with different behaviors, and genome-wide association studies in humans are promising and complementary approaches to understanding the heritable aspects of complex behaviors. To connect genes to behaviors requires measuring behavioral differences, locating correlated genetic changes, determining when, where, and how these candidate genes act, and designing causative confirmatory experiments. This area of research has implications from basic discovery science to human mental health.

Phenotypic plasticity is defined as the property of organisms to produce distinct phenotypes in response to environmental variation. While for more than a century, biologists have proposed this organismal feature to play an important role in evolution and the origin of novelty, the idea has remained contentious. Plasticity is found in all domains of life, but only recently has there been an increase in empirical studies. This contribution is intended as a fresh view and will discuss current and future challenges of plasticity research, and the need to identify associated molecular mechanisms. After a brief summary of conceptual, theoretical, and historical aspects, some of which were responsible for confusion and contention, I will formulate three major research directions and predictions for the role of plasticity as a facilitator of novelty. These predictions result in a four-step model that, when properly filled with molecular mechanisms, will reveal plasticity as a major factor of evolution. Such mechanistic insight must be complemented with comparative investigations to show that plasticity has indeed created novelty and innovation. Together, such studies will help develop a true developmental evolutionary biology.

Key Points

Key Points

Key Points

Key Points

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 x 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 x 10³ CFU (50 LD₅₀) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Antimicrobial peptides play an important role in host defense against Vibrio cholerae. Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carR^r and carR^s alleles, respectively, and replaced the carR^s allele of a sensitive strain with the carR^r allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarR^s protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca²⁺ but increased with a rise in pH. The downregulation of almEFG in CarR^s strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.