TargeTron inactivation of plasmid-regulated Chlamydia trachomatis CT084 results in a nonlytic phenotype.

Chlamydia trachomatis is an obligate intracellular bacterium that causes blinding trachoma and sexually transmitted disease. The chlamydial plasmid is a critical virulence factor in the pathogenesis of these diseases. Plasmid gene protein 4 (Pgp4) plays a major role in chlamydial virulence by regulating the expression of both chromosomal genes and Pgp3. Despite the importance of Pgp4 in mediating lytic exit from host cells the pathogenic mechanism by which it functions is unknown. CT084 is a highly conserved chromosomal gene with homology to phospholipase D. We showed CT084 expression is regulated by Pgp4 and expressed late in the chlamydial developmental cycle. To investigate the function of CT084 in chlamydial lytic exit from infected cells, we made a CT084 null strain (ct084::bla) by using Targetron. The ct084::bla strain grew normally in vitro compared to wild-type strain; however, the strain did not lyse infected cells and produced significantly less and smaller plaques. Collectively, our finding shows Pgp4-regulated CT084-mediated chlamydia lytic exit from infected host cells.

Transcriptional profiling of immune responses in NHPs after low-dose, VSV-based vaccination against Marburg virus.

Infection with Marburg virus (MARV), the causative agent of Marburg virus disease (MVD), results in haemorrhagic disease and high case fatality rates (>40%) in humans. Despite its public health relevance, there are no licensed vaccines or therapeutics to prevent or treat MVD. A vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) is currently in clinical development. Previously, a single 10 million PFU dose of VSV-MARV administered 1-5 weeks before lethal MARV challenge conferred uniform protection in nonhuman primates (NHPs), demonstrating fast-acting potential. Additionally, our group recently demonstrated that even a low dose VSV-MARV (1000 PFU) protected NHPs when given 7 days before MARV challenge. In this study, we longitudinally profiled the transcriptional responses of NHPs vaccinated with this low dose of VSV-MARV either 14 or 7 days before lethal MARV challenge. NHPs vaccinated 14 days before challenge presented with transcriptional changes consistent with an antiviral response before challenge. Limited gene expression changes were observed in the group vaccinated 7 days before challenge. After challenge, genes related to lymphocyte-mediated immunity were only observed in the group vaccinated 14 days before challenge, indicating that the length of time between vaccination and challenge influenced gene expression. Our results indicate that a low dose VSV-MARV elicits distinct immune responses that correlate with protection against MVD. A low dose of VSV-MARV should be evaluated in clinical rails as it may be an option to deliver beneficial public health outcomes to more people in the event of future outbreaks.

Atypical Ebola Virus Disease in a Rhesus Macaque.

Ebola virus (EBOV)-Makona infected more than 30 000 people from 2013 to 2016 in West Africa, among them many health care workers including foreign nationals. Most of the infected foreign nationals were evacuated and treated in their respective home countries, resulting in detailed reports of the acute disease following EBOV infection as well as descriptions of symptoms now known as post-Ebola syndrome, which occurred months after the infection. Symptoms associated with this syndrome include uveitis and neurological manifestations. In 1 of our EBOV-Makona nonhuman primate (NHP) studies, 1 NHP was euthanized on day 28 after infection having completely recovered from the acute disease. During convalescence, this NHP developed neurological signs and acute respiratory distress requiring euthanasia. The organ tropism had changed with high virus titers in lungs, brain, eye, and reproductive organs but no virus in the typical target organs for acute EBOV infection. This in part reflects sequelae described for EBOV survivors albeit developing quicker after recovery from acute disease.

TargeTron Inactivation of Chlamydia trachomatis gseA Results in a Lipopolysaccharide 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid-Deficient Strain That Is Cytotoxic for Cells.

All members of the family Chlamydiaceae have lipopolysaccharides (LPS) that possess a shared carbohydrate trisaccharide antigen, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that is functionally uncharacterized. A single gene, genus-specific epitope (gseA), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a gseA-null strain (L2ΔgseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2ΔgseA lacked Kdo. L2ΔgseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating that core lipid A was retained by the mutant. The mutant strain produced a similar number of inclusions as the parental strain but yielded lower numbers of infectious elementary bodies. Transmission electron microscopy of L2ΔgseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2ΔgseA-infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide cross-linking, suggesting that the elementary body outer membrane structure was affected by the lack of Kdo. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2ΔgseA was significantly more cytotoxic to host cells than the wild type. The cytotoxic phenotype may result from an altered outer membrane biogenesis structure and/or function or, conversely, from a direct pathobiological effect of Kdo on an unknown host cell target. These findings implicate a previously unrecognized role for Kdo in host cell interactions that facilitates postinfection host cell survival.

Chlamydia evasion of neutrophil host defense results in NLRP3 dependent myeloid-mediated sterile inflammation through the purinergic P2X7 receptor.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis.

Transcriptomic profiling of the digestive tract of the rat flea, Xenopsylla cheopis, following blood feeding and infection with Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a highly lethal pathogen transmitted by the bite of infected fleas. Once ingested by a flea, Y. pestis establish a replicative niche in the gut and produce a biofilm that promotes foregut colonization and transmission. The rat flea Xenopsylla cheopis is an important vector to several zoonotic bacterial pathogens including Y. pestis. Some fleas naturally clear themselves of infection; however, the physiological and immunological mechanisms by which this occurs are largely uncharacterized. To address this, RNA was extracted, sequenced, and distinct transcript profiles were assembled de novo from X. cheopis digestive tracts isolated from fleas that were either: 1) not fed for 5 days; 2) fed sterile blood; or 3) fed blood containing ~5x108 CFU/ml Y. pestis KIM6+. Analysis and comparison of the transcript profiles resulted in identification of 23 annotated (and 11 unknown or uncharacterized) digestive tract transcripts that comprise the early transcriptional response of the rat flea gut to infection with Y. pestis. The data indicate that production of antimicrobial peptides regulated by the immune-deficiency pathway (IMD) is the primary flea immune response to infection with Y. pestis. The remaining infection-responsive transcripts, not obviously associated with the immune response, were involved in at least one of 3 physiological themes: 1) alterations to chemosensation and gut peristalsis; 2) modification of digestion and metabolism; and 3) production of chitin-binding proteins (peritrophins). Despite producing several peritrophin transcripts shortly after feeding, including a subset that were infection-responsive, no thick peritrophic membrane was detectable by histochemistry or electron microscopy of rat flea guts for the first 24 hours following blood-feeding. Here we discuss the physiological implications of rat flea infection-responsive transcripts, the function of X. cheopis peritrophins, and the mechanisms by which Y. pestis may be cleared from the flea gut.

Therapeutic responses to Roseomonas mucosa in atopic dermatitis may involve lipid-mediated TNF-related epithelial repair.

Dysbiosis of the skin microbiota is increasingly implicated as a contributor to the pathogenesis of atopic dermatitis (AD). We previously reported first-in-human safety and clinical activity results from topical application of the commensal skin bacterium Roseomonas mucosa for the treatment of AD in 10 adults and 5 children older than 9 years of age. Here, we examined the potential mechanism of action of R. mucosa treatment and its impact on children with AD less than 7 years of age, the most common age group for children with AD. In 15 children with AD, R. mucosa treatment was associated with amelioration of disease severity, improvement in epithelial barrier function, reduced Staphylococcus aureus burden on the skin, and a reduction in topical steroid requirements without severe adverse events. Our observed response rates to R. mucosa treatment were greater than those seen in historical placebo control groups in prior AD studies. Skin improvements and colonization by R. mucosa persisted for up to 8 months after cessation of treatment. Analyses of cellular scratch assays and the MC903 mouse model of AD suggested that production of sphingolipids by R. mucosa, cholinergic signaling, and flagellin expression may have contributed to therapeutic impact through induction of a TNFR2-mediated epithelial-to-mesenchymal transition. These results suggest that a randomized, placebo-controlled trial of R. mucosa treatment in individuals with AD is warranted and implicate commensals in the maintenance of the skin epithelial barrier.

Bacillus paranthracis Isolate from Blood of Fatal Ebola Virus Disease Case.

A Bacillus paranthracis isolate was cultured from the blood of a fatal Ebola virus disease (EVD) case in Liberia and was identified by whole genome sequencing. Although B. paranthracis has only recently been described and is poorly characterized, this case may represent the bacterial co-infection of an EVD patient.

Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons.

BACKGROUND: La Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection. METHODS: Human cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus. RESULTS: Overall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability. CONCLUSIONS: These findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.

Identification of rare HIV-1-infected patients with extreme CD4+ T cell decline despite ART-mediated viral suppression.

BACKGROUND: The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here, we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS: EXID's clinical and immunological characteristics were compared to immunological responders (IRs), immunological nonresponders (INRs), healthy controls (HCs), and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS: EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/µl, compared with CD4+ increases of 193 cells/µl and 427 cells/µl in INR and IR, respectively. EXID had reduced naive CD4+ T cells, but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR, but the IL-7 axis was profoundly perturbed compared with HC, IR, INR, and ICL. Genes involved in T cell and monocyte/macrophage function, autophagy, and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC, while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS: EXID is a distinct immunological outcome compared with previously described INR. Anti-CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID.

The Ability to Acquire Iron Is Inversely Related to Virulence and the Protective Efficacy of Francisella tularensis Live Vaccine Strain.

Francisella tularensis is a highly infectious bacterial pathogen that causes the potentially fatal disease tularemia. The Live Vaccine Strain (LVS) of F. tularensis subsp. holarctica, while no longer licensed as a vaccine, is used as a model organism for identifying correlates of immunity and bacterial factors that mediate a productive immune response against F. tularensis. Recently, it was reported that two biovars of LVS differed in their virulence and vaccine efficacy. Genetic analysis showed that they differ in ferrous iron homeostasis; lower Fe2+ levels contributed to increased resistance to hydrogen peroxide in the vaccine efficacious LVS biovar. This also correlated with resistance to the bactericidal activity of interferon γ-stimulated murine bone marrow-derived macrophages. We have extended these findings further by showing that a mutant lacking bacterioferritin stimulates poor protection against Schu S4 challenge in a mouse model of tularemia. Together these results suggest that the efficacious biovar of LVS stimulates productive immunity by a mechanism that is dependent on its ability to limit the toxic effects of oxidative stress by maintaining optimally low levels of intracellular Fe2+.

Draft Genome Sequences of the Avirulent Coxiella burnetii Dugway 7D77-80 and Dugway 7E65-68 Strains Isolated from Rodents in Dugway, Utah.

Here, we present the draft genome sequences of the Coxiella burnetii Dugway 7D77-80 and Dugway 7E65-68 strains, which were isolated from rodents in Dugway, UT, in the 1950s. The strains reside in a distinct genomic group of C. burnetii and are considered avirulent despite having the largest genomes of the Coxiella genus.

Draft Genome Sequences of Historical Strains of Coxiella burnetii Isolated from Cow's Milk and a Goat Placenta.

Here, we report draft genome sequences of historical strains of Coxiella burnetii derived from cow's milk and the placenta of a goat that had aborted. The California and Ohio milk strains display a different sequence type than do contemporary milk strains.

Draft Genome Sequences of Three Coxiella burnetii Strains Isolated from Q Fever Patients.

In the current study, we determined the draft genome sequences of three Coxiella burnetii human disease isolates. The Coxiella burnetii Turkey (RSA315) and Dyer (RSA345) strains were isolated from acute Q fever patients, while the Ko (Q229) strain was isolated from a Q fever endocarditis patient.

Whole-Genome Sequence of Coxiella burnetii Nine Mile RSA439 (Phase II, Clone 4), a Laboratory Workhorse Strain.

Here, we report the whole-genome sequence of Coxiella burnetii Nine Mile RSA439 (phase II, clone 4), a laboratory strain used extensively to investigate the biology of this intracellular bacterial pathogen. The genome consists of a 1.97-Mb chromosome and a 37.32-kb plasmid.

Contrasting antibody responses to intrasubtype superinfection with CRF02_AG.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.

Treatment as Prevention: Characterization of Partner Infections in the HIV Prevention Trials Network 052 Trial.

HIV Prevention Trials Network 052 demonstrated that antiretroviral therapy (ART) prevents HIV transmission in serodiscordant couples. HIV from index-partner pairs was analyzed to determine the genetic linkage status of partner infections. Forty-six infections were classified as linked, indicating that the index was the likely source of the partner's infection. Lack of viral suppression and higher index viral load were associated with linked infection. Eight linked infections were diagnosed after the index started ART: 4 near the time of ART initiation and 4 after ART failure. Linked infections were not observed when the index participant was stably suppressed on ART.

Molluscum Contagiosum Virus Transcriptome in Abortively Infected Cultured Cells and a Human Skin Lesion.

UNLABELLED: Molluscum contagiosum virus (MOCV), the only circulating human-specific poxvirus, has a worldwide distribution and causes benign skin lesions that may persist for months in young children and severe infections in immunosuppressed adults. Studies of MOCV are restricted by the lack of an efficient animal model or a cell culture replication system. We used next-generation sequencing to analyze and compare polyadenylated RNAs from abortive MOCV infections of several cell lines and a human skin lesion. Viral RNAs were detected for 14 days after MOCV infection of cultured cells; however, there was little change in the RNA species during this time and a similar pattern occurred in the presence of an inhibitor of protein synthesis, indicating a block preventing postreplicative gene expression. Moreover, a considerable number of MOCV RNAs mapped to homologs of orthopoxvirus early genes, but few did so to homologs of intermediate or late genes. The RNAs made during in vitro infections represent a subset of RNAs detected in human skin lesions which mapped to homologs of numerous postreplicative as well as early orthopoxvirus genes. Transfection experiments using fluorescent protein and luciferase reporters demonstrated that vaccinia virus recognized MOCV intermediate and late promoters, indicating similar gene regulation. The specific recognition of the intermediate promoter in MOCV-infected cells provided evidence for the synthesis of intermediate transcription factors, which are products of early genes, but not for late transcription factors. Transcriptome sequencing (RNA-seq) and reporter gene assays may be useful for testing engineered cell lines and conditions that ultimately could provide an in vitro replication system. IMPORTANCE: The inability to propagate molluscum contagiosum virus, which causes benign skin lesions in young children and more extensive infections in immunosuppressed adults, has constrained our understanding of the biology of this human-specific virus. In the present study, we characterized the RNAs synthesized in abortively infected cultured cells and a human skin lesion by next-generation sequencing. These studies provided an initial transcription map of the MOCV genome, suggested temporal regulation of gene expression, and indicated that the in vitro replication block occurs prior to intermediate and late gene expression. RNA-seq and reporter assays, as described here, may help to further evaluate MOCV gene expression and define conditions that could enable MOCV replication in vitro.

Deciphering poxvirus gene expression by RNA sequencing and ribosome profiling.

UNLABELLED: The more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome. IMPORTANCE: This report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological advances allowing examination of the translated regions of mRNAs including start sites at single-nucleotide resolution. Vaccinia virus ribosome-associated mRNA sequences were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h after infection. The ribosome profiling approach was particularly valuable for poxviruses because of the close spacing of approximately 200 open reading frames and extensive transcriptional read-through resulting in overlapping mRNAs. The expression of intermediate and late genes, in particular, was visualized with unprecedented clarity and quantitation. We also identified novel putative translation initiation sites that were mostly associated with short protein coding sequences. The results provide a framework for further studies of poxvirus gene expression.

Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae.

Infections caused by drug-resistant bacteria are a major problem worldwide. Carbapenem-resistant Klebsiella pneumoniae, most notably isolates classified as multilocus sequence type (ST) 258, have emerged as an important cause of hospital deaths. ST258 isolates are predominantly multidrug resistant, and therefore infections caused by them are difficult to treat. It is not known why the ST258 lineage is the most prevalent cause of multidrug-resistant K. pneumoniae infections in the United States and other countries. Here we tested the hypothesis that carbapenem-resistant ST258 K. pneumoniae is a single genetic clone that has disseminated worldwide. We sequenced to closure the genomes of two ST258 clinical isolates and used these genomes as references for comparative genome sequencing of 83 additional clinical isolates recovered from patients at diverse geographic locations worldwide. Phylogenetic analysis of the SNPs in the core genome of these isolates revealed that ST258 K. pneumoniae organisms are two distinct genetic clades. This unexpected finding disproves the single-clone hypothesis. Notably, genetic differentiation between the two clades results from an ∼ 215-kb region of divergence that includes genes involved in capsule polysaccharide biosynthesis. The region of divergence appears to be a hotspot for DNA recombination events, and we suggest that this region has contributed to the success of ST258 K. pneumoniae. Our findings will accelerate research on novel diagnostic, therapeutic, and vaccine strategies designed to prevent and/or treat infections caused by multidrug resistant K. pneumoniae.