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Publications

Fitting Pieces Into the Puzzle of Pseudomonas aeruginosa Type III Secretion System Gene Expression

Abstract
Type III secretion systems (T3SS) are widely distributed in Gram-negative microorganisms and critical for host-pathogen and host-symbiont interactions with plants and animals. Central features of the T3SS are a highly conserved set of secretion and translocation genes and contact dependence wherein host-pathogen interactions trigger effector protein delivery and serve as an inducing signal for T3SS gene expression. In addition to these conserved features, there are pathogen-specific properties that include a unique repertoire of effector genes and mechanisms to control T3SS gene expression. The Pseudomonas aeruginosa T3SS serves as a model system to understand transcriptional and posttranscriptional mechanisms involved in the control of T3SS gene expression. The central regulatory feature is a partner-switching system that controls the DNA-binding activity of ExsA, the primary regulator of T3SS gene expression. Superimposed upon the partner-switching mechanism are cyclic AMP and cyclic di-GMP signaling systems, two-component systems, global regulators, and RNA-binding proteins that have positive and negative effects on ExsA transcription and/or synthesis. In the present review, we discuss advances in our understanding of how these regulatory systems orchestrate the activation of T3SS gene expression in the context of acute infections and repression of the T3SS as P. aeruginosa adapts to and colonizes the cystic fibrosis airways.

Authors:
Emily A Williams McMackin, Louise Djapgne, Jodi M Corley, Timothy L Yahr


Oligodendrocytes that survive acute coronavirus infection induce prolonged inflammatory responses in the CNS

 

Abstract
Neurotropic strains of mouse hepatitis virus (MHV), a coronavirus, cause acute and chronic demyelinating encephalomyelitis with similarities to the human disease multiple sclerosis. Here, using a lineage-tracking system, we show that some cells, primarily oligodendrocytes (OLs) and oligodendrocyte precursor cells (OPCs), survive the acute MHV infection, are associated with regions of demyelination, and persist in the central nervous system (CNS) for at least 150 d. These surviving OLs express major histocompatibility complex (MHC) class I and other genes associated with an inflammatory response. Notably, the extent of inflammatory cell infiltration was variable, dependent on anatomic location within the CNS, and without obvious correlation with numbers of surviving cells. We detected more demyelination in regions with larger numbers of T cells and microglia/macrophages compared to those with fewer infiltrating cells. Conversely, in regions with less inflammation, these previously infected OLs more rapidly extended processes, consistent with normal myelinating function. Together, these results show that OLs are inducers as well as targets of the host immune response and demonstrate how a CNS infection, even after resolution, can induce prolonged inflammatory changes with CNS region-dependent impairment in remyelination.

Authors:
Ruangang Pan, Qinran Zhang, Scott M Anthony, Yu Zhou, Xiufen Zou, Martin Cassell, Stanley Perlman


TLR2 on Blood Monocytes Senses Dengue Virus Infection and Its Expression Correlates With Disease Pathogenesis

Lead author is Postdoctoral Student Scholar  Alberto Aguilar-Briseño, PhD who is in the Wendy Maury, PhD lab. 

 

Abstract

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis.

Authors:
José A Aguilar-Briseño, Vinit Upasani, Bram M Ter Ellen, Jill Moser, Mindaugas Pauzuolis 1, Mariana Ruiz-Silva, Sothy Heng , Denis Laurent , Rithy Choeung , Philippe Dussart , Tineke Cantaert , Jolanda M Smit , Izabela A Rodenhuis-Zybe


Hfq and sRNA 179 Inhibit Expression of the Pseudomonas Aeruginosa cAMP-Vfr and Type III Secretion Regulons

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen causing skin and soft tissue, respiratory, and bloodstream infections. The type III secretion system (T3SS) is one important virulence factor. Production of the T3SS is controlled by ExsA, a transcription factor that activates expression of the entire T3SS regulon. Global regulators including Vfr, RsmA, and Hfq also contribute to regulation of the T3SS. Vfr is a cAMP-responsive transcription factor that activates exsA transcription. RsmA, an RNA-binding protein, inversely controls expression of the T3SS and the type VI secretion system (T6SS). Hfq is an RNA chaperone that functions by stabilizing small noncoding RNAs (sRNAs) and/or facilitating base pairing between sRNAs and mRNA targets. A previous study identified sRNA 1061, which directly targets the exsA mRNA and likely inhibits ExsA synthesis. In this study, we screened an sRNA expression library and identified sRNA 179 as an Hfq-dependent inhibitor of T3SS gene expression. Further characterization revealed that sRNA 179 inhibits the synthesis of both ExsA and Vfr. The previous finding that RsmA stimulates ExsA and Vfr synthesis suggested that sRNA 179 impacts the Gac/Rsm system. Consistent with that idea, the inhibitory activity of sRNA 179 is suppressed in a mutant lacking rsmY and rsmZ, and sRNA 179 expression stimulates rsmY transcription. RsmY and RsmZ are small noncoding RNAs that sequester RsmA from target mRNAs. Our combined findings show that Hfq and sRNA 179 indirectly regulate ExsA and Vfr synthesis by reducing the available pool of RsmA, leading to reduced expression of the T3SS and cAMP-Vfr regulons.IMPORTANCE Control of gene expression by small noncoding RNA (sRNA) is well documented but underappreciated. Deep sequencing of mRNA preparations from Pseudomonas aeruginosa suggests that >500 sRNAs are generated. Few of those sRNAs have defined roles in gene expression. To address that knowledge gap, we constructed an sRNA expression library and identified sRNA 179 as a regulator of the type III secretion system (T3SS) and the cAMP-Vfr regulons. The T3SS- and cAMP-Vfr-controlled genes are critical virulence factors. Increased understanding of the signals and regulatory mechanisms that control these important factors will enhance our understanding of disease progression and reveal potential approaches for therapeutic intervention.

Authors:
Kayley H Janssen, Jodi M Corley, Louise Djapgne, J T Cribbs, Deven Voelker, Zachary Slusher, Robert Nordell, Elizabeth E Regulski, Barbara I Kazmierczak, Emily Williams McMackin, Timothy L Yahr


Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment

Summary

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Authors:
Jing Sun, Zhen Zhuang, Jian Zheng...Paul McCray Jr., Stanley Perlman, Jincun Zhao


Key Positions of HIV-1 Env and Signatures of Vaccine Efficacy Show Gradual Reduction of Population Founder Effects at the Clade and Regional Levels

Abstract

HIV-1 group M was transmitted to humans nearly one century ago. The virus has since evolved to form distinct clades, which spread to different regions of the world. The envelope glycoproteins (Envs) of HIV-1 have rapidly diversified in all infected populations. We examined whether key antigenic sites of Env and signatures of vaccine efficacy are evolving toward similar or distinct structural forms in different populations worldwide. Patterns of amino acid variants that emerged at each position of Env were compared between diverse HIV-1 clades and isolates from different geographic regions. Interestingly, at each Env position, the amino acid in the clade ancestral or regional-founder virus was replaced by a unique frequency distribution (FD) of amino acids. FDs are highly conserved in populations from different regions worldwide and in paraphyletic and monophyletic subclade groups. Remarkably, founder effects of Env mutations at the clade and regional levels have gradually decreased during the pandemic by evolution of each site toward the unique combination of variants. Therefore, HIV-1 Env is evolving at a population level toward well-defined "target" states; these states are not specific amino acids but rather specific distributions of amino acid frequencies. Our findings reveal the powerful nature of the forces that guide evolution of Env and their conservation across different populations. Such forces have caused a gradual decrease in the interpopulation diversity of Env despite an increasing intrapopulation diversity.IMPORTANCE The Env protein of HIV-1 is the primary target in AIDS vaccine design. Frequent mutations in the virus increase the number of Env forms in each population, limiting the efficacy of AIDS vaccines. Comparison of newly emerging forms in different populations showed that each position of Env is evolving toward a specific combination of amino acids. Similar changes are occurring in different HIV-1 subtypes and geographic regions toward the same position-specific combinations of amino acids, often from distinct ancestral sequences. The predictable nature of HIV-1 Env evolution, as shown here, provides a new framework for designing vaccines that are tailored to the unique combination of variants expected to emerge in each virus subtype and geographic region.

Authors:
Changze Han, Jacklyn Johnson, Rentian Dong, Raghavendranath Kandula, Alexa Kort, Maria Wong, Tianbao Yang, Patrick J Breheny, Grant D Brown, Hillel Haim


Infection-induced plasmablasts are a nutrient sink that impairs humoral immunity to malaria

Abstract

Plasmodium parasite–specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.

Authors:
Vijay R, Guthmiller JJ, Sturtz AJ, Surette FA, Rogers KJ, Sompallae RR, Li F, Pope RL, Chan JA, de Labastida Rivera F, Andrew D, Webb L, Maury WJ, Xue HH, Engwerda CR, McCarthy JS, Boyle MJ, Butler NS


Acute Plasmodium Infection Promotes Interferon-Gamma-Dependent Resistance to Ebola Virus Infection 

Abstract

During the 2013-2016 Ebola virus (EBOV) epidemic, a significant number of patients admitted to Ebola treatment units were co-infected with Plasmodium falciparum, a predominant agent of malaria. However, there is no consensus on how malaria impacts EBOV infection. The effect of acute Plasmodium infection on EBOV challenge was investigated using mouse-adapted EBOV and a biosafety level 2 (BSL-2) model virus. We demonstrate that acute Plasmodium infection protects from lethal viral challenge, dependent upon interferon gamma (IFN-γ) elicited as a result of parasite infection. Plasmodium-infected mice lacking the IFN-γ receptor are not protected. Ex vivo incubation of naive human or mouse macrophages with sera from acutely parasitemic rodents or macaques programs a proinflammatory phenotype dependent on IFN-γ and renders cells resistant to EBOV infection. We conclude that acute Plasmodium infection can safeguard against EBOV by the production of protective IFN-γ. These findings have implications for anti-malaria therapies administered during episodic EBOV outbreaks in Africa.

Authors:
Rogers KJ, Shtanko O, Vijay R, Mallinger LN, Joyner CJ, Galinski MR, Butler NS, Maury W


The High Content of Fructose in Human Semen Competitively Inhibits Broad and Potent Antivirals that Target High-mannose Glycans

ABSTRACT

Semen is the primary transmission vehicle for various pathogenic viruses. Initial steps of transmission, including cell attachment and entry, likely occur in the presence of semen. However, the unstable nature of human seminal plasma and its toxic effects on cells in culture limit the in vitro study of virus infection and inhibition in this medium. We found that whole semen significantly reduces the potency of antibodies and microbicides that target glycans on the envelope glycoproteins (Envs) of HIV-1. The extraordinarily high concentration of the monosaccharide fructose in semen contributes significantly to the effect by competitively inhibiting binding of ligands to α1,2-linked mannose residues on Env. Infection and inhibition in whole human semen are accurately mimicked by a stable synthetic simulant of seminal fluid we formulated. Our findings indicate that, in addition to the protein content of biological secretions, their small-solute composition impacts the potency of antiviral microbicides and mucosal antibodies.

Authors:
Johnson J, Flores MG, Rosa J, Han C, Salvi AM, DeMali KA, Jagnow JR, Sparks A, Haim H


Staphylococcal Virulence Factors on the Skin of Atopic Dermatitis Patients

Abstract

Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, infective endocarditis, osteoarticular, pleuropulmonary, and device-related infections. Virulence factors secreted by S. aureus, including superantigens and cytotoxins, play significant roles in driving disease. The ability to identify virulence factors present at the site of infection will be an important tool in better identifying and understanding how specific virulence factors contribute to disease. Previously, virulence factor production has been determined by culturing S. aureus isolates and detecting the mRNA of specific virulence factors. We demonstrated for the first time that virulence factors can be directly detected at the protein level from human samples, removing the need to first culture isolated bacteria. Superantigens and cytotoxins were detected and quantified with a Western dot blot assay by using reconstituted skin swabs obtained from patients with atopic dermatitis. This methodology will significantly enhance our ability to investigate the complex host-microbe environment and the effects various therapies have on virulence factor production. Overall, the ability to directly quantify virulence factors present at the site of infection or colonization will enhance our understanding of S. aureus-related diseases and help identify optimal treatments.IMPORTANCE For the first time, we show that secreted staphylococcal virulence factors can be quantified at the protein level directly from skin swabs obtained from the skin of atopic dermatitis patients. This technique eliminates the need to culture Staphylococcus aureus and then test the strain's potential to produce secreted virulence factors. Our methodology shows that secreted virulence factors are present on the skin of atopic patients and provides a more accurate means of evaluating the physiological impact of S. aureus in inflammatory diseases such as atopic dermatitis.

Authors:
Moran MC, Cahill MP, Brewer MG, Yoshida T, Knowlden, Perez-Nazario, Schlievert PM, Beck LA


High Prevalence of Staphylococcus aureus Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates

Abstract

BACKGROUND:

Staphylococcus aureus is a highly prevalent respiratory pathogen in cystic fibrosis (CF). It is unclear how this organism establishes chronic infections in CF airways. We hypothesized that S. aureus isolates from patients with CF would share common virulence properties that enable chronic infection.

METHODS:

77 S. aureus isolates were obtained from 45 de-identified patients with CF at the University of Iowa. We assessed isolates phenotypically and used genotyping assays to determine the presence or absence of 18 superantigens (SAgs).

RESULTS:

We observed phenotypic diversity among S. aureus isolates from patients with CF. Genotypic analysis for SAgs revealed 79.8% of CF clinical isolates carried all six members of the enterotoxin gene cluster (EGC). MRSA and MSSA isolates had similar prevalence of SAgs. We additionally observed that EGC SAgs were prevalent in S. aureus isolated from two geographically distinct CF centers.

CONCLUSIONS:

S. aureus SAgs belonging to the EGC are highly prevalent in CF clinical isolates. The greater prevalence in these SAgs in CF airway specimens compared to skin isolates suggests that these toxins confer selective advantage in the CF airway.

Authors:
Fischer AJ, Kilgore SH, Singh SB, Allen PD, Hansen AR, Limoli DH, Schlievert PM.


Effect of non-absorbent intravaginal menstrual/contraceptive products on Staphylococcus aureus and production of the superantigen TSST-1

Abstract

Tampons are associated with toxic shock syndrome (mTSS). One reason for this association is oxygen introduction within tampons into the anaerobic vagina. Oxygen is required for Staphylococcus aureus to produce TSS toxin-1 (TSST-1). There have been changes in use of medical devices to control menstrual flow, including increased use of menstrual discs and cups. These devices composed of solid, flexible materials do not absorb menstrual fluid and thus do not trap oxygen. This study evaluates tampons and non-absorbent devices for effect on S. aureus and TSST-1 production. There are three in vitro tests to evaluate devices for effect on TSST-1 production: (1) stationary flask, (2) shake flask, and (3) tampon sac. In this study, 100% rayon and 100% cotton tampons with three absorbencies, contraceptive diaphragms, and menstrual discs and cups were tested for effect on S. aureus growth and TSST-1 production. Product composition did not affect bacterial growth or TSST-1 production. Tampons showed no effect on S. aureus growth compared with no-tampon controls, but tampons showed enhanced TSST-1 production as a function of trapped oxygen in stationary cultures and tampon sacs but not in shake flasks. The non-absorbent devices showed no enhanced S. aureus growth or TSST-1 production compared with no-device controls. These studies are consistent with the association of tampons with mTSS as a function of absorbency, but they suggest the occasional association of mTSS with non-absorbent devices may be coincidental as opposed to co-causative.

Authors:
Schlievert PM


Pre-existing neutralizing antibodies prevent CD8 T cell-mediated immunopathology following respiratory syncytial virus infection

Abstract

Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.

Authors:
Schmidt M, Meyerholz D, Varga S


You Shall Not Pass: Memory CD8 T Cells in Liver-Stage Malaria

Highlights

Novel discoveries about the role of memory CD8 T cells in mediating immunity to liver-stage malaria are informing methods of resolving historical issues of malaria vaccine efficacy and implementation.

Liver resident memory (Trm) cells are essential to mediate sterilizing immunity against liver-stage malaria after whole-parasite immunization in mouse models. Circulating memory CD8 T cell contributions to protection remain undefined.

Mechanisms of liver Trm cell generation, maintenance, and protective function are active areas of investigation.

The unique hepatic microenvironment appears to dictate phenotypic characteristics and motility dynamics of local liver Trm cells at homeostasis.

Authors:
Lefebvre M, Harty, J


The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells

Abstract

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing wild-type (WT) SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide (LPS)-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-a, IFN-b, and TNF-a mRNA levels induced by Sendai virus (SeV) infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in LPS-treated or SeV-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-β mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1’s nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

Authors:
Qin Z, Bonifati S, St. Gelais C, Li T-W, Kim S-H, Antonucci, J, Mahboubi B, Yount J, Xiong Y, Kim B, Wu L.


The lipid membrane of HIV-1 stabilizes the viral envelope glycoproteins and modulates their sensitivity to antibody neutralization.

Abstract

The envelope glycoproteins (Envs) of HIV-1 are embedded in the cholesterol-rich lipid membrane of the virus. Chemical depletion of cholesterol from HIV-1 particles inactivates their infectivity. We observed that diverse HIV-1 strains exhibit a range of sensitivities to such treatment. Differences in sensitivity to cholesterol depletion could not be explained by variation in Env components known to interact with cholesterol, including the cholesterol-recognition motif and cytoplasmic tail of gp41. Using antibody-binding assays, measurements of virus infectivity and analyses of lipid membrane order, we found that depletion of cholesterol from HIV-1 particles decreases the conformational stability of Env. It enhances exposure of partially cryptic epitopes on the trimer and increases sensitivity to structure-perturbing treatments such as antibodies and cold denaturation. Substitutions in the cholesterol-interacting motif of gp41 induced similar effects as depletion of cholesterol. Surface-acting agents, which are incorporated into the virus lipid membrane, caused similar effects as disruption of the Env-cholesterol interaction. Furthermore, substitutions in gp120 that increased structural stability of Env (i.e., induced a "closed" conformation of the trimer) increased virus resistance to cholesterol depletion and to the surface-acting agents. Collectively, these results indicate a critical contribution of the viral membrane to the stability of the Env trimer and to neutralization resistance against antibodies. Our findings suggest that the potency of poorly neutralizing antibodies, which are commonly elicited in vaccinated individuals, may be markedly enhanced by altering the lipid composition of the viral membrane.

Authors:
Salimi H, Johnson J Flores MG, Zhang MS, O'Malley YQ, Houtman JC, Schlievert PM, Haim H


IL-4/IL-13 polarization of macrophages enhances Ebola virus glycoprotein-dependent infection.

Abstract


Ebolavirus (EBOV) outbreaks, while sporadic, cause tremendous morbidity and mortality. No therapeutics or vaccines are currently licensed; however, a vaccine has shown promise in clinical trials. A critical step towards development of effective therapeutics is a better understanding of factors that govern host susceptibility to this pathogen. As macrophages are an important cell population targeted during virus replication, we explore the effect of cytokine polarization on macrophage infection.

Authors:
Rogers KJ, Brunton B, Mallinger L, Bohan D, Sevcik KM, Chen J, Ruggio N, Maury W


Control of Lymphocyte Fate, Infection, and Tumor Immunity by TCF-1

Highlights

T cell factor-1 (TCF-1) acts as a transcription factor and histone deacetylase (HDAC) in both mouse and humans to shape innate and adaptive immunity.

Expression of TCF-1 is necessary for the development of ILC progenitor cells in mouse bone marrow.

In murine T cell development, TCF-1 is critical for ETP development, commitment to the CD4+ T cell lineage, and stabilization of CD8+ T cells by suppressing alternative fate differentiation.

Increased TCF-1 expression is important for the development of central memory CD8+ T cells that provide long-term protection following acute viral infection in mice.

TCF-1 is critical for the development of Tex-stem cells that replenish Tex-term cells following chronic viral infection and in response to tumor formation. The presence of Tex-stem cells can be predictive of good prognosis in various cancer types and help to mediate patient responses to ICB.

Authors:
Raghu D, Xue HH, Mielke LA.


Interspecies interactions induce exploratory motility in Pseudomonas aeruginosa

Abstract

Microbes often live in multispecies communities where interactions among community members impact both the individual constituents and the surrounding environment. Here, we developed a system to visualize interspecies behaviors at initial encounters. By imaging two prevalent pathogens known to be coisolated from chronic illnesses, Pseudomonas aeruginosa and Staphylococcus aureus, we observed P. aeruginosa can modify surface motility in response to secreted factors from S. aureus. Upon sensing S. aureus, P. aeruginosa transitioned from collective to single-cell motility with an associated increase in speed and directedness - a behavior we refer to as 'exploratory motility'. Explorer cells moved preferentially towards S. aureus and invaded S. aureus colonies through the action of the type IV pili. These studies reveal previously undescribed motility behaviors and lend insight into how P. aeruginosa senses and responds to other species. Identifying strategies to harness these interactions may open avenues for new antimicrobial strategies.

Authors:
Dominique LH , Warren EA, Yarrington KD, Donegan NP, Cheung AL, O'Toole G


Glycerol monolaurate induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters

Abstract

Glycerol monolaurate (GML) is a monoglyceride with potent antimicrobial properties that suppresses T cell receptor (TCR)-induced signaling and T cell effector function. Actin rearrangement is needed for the interaction of T cells with antigen-presenting cells and for migration to sites of infection. Because of the critical role actin rearrangement plays in T cell effector function, we analyzed the effect of GML on the rearrangement of the actin cytoskeleton after TCR activation. We found that GML-treated human T cells were less adherent than untreated T cells and did not form actin ring structures but instead developed numerous inappropriate actin-mediated filopodia. The formation of these filopodia was not due to disruption of TCR-proximal regulators of actin or microtubule polymerization. Instead, total internal reflection fluorescence microscopy demonstrated mislocalization of actin nucleation protein Arp2 microclusters, but not those containing the adaptor proteins SLP-76 and WASp, or the actin nucleation protein ARPC3, which are necessary for TCR-induced actin rearrangement. Additionally, SLP-76 microclusters colocalized with WASp and WAVE microclusters but not with LAT. Together, our data suggest that GML alters actin cytoskeletal rearrangements and identify diverse functions for GML as a T cell-suppressive agent.

Authors:
Zhang MS, Tran PM, Wolff AJ, Tremblay MM, Fosdick MG, Houtman JCD


Interspecies interactions induce exploratory motility in Pseudomonas aeruginosa

Abstract

Microbes often live in multispecies communities where interactions among community members impact both the individual constituents and the surrounding environment. Here, we developed a system to visualize interspecies behaviors at initial encounters. By imaging two prevalent pathogens known to be coisolated from chronic illnesses, Pseudomonas aeruginosa and Staphylococcus aureus, we observed P. aeruginosa can modify surface motility in response to secreted factors from S. aureus. Upon sensing S. aureus, P. aeruginosa transitioned from collective to single-cell motility with an associated increase in speed and directedness - a behavior we refer to as 'exploratory motility'. Explorer cells moved preferentially towards S. aureus and invaded S. aureus colonies through the action of the type IV pili. These studies reveal previously undescribed motility behaviors and lend insight into how P. aeruginosa senses and responds to other species. Identifying strategies to harness these interactions may open avenues for new antimicrobial strategies.

Authors:
Limoli DH, Warren EA, Yarrington KD, Donegan NP, Cheung AL, O'Toole G


Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

Abstract

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.

Authors:
Krishnamurthy S, Wohlford-Lenane C, Kandimalla S.,...McCray PB Jr.


Glycerol Monolaurate Contributes to the Antimicrobial and Antiinflammatory Activity of Human Milk

Abstract

Human milk has antimicrobial compounds and immunomodulatory activities. We investigated glycerol monolaurate (GML) in human milk versus bovine milk and infant formula for antimicrobial and anti-inflammatory activities. Human milk contained approximately 3000 µg/ml of GML, compared to 150 μg/ml in bovine milk and none in infant formula. For bacteria tested (Staphylococcus aureus, Bacillus subtilis, Clostridium perfringens, Escherichia coli), except Enterococcus faecalis, human milk was more antimicrobial than bovine milk and formula. The Enterococcus faecalis strain, which was not inhibited, produced reutericyclin, which is an analogue of GML and functions as a growth stimulant in bacteria that produce it. Removal of GML and other lipophilic molecules from human milk by ethanol extraction resulted in a loss of antibacterial activity, which was restored by re-addition of GML. GML addition caused bovine milk to become antimicrobial. Human milk but not bovine milk or formula inhibited superantigen and bacterial-induced IL-8 production by model human epithelial cells. GML may contribute beneficially to human milk compared to bovine milk or infant formula.

 

Authors:
Schlievert PM, Kilgore SH, Seo KS, Leung DYM


Cutting Edge: Tcf1 Instructs T Follicular Helper Cell Differentiation by Repressing Blimp1 in Response to Acute Viral Infection

Abstract

Differentiation of T follicular helper (TFH) cells is regulated by a complex transcriptional network, with mutually antagonistic Bcl6–Blimp1 as a core regulatory axis. It is well established that Tcf1 acts upstream of Bcl6 for its optimal induction to program TFH cell differentiation. In this study, we show that whereas genetic ablation of Tcf1 in mice greatly diminished TFH cells in response to viral infection, compound deletion of Blimp1 with Tcf1 restored TFH cell frequency, numbers, and generation of germinal center B cells. Aberrant upregulation of T-bet and Id2 in Tcf1-deficient TFH cells was also largely rectified by ablating Blimp1. Tcf1 chromatin immunoprecipitation sequencing in TFH cells identified two strong Tcf1 binding sites in the Blimp1 gene at a 24-kb upstream and an intron-3 element. Deletion of the intron-3 element, but not the 24-kb upstream element, compromised production of TFH cells. Our data demonstrate that Tcf1-mediated Blimp1 repression is functionally critical for safeguarding TFHcell differentiation.

Authors:
Shao P, Li F, Wang J, Chen X, Liu C and Xue HH


A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile

Abstract
Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712 CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis.IMPORTANCE Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (P xyl ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

Authors:
Müh U, Pannullo AG, Weiss DS, Ellermeier CD


Monocyte-Derived CD11c+ Cells Acquire Plasmodium from Hepatocytes to Prime CD8 T Cell Immunity to Liver-Stage Malaria

Abstract

Plasmodium sporozoites inoculated by mosquitoes migrate to the liver and infect hepatocytes prior to release of merozoites that initiate symptomatic blood-stage malaria. Plasmodium parasites are thought to be restricted to hepatocytes throughout this obligate liver stage of development, and how liver-stage-expressed antigens prime productive CD8 T cell responses remains unknown. We found that a subset of liver-infiltrating monocyte-derived CD11c+ cells co-expressing F4/80, CD103, CD207, and CSF1R acquired parasites during the liver stage of malaria, but only after initial hepatocyte infection. These CD11c+ cells found in the infected liver and liver-draining lymph nodes exhibited transcriptionally and phenotypically enhanced antigen-presentation functions and primed protective CD8 T cell responses against Plasmodium liver-stage-restricted antigens. Our findings highlight a previously unrecognized aspect of Plasmodium biology and uncover the fundamental mechanism by which CD8 T cell responses are primed against liver-stage malaria antigens.

Authors:
Kurup SP, Anthony SM, Hancox LS, Vijay R, Pewe LL, Moioffer SJ, Sompallae R, Janse CJ, Khan SM, Harty JT.


Staphylococcal Superantigens Stimulate Epithelial Cells through CD40 To Produce Chemokines

Mucosal and skin tissues form barriers to infection by most bacterial pathogens. Staphylococcus aureus causes diseases across these barriers in part dependent on the proinflammatory properties of superantigens. We showed, through use of a CRISPR-Cas9 CD40 knockout, that the superantigens toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxins (SEs) B and C stimulated chemokine production from human vaginal epithelial cells (HVECs) through human CD40. This response was enhanced by addition of antibodies against CD40 through an unknown mechanism. TSST-1 was better able to stimulate chemokine (IL-8 and MIP-3α) production by HVECs than SEB and SEC, suggesting this is the reason for TSST-1's exclusive association with menstrual TSS. A mutant of TSST-1, K121A, caused TSS in a rabbit model when administered vaginally but not intravenously, emphasizing the importance of the local vaginal environment. Collectively, our data suggested that superantigens facilitate infections by disruption of mucosal barriers through their binding to CD40, with subsequent expression of chemokines. The chemokines facilitate TSS and possibly other epithelial conditions after attraction of the adaptive immune system to the local environment.IMPORTANCE Menstrual toxic shock syndrome (TSS) is a serious infectious disease associated with vaginal colonization by Staphylococcus aureus producing the exotoxin TSS toxin 1 (TSST-1). We show that menstrual TSS occurs after TSST-1 interaction with an immune costimulatory molecule called CD40 on the surface of vaginal epithelial cells. Other related toxins, where the entire family is called the superantigen family, bind to CD40, but not with a high-enough apparent affinity to cause TSS; thus, TSST-1 is the only exotoxin superantigen associated. Once the epithelial cells become activated by TSST-1, they produce soluble molecules referred to as chemokines, which in turn facilitate TSST-1 activation of T lymphocytes and macrophages to cause the symptoms of TSS. Identification of small-molecule inhibitors of the interaction of TSST-1 with CD40 may be useful so that they may serve as additives to medical devices, such as tampons and menstrual cups, to reduce the incidence of menstrual TSS.

Authors:
Schlievert PM, Cahill MP, Hostager BS, Brosnahan AJ, Klingelhutz AJ, Gourronc FA, Bishop GA, Leung DYM.


Article Chlamydia trachomatis CT229 Subverts Rab GTPase-Dependent CCV Trafficking Pathways to Promote Chlamydial Infection

Chlamydial infection requires the formation of a membrane-bound vacuole, termed the inclusion, that undergoes extensive interactions with select host organelles. The importance of the Inc protein CT229 in the formation and maintenance of the chlamydial inclusion was recently highlighted by studies demonstrating that its absence during infection results in reduced bacterial replication, premature inclusion lysis, and host cell death. Previous reports have indicated that CT229 binds Rab GTPases; however, the physiological implications of this interaction are unknown. Here, we show that CT229 regulates host multivesicular trafficking by recruiting multiple Rab GTPases and their cognate effectors to the inclusion. We demonstrate that CT229 specifically modulates clathrin-coated vesicle trafficking and regulates the trafficking of transferrin and the mannose-6-phosphate receptor, both of which are crucial for proper chlamydial development. This study highlights CT229 as a master regulator of multiple host vesicular trafficking pathways essential for chlamydial infection.

Authors:
Faris R, Merling M, Andersen SE, Dooley CA, Hackstadt T, Weber MM.

Cell Rep. 2019 Mar 19;26(12):3380-3390.e5. doi: 10.1016/j.celrep.2019.02.079.

Chlamydia trachomatis CT229 Subverts Rab GTPase-Dependent CCV Trafficking Pathways to Promote Chlamydial Infection.

Faris R, Merling M, Andersen SE, Dooley CA, Hackstadt T, Weber MM.


Toxins and Superantigens of Group A Streptococci.

Group A Streptococcus is a human-restricted bacterial pathogen that produces an impressive arsenal of virulence factors. The secreted virulence factors are major mediators of tissue damage and toxicity seen during active infection. In this article, we focus on the biology of the true secreted exotoxins of the group A Streptococcus, as well as their roles in the pathogenesis of human disease.

Authors:
Shannon BA, McCormick JK, Schlievert PM


Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells

Tcf1 and Lef1 have versatile functions in regulating T cell development and differentiation, but intrinsic requirements for these factors in regulatory T (T reg) cells remain to be unequivocally defined. Specific ablation of Tcf1 and Lef1 in T reg cells resulted in spontaneous multi-organ autoimmunity that became more evident with age. 

Authors:
Xing S, Gai X, Shao P....Xue HH


Glycerol Monolaurate (GML) and a Nonaqueous Five-Percent GML Gel Kill Bacillus and Clostridium Spores

Glycerol monolaurate is a broadly antimicrobial fatty acid monoester, killing bacteria, fungi, and enveloped viruses. The compound kills stationary-phase cultures of Bacillus anthracis, suggesting that the molecule may kill spores. In this study, we examined the ability of glycerol monolaurate alone or solubilized in a nonaqueous gel to kill vegetative cells and spores of aerobic B. anthracis, B. subtilis, and B. cereus and anaerobic Clostridium perfringens and Clostridium (Clostridioides) difficile.

Authors:
Schlievert PM, Kilgore SH, Kaus GM, Ho TD, Ellermeier CD.

mSphere. 2018 Nov 21;3(6). pii: e00597-18. doi: 10.1128/mSphereDirect.00597-18.


TRAF3 as a Multifaceted Regulator of B Lymphocyte Survival and Activation

The adaptor protein TNF receptor-associated factor 3 (TRAF3) serves as a powerful negative regulator in multiple aspects of B cell biology. Early in vitro studies in transformed cell lines suggested the potential of TRAF3 to inhibit signaling by its first identified binding receptor, CD40. 

Authors:
Bishop GA, Stunz LL, Hostager BS


Nlrp12 Mediates Adverse Neutrophil Recruitment during Influenza Virus Infection

Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation.

Authors:
Hornick EE, Banoth B, Miller AM...Legge KL, Bishop GA...


Repeated Antigen Exposure Extends the Durability of Influenza-Specific Lung-Resident Memory CD8+ T Cells and Heterosubtypic Immunity

Lung-resident primary memory CD8+ T cell populations (Trm) induced by a single influenza infection decline within months, rendering the host susceptible to new heterosubtypic influenza infections.

Authors:
Braeckel-Budmir, NV, Varga SM, Badovinac VP, Harty JT


RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression

Authors:
Janssen KH, Diaz MR, Gode CJ, Wolfgang MC, Yahr TL


Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia.

Authors:
Kinkead LC, Whitmore LC, McCracken JM, Fletcher JR, Ketelsen BB, Kaufman JW, Jones BD, Weiss DS, Barker JH, Allen LH


Nasal priming by a murine coronavirus provides protective immunity against lethal heterologous virus pneumonia.

The nasal mucosa is an important component of mucosal immunity. Immunogenic particles in inspired air are known to activate the local nasal mucosal immune system and can lead to sinonasal inflammation; however, little is known about the effect of this activation on the lung immune environment.

Authors:
Hua X, Vijay R, Channappanvar R, Athmer J, Meyerholz DK, Pagedar N, Tilley S, Perlman S

JCI Insight. 2018 Jun 7;3(11). pii: 99025. doi: 10.1172/jci.insight.99025. [Epub ahead of print]