Genetic and Other Determinants for the Severity of Coccidioidomycosis: A Clinician's Perspective.

The endemic fungal infection, coccidioidomycosis, occurs after inhalation of one or very few Coccidioides spp. spores. Infections produce diverse clinical manifestations, ranging from insignificant to extremely destructive, even fatal. Approaches to understanding this range of consequences have traditionally categorized patients into a small number of groups (asymptomatic, uncomplicated self-limited, fibro-cavitary, and extra-thoracic disseminated) and then looked for immunologic differences among them. Recently, variants within genes of innate pathways have been found to account, in part, for infections that result in disseminated disease. This discovery raises the very attractive theory that, in patients without severe immunosuppression, much of the disease spectrum can be accounted for by various combinations of such deleterious variants in innate pathways. In this review, we summarize what is known about genetic determinants that are responsible for the severity of coccidioidal infections and how complex innate genetic differences among different people might account for the spectrum of disease observed clinically.

Reactivation of Coccidioidomycosis in a Mouse Model of Asymptomatic Controlled Disease.

The majority of human coccidioidomycosis infections are asymptomatic or self-limited but may have sequestered spherules in highly structured granulomas. Under immunosuppression, reactivation of fungal growth can result in severe disease. B6D2F1 mice asymptomatically infected with C. posadasii strain 1038 were immunosuppressed with dexamethasone (DXM) in drinking water. Treated mice died 16−25 days later, while untreated mice survived (p < 0.001). Flow cytometry of lung granulomas on days 5, 10, 15, and 20 of DXM treatment showed immune cell populations decreased 0.5−1 log compared with untreated mice though neutrophils and CD19+IgD−IgM− cells rebounded by day 20. Histopathology demonstrated loss of granuloma structure by day 5 and increasing spherules through day 20. On day 20, T-cells were nearly absent and disorganized pyogranulomatous lesions included sheets of plasma cells and innumerable spherules. Mice given DXM for 14 days then stopped (DXM stop) survived 6 weeks (9/10). Lung fungal burdens were significantly lower (p = 0.0447) than mice that continued treatment (DXM cont) but higher than untreated mice. Histopathologically, DXM stop mice did not redevelop controlled granulomas by sacrifice, though T-cells were densely scattered throughout the lesions. This demonstrates a mouse model suitable for further study to understand the immunologic components responsible for maintenance control of coccidioidomycosis.

Immunogenetics associated with severe coccidioidomycosis.

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, less than 1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled patients with DCM and performed whole-exome sequencing. In an exploratory set of 67 patients with DCM, 2 had haploinsufficient STAT3 mutations, and defects in ß-glucan sensing and response were seen in 34 of 67 cases. Damaging CLEC7A and PLCG2 variants were associated with impaired production of ß-glucan-stimulated TNF-α from PBMCs compared with healthy controls. Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were overrepresented in DCM, including CLEC7A Y238* and PLCG2 R268W. A validation cohort of 111 patients with DCM confirmed the PLCG2 R268W, CLEC7A I223S, and CLEC7A Y238* variants. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived hydrogen peroxide [H2O2] in transfected cells. Heterozygous DUOX1 or DUOXA1 variants that impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired ß-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Vaccines to Prevent Coccidioidomycosis: A Gene-Deletion Mutant of Coccidioides Posadasii as a Viable Candidate for Human Trials.

Coccidioidomycosis is an endemic fungal infection that is reported in up to 20,000 persons per year and has an economic impact close to $1.5 billion. Natural infection virtually always confers protection from future exposure, and this suggests that a preventative vaccine strategy is likely to succeed. We here review progress toward that objective. There has been ongoing research to discover a coccidioidal vaccine over the past seven decades, including one phase III clinical trial, but for reasons of either efficacy or feasibility, a safe and effective vaccine has not yet been developed. This review first summarizes the past research to develop a coccidioidal vaccine. It then details the evidence that supports a live, gene-deletion vaccine candidate as suitable for further development as both a veterinary and a human clinical product. Finally, a plausible vaccine development plan is described which would be applicable to this vaccine candidate and also useful to other future candidates. The public health and economic impact of coccidioidomycosis fully justifies a public private partnership for vaccine development, and the development of a vaccine for this orphan disease will likely require some degree of public funding.

Mouse Model of a Human STAT4 Point Mutation That Predisposes to Disseminated Coccidiomycosis.

STAT4 plays a critical role in the generation of both innate and adaptive immune responses. In the absence of STAT4, Th1 responses, critical for resistance to fungal disease, do not occur. Infection with the dimorphic fungus, Coccidioides, is a major cause of community-acquired pneumonia in the endemic regions of Arizona and California. In some people and often for unknown reasons, coccidioidal infection results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis, prompting genetic investigation. All affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4E626G/+ ). A knockin mouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-type mice. Stat4E626G/+ T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells from Stat4E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4E626G/+ mice failed to produce IFN-γ and related cytokines in the lung and to accumulate activated adaptive immune cells in mediastinal lymph nodes. Therefore, defective early induction of IFN-γ and adaptive responses by STAT4 prevents normal control of coccidioidomycosis in both mice and humans.

Δcps1 vaccine protects dogs against experimentally induced coccidioidomycosis.

Coccidioidomycosis is a significant health problem of dogs and humans in endemic regions, especially California and Arizona in the U.S. Both species would greatly benefit from a vaccine to prevent this disease. A live avirulent vaccine candidate, Δcps1, was tested for tolerability and efficacy to prevent pulmonary coccidioidomycosis in a canine challenge model. Vaccine injection-site reactions were transient and there were no systemic effects observed. Six of seven vaccine sites tested and all draining lymph nodes were sterile post-vaccination. Following infection with Coccidioides posadasii, strain Silveira, arthroconidia into the lungs, dogs given primary and booster vaccinations had significantly reduced lung fungal burdens (P = 0.0003) and composite disease scores (P = 0.0002) compared to unvaccinated dogs. Dogs vaccinated once had fungal burdens intermediate between those given two doses or none, but disease scores were not significantly different from unvaccinated (P = 0.675). Δcps1 was well-tolerated in the dogs and it afforded a high level of protection when given as prime and boost. These results drive the Δcps1 vaccine toward a licensed veterinary vaccine and support continued development of this vaccine to prevent coccidioidomycosis in humans.

TNFα Blockade Inhibits Both Initial and Continued Control of Pulmonary Coccidioides.

Tumor necrosis factor alpha (TNFα) is a pluripotent cytokine that is important in many infections, though its role in Coccidioides infection remains poorly understood. The need to understand TNFα in Coccidioides infection has increased recently with the widespread use of TNFα inhibitors for a wide variety of autoimmune conditions. Here, we couple the newly developed Coccidioides infection model using strain Cp1038 and C57BL/6 × DBA/2J F1 (B6D2F1) mice. B6D2F1 mice develop long-lasting control of Cp1038. Treatment of B6D2F1 mice with anti-TNFα antibodies permits significant fungal proliferation and death. Additionally, we show that antibody treatment limited to the first 2 weeks of infection was sufficient to induce this same loss of fungal control. Importantly, anti-TNFα antibody treatment initiated after fungal control leads to a loss of host control. These results highlight the importance of TNFα in both the initial control of murine Coccidioides and ongoing suppression of the fungal disease.

Vaccine Protection of Mice With Primary Immunodeficiencies Against Disseminated Coccidioidomycosis.

Disseminated coccidioidomycosis (DCM), often a severe and refractory disease leading to poor outcomes, is a risk for people with certain primary immunodeficiencies (PID). Several DCM-associated PID (STAT4, STAT3, IFNγ, and Dectin-1) are modeled in mice. To determine if vaccination could provide these mice protection, mice with mutations in Stat4, Stat3, Ifngr1, Clec7a (Dectin-1), and Rag-1 (T- and B-cell deficient) knockout (KO) mice were vaccinated with the live, avirulent, Δcps1 vaccine strain and subsequently challenged intranasally with pathogenic Coccidioides posadasii Silveira strain. Two weeks post-infection, vaccinated mice of all strains except Rag-1 KO had significantly reduced lung and spleen fungal burdens (p<0.05) compared to unvaccinated control mice. Splenic dissemination was prevented in most vaccinated immunodeficient mice while all unvaccinated B6 mice and the Rag-1 KO mice displayed disseminated disease. The mitigation of DCM by Δcps1 vaccination in these mice suggests that it could also benefit humans with immunogenetic risks of severe disease.

A Chronic Murine Disease Model of Coccidioidomycosis Using Coccidioides posadasii, Strain 1038.

Murine infections with most Coccidioides spp. strains are lethal by 3 weeks, limiting the study of immune responses. Coccidioides posadasii, strain 1038 (Cp1038), while slowly lethal, resulted in protracted survival of C57BL/6 (B6) mice. In resistant (B6D2)F1/J mice, lung fungal burdens stabilized by week 4 without progression through week 16, better modeling human coccidioidal infections after their immunologic control. Immunodeficient tumor necrosis factor (Tnf) α knockout (KO) and interferon (Ifn) γ receptor 1 (Ifn-γr1) KO mice survived a median of 22.5 and 34 days, compared with 70 days in B6 mice (P = .001 and P < .01, respectively), though 14-day lung fungal burden studies showed little difference between Ifn-γr1 KO and B6 mice. B6 mice showed peak concentrations of key inflammatory lung cytokines, including interleukin 6, 23, and 17A, Tnf-α, and Ifn-γ, only after 4 weeks of infection. The slower progression in B6 and the acquired fungal burden stability in B6D2 mice after Cp1038 infection greatly increases the array of possible immunologic studies.

Viable spores of Coccidioides posadasii Δcps1 are required for vaccination and provide long lasting immunity.

Coccidioidomycosis is a systemic fungal infection for which a vaccine has been sought for over fifty years. The avirulent Coccidioides posadasii strain, Δcps1, which is missing a 6 kb gene, showed significant protection in mice. These studies explore conditions of protection in mice and elucidate the immune response. Mice were vaccinated with different doses and viability states of Δcps1 spores, challenged with virulent C. posadasii, and sacrificed at various endpoints, dependent on experimental objectives. Tissues from vaccinated mice were harvested for in vitro elucidation of immune response. Vaccination with viable Δcps1 spores was required for protection from lethal challenge. Viable spore vaccination produced durable immunity, lasting at least 6 months, and prolonged survival (≥6 months). The C. posadasii vaccine strain also protected mice against C. immitis (survival ≥ 6 months). Cytokines from infected lungs of vaccinated mice in the first four days after Cp challenge showed significant increases of IFN-γ, as did stimulated CD4+ spleen cells from vaccinated mice. Transfer of CD4+ cells, but not CD8+ or B cells, reduced fungal burdens following challenge. IFN-γ from CD4+ cells in vaccinated mice indicates a Th1 response, which is critical for host control of coccidioidomycosis.

Adaptive Immunity to Francisella tularensis and Considerations for Vaccine Development.

Francisella tularensis is an intracellular bacterium that causes the disease tularemia. There are several subspecies of F. tularensis whose ability to cause disease varies in humans. The most virulent subspecies, tularensis, is a Tier One Select Agent and a potential bioweapon. Although considerable effort has made to generate efficacious tularemia vaccines, to date none have been licensed for use in the United States. Despite the lack of a tularemia vaccine, we have learned a great deal about the adaptive immune response the underlies protective immunity. Herein, we detail the animal models commonly used to study tularemia and their recapitulation of human disease, the field's current understanding of vaccine-mediated protection, and discuss the challenges associated with new vaccine development.

A Natural Mouse Model for Neisseria Colonization.

Commensals are important for the proper functioning of multicellular organisms. How a commensal establishes persistent colonization of its host is little understood. Studies of this aspect of microbe-host interactions are impeded by the absence of an animal model. We have developed a natural small animal model for identifying host and commensal determinants of colonization and of the elusive process of persistence. Our system couples a commensal bacterium of wild mice, Neisseria musculi, with the laboratory mouse. The pairing of a mouse commensal with its natural host circumvents issues of host restriction. Studies are performed in the absence of antibiotics, hormones, invasive procedures, or genetic manipulation of the host. A single dose of N. musculi, administered orally, leads to long-term colonization of the oral cavity and gut. All mice are healthy. Susceptibility to colonization is determined by host genetics and innate immunity. For N. musculi, colonization requires the type IV pilus. Reagents and powerful tools are readily available for manipulating the laboratory mouse, allowing easy dissection of host determinants controlling colonization resistance. N. musculi is genetically related to human-dwelling commensal and pathogenic Neisseria and encodes host interaction factors and vaccine antigens of pathogenic Neisseria Our system provides a natural approach for studying Neisseria-host interactions and is potentially useful for vaccine efficacy studies.

The Commensal Neisseria musculi Modulates Host Innate Immunity To Promote Oral Colonization.

Neisseria musculi, isolated from the oral cavity of wild-caught mice, does not colonize most inbred mouse strains. N. musculi does weakly (50%) colonize C57BL/6J (B6) mice but readily colonizes CAST/EiJ (CAST) mice. In this study, we examined whether differences in the CAST and B6 host response could elucidate mechanisms governing N. musculi colonization. In vivo stimulation of B6 or CAST splenocytes with wild type (WT) Neisseria or Escherichia coli LPS showed that CAST mice had a blunted inflammatory response, producing significantly lower levels of IL-6 than B6 mice. The use of specific genetic knockouts highlighted a need for an intact innate immune system to prevent colonization. B6-RAG-1-/- mice were colonized at a similar rate as WT B6 mice, whereas B6-MyD88-/- and TLR4-/- mice were readily colonized like CAST (100%) mice. Sequence analysis revealed a unique point mutation in TLR4 in CAST mice. However, crosses to TLR4-/- mice and analysis of recombinant inbred Collaborative Cross mice showed that TLR4 from CAST mice was not sufficient to allow Neisseria colonization. In vitro stimulation of B6 bone marrow-derived macrophages or splenocytes with WT Neisseria yielded low levels of IL-6 compared with LPS stimulation. Surprisingly, UV-inactivated Neisseria induced high levels of IL-6, suggesting suppression of IL-6 production is an active bacterial process. Consistent with a critical role for IL-6 in preventing colonization, mice deficient for the IL-6 receptor were efficiently colonized, indicating host IL-6 production plays a critical role in determining host colonization susceptibility.

Efficacy of Resistance to Francisella Imparted by ITY/NRAMP/SLC11A1 Depends on Route of Infection.

Natural resistance-associated macrophage protein (NRAMP) encoded by the Slc11a1 gene is a membrane-associated transporter of divalent metal ions. Murine Slc11a1 has two known alleles, a functional Slc11a1Gly169, which is found in DBA2/J, NOD/LtJ, and 129p3/J and related mouse strains, and a non-functional Slc11a1Asp169, that is found in C56Bl/6J (B6) and BALB/cJ mice. B6 mice congenic for Slc11a1Gly169 (B6-Slc11a1G169 ) are markedly resistant to the intracellular pathogens Salmonella, Leishmania, and Mycobacterium tuberculosis. We examined the host cell response and replication of Francisella in B6-Slc11a1G169 mice. Bone marrow-derived macrophages from either B6-Slc11a1G169 or B6 mice were both effectively invaded by Francisella live vaccine strain (LVS). However, at 16 hours post-infection (hpi), the number of LVS bacteria recovered from B6 macrophages had increased roughly 100-fold, while in B6-Slc11a1G169 mice the number decreased 10-fold. When the mice were challenged intranasally (i.n.) B6 mice lost significant amounts (~15%) of weight, where as B6-Slc11a1G169 mice lost no weight. Three days after infection in B6-Slc11a1G169 mice, we failed to recover viable Francisella from the lungs, livers, or spleens. By contrast, B6 mice had bacterial burdens approaching 1 × 106 CFU/organ in all three organs. To further examine the degree of resistance imparted by Slc11a1Gly169 expression, we challenged mice deficient in TLR2, TLR4, and TLR9, but expressing the functional Slc11a1 (B6-Slc11a1G169Tlr2/4/9-/- ). Surprisingly, B6-Slc11a1G169Tlr2/4/9-/- mice had no notable weight loss. Eighty percent of B6-Slc11a1G169Tlr2/4/9-/- mice yielded no detectable Francisella in any organ tested. Additionally, Slc11a1G169 produced little detectable cytokine either in the lung or serum compared to B6 mice. Mice expressing Slc11a1Gly169 survived even high doses (~80 LD50) of LVS inoculation. These data taken together serve to highlight that functional Slc11a1Gly169 can compensate the lack of TLR2/4/9. Thus Slc11a1 is a critical player in murine resistance to pulmonary Francisella infection, but not footpad infection.

Distinct innate responses are induced by attenuated Salmonella enterica serovar Typhimurium mutants.

Upon bacterial infection the host cells generate a wide variety of cytokines. Genetic attenuation of bacterial physiological pathogens can be accomplished not only by disruption of normal bacterial processes, but also by the loss of the ability to redirect the host immune system. We examined nine attenuated Salmonella Typhimurium mutants for their ability to replicate as well as the cytokines produced after infection of Bone Marrow Derived Macrophages (BMDM). Infection of BMDM with attenuated Salmonella mutants led to host cytokine patterns distinct from those that followed WT infection. Surprisingly, each bacterial mutant had a unique cytokine signature. Because some of the mutants induced an IL-10 response not seen in WT, we examined the role of IL-10 on Salmonella replication. Surprisingly, addition of IL-10 before or concurrent with infection restricted growth of WT Salmonella in BMDM. Bacterial attenuation is not a single process and results in attenuated host responses, which result in unique patterns for each attenuated mutants.

Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis.

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endotoxic moiety of LPS, is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria making it a critical factor for bacterial adaptation. One way PA modifies its lipid A is through the addition of laurate and 2-hydroxylaurate. This secondary or late acylation is carried out by the acyltransferase, HtrB (LpxL). Analysis of the PA genome revealed the presence of two htrB homologs, PA0011 (htrB1) and PA3242 (htrB2). In this study, we were able to show that each gene identified is responsible for site-specific modification of lipid A. Additionally, deletions of either gene altered resistance to specific classes of antibiotics, cationic antimicrobial peptides and increased membrane permeability suggesting a role for these enzymes in maintaining optimal membrane organization and integrity.

The prrF-encoded small regulatory RNAs are required for iron homeostasis and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part of P. aeruginosa's iron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2 mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identified phuS, encoding a heme binding protein involved in heme acquisition, and vreR, encoding a previously identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-mediated heme regulation. Finally, we showed that the prrF locus encoding the PrrF and PrrH sRNAs is required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2 deletion mutant protects against future challenge with wild-type P. aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P. aeruginosa virulence.

Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei.

The cell cycle consists of an orderly sequence of events, whose purpose is to faithfully replicate and segregate cellular components. Many events in the cell cycle are triggered by protein kinases and counteracting phosphoprotein phosphatases (PPP). In Trypanosoma brucei, RNAi has been used to characterize numerous regulatory kinases, while the role of protein phosphatases has primarily been deduced with inhibitors such as okadaic acid and calyculin. In the present study, we identify for the first time a protein phosphatase 2A family member (TbPP2A-1) whose knockdown with RNAi phenocopies the effects of okadaic acid (OKA). In bloodstream forms (BF) and insect stage procyclic forms (PF) RNAi of TbPP2A-1 generates a cell population characterized by: an inhibition of cell growth, a block in cytokinesis; continued synthesis of nuclear DNA leading to aneuploidy; continued mitosis leading to cells with N>2, and an unusual phenotype where number of kinetoplasts (and flagella) is less than the number of nuclei. An engineered cell line was constructed to further study TbPP2A-1 and to facilitate the discovery of other cell cycle regulatory genes.

Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock.

Inflammatory caspases, such as caspase-1 and -11, mediate innate immune detection of pathogens. Caspase-11 induces pyroptosis, a form of programmed cell death, and specifically defends against bacterial pathogens that invade the cytosol. During endotoxemia, however, excessive caspase-11 activation causes shock. We report that contamination of the cytoplasm by lipopolysaccharide (LPS) is the signal that triggers caspase-11 activation in mice. Specifically, caspase-11 responds to penta- and hexa-acylated lipid A, whereas tetra-acylated lipid A is not detected, providing a mechanism of evasion for cytosol-invasive Francisella. Priming the caspase-11 pathway in vivo resulted in extreme sensitivity to subsequent LPS challenge in both wild-type and Tlr4-deficient mice, whereas Casp11-deficient mice were relatively resistant. Together, our data reveal a new pathway for detecting cytoplasmic LPS.