The outcome of Cryptococcus neoformans intracellular pathogenesis in human monocytes

<p>Abstract</p> <p>Background</p> <p><it>Cryptococcus neoformans </it>is an encapsulated yeast that is a facultative intracellular pathogen. The interaction between macrophages and <it>C. neoformans </it>is critical for extrapulmonary dissemination of this pathogenic yeast. <it>C. neoformans </it>can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. However, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. In this study we extended studies of <it>C. neoformans</it>-macrophage cellular interaction/s to human peripheral blood monocytes.</p> <p>Results</p> <p>This study demonstrated that <it>C. neoformans </it>can shed polysaccharide within human monocytes, spread from cell to cell, and be extruded from them. Furthermore, human monocytes responded to ingestion of <it>C. neoformans </it>with cell cycle progression from G1 to S.</p> <p>Conclusion</p> <p>Similarities between mouse and human cells support the suitability of mouse cells for the study of intracellular pathogenesis mechanisms. Given that these hosts diverged over 70 million years ago, the similar pathogenic strategies for <it>C. neoformans </it>in murine and human cells supports the hypothesis that the mechanism that underlies the mammalian intracellular pathogenesis of <it>C. neoformans </it>originated from interactions with a third host, possibly soil amoeboid predators, before the mammalian radiation.</p

Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

The use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two different Streptococcus pneumoniae serotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotype in vitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells

Extracellular Vesicles from Different Pneumococcal Serotypes Are Internalized by Macrophages and Induce Host Immune Responses

Bacterial extracellular vesicles are membranous ultrastructures released from the cell surface. They play important roles in the interaction between the host and the bacteria. In this work, we show how extracellular vesicles produced by four different serotypes of the important human pathogen, Streptococcus pneumoniae, are internalized by murine J774A.1 macrophages via fusion with the membrane of the host cells. We also evaluated the capacity of pneumococcal extracellular vesicles to elicit an immune response by macrophages. Macrophages treated with the vesicles underwent a serotype-dependent transient loss of viability, which was further reverted. The vesicles induced the production of proinflammatory cytokines, which was higher for serotype 1 and serotype 8-derived vesicles. These results demonstrate the biological activity of extracellular vesicles of clinically important pneumococcal serotypes

The identification of Staphylococcus aureus factors required for pathogenicity and growth in human blood

Staphylococcus aureus is a human commensal but also has devastating potential as an opportunist pathogen. S. aureus bacteraemia is often associated with an adverse outcome. To identify potential targets for novel control approaches we have identified S. aureus components that are required for growth on human blood. An ordered transposon mutant library was screened, identifying 9 genes involved specifically in haemolysis or growth on human blood agar compared to the parental strain. Three genes (purA, purB and pabA) were subsequently found to be required for pathogenesis in the zebrafish embryo infection model. The pabA growth defect was specific to the red blood cell component of human blood, showing no growth difference compared to the parental strain on human serum, human plasma, sheep or horse blood. PabA is required in the tetrahydrofolate (THF) biosynthesis pathway. The pabA growth defect was found to be due to a combination of loss of THF-dependent dTMP production by the enzyme ThyA and an increased demand for pyrimidines in human blood. Our work highlights pabA and the pyrimidine salvage pathway as potential targets for novel therapeutics and suggests a previously undefined role for a human blood factor in the activity of sulphonamide antibiotics

Antibody Responses in HIV-Infected Patients With Advanced Immunosuppression and Asymptomatic Cryptococcal Antigenemia.

BACKGROUND: There are no host biomarkers of risk for HIV-associated cryptococcal meningitis (CM) except CD4+ T-cell deficiency. At present, serum cryptococcal antigen (CrAg) screening of those with CD4 <100 cells/µL is used to identify persons at risk for HIV-associated CM. We determined if plasma antibody profiles could discriminate CrAg+ from CrAg- patients. METHODS: We performed serological analyses of 237 HIV-infected asymptomatic Zimbabwean patients with CD4 <100 cells/µL; 125 CrAg- and CrAg+ but cerebrospinal fluid CrAg- by CrAg lateral flow assay. We measured plasma immunoglobulin M (IgM), immunoglobulin G (IgG) 1, and IgG2 concentrations by Luminex, and titers of Cryptococcus neoformans (Cn) glucuronoxylomannan (GXM) polysaccharide and naturally occurring Laminarin (natural Lam, a β-(1-3)-glucan linked polysaccharide)-binding IgM and IgG by enzyme-linked immunosorbent assay. RESULTS: GXM-IgG, -IgM, and -IgG2 levels were significantly higher in CrAg+ patients, whereas natural Lam-IgM and Lam-IgG were higher in CrAg- patients before and after adjustment for age, sex, and CD4 T-cell count, despite overlap of values. To address this variability and better discriminate the groups, we used Akaike Information Criteria to select variables that independently predicted CrAg+ status and included them in a receiver operating characteristic curve to predict CrAg status. By inclusion of CD4, GXM-IgG, GXM-IgM, and Lam-IgG, -IgG2, and -IgM, this model had an 80.4% probability (95% confidence interval, 0.75-0.86) of predicting CrAg+ status. CONCLUSIONS: Statistical models that include multiple serological variables may improve the identification of patients at risk for CM and inform new directions in research on the complex role that antibodies may play in resistance and susceptibility to CM

COVID-19 Convalescent Plasma Therapy: Long-term Implications

BACKGROUND: The long-term effect of coronavirus disease 2019 (COVID-19) acute treatments on postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) is unknown. The CONTAIN-Extend study explores the long-term impact of COVID-19 convalescent plasma (CCP) therapy on postacute sequelae of SARS-CoV-2 infection (PASC) symptoms and general health 18 months following hospitalization. METHODS: The CONTAIN-Extend study examined 281 participants from the original CONTAIN COVID-19 trial (CONTAIN-RCT, NCT04364737) at 18 months post-hospitalization for acute COVID-19. Symptom surveys, global health assessments, and biospecimen collection were performed from November 2021 to October 2022. Multivariable logistic and linear regression estimated associations between the randomization arms and self-reported symptoms and Patient-Reported Outcomes Measurement Information System (PROMIS) scores and adjusted for covariables, including age, sex, race/ethnicity, disease severity, and CONTAIN enrollment quarter and sites. RESULTS: There were no differences in symptoms or PROMIS scores between CCP and placebo (adjusted odds ratio [aOR] of general symptoms, 0.95; 95% CI, 0.54-1.67). However, females (aOR, 3.01; 95% CI, 1.73-5.34), those 45-64 years (aOR, 2.55; 95% CI, 1.14-6.23), and April-June 2020 enrollees (aOR, 2.39; 95% CI, 1.10-5.19) were more likely to report general symptoms and have poorer PROMIS physical health scores than their respective reference groups. Hispanic participants (difference, -3.05; 95% CI, -5.82 to -0.27) and Black participants (-4.48; 95% CI, -7.94 to -1.02) had poorer PROMIS physical health than White participants. CONCLUSIONS: CCP demonstrated no lasting effect on PASC symptoms or overall health in comparison to the placebo. This study underscores the significance of demographic factors, including sex, age, and timing of acute infection, in influencing symptom reporting 18 months after acute hypoxic COVID-19 hospitalization

Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Mycobacterium tuberculosis Infection

ABSTRACT Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF−/−) are highly susceptible to infection with Mycobacterium tuberculosis, and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis. However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4+ T cells as the infection progresses. M. tuberculosis-specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis

Phage-derived protein induces increased platelet activation and is associated with mortality in patients with invasive pneumococcal disease

To improve our understanding about the severity of invasive pneumococcal disease (IPD), we investigated the association between the genotype of Streptococcus pneumoniae and disease outcomes for 349 bacteremic patients. A pneumococcal genome-wide association study (GWAS) demonstrated a strong correlation between 30-day mortality and the presence of the phage-derived gene pblB, encoding a platelet-binding protein whose effects on platelet activation were previously unknown. Platelets are increasingly recognized as key players of the innate immune system, and in sepsis, excessive platelet activation contributes to microvascular obstruction, tissue hypoperfusion, and finally multiorgan failure, leading to mortality. Our in vitro studies revealed that pblB expression was induced by fluoroquinolones but not by the beta-lactam antibiotic penicillin G. Subsequently, we determined pblB induction and platelet activation by incubating whole blood with the wild type or a pblB knockout mutant in the presence or absence of antibiotics commonly administered to our patient cohort. pblB-dependent enhancement of platelet activation, as measured by increased expression of the ɑ-granule protein P-selectin, the binding of fibrinogen to the activated ɑ IIbβ3 receptor, and the formation of platelet-monocyte complex occurred irrespective of antibiotic exposure. In conclusion, the presence of pblB on the pneumococcal chromosome potentially leads to increased mortality in patients with an invasive S. pneumoniae infection, which may be explained by enhanced platelet activation. This study highlights the clinical utility of a bacterial GWAS, followed by functional characterization, to identify bacterial factors involved in disease severity. IMPORTANCE The exact mechanisms causing mortality in invasive pneumococcal disease (IPD) patients are not completely understood. We examined 349 patients with IPD and found in a bacterial genome-wide association study (GWAS) that the presence of the phage-derived gene pblB was associated with mortality in the first 30 days after hospitalization. Although pblB has been extensively studied in Streptococcus mitis, its consequence for the interaction between platelets and Streptococcus pneumoniae is largely unknown. Platelets are important in immunity and inflammation, and excessive platelet activation contributes to microvascular obstruction and multiorgan failure, leading to mortality. We therefore developed this study to assess whether the expression of pblB might increase the risk of death for IPD patients through its effect on enhanced platelet activation. This study also shows the value of integrating extensive bacterial genomics and clinical data in predicting and understanding pathogen virulence, which in turn will help to improve prognosis and therapy