Cryptides Identified in Human Apolipoprotein B as New Weapons to Fight Antibiotic Resistance in Cystic Fibrosis Disease.

Chronic respiratory infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients, and are characterized by the development of multidrug resistance (MDR) phenotype and biofilm formation, generally recalcitrant to treatment with conventional antibiotics. Hence, novel effective strategies are urgently needed. Antimicrobial peptides represent new promising therapeutic agents. Here, we analyze for the first time the efficacy of three versions of a cryptide identified in human apolipoprotein B (ApoB, residues 887-922) towards bacterial strains clinically isolated from CF patients. Antimicrobial and anti-biofilm properties of ApoB-derived cryptides have been analyzed by broth microdilution assays, crystal violet assays, confocal laser scanning microscopy and scanning electron microscopy. Cell proliferation assays have been performed to test cryptide effects on human host cells. ApoB-derived cryptides have been found to be endowed with significant antimicrobial and anti-biofilm properties towards Pseudomonas and Burkholderia strains clinically isolated from CF patients. Peptides have been also found to be able to act in combination with the antibiotic ciprofloxacin, and they are harmless when tested on human bronchial epithelial mesothelial cells. These findings open interesting perspectives to cryptide applicability in the treatment of chronic lung infections associated with CF disease.

Resolving the pathogenicity factors of a novel opportunistic fungus Schizophyllum commune at molecular level.

Schizophyllum commune is a well-known mushroom forming fungi which is an edible one due to its nutritive value. It exhibits a special wood degrading mechanism to grow in decay matters by releasing a series of enzymes. These enzymes might make them an opportunistic pathogen which has been reported to infect various animals and human beings too. Although these fungi were identified as human and animal pathogens, their mechanisms of pathogenesis and the key virulence factors involved in disease establishment are not known. In this study, we reported this fungal infection in freshwater fish for the first time and its morphological features. Further, we employed RNA-seq technique to identify the major virulence factors involved in the pathogenesis in fish and the network of interaction between the identified virulence factors were analysed. Also, we confirmed the virulence roles of this fungus during infection by qRT-PCR analysis. This study emphasizes the virulence nature of the common mushroom forming food fungus and the involvement of enzymes such as phosphoinositide phospholipase C, hexosaminidase and few toxins such as pesticidal and insecticidal crystal proteins which opened a new avenue in the virulence nature of edible mushrooms.

Digging through the Obstruction: Insight into the Epithelial Cell Response to Respiratory Virus Infection in Patients with Cystic Fibrosis.

Respiratory virus infections are common but generally self-limiting infections in healthy individuals. Although early clinical studies reported low detection rates, the development of molecular diagnostic techniques by PCR has led to an increased recognition that respiratory virus infections are associated with morbidity and acute exacerbations of chronic lung diseases, such as cystic fibrosis (CF). The airway epithelium is the first barrier encountered by respiratory viruses following inhalation and the primary site of respiratory viral replication. Here, we describe how the airway epithelial response to respiratory viral infections contributes to disease progression in patients with CF and other chronic lung diseases, including the role respiratory viral infections play in bacterial acquisition in the CF patient lung.

Relation between neonatal malnutrition and gene expression: inflammasome function in infections caused by Candida Albicans.

PURPOSE: To investigate the effects of neonatal malnutrition followed by nutritional replacement on the signaling mechanisms developed by the inflammasome complex by analyzing the expression of the targeted TLR2, TLR4, NLRP3, caspase-1 and release of IL-1ß and IL-18 by alveolar macrophages infected in vitro with Candida albicans. METHODS: Male Wistar rats (n = 24), 90-120 days, were suckled by mothers whose diet during lactation contained 17 % protein in the nourish group and 8 % protein in the malnourished group. After weaning, both groups were fed a normal protein diet. Macrophages were obtained after tracheostomy, through the collection of bronchoalveolar lavage fluid. The quantification of the expression levels of targets (TLR2, TLR4, NLRP3 and caspase-1) was performed by real-time RT-PCR. Production of cytokines was performed by ELISA. RESULTS: The malnourished animals during lactation showed reduced body weight from the fifth day of life, remaining until adulthood. Further, the model applied malnutrition induced a lower expression of TLR4 and caspase-1. The quantification of the TLR2 and NLRP3, as well as the release of IL-1ß and IL-18, was not different between groups of animals nourished and malnourished. The system challenged with Candida albicans showed high expression levels of all targets in the study. CONCLUSIONS: The tests demonstrate nutritional restriction during critical periods of development, although nutritional supplementation may compromise defense patterns in adulthood in a timely manner, preserving distinct signaling mechanism, so that the individual does not become widely vulnerable to infections by opportunistic pathogens.

Lung Infections in Systemic Rheumatic Disease: Focus on Opportunistic Infections.

Systemic rheumatic diseases have significant morbidity and mortality, due in large part to concurrent infections. The lung has been reported among the most frequent sites of infection in patients with rheumatic disease, who are susceptible to developing pneumonia sustained both by common pathogens and by opportunistic microorganisms. Patients with rheumatic disease show a peculiar vulnerability to infectious complications. This is due in part to intrinsic disease-related immune dysregulation and in part to the immunosuppressive treatments. Several therapeutic agents have been associated to a wide spectrum of infections, complicating the management of rheumatic diseases. This review discusses the most frequent pulmonary infections encountered in rheumatic diseases, focusing on opportunistic agents, consequent diagnostic challenges and appropriate therapeutic strategies.

cPLA2α-mediated actin rearrangements downstream of the Akt signaling is required for Cronobacter sakazakii invasion into brain endothelial cells.

Cronobacter sakazakii (C. sakazakii) is an opportunistic pathogen that causes sepsis and meningitis in neonate. The molecular mechanism involved in the pathogenesis of C. sakazakii meningitis remains unclear. In this study, we found that C. sakazakii invasion was significantly decreased in human brain microvascular endothelial cells (HBMEC) treated with cytosolic phospholipases A(2)α (cPLA(2)α) inhibitor. Increased phosphorylation of cPLA(2)α was observed in HBMEC infected with C. sakazakii, which was prevented by treatment with cPLA(2)α inhibitor. cPLA(2)α knockdown in HBMEC significantly attenuated C. sakazakii invasion into HBMEC. Immunofluorescence demonstrated that the rearrangements of actin filaments in HBMEC induced by C. sakazakii were effectively blocked by either treatment with cPLA(2)α inhibitor or transfection with cPLA(2)α siRNA. Interestingly, we found that C. sakazakii infection promoted the aggregation of phosphorylated cPLA(2)α, which was associated with depolymerized actin filaments in HBMEC. Furthermore, our data revealed that cPLA(2)α acts downstream of Akt signaling pathway in HBMEC stimulated with C. sakazakii. Taken together, our results illustrated that cPLA(2)α-mediated actin filament rearrangements downstream of Akt activation is required for C. sakazakii invasion into brain endothelial cells.

Idiopathic CD4+ T-cell lymphocytopenia is associated with impaired membrane expression of the chemokine receptor CXCR4.

Idiopathic CD4(+) T-cell lymphocytopenia (ICL) is a rare acquired T-cell immunodeficiency of unknown pathogenic basis. Six adults with ICL who developed opportunistic infections were investigated using extensive immunophenotyping analysis and functional evaluation of the chemokine receptor CXCR4. For all 6 patients studied, a profound defect in CXCR4 expression was detected at the surface of CD4(+) T lymphocytes, in association with an abnormal intracellular accumulation of CXCR4 and of its natural ligand, the chemokine CXCL12. For all patients studied, CD4(+) T-cell chemotactic response toward CXCL12 was decreased, whereas sensitivity to CXCL8 was preserved. CXCR4 recovery after ligand-induced endocytosis was impaired in ICL CD4(+) T cells. Upon in vitro addition of interleukin-2 (IL-2), membrane expression of CXCR4 returned to normal levels in 5 of 6 patients, whereas intracellular accumulation of CXCR4 and CXCL12 disappeared. Upon therapeutic administration of IL-2, CD4(+) T-cell count and membrane CXCR4 expression and function improved over time in 3 of 4 patients treated. Therefore, our data indicate that ICL is associated with defective surface expression of CXCR4, which may be reversed by IL-2.

MDA5 signaling induces type 1 IFN- and IL-1-dependent lung vascular permeability which protects mice from opportunistic fungal infection.

Lungs balance threat from primary viral infection, secondary infection, and inflammatory damage. Severe pulmonary inflammation induces vascular permeability, edema, and organ dysfunction. We previously demonstrated that poly(I:C) (pICLC) induced type 1 interferon (t1IFN) protected mice from Cryptococcus gattii (Cg) via local iron restriction. Here we show pICLC increased serum protein and intravenously injected FITC-dextran in the lung airspace suggesting pICLC induces vascular permeability. Interestingly, pICLC induced a pro-inflammatory signature with significant expression of IL-1 and IL-6 which depended on MDA5 and t1IFN. Vascular permeability depended on MDA5, t1IFN, IL-1, and IL-6. T1IFN also induced MDA5 and other MDA5 signaling components suggesting that positive feedback contributes to t1IFN dependent expression of the pro-inflammatory signature. Vascular permeability, induced by pICLC or another compound, inhibited Cg by limiting iron. These data suggest that pICLC induces t1IFN which potentiates pICLC-MDA5 signaling increasing IL-1 and IL-6 resulting in leakage of antimicrobial serum factors into lung airspace. Thus, induced vascular permeability may act as an innate defense mechanism against opportunistic fungal infection, such as cryptococcosis, and may be exploited as a host-directed therapeutic target.

CD34+CD10+CD19- Cells in Patients with Unhealthy Alcohol Use Stimulate the M2b Monocyte Polarization.

M2b monocytes commonly isolated from patients with unhealthy alcohol use (Alc) have been described as cells that make the host susceptible to opportunistic infections. CD34+CD10+CD19- cells are multilineage progenitors of CD19+ cells, and we show that the effect of these cells from the peripheral blood on M2b monocyte polarization differed between healthy donors and Alc in this study. In healthy donors, these cells consistently differentiated into high-mobility group box-1 (HMGB1)-nonproducing cells (CD19+ cells) in response to retinoic acid (RA). However, owing to the lack of expression of RA receptor (RAR), these cells from Alc failed to differentiate into CD19+ cells under the same RA stimulation. Conditioned medium (CM) of these cells from Alc induced the polarization of M2b monocytes, which increases the susceptibility of hosts to opportunistic infections in Alc. When the alcoholic individuals were subjected to 2 weeks of abstinence from alcohol, these cells from Alc recovered their RAR expression and differentiated into CD19+ cells. Moreover, the CM of these cells from Alc after abstinence lost its ability to induce M2b monocyte polarization. These results indicate that these cells from Alc have different properties from those of healthy donors. In Alc, these cells without RAR stimulate M2b monocyte polarization through the production of HMGB1.

Murine response to the opportunistic bacterium Pseudomonas aeruginosa infection in gut dysbiosis caused by 5-fluorouracil chemotherapy-induced mucositis.

AIMS: This manuscript aims to explain the relationship between mucositis caused by 5-FU over gut bacterial species and susceptibility to opportunistic infection caused by P. aeruginosa. MAIN METHODS: BALB/c mice were intraperitoneally treated with PBS or 5-FU. Bodyweight and faecal consistency were checked daily. Mice faecal DNA was extracted, and bacterial phylogenetic groups were analysed using qPCR or high-throughput sequencing. Immunofluorescence was used to evaluate BMDM activation by mice-treated faecal content. Mice were challenged intratracheally with virulent P. aeruginosa, and the CFU and histology were analysed. Faecal microbiota were transplanted to evaluate the gut microbiota and resistance to pulmonary P. aeruginosa recovery. KEY FINDINGS: The animals treated with 5-FU presented mucositis with great weight loss, altered faecal consistency, bacterial gut dysbiosis and histological changes in the intestinal mucosa. Mice under 5-FU treatment were more susceptible to lung infection by the bacteria P. aeruginosa and had more extensive tissue damage during their lung infection with greater pro-inflammatory gene expression. It was observed that the mucositis remained in the groups with 5-FU even with the FMT. The results caused by mucositis in animals that received allogeneic FMT were reversed, however, with a decrease in P. aeruginosa susceptibility in animals treated with 5-FU and allogeneic FMT compared to animals treated with 5-FU and autologous FMT. SIGNIFICANCE: Treatment with 5-FU in a murine model makes it more susceptible to pulmonary infection by the bacterium P. aeruginosa, FMT offers an opportunity to protect against this susceptibility to infection.

The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T-mediated virulence.

Pseudomonas aeruginosa, an important cause of opportunistic infections in humans, delivers bacterial cytotoxins by type III secretion directly into the host cell cytoplasm, resulting in disruption of host cell signaling and host innate immunity. However, little is known about the fate of the toxins themselves following injection into the host cytosol. Here, we show by both in vitro and in vivo studies that the host ubiquitin ligase Cbl-b interacts with the type III-secreted effector exotoxin T (ExoT) and plays a key role in vivo in limiting bacterial dissemination mediated by ExoT. We demonstrate that, following polyubiquitination, ExoT undergoes regulated proteasomal degradation in the host cell cytosol. ExoT interacts with the E3 ubiquitin ligase Cbl-b and Crk, the substrate for the ExoT ADP ribosyltransferase (ADPRT) domain. The efficiency of degradation is dependent upon the activity of the ADPRT domain. In mouse models of acute pneumonia and systemic infection, Cbl-b is specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl plays no detectable role. To the best of our knowledge, this represents the first identification of a mammalian gene product that is specifically required for in vivo resistance to disease mediated by a type III-secreted effector.

Iron Assimilation during Emerging Infections Caused by Opportunistic Fungi with emphasis on Mucorales and the Development of Antifungal Resistance.

Iron is a key transition metal required by most microorganisms and is prominently utilised in the transfer of electrons during metabolic reactions. The acquisition of iron is essential and becomes a crucial pathogenic event for opportunistic fungi. Iron is not readily available in the natural environment as it exists in its insoluble ferric form, i.e., in oxides and hydroxides. During infection, the host iron is bound to proteins such as transferrin, ferritin, and haemoglobin. As such, access to iron is one of the major hurdles that fungal pathogens must overcome in an immunocompromised host. Thus, these opportunistic fungi utilise three major iron acquisition systems to overcome this limiting factor for growth and proliferation. To date, numerous iron acquisition pathways have been fully characterised, with key components of these systems having major roles in virulence. Most recently, proteins involved in these pathways have been linked to the development of antifungal resistance. Here, we provide a detailed review of our current knowledge of iron acquisition in opportunistic fungi, and the role iron may have on the development of resistance to antifungals with emphasis on species of the fungal basal lineage order Mucorales, the causative agents of mucormycosis.

[Functional immunoassays in the setting of infectious risk and immunosuppressive therapy of non-HIV immunocompromised patients]. / Place des tests immunitaires fonctionnels dans la prise en charge du risque infectieux et de la gestion des thérapies immunosuppressives chez les patients immunodéprimés non-VIH.

The holistic approach of the human immune system is based on the study of its components collectively driving a functional response to an immunogenic stimulus. To appreciate a specific immune dysfunction, a condition is mimicked ex vivo and the immune response induced is assessed. The application field of such assays are broad and expanding, from the diagnosis of primary and secondary immunodeficiencies, immunotherapy for cancer to the management of patients at-risk for infections and vaccination. These assays are immune monitoring tools that may contribute to a personalised and precision medicine. The purpose of this review is to describe immune functional assays available in the setting of non-HIV acquired immune deficiency. First, we will address the use of theses assays in the diagnosis of opportunistic infections such as viral reactivation. Secondly, we will report the usefulness of these assays to assess vaccine efficacy and to manage immunosuppressive therapies.

Gastric Ulceration and Immune Suppression in Weaned Piglets Associated with Feed-Borne Bacillus cereus and Aspergillus fumigatus.

As a multifactorial cause, gastric ulceration-mediated diarrhea is widely prevalent in the weaned piglets, impairing pig health and economic benefits. With full implementation of antibiotic stewardship programs in China, Bacillus cereus (B. cereus) and Aspergillus fumigatus (A. fumigatus) were identified frequently in porcine feedstuffs and feeds of the animal industry. Association between feed-borne B. cereus and frequent diarrhea remains unclear. In the present study, we conducted a survey of B. cereus and A. fumigatus from feeds and feedstuffs in pig farms during hot season. Interestingly, B. cereus, B. subtilis, B. licheniformis and B. thuringinesis were isolated and identified from piglets' starter meals to sow feeds, accounting for 56.1%, 23.7%, 13.7% and 6.5%, respectively. Obviously, both B. cereus and B. subtili were dominant contaminants in the survey. In an in vitro study, Deoxynivalenol (DON) contents were determined in a dose-dependent manner post fermentation with B. cereus (405 and DawuC). Subsequently, 36 weaned piglets were randomly assigned to four groups and the piglets simultaneously received the combination of virulent B. cereus (Dawu C) and A. fumigatus while animals were inoculated with B. cereus (Dawu C), A. fumigatus or PBS as the control group. Clinically, piglets developed yellow diarrhea on day 5 and significant reductions of relative body weight were observed in the B. cereus group, and co-infection group. More importantly, IgG titers against Classical swine fever virus (CSFV) and Porcine epidemic diarrhea (PED) were reduced dramatically during 14-day observation in co-infection group, the B. cereus (Dawu C) group or the A. fumigatus group. However, lower Foot and mouth disease (FMD) -specific antibodies were reduced on day 7 compared to those of the control group. Additionally, lower lymphocyte proliferations were found in the B. cereus group and the co-infection group compared to the control group. Postmortem, higher lesions of gastric ulceration were observed in the B. cereus group and the co-infection group from day 7 to day 14 compared with those of the A. fumigatus group and the control group. Compared to the A. fumigatus group, higher DON contents were detected in the stomach inoculated with B. cereus and the co-infection with A. fumigatus. In conclusion, our data support the hypothesis that B. cereus might be associated with severe diarrhea by inducing gastric ulcerations and A. fumigatus might aggravate immune suppression, threating a sustainable swine industry. It is urgently needed to control feed-borne B. cereus contamination.

Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.

IKBKG (nuclear factor-kappa B essential modulator) mutation can be associated with opportunistic infection without impairing Toll-like receptor function.

BACKGROUND: Patients with hypomorphic nuclear factor-kappaB essential modulator (NEMO) mutations have extensive phenotypic variability that can include atypical infectious susceptibility. OBJECTIVE: This study may provide important insight into immunologic mechanisms of host defense. METHODS: Immunologic evaluation, including studies of Toll-like receptor (TLR) function, was performed in a 6-month-old boy with normal ectodermal development who was diagnosed with Pneumocystis pneumonia and cytomegalovirus sepsis. RESULTS: Genomic and cDNA sequencing demonstrated a novel NEMO missense mutation, 337G->A, predicted to cause a D113N (aspartic acid to asparagine) substitution in the first coiled-coil region of the NEMO protein. Quantitative serum immunoglobulins, lymphocyte subset numbers, and mitogen-induced lymphocyte proliferation were essentially normal. The PBMC responses to TLR ligands were also surprisingly normal, whereas natural killer cell cytolytic activity, T-cell proliferative responses to specific antigens, and T-cell receptor-induced NF-kappaB activation were diminished. CONCLUSION: Unlike the unique NEMO mutation described here, the most commonly reported mutations are clustered at the 3' end in the tenth exon, which encodes a zinc finger domain. Because specific hypomorphic variants of NEMO are associated with distinctive phenotypes, this particular NEMO mutation highlights a dispensability of the region including amino acid 113 for TLR signaling and ectodysplasin A receptor function. This region is required for certain immunoreceptor functions as demonstrated by his susceptibility to infections as well as natural killer cell and T-cell defects.

Pathogen-Specific T Cells Beyond CMV, EBV and Adenovirus.

PURPOSE OF REVIEW: Infectious diseases contribute significantly to morbidity and mortality in recipients of allogeneic haematopoietic stem cell transplantation (aHSCT), particularly in the era of highly immunosuppressive transplant regimens and alternate donor transplants. Delayed cellular immune recovery is a major mechanism for the increased risk in these patients. Adoptive cell therapy with ex vivo manipulated pathogen-specific T cells (PSTs) is increasingly taking its place as a treatment strategy using donor-derived or third party-banked cells. RECENT FINDINGS: The majority of clinical trial data in the form of early-phase studies has been in the prophylaxis or treatment of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV). Advancements in methods to select and enrich PSTs offer the opportunity to target the less common viral pathogens as well as fungi with this technology. Early clinical studies of PSTs targeting polyomaviruses (BK virus and JC virus), human herpesvirus 6 (HHV6), varicella zoster virus (VZV) and Aspergillus spp. have shown promising results in small numbers of patients. Other potential targets include herpes simplex virus (HSV), respiratory viruses and other invasive fungal species. In this review, we describe the burden of disease of this wider spectrum of pathogens, the progress in the development of manufacturing capability, early clinical results and the opportunities and challenges for implementation in the clinic.