Antiviral and Anti-Inflammatory Therapeutic Effect of RAGE-Ig Protein against Multiple SARS-CoV-2 Variants of Concern Demonstrated in K18-hACE2 Mouse and Syrian Golden Hamster Models.

SARS-CoV-2 variants of concern (VOCs) continue to evolve and reemerge with chronic inflammatory long COVID sequelae, necessitating the development of anti-inflammatory therapeutic molecules. Therapeutic effects of the receptor for advanced glycation end products (RAGE) were reported in many inflammatory diseases. However, a therapeutic effect of RAGE in COVID-19 has not been reported. In the present study, we investigated whether and how the RAGE-Ig fusion protein would have an antiviral and anti-inflammatory therapeutic effect in the COVID-19 system. The protective therapeutic effect of RAGE-Ig was determined in vivo in K18-hACE2 transgenic mice and Syrian golden hamsters infected with six VOCs of SARS-CoV-2. The underlying antiviral mechanism of RAGE-Ig was determined in vitro in SARS-CoV-2-infected human lung epithelial cells (BEAS-2B). Following treatment of K18-hACE2 mice and hamsters infected with various SARS-CoV-2 VOCs with RAGE-Ig, we demonstrated (1) significant dose-dependent protection (i.e., greater survival, less weight loss, lower virus replication in the lungs); (2) a reduction of inflammatory macrophages (F4/80+/Ly6C+) and neutrophils (CD11b+/Ly6G+) infiltrating the infected lungs; (3) a RAGE-Ig dose-dependent increase in the expression of type I IFNs (IFN-α and IFN-ß) and type III IFN (IFNλ2) and a decrease in the inflammatory cytokines (IL-6 and IL-8) in SARS-CoV-2-infected human lung epithelial cells; and (4) a dose-dependent decrease in the expression of CD64 (FcgR1) on monocytes and lung epithelial cells from symptomatic COVID-19 patients. Our preclinical findings revealed type I and III IFN-mediated antiviral and anti-inflammatory therapeutic effects of RAGE-Ig protein against COVID-19 caused by multiple SARS-CoV-2 VOCs.

A multi-epitope/CXCL11 prime/pull coronavirus mucosal vaccine boosts the frequency and the function of lung-resident memory CD4+ and CD8+ T cells and enhanced protection against COVID-19-like symptoms and death caused by SARS-CoV-2 infection.

IMPORTANCE: Although the current rate of SARS-CoV-2 infections has decreased significantly, COVID-19 still ranks very high as a cause of death worldwide. As of October 2023, the weekly mortality rate is still at 600 deaths in the United States alone, which surpasses even the worst mortality rates recorded for influenza. Thus, the long-term outlook of COVID-19 is still a serious concern outlining the need for the next-generation vaccine. This study found that a prime/pull coronavirus vaccine strategy increased the frequency of functional SARS-CoV-2-specific CD4+ and CD8+ memory T cells in the lungs of SARS-CoV-2-infected triple transgenic HLA-DR*0101/HLA-A*0201/hACE2 mouse model, thereby resulting in low viral titer and reduced COVID-19-like symptoms.

Simian Varicella Virus: Molecular Virology and Mechanisms of Pathogenesis.

Simian varicella virus (SVV) was first isolated in 1966 from African green monkeys (Cercopithecus aethiops) imported from Nairobi, Kenya, to the Liverpool School of Tropical Medicine in the United Kingdom (UK) (Clarkson et al., Arch Gesamte Virusforsch 22:219-234, 1967). SVV infection caused severe disease that resulted in a 56% case fatality rate (CFR) in the imported animals within 48 h of the appearance of a varicella-like rash (Clarkson et al., Arch Gesamte Virusforsch 22:219-234, 1967; Hemme et al., Am J Trop Med Hyg 94:1095-1099, 2016). The deceased animals presented with fever, widespread vesicular rash, and multiple hemorrhagic foci throughout the lungs, liver, and spleen (Clarkson et al., Arch Gesamte Virusforsch 22:219-234, 1967). This outbreak was quickly followed by a second outbreak in 47 patas monkeys (Erythrocebus patas) imported from Chad and Nigeria by Glaxo Laboratories (London, England, UK), which quickly spread within the facility (McCarthy et al., Lancet 2:856-857, 1968).

SARS-CoV-2 acquisition and immune pathogenesis among school-aged learners in four diverse schools.

BACKGROUND: Understanding SARS-CoV-2 infection in children is necessary to reopen schools safely. METHODS: We measured SARS-CoV-2 infection in 320 learners [10.5 ± 2.1 (sd); 7-17 y.o.] at four diverse schools with either remote or on-site learning. Schools A and B served low-income Hispanic learners; school C served many special-needs learners, and all provided predominantly remote instruction. School D served middle- and upper-income learners, with predominantly on-site instruction. Testing occurred in the fall (2020), and 6-8 weeks later during the fall-winter surge (notable for a tenfold increase in COVID-19 cases). Immune responses and mitigation fidelity were also measured. RESULTS: We found SARS-CoV-2 infections in 17 learners only during the surge. School A (97% remote learners) had the highest infection (10/70, 14.3%, p < 0.01) and IgG positivity rates (13/66, 19.7%). School D (93% on-site learners) had the lowest infection and IgG positivity rates (1/63, 1.6%). Mitigation compliance [physical distancing (mean 87.4%) and face-covering (91.3%)] was remarkably high at all schools. Documented SARS-CoV-2-infected learners had neutralizing antibodies (94.7%), robust IFN-γ + T cell responses, and reduced monocytes. CONCLUSIONS: Schools can implement successful mitigation strategies across a wide range of student diversity. Despite asymptomatic to mild SARS-CoV-2 infection, children generate robust humoral and cellular immune responses. IMPACT: Successful COVID-19 mitigation was implemented across a diverse range of schools. School-associated SARS-CoV-2 infections reflect regional rates rather than remote or on-site learning. Seropositive school-aged children with asymptomatic to mild SARS-CoV-2 infections generate robust humoral and cellular immunity.

Transcriptome profile of Corynebacterium pseudotuberculosis in response to iron limitation.

BACKGROUND: Iron is an essential micronutrient for the growth and development of virtually all living organisms, playing a pivotal role in the proliferative capability of many bacterial pathogens. The impact that the bioavailability of iron has on the transcriptional response of bacterial species in the CMNR group has been widely reported for some members of the group, but it hasn't yet been as deeply explored in Corynebacterium pseudotuberculosis. Here we describe for the first time a comprehensive RNA-seq whole transcriptome analysis of the T1 wild-type and the Cp13 mutant strains of C. pseudotuberculosis under iron restriction. The Cp13 mutant strain was generated by transposition mutagenesis of the ciuA gene, which encodes a surface siderophore-binding protein involved in the acquisition of iron. Iron-regulated acquisition systems are crucial for the pathogenesis of bacteria and are relevant targets to the design of new effective therapeutic approaches. RESULTS: Transcriptome analyses showed differential expression in 77 genes within the wild-type parental T1 strain and 59 genes in Cp13 mutant under iron restriction. Twenty-five of these genes had similar expression patterns in both strains, including up-regulated genes homologous to the hemin uptake hmu locus and two distinct operons encoding proteins structurally like hemin and Hb-binding surface proteins of C. diphtheriae, which were remarkably expressed at higher levels in the Cp13 mutant than in the T1 wild-type strain. These hemin transport protein genes were found to be located within genomic islands associated with known virulent factors. Down-regulated genes encoding iron and heme-containing components of the respiratory chain (including ctaCEF and qcrCAB genes) and up-regulated known iron/DtxR-regulated transcription factors, namely ripA and hrrA, were also identified differentially expressed in both strains under iron restriction. CONCLUSION: Based on our results, it can be deduced that the transcriptional response of C. pseudotuberculosis under iron restriction involves the control of intracellular utilization of iron and the up-regulation of hemin acquisition systems. These findings provide a comprehensive analysis of the transcriptional response of C. pseudotuberculosis, adding important understanding of the gene regulatory adaptation of this pathogen and revealing target genes that can aid the development of effective therapeutic strategies against this important pathogen.

Cross-protection induced by highly conserved human B, CD4+, and CD8+ T-cell epitopes-based vaccine against severe infection, disease, and death caused by multiple SARS-CoV-2 variants of concern.

Background: The coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has decreased significantly, the long-term outlook of COVID-19 remains a serious cause of morbidity and mortality worldwide, with the mortality rate still substantially surpassing even that recorded for influenza viruses. The continued emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, has prolonged the COVID-19 pandemic and underscores the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods: We designed a multi-epitope-based coronavirus vaccine that incorporated B, CD4+, and CD8+ T- cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-variant SARS-CoV-2 vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results: The pan-variant SARS-CoV-2 vaccine (i) is safe , (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells , and (iii) provides robust protection against morbidity and virus replication. COVID-19-related lung pathology and death were caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2), and Omicron (B.1.1.529). Conclusion: A multi-epitope pan-variant SARS-CoV-2 vaccine bearing conserved human B- and T- cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that facilitated virus clearance, and reduced morbidity, COVID-19-related lung pathology, and death caused by multiple SARS-CoV-2 VOCs.

A Broad-Spectrum Multi-Antigen mRNA/LNP-Based Pan-Coronavirus Vaccine Induced Potent Cross-Protective Immunity Against Infection and Disease Caused by Highly Pathogenic and Heavily Spike-Mutated SARS-CoV-2 Variants of Concern in the Syrian Hamster Model.

The first-generation Spike-alone-based COVID-19 vaccines have successfully contributed to reducing the risk of hospitalization, serious illness, and death caused by SARS-CoV-2 infections. However, waning immunity induced by these vaccines failed to prevent immune escape by many variants of concern (VOCs) that emerged from 2020 to 2024, resulting in a prolonged COVID-19 pandemic. We hypothesize that a next-generation Coronavirus (CoV) vaccine incorporating highly conserved non-Spike SARS-CoV-2 antigens would confer stronger and broader cross-protective immunity against multiple VOCs. In the present study, we identified ten non-Spike antigens that are highly conserved in 8.7 million SARS-CoV-2 strains, twenty-one VOCs, SARS-CoV, MERS-CoV, Common Cold CoVs, and animal CoVs. Seven of the 10 antigens were preferentially recognized by CD8+ and CD4+ T-cells from unvaccinated asymptomatic COVID-19 patients, irrespective of VOC infection. Three out of the seven conserved non-Spike T cell antigens belong to the early expressed Replication and Transcription Complex (RTC) region, when administered to the golden Syrian hamsters, in combination with Spike, as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) (i.e., combined mRNA/LNP-based pan-CoV vaccine): (i) Induced high frequencies of lung-resident antigen-specific CXCR5+CD4+ T follicular helper (TFH) cells, GzmB+CD4+ and GzmB+CD8+ cytotoxic T cells (TCYT), and CD69+IFN-γ+TNFα+CD4+ and CD69+IFN-γ+TNFα+CD8+ effector T cells (TEFF); and (ii) Reduced viral load and COVID-19-like symptoms caused by various VOCs, including the highly pathogenic B.1.617.2 Delta variant and the highly transmittable heavily Spike-mutated XBB1.5 Omicron sub-variant. The combined mRNA/LNP-based pan-CoV vaccine could be rapidly adapted for clinical use to confer broader cross-protective immunity against emerging highly mutated and pathogenic VOCs.

Glycoconjugates in New World species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts.

BACKGROUND: Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review. SCOPE OF THE REVIEW: The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species. MAJOR CONCLUSIONS: LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection. GENERAL SIGNIFICANCE: Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host-parasite interaction. This article is part of a Special Issue entitled Glycoproteomics.

Cross-Protection Induced by Highly Conserved Human B, CD4+, and CD8+ T Cell Epitopes-Based Coronavirus Vaccine Against Severe Infection, Disease, and Death Caused by Multiple SARS-CoV-2 Variants of Concern.

Background: The Coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of SARS-CoV-2 infections has decreased significantly; the long-term outlook of COVID-19 remains a serious cause of high death worldwide; with the mortality rate still surpassing even the worst mortality rates recorded for the influenza viruses. The continuous emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, have prolonged the COVID-19 pandemic and outlines the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods: In the present study, we designed a multi-epitope-based Coronavirus vaccine that incorporated B, CD4+, and CD8+ T cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-Coronavirus vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results: The Pan-Coronavirus vaccine: (i) is safe; (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells; and (iii) provides robust protection against virus replication and COVID-19-related lung pathology and death caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2) and Omicron (B.1.1.529). Conclusions: A multi-epitope pan-Coronavirus vaccine bearing conserved human B and T cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that cleared the virus, and reduced COVID-19-related lung pathology and death caused by multiple SARS-CoV-2 VOCs.

Identification of a fibrinogen-related protein (FBN9) gene in neotropical anopheline mosquitoes.

BACKGROUND: Malaria has a devastating impact on worldwide public health in many tropical areas. Studies on vector immunity are important for the overall understanding of the parasite-vector interaction and for the design of novel strategies to control malaria. A member of the fibrinogen-related protein family, fbn9, has been well studied in Anopheles gambiae and has been shown to be an important component of the mosquito immune system. However, little is known about this gene in neotropical anopheline species. METHODS: This article describes the identification and characterization of the fbn9 gene partial sequences from four species of neotropical anopheline primary and secondary vectors: Anopheles darlingi, Anopheles nuneztovari, Anopheles aquasalis, and Anopheles albitarsis (namely Anopheles marajoara). Degenerate primers were designed based on comparative analysis of publicly available Aedes aegypti and An. gambiae gene sequences and used to clone putative homologs in the neotropical species. Sequence comparisons and Bayesian phylogenetic analyses were then performed to better understand the molecular diversity of this gene in evolutionary distant anopheline species, belonging to different subgenera. RESULTS: Comparisons of the fbn9 gene sequences of the neotropical anophelines and their homologs in the An. gambiae complex (Gambiae complex) showed high conservation at the nucleotide and amino acid levels, although some sites show significant differentiation (non-synonymous substitutions). Furthermore, phylogenetic analysis of fbn9 nucleotide sequences showed that neotropical anophelines and African mosquitoes form two well-supported clades, mirroring their separation into two different subgenera. CONCLUSIONS: The present work adds new insights into the conserved role of fbn9 in insect immunity in a broader range of anopheline species and reinforces the possibility of manipulating mosquito immunity to design novel pathogen control strategies.

Single-cell profiling of T and B cell repertoires following SARS-CoV-2 mRNA vaccine.

mRNA vaccines for SARS-CoV-2 have shown exceptional clinical efficacy, providing robust protection against severe disease. However, our understanding of transcriptional and repertoire changes following full vaccination remains incomplete. We used scRNA-Seq and functional assays to compare humoral and cellular responses to 2 doses of mRNA vaccine with responses observed in convalescent individuals with asymptomatic disease. Our analyses revealed enrichment of spike-specific B cells, activated CD4+ T cells, and robust antigen-specific polyfunctional CD4+ T cell responses following vaccination. On the other hand, although clonally expanded CD8+ T cells were observed following both vaccination and natural infection, CD8+ T cell responses were relatively weak and variable. In addition, TCR gene usage was variable, reflecting the diversity of repertoires and MHC polymorphism in the human population. Natural infection induced expansion of CD8+ T cell clones that occupy distinct clusters compared to those induced by vaccination and likely recognize a broader set of viral antigens of viral epitopes presented by the virus not seen in the mRNA vaccine. Our study highlights a coordinated adaptive immune response in which early CD4+ T cell responses facilitate the development of the B cell response and substantial expansion of effector CD8+ T cells, together capable of contributing to future recall responses.

Acute SARS-CoV-2 infection is associated with an expansion of bacteria pathogens in the nose including Pseudomonas Aeruginosa.

Much of the research conducted on SARS-CoV-2 and COVID-19 has focused on the systemic host response, especially that generated by severely ill patients. Very few studies have investigated the impact of acute SARS-CoV-2 within the nasopharynx, the site of initial infection and viral replication. In this study we profiled changes in the nasal microbial communities as well as in host transcriptional profile during acute SARS-CoV-2 infection using 16S amplicon sequencing and RNA sequencing. These analyses were coupled to viral genome sequencing. Our microbiome analysis revealed that the nasal microbiome of COVID patients was unique and was marked by an expansion of bacterial pathogens. Some of these microbes (i.e. Acinetobacter ) were shared with COVID negative health care providers from the same medical center but absent in COVID negative outpatients seeking care at the same institutions suggesting acquisition of nosocomial respiratory pathogens. Specifically, we report a distinct increase in the prevalence and abundance of the pathogen Pseudomonas aeruginosa in COVID patients that correlated with viral RNA load. These data suggest that the inflammatory environment caused by SARS-CoV-2 infection and potentially exposure to the hospital environment leads to an expansion of bacterial pathogens in the nasal cavity that could contribute to increased incidence of secondary bacterial infections. Additionally, we observed a robust host transcriptional response in the nasal epithelia of COVID patients, indicative of an antiviral innate immune repones and neuronal damage. Finally, analysis of viral genomes did not reveal an association between viral loads and viral sequences.

Acute SARS-CoV-2 infection is associated with an increased abundance of bacterial pathogens, including Pseudomonas aeruginosa in the nose.

Research conducted on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and coronavirus disease 2019 (COVID-19) generally focuses on the systemic host response, especially that generated by severely ill patients, with few studies investigating the impact of acute SARS-CoV-2 at the site of infection. We show that the nasal microbiome of SARS-CoV-2-positive patients (CoV+, n = 68) at the time of diagnosis is unique when compared to CoV- healthcare workers (n = 45) and CoV- outpatients (n = 21). This shift is marked by an increased abundance of bacterial pathogens, including Pseudomonas aeruginosa, which is also positively associated with viral RNA load. Additionally, we observe a robust host transcriptional response in the nasal epithelia of CoV+ patients, indicative of an antiviral innate immune response and neuronal damage. These data suggest that the inflammatory response caused by SARS-CoV-2 infection is associated with an increased abundance of bacterial pathogens in the nasal cavity that could contribute to increased incidence of secondary bacterial infections.

Differential dynamics of peripheral immune responses to acute SARS-CoV-2 infection in older adults.

In this study, peripheral blood mononuclear cells from young and old patients with COVID-19 were examined phenotypically, transcriptionally and functionally to reveal age-, time- and severity-specific adaptations. Gene signatures within memory B cells and plasmablasts correlated with reduced frequency of antigen-specific B cells and neutralizing antibodies in older patients with severe COVID-19. Moreover, these patients exhibited exacerbated T cell lymphopenia, which correlated with lower plasma interleukin-2, and diminished antigen-specific T cell responses. Single-cell RNA sequencing revealed augmented signatures of activation, exhaustion, cytotoxicity and type I interferon signaling in memory T and natural killer cells with age. Although cytokine storm was evident in both age groups, older individuals exhibited elevated levels of myeloid cell recruiting factors. Furthermore, we observed redistribution of monocyte and dendritic cell subsets and emergence of a suppressive phenotype with severe disease, which was reversed only in young patients over time. This analysis provides new insights into the impact of aging on COVID-19.

SARS-CoV-2 Acquisition and Immune Pathogenesis Among School-Aged Learners in Four Diverse Schools.

BACKGROUND: Understanding SARS-CoV-2 infection in children is necessary to reopen schools safely. METHODS: We measured SARS-CoV-2 infection in 320 learners [10.5 ± 2.1(sd); 7-17 y.o.] at four diverse schools with either remote or on-site learning. Schools A and B served low-income Hispanic learners; school C served many special-needs learners; and all provided predominantly remote instruction. School D served middle- and upper-income learners, with predominantly on-site instruction. Testing occurred in the fall (2020), and 6-8 weeks later during the fall-winter surge (notable for a tenfold increase in COVID-19 cases). Immune responses and mitigation fidelity were also measured. RESULTS: We found SARS-CoV-2 infections in 17 learners only during the surge. School A (97% remote learners) had the highest infection (10/70, 14.3%, p<0.01) and IgG positivity rates (13/66, 19.7%). School D (93% on-site learners) had the lowest infection and IgG positivity rates (1/63, 1.6%). Mitigation compliance [physical distancing (mean 87.4%) and face covering (91.3%)] was remarkably high at all schools. Documented SARS-CoV-2-infected learners had neutralizing antibodies (94.7%), robust IFN-γ+ T cell responses, and reduced monocytes. CONCLUSION: Schools can implement successful mitigation strategies across a wide range of student diversity. Despite asymptomatic to mild SARS-CoV-2 infection, children generate robust humoral and cellular immune responses. KEY POINTS: Successful COVID-19 mitigation was implemented across a diverse range of schools.School-associated SARS-CoV-2 infections reflect regional rates rather than remote or on-site learning.Seropositive school-aged children with asymptomatic to mild SARS-CoV-2 infections generate robust humoral and cellular immunity.

Mutation of the Surface Layer Protein SlpB Has Pleiotropic Effects in the Probiotic Propionibacterium freudenreichii CIRM-BIA 129.

Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese-ripening starter, and currently considered as an emerging probiotic. As an example, the P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties. Its consumption accordingly exerts healing effects in different animal models of colitis, suggesting a potent role in the context of inflammatory bowel diseases. This anti-inflammatory effect depends on surface layer proteins (SLPs). SLPs may be involved in key functions in probiotics, such as persistence within the gut, adhesion to host cells and mucus, or immunomodulation. Several SLPs coexist in P. freudenreichii CIRM-BIA 129 and mediate immunomodulation and adhesion. A mutant P. freudenreichii CIRM-BIA 129ΔslpB (CB129ΔslpB) strain was shown to exhibit decreased adhesion to intestinal epithelial cells. In the present study, we thoroughly analyzed the impact of this mutation on cellular properties. Firstly, we investigated alterations of surface properties in CB129ΔslpB. Surface extractable proteins, surface charges (ζ-potential) and surface hydrophobicity were affected by the mutation. Whole-cell proteomics, using high definition mass spectrometry, identified 1,288 quantifiable proteins in the wild-type strain, i.e., 53% of the theoretical proteome predicted according to P. freudenreichii CIRM-BIA 129 genome sequence. In the mutant strain, we detected 1,252 proteins, including 1,227 proteins in common with the wild-type strain. Comparative quantitative analysis revealed 97 proteins with significant differences between wild-type and mutant strains. These proteins are involved in various cellular process like signaling, metabolism, and DNA repair and replication. Finally, in silico analysis predicted that slpB gene is not part of an operon, thus not affecting the downstream genes after gene knockout. This study, in accordance with the various roles attributed in the literature to SLPs, revealed a pleiotropic effect of a single slpB mutation, in the probiotic P. freudenreichii. This suggests that SlpB may be at a central node of cellular processes and confirms that both nature and amount of SLPs, which are highly variable within the P. freudenreichii species, determine the probiotic abilities of strains.

Transcriptome analysis of Corynebacterium pseudotuberculosis biovar Equi in two conditions of the environmental stress.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses.

Leishmania infantum lipophosphoglycan induced-Prostaglandin E2 production in association with PPAR-γ expression via activation of Toll like receptors-1 and 2.

Lipophosphoglycan (LPG) is a key virulence factor expressed on the surfaces of Leishmania promastigotes. Although LPG is known to activate macrophages, the underlying mechanisms resulting in the production of prostaglandin E2 (PGE2) via signaling pathways remain unknown. Here, the inflammatory response arising from stimulation by Leishmania infantum LPG and/or its lipid and glycan motifs was evaluated with regard to PGE2 induction. Intact LPG, but not its glycan and lipid moieties, induced a range of proinflammatory responses, including PGE2 and nitric oxide (NO) release, increased lipid droplet formation, and iNOS and COX2 expression. LPG also induced ERK-1/2 and JNK phosphorylation in macrophages, in addition to the release of PGE2, MCP-1, IL-6, TNF-α and IL-12p70, but not IL-10. Pharmacological inhibition of ERK1/2 and PKC affected PGE2 and cytokine production. Moreover, treatment with rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ), also modulated the release of PGE2 and other proinflammatory mediators. Finally, we determined that LPG-induced PPAR-γ signaling occurred via TLR1/2. Taken together, these results reinforce the role played by L. infantum-derived LPG in the proinflammatory response seen in Leishmania infection.

Two biochemically distinct lipophosphoglycans from Leishmania braziliensis and Leishmania infantum trigger different innate immune responses in murine macrophages.

BACKGROUND: The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units. METHODS: Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs (ERK, p38 and JNK) and NF-kB activation were evaluated. RESULTS: Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1ß, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB. CONCLUSION: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions.