Streptococcus suis serotype 2 is an important encapsulated bacterial swine pathogen and zoonotic agent for which no effective vaccine exists. The interaction with B cells and the humoral response ...against S. suis are poorly understood despite their likely relevance for a potential vaccine. We evaluated germinal center (GC) B cell kinetics, as well as the production and role of S. suis-specific antibodies following infections in a mouse model. We found that mice infected with S. suis developed GC that peaked 13-21 days post-infection. GC further increased and persisted upon periodic reinfection that mimics real life conditions in swine farms. Anti-S. suis IgM and several IgG subclasses were produced, but antibodies against the S. suis capsular polysaccharide (CPS) were largely IgM. Interestingly, depletion of total IgG from the wild-type mice sera had no effect on bacterial killing by opsonophagocytosis in vitro. Somatic hypermutation and isotype switching were dispensable for controlling the infection or anti-CPS IgM production. However, T cell-deficient (Tcrb-/-) mice were unable to control bacteremia, produce optimal anti-CPS IgM titers, or elicit antibodies with opsonophagocytic activity. SAP deficiency, which prevents GC formation but not extrafollicular B cell responses, ablated anti S. suis-IgG production but maintained IgM production and eliminated the infection. In contrast, B cell deficient mice were unable to control bacteremia. Collectively, our results indicate that the antibody response plays a large role in immunity against S. suis, with GC-independent but T cell-dependent germline IgM being the major effective antibody specificities. Our results further highlight the importance IgM, and potentially anti-CPS antibodies, in clearing S. suis infections and provide insight for future development of S. suis vaccines.
is an encapsulated bacterium causing severe diseases in swine. Here, we compared the protective properties of the capsular polysaccharide (CPS) of different
serotypes by using serotype-switched ...mutants in a mouse model of infection. CPS structure influenced bacterial survival in mice, antibody binding, and antibody-mediated bacterial killing. The CPS of serotypes 3, 4 and 14 allowed more antibody binding and bacterial elimination than the CPS of serotypes 2, 7 and 9. Results suggest that the different CPS structures of
provide varying levels of protection by influencing antigen availability and elimination by the host immune system.
Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using ...mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.
Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While ...the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian beta-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.
The mold Aspergillus fumigatus causes invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetylgalactosamine ...(GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-D-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster gene agd3 encodes a protein containing a deacetylase domain. Because deacetylation of N-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3 mutant in the presence of culture supernatants of the GAG-deficient Δuge3 mutant rescued the biofilm defect of the Δagd3 mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of the Pezizomycotina subphylum of the Ascomycota including a number of plant-pathogenic fungi and a single basidiomycete species,Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation.
This study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatus virulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.
Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both ...contain 1,4-linked N-acetyl-D-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h. In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.
Objectives: In COVID-19 patients, bacterial and fungal pulmonary coinfections, such as Streptococcus pneumoniae, 'Staphylococcus aureus', 'Haemophilus influenzae', or 'Aspergillus', have been ...reported, but to our knowledge, no case has been reported due to 'Pasteurella multocida'. Patients and methods: We describe three cases of 'Pasteurella multocida' coinfections occurring during the 4th wave of COVID-19 in Martinique (French West Indies). Results: All three cases were fatal; thus, 'Pasteurella multocida' has to be considered as a potentially severe coinfection agent. Conclusions: Alteration of the epithelial-endothelial barrier due to a SARS-CoV-2 infection probably promotes the expression of a 'Pasteurella' infection. In addition, the SARS-CoV-2 infection induced immunosuppression, and an inflammatory cascade could explain the infection's severity. The use of corticosteroids, which are part of the first-line therapeutic arsenal against COVID-19, may also promote the pathogenicity of this agent.
Deacetylation of chitin by chitin deacetylases (Cda) results in the formation of chitosan. Chitosan, a polymer of β1,4 linked glucosamine, plays multiple roles in the function of the fungal cell ...wall, including virulence and evasion of host immune responses. In this study, the roles of chitosan and putative
s in cell wall structure and virulence of
were investigated. Low levels of chitosan were found in the conidial and cell wall of
. Seven putative
genes were identified, disrupted and the phenotype of the single mutants and the septuple mutants were investigated. No alterations in fungal cell wall chitosan levels, changes in fungal growth or alterations in virulence were detected in the single or septuple Δ
mutant strains. Collectively, these results suggest that chitosan is a minority component of the
cell wall, and that the seven candidate Cda proteins do not play major roles in fungal cell wall synthesis or virulence. However, Cda2 is involved in conidiation, suggesting that this enzyme may play a role in N-acetyl-glucosamine metabolism.
Bacterial and/or viral co-infections are very common in swine production and cause severe economic losses.
,
and
are pathogenic bacteria that may be found simultaneously in the respiratory tracts of ...pigs. In the present study, the interactions of
with epithelial and phagocytic cells in the presence or absence of a pre-infection with
and/or
were studied. Results showed relatively limited interactions between these pathogens. A previous infection with one or both mycoplasmas did not influence the adhesion or invasion properties of
in epithelial cells or its resistance to phagocytosis (including intracellular survival) by macrophages and dendritic cells. The most important effect observed during the co-infection was a clear increment in toxicity for the cells. An increase in the relative expression of the pro-inflammatory cytokines IL-6 and CXCL8 was also observed; however, this was the consequence of an additive effect due to the presence of different pathogens rather than a synergic effect. It may be hypothesized that if one or both mycoplasmas are present along with
in the lower respiratory tract at the same time, then increased damage to epithelial cells and phagocytes, as well as an increased release of pro-inflammatory cytokines, may eventually enhance the invasive properties of
. However, more studies should be carried out to confirm this hypothesis.
is a zoonotic pathogen affecting pigs and humans. This bacterium causes severe economic losses in the swine industry and poses a serious threat to public health and food safety. There is no effective ...commercial vaccine available for pigs or humans. In this study, we applied the biopolymer particle (BP) vaccine technology to incorporate seven conserved
antigens (38 kDa protein (38), enolase (Enol), SSU1915, SSU1355, SSU0185, SSU1215, and SSU1773 (SSU1 and SSU2)). Two combinations of these antigens (38 and Enol; all SSU antigens designated as SSU1 and SSU2) were engineered to mediate production of BPs coated with either antigens 38 and Enol or SSU1 and SSU2 inside recombinant
. The isolated and purified empty BPs, 38-BP-Enol and SSU1-BP-SSU2, showed size ranges of 312-428 nm and 292-344 nm with and without the QuilA
adjuvant, respectively, and all showed a negative surface charge. Further characterization of purified BPs confirmed the presence of the expected antigen-comprising fusion proteins as assessed by tryptic peptide fingerprinting analysis using quadrupole time-of-flight mass spectrometry and immunoblotting. Vaccination with 38-BP-Enol and SSU1-BP-SSU2 formulated with and without QuilA
adjuvant induced significant antigen-specific humoral immune responses in mice. Antigen-coated BPs induced significant and specific Ig (IgM + IgG) and IgG immune responses (1.0 × 10
-1.0 × 10
) when compared with mice vaccinated with empty BPs. Functionality of the immune response was confirmed in challenge experiments using an acute murine
infection model, which showed 100% survival of the 38-BP-Enol and SSU1-BP-SSU2 vaccinated mice compared to 70% survival when vaccinated with empty BPs. Overall, our data suggest that
antigen-coated BPs could be developed into particulate vaccines that induce protective immunity against
infections.
PDF
