Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3.

Staphylococcal protein A (SpA) is a multifunctional, highly conserved virulence factor of Staphylococcus aureus. By binding the Fc portion of all human IgG subclasses apart from IgG3, SpA interferes with antibody and complement deposition on the bacterial surface, impairing staphylococcal clearance by phagocytosis. Because of its anti-opsonic properties, SpA is not investigated as a surface antigen to mediate bacterial phagocytosis. Herein we investigate human sera for the presence of SpA-opsonizing antibodies. The screening revealed that sera containing IgG3 against SpA were able to correctly opsonize the target and drive Fcγ receptor-mediated interactions and phagocytosis. We demonstrated that IgG3 Fc is significantly more efficient in inducing phagocytosis of SpA-expressing S. aureus as compared to IgG1 Fc in an assay resembling physiological conditions. Furthermore, we show that the capacity of SpA antibodies to induce phagocytosis depends on the specific epitope recognized by the IgGs on SpA molecules. Overall, our results suggest that anti-SpA IgG3 antibodies could favor the anti-staphylococcal response in humans, paving the way towards the identification of a correlate of protection against staphylococcal infections.

Dissecting the Human Response to Staphylococcus aureus Systemic Infections.

Staphylococcus aureus is a common human commensal and the leading cause of diverse infections. To identify distinctive parameters associated with infection and colonization, we compared the immune and inflammatory responses of patients with a diagnosis of invasive S. aureus disease to healthy donors. We analyzed the inflammatory responses founding a pattern of distinctive cytokines significantly higher in the patients with invasive disease. The measure of antibody levels revealed a wide antibody responsiveness from all subjects to most of the antigens, with significantly higher response for some antigens in the invasive patients compared to control. Moreover, functional antibodies against toxins distinctively associated with the invasive disease. Finally, we examined the genomic variability of isolates, showing no major differences in genetic distribution compared to a panel of representative strains. Overall, our study shows specific signatures of cytokines and functional antibodies in patients with different primary invasive diseases caused by S. aureus. These data provide insight into human responses towards invasive staphylococcal infections and are important for guiding the identification of novel preventive and therapeutic interventions against S. aureus.

Virulence Gene Expression of Staphylococcus aureus in Human Skin.

Staphylococcus aureus is the main cause of human skin and soft tissue infections. However, S. aureus pathogenicity within the skin is not fully characterized. Here, we implemented an S. aureus cutaneous infection model using human skin explants and performed a time-course infection to study the gene expression profile of a large panel of virulence-related factors of S. aureus USA300 LAC strain, by high-throughput RT-PCR. We pinpointed the genes that were differentially regulated by the bacteria in the skin tissues and identified 12 virulence factors that were upregulated at all time points assessed. Finally, using confocal microscopy, we show that the expression of alpha-hemolysin by S. aureus varies dependent on the skin niche and that the bacteria preferentially accumulates inside sweat glands and ducts. Taken together, our study gives insights about the pathogenic lifestyle of S. aureus within human skin tissues, which may contribute for the development of anti-S. aureus therapeutic strategies.

Human Organotypic Models for Anti-infective Research.

The use of human organotypic models for biomedical research is experiencing a significant increase due to their biological relevance, the possibility to perform high-throughput analyses, and their cost efficiency. In the field of anti-infective research, comprising the search for novel antipathogenic treatments including vaccines, efforts have been made to reduce the use of animal models. That is due to two main reasons: unreliability of data obtained with animal models and the increasing willingness to reduce the use of animals in research for ethical reasons. Human three-dimensional (3-D) models may substitute and/or complement in vivo studies, to increase the translational value of preclinical data. Here, we provide an overview of recent studies utilizing human organotypic models, resembling features of the cervix, intestine, lungs, brain, and skin in the context of anti-infective research. Furthermore, we focus on the future applications of human skin models and present methodological protocols to culture human skin equivalents and human skin explants.

Human Three-Dimensional Models for Studying Skin Pathogens.

Skin is the most exposed surface of the human body, separating the microbe-rich external environment, from the sterile inner part. When skin is breached or its homeostasis is perturbed, bacterial, fungal and viral pathogens can cause local infections or use the skin as an entry site to spread to other organs. In the last decades, it has become clear that skin provides niches for permanent microbial colonization, and it actively interacts with microorganisms. This crosstalk promotes skin homeostasis and immune maturation, preventing expansion of harmful organisms. Skin commensals, however, are often found to be skin most prevalent and dangerous pathogens. Despite the medical interest, mechanisms of colonization and invasion for most skin pathogens are poorly understood. This limitation is due to the lack of reliable skin models. Indeed, animal models do not adequately mimic neither the anatomy nor the immune response of human skin. Human 3D skin models overcome these limitations and can provide new insights into the molecular mechanisms of microbial pathogenesis. Herein, we address the strengths and weaknesses of different types of human skin models and we review the main findings obtained using these models to study skin pathogens.

The Gauze technique: a simple method of dissection in laparoscopic inguinal hernia repair.

BACKGROUND: Hernia repair is one of the most frequent operation in general surgery. The aim of this is study is to present a new preperitoneal atraumatic dissection of peritoneal flap during laparoscopic transabdominal preperitoneal inguinal hernia repair. METHODS: From January 2019 to December 2019, 30 patients (19 male, 11 female) attended TAPP inguinal hernia repairs. The inclusion's criteria were: patients from 32 to 88 years; male and woman; bilateral inguinal hernia. RESULTS: The median age of patients was 46,46 (32-88). The median time of operation was 63,8 (45-100) minutes. All procedures were performed without complications and convertions. CONCLUSIONS: Based on our experience the soft technique is a safe and feasible way to create a preperitoneal flap. Operating time is drastically reduced and the learning curve is reduced even for less experienced surgeons.

Absence of Protein A Expression Is Associated With Higher Capsule Production in Staphylococcal Isolates.

Staphylococcus aureus is a major human pathogen, and a leading cause of soft tissue and blood stream infections. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors through which the bacterium avoids host defenses, where the staphylococcal protein A (SpA) plays a major role thanks to its IgG binding activities. Moreover, SpA has recently been proposed as a promising vaccine antigen. In this study, we evaluated the expression of SpA in a collection of staphylococcal strains, about 7% of which did not express SpA (SpA- strains), despite the presence of the gene. By a comparative genomic analysis, we identified that a mutation in the spa 5' UTR sequence affecting the RBS is responsible for the loss of SpA in a subset of SpA- strains. Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Our data suggest that the SpA- phenotype has occurred in geographically distinct strains through different molecular mechanisms including both mutation, leading likely to translation alterations, and transcriptional deregulation. Furthermore, we provide evidence that SpA- strains are highly susceptible to phagocytic uptake mediated by anti-capsule antibodies. These data suggest that S. aureus may alter its virulence factor expression pattern as an adaptation to the host or environment. Vaccination strategies targeting both SpA and capsule could therefore result in broader coverage against staphylococcal isolates than SpA alone.

SEEG initiative estimates of Brazilian greenhouse gas emissions from 1970 to 2015.

This work presents the SEEG platform, a 46-year long dataset of greenhouse gas emissions (GHG) in Brazil (1970-2015) providing more than 2 million data records for the Agriculture, Energy, Industry, Waste and Land Use Change Sectors at national and subnational levels. The SEEG dataset was developed by the Climate Observatory, a Brazilian civil society initiative, based on the IPCC guidelines and Brazilian National Inventories embedded with country specific emission factors and processes, raw data from multiple official and non-official sources, and organized together with social and economic indicators. Once completed, the SEEG dataset was converted into a spreadsheet format and shared via web-platform that, by means of simple queries, allows users to search data by emission sources and country and state activities. Because of its effectiveness in producing and making available data on a consistent and accessible basis, SEEG may significantly increase the capacity of civil society, scientists and stakeholders to understand and anticipate trends related to GHG emissions as well as its implications to public policies in Brazil.

Prevalence of M75 Streptococcus pyogenes Strains Harboring slaA Gene in Patients Affected by Pediatric Obstructive Sleep Apnea Syndrome in Central Italy.

Recently we reported an association between pediatric obstructive sleep apnea syndrome (OSAS) and Group A streptococcus (GAS) sub-acute chronic tonsil colonization. We showed that GAS may contribute to tonsil hyperplasia via a streptolysin O (SLO)-dependent cysteinyl leukotrienes (CysLTs) production, which can trigger T and B cell proliferation. In the present study, we characterized the GAS strains isolated from pediatric OSAS patients in comparison with a panel of age and sex matched GAS strains unrelated to OSAS, but isolated in the same area and during the same period ranging from 2009 to 2013. We found that slaA gene, previously reported to be associated to CysLTs production pathway, was significantly associated to GAS OSAS strains. Moreover, the most numerous group (32%) of the GAS OSAS strains belonged to M75 type, and 6 out of 7 of these strains harbored the slaA gene. Multilocus Sequence Typing (MLST) experiments demonstrated that the clone emm75/ST49/ smeZ, slaA was associated to OSAS cases. In conclusion, we found an association between slaA gene and the GAS OSAS strains, and we showed that the clone emm75/ST49 harboring genes smeZ and slaA was exclusively isolated from patients affected by OSAS, thus suggesting that this genotype might be associated to the pathogenesis of OSAS, although further studies are needed to elucidate the possible role of SlaA in tonsil hypertrophy development.

Staphylococcus aureus-dependent septic arthritis in murine knee joints: local immune response and beneficial effects of vaccination.

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use.

Paediatric obstructive sleep apnoea syndrome (OSAS) is associated with tonsil colonisation by Streptococcus pyogenes.

The involvement of pathogenic bacteria in obstructive sleep apnoea syndrome (OSAS) has yet to be elucidated. We investigated the possible role of group A streptococcus (GAS) in OSAS pathogenesis. In 40 tonsillectomized patients affected by OSAS and 80 healthy controls, significant (p < 0.0001) association of GAS with paediatric OSAS was found. Supernatant from streptolysin O (SLO)-producing GAS induced production of cysteinyl leukotrienes (CysLTs) in tonsil mononuclear cells (TMCs). CysLTs-treated TMCs showed significant (p < 0.05) proliferation of CD4+ T, CD19+ and CD19+CD27+CD38+ B lymphocytes. We discovered a SLO-dependent activation of CysLTs production through a pathway involving TOLL-like receptor 4 (TLR4), TIR-domain-containing adapter-inducing interferon-ß (TRIF), Myeloid differentiation primary response gene 88 (MyD88), and p38 MAP Kinase. In conclusion, we hypothesise that GAS may contribute to paediatric tonsillar hyperplasia through CysLTs production induced by SLO, and this might explain its association with OSAS.

Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF⁻ B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

Protective activity of the CnaBE3 domain conserved among Staphylococcus aureus Sdr proteins.

Staphylococcus aureus is an opportunistic pathogen, commensal of the human skin and nares, but also responsible for invasive nosocomial as well as community acquired infections. Staphylococcus aureus adheres to the host tissues by means of surface adhesins, such as SdrC, SdrD, and SdrE proteins. The Sdr family of proteins together with a functional A domain, contain respectively two, three or five repeated sequences called B motifs which comprise the CnaB domains. SdrD and SdrE proteins were reported to be protective in animal models against invasive diseases or lethal challenge with human clinical S. aureus isolates. In this study we identified a 126 amino acid sequence containing a CnaB domain, conserved among the three Sdr proteins. The three fragments defined here as CnaBC2, D5 and E3 domains even though belonging to phylogenetically distinct strains, displayed high sequence similarity. Based on the sequence conservation data, we selected the CnaBE3 domain for further analysis and characterization. Polyclonal antibodies raised against the recombinant CnaBE3 domain recognized SdrE, SdrC and SdrD proteins of different S. aureus lineages. Moreover, we demonstrated that the CnaBE3 domain was expressed in vivo during S. aureus infections, and that immunization of this domain alone significantly reduces the bacterial load in mice challenged with S. aureus. Furthermore, we show that the reduction of bacteria by CnaBE3 vaccination is due to functional antibodies. Finally, we demonstrated that the region of the SdrE protein containing the CnaBE3 domain was resistant to trypsin digestion, a characteristic often associated with the presence of an isopeptide bond.

The ancillary protein 1 of Streptococcus pyogenes FCT-1 pili mediates cell adhesion and biofilm formation through heterophilic as well as homophilic interactions.

Gram-positive pili are known to play a role in bacterial adhesion to epithelial cells and in the formation of biofilm microbial communities. In the present study we undertook the functional characterization of the pilus ancillary protein 1 (AP1_M6) from Streptococcus pyogenes isolates expressing the FCT-1 pilus variant, known to be strong biofilm formers. Cell binding and biofilm formation assays using S. pyogenes in-frame deletion mutants, Lactococcus expressing heterologous FCT-1 pili and purified recombinant AP1_M6, indicated that this pilin is a strong cell adhesin that is also involved in bacterial biofilm formation. Moreover, we show that AP1_M6 establishes homophilic interactions that mediate inter-bacterial contact, possibly promoting bacterial colonization of target epithelial cells in the form of three-dimensional microcolonies. Finally, AP1_M6 knockout mutants were less virulent in mice, indicating that this protein is also implicated in GAS systemic infection.

Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case.

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.

Environmental acidification drives S. pyogenes pilus expression and microcolony formation on epithelial cells in a FCT-dependent manner.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a gram-positive human pathogen responsible for a diverse variety of diseases, including pharyngitis, skin infections, invasive necrotizing fasciitis and autoimmune sequelae. We have recently shown that GAS cell adhesion and biofilm formation is associated with the presence of pili on the surface of these bacteria. GAS pilus proteins are encoded in the FCT (Fibronectin-Collagen-T antigen) genomic region, of which nine different variants have been identified so far. In the present study we undertook a global analysis of GAS isolates representing the majority of FCT-variants to investigate the effect of environmental growth conditions on their capacity to form multicellular communities. For FCT-types 2, 3, 5 and 6 and a subset of FCT-4 strains, we observed that acidification resulting from fermentative sugar metabolism leads to an increased ability of the bacteria to form biofilm on abiotic surfaces and microcolonies on epithelial cells. The higher biofilm forming capacity at low environmental pH was directly associated with an enhanced expression of the genes encoding the pilus components and of their transcription regulators. The data indicate that environmental pH affects the expression of most pilus types and thereby the formation of multicellular cell-adhering communities that assist the initial steps of GAS infection.

Typing of the pilus-protein-encoding FCT region and biofilm formation as novel parameters in epidemiological investigations of Streptococcus pyogenes isolates from various infection sites.

Streptococcus pyogenes is an important human pathogen for which an association between infection site and selected epidemiological or functional markers has previously been suggested. However, the studies involved often used strains with an insufficiently defined clinical background and laboratory history. Thus, the major goal of the present study was to investigate these relationships in 183 prospectively collected, well-defined, low-passage isolates from a North-East German centre for tertiary care. For each isolate the clinical background (91 respiratory, 71 skin and 21 invasive isolates) and antibiotic-resistance pattern was recorded. All isolates were classified according to their emm type, antibiotic-resistance and PFGE pattern ( SmaI restriction analysis of genomic DNA). As novel discriminatory methods we performed a PCR-based typing of the pilus-protein-encoding FCT region (FCT) and biofilm-formation phenotyping in various culture media. Forty-one isolates were found to be resistant to at least one of the tested antibiotics. emm typing revealed emm28, emm12 , emm1, emm4, emm89 and emm2 as the most frequent types in our collection. The novel FCT typing showed isolates encoding FCT types 4 and 2 to be the most common. Overall 113 strains with unique combinations of emm and FCT types, antibiotic-resistance and PFGE patterns were identified. The majority of all isolates revealed an association of biofilm-formation capacity with growth media. Comparing all results for potential associations, no correlation could be established between the anatomical site of isolation and the emm or the FCT type. There was no relationship between biofilm formation and emm type, antibiotic-resistance or PFGE patterns. However, a novel association between biofilm formation and FCT type became obvious among strains from our collection.

Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.