Immunological studies on staphylococcal superantigen-like proteins.

The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded within the Staphylococcus aureus genome, whose function remains unknown. The ability of SSL7, and a closely related paralog SSL9, to interact with cells of the immune system was investigated. Within the populations of human white blood cells, both SSLs interact selectively with monocytes, via specific but separate binding sites, leading to rapid uptake of SSLs. In addition, SSLs are rapidly taken up by dendritic cells (DC), but not macrophages, and target the mannose-receptor dependent endosomal antigen processing pathway. The effect of these proteins on the functional capacity of antigen presenting cells to uptake and present antigens to T cells was also determined. Neither SSL was toxic to DCs and the presence of SSL protein did not inhibit the antigen presenting cell activity, in terms of stimulation of either allogeneic or recall T cell responses. The immunological response to the SSL proteins in the normal human population was investigated. More than 30% of healthy normal subjects tested showed T cell responses to both SSL7 and SSL9. Moreover, almost all individuals had specific non- cross reacting antibodies against this family of proteins. In order to identify the SSL receptor(s), affinity chromatography techniques were used to isolate and identify the receptor(s) from selected cell lines. A single specific protein band that may represent the putative SSL receptor was observed, and mass spectrometry identified a candidate binding protein as heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa, GRP78 (BiP)). The presence of eleven members of this protein family within the pathogenicity island SaPIn2 in almost all S. aureus strains tested so far, suggest that these proteins have important non-redundant biological functions as agents of host/pathogen interactions. The data presented in this thesis further suggests that this function may involve targeting the host antigen presenting cell

GPVI and GPIbα Mediate Staphylococcal Superantigen-Like Protein 5 (SSL5) Induced Platelet Activation and Direct toward Glycans as Potential Inhibitors

Background Staphylococcus aureus (S. aureus) is a common pathogen capable of causing life-threatening infections. Staphylococcal superantigen-like protein 5 (SSL5) has recently been shown to bind to platelet glycoproteins and induce platelet activation. This study investigates further the interaction between SSL5 and platelet glycoproteins. Moreover, using a glycan discovery approach, we aim to identify potential glycans to therapeutically target this interaction and prevent SSL5-induced effects. Methodology/Principal Findings In addition to platelet activation experiments, flow cytometry, immunoprecipitation, surface plasmon resonance and a glycan binding array, were used to identify specific SSL5 binding regions and mediators. We independently confirm SSL5 to interact with platelets via GPIbα and identify the sulphated-tyrosine residues as an important region for SSL5 binding. We also identify the novel direct interaction between SSL5 and the platelet collagen receptor GPVI. Together, these receptors offer one mechanistic explanation for the unique functional influences SSL5 exerts on platelets. A role for specific families of platelet glycans in mediating SSL5-platelet interactions was also discovered and used to identify and demonstrate effectiveness of potential glycan based inhibitors in vitro. Conclusions/Significance These findings further elucidate the functional interactions between SSL5 and platelets, including the novel finding of a role for the GPVI receptor. We demonstrate efficacy of possible glycan-based approaches to inhibit the SSL5-induced platelet activation. Our data warrant further work to prove SSL5-platelet effects in viv

Undiagnosed Active Pulmonary Tuberculosis among Pilgrims during the 2015 Hajj Mass Gathering: A Prospective Cross-sectional Study

Mass gatherings pose a risk for tuberculosis (TB) transmission and reactivation of latent TB infection. The annual Hajj pilgrimage attracts 2 million pilgrims many from high TB-endemic countries. We evaluated the burden of undiagnosed active pulmonary TB in pilgrims attending the 2015 Hajj mass gathering. We conducted a prospective cross-sectional study in Mecca, Kingdom of Saudi Arabia, for nonhospitalized adult pilgrims from five high TB-endemic countries. Enrollment criteria were the presence of a cough and the ability to produce a sputum sample. Sputum samples were processed using the Xpert MTB-RIF assay. Data were analyzed for drug-resistant TB, risk factors, and comorbidities by the country of origin. Of 1,164 consenting pilgrims enrolled from five countries: Afghanistan (316), Bangladesh (222), Nigeria (176), Pakistan (302), and South Africa (148), laboratory results were available for 1,063 (91.3%). The mean age of pilgrims was 54.5 (range = 18–94 years) with a male to female ratio of 2.6:1; 27.7% had an underlying comorbidity, with hypertension and diabetes being the most common, 20% were smokers, and 2.8% gave a history of previous TB treatment. Fifteen pilgrims (1.4%) had active previously undiagnosed drug-sensitive pulmonary TB (Afghanistan [12; 80%], Pakistan [2; 13.3%], and Nigeria [1; 6.7%]). No multidrug-resistant TB cases were detected. Pilgrims from high TB-endemic Asian and African countries with undiagnosed active pulmonary TB pose a risk to other pilgrims from over 180 countries. Further studies are required to define the scale of the TB problem during the Hajj mass gathering and the development of proactive screening, treatment and prevention guidelines

Assessment of household use of iodized salt and adequacy of salt iodization: a cross-sectional National Study in Saudi Arabia

Abstract Objectives This study was conducted to assess household coverage with iodized salt in Saudi Arabia, and to determine adequacy of salt iodization. Methods A school-based cross-sectional study using WHO 30-cluster survey methodology. Results Analysis of 4242 salt samples using qualitative rapid test kit (RTK) revealed that 68.7% (95% CI 67.3–70.1%) were iodized with significant regional differences (p < 0.001). The highest iodized salt samples came from Makkah (82.3%), Riyadh (81.1%) and Maddinah (76.2%) regions, while the least iodized salt samples came from Hail (31.3%), Baha (53.0%), and Northern Borders (57.5%) regions. The national weighted proportion of households consuming iodizes salt was 69.8% (95% CI 69.4–71.2), which is below the Universal Salt Iodization (USI) goal (≥90% coverage). For validation, a quantitative iodometric titration method was used to analyze 775 representative salt samples screened iodized by RTK; iodine content of ≥15 ppm was found in 95.2% (95% CI 93.9–96.5) of samples with median iodine content 51 ppm (mean 50.4 ± 21.8). More than 70% of the iodized salt samples contained iodine concentration higher than the recommended national level (15–40 ppm). Conclusions The study revealed inadequate consumption of iodized salt among Saudi households and explored marked regional heterogeneity. The majority of iodized salt samples contained iodine concentration more than the recommended level. These findings imply the need to launch a public awareness campaign on use of iodized salt. Legislation to ban production and sale of non-iodized salt sale for human consumption might be considered. A well-functioning monitoring system at factory level and surveillance system are crucially needed to ensure proper salt iodization and intake

Structural Relationships and Cellular Tropism of Staphylococcal Superantigen-Like Proteins

The staphylococcal superantigen-like proteins (SSLs) are a family of polymorphic paralogs encoded in the Staphylococcus aureus genome whose function is unknown. The crystal structure of SSL7 was determined and compared to that of SSL5 and that of a classical superantigen, streptococcal pyrogenic exotoxin. Although the overall architecture of the superantigen family is retained in both SSL7 and SSL5, there are significant differences in the structures which suggest that the characteristic major histocompatibility complex binding site of superantigens has been lost. To complement these data, the abilities of SSL7 and a closely related paralog, SSL9, to interact with cells of the immune system were investigated. In populations of human white blood cells, both SSLs interacted selectively with monocytes via specific saturable but separate binding sites, which led to rapid uptake of the SSLs. In addition, SSLs were rapidly taken up by dendritic cells, but not by macrophages, into the same endosomal compartment as dextran. The ability of these secreted proteins to target antigen-presenting cells may enhance a misplaced antibody response against the proteins, which may facilitate bacterial colonization rather than contribute to host protection. Like classical superantigens, therefore, SSLs may distract the host's immune system, but they may do so via entirely different molecular mechanisms

Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1

Infection by Staphylococcus aureus can result in severe conditions such as septicemia, toxic shock, pneumonia, and endocarditis with antibiotic resistance and persistent nasal carriage in normal individuals being key drivers of the medical impact of this virulent pathogen. In both virulent infection and nasal colonization, S. aureus encounters the host immune system and produces a wide array of factors that frustrate host immunity. One in particular, the prototypical staphylococcal superantigen-like protein SSL7, potently binds IgA and C5, thereby inhibiting immune responses dependent on these major immune mediators. We report here the three-dimensional structure of the complex of SSL7 with Fc of human IgA1 at 3.2 Å resolution. Two SSL7 molecules interact with the Fc (one per heavy chain) primarily at the junction between the Cα2 and Cα3 domains. The binding site on each IgA chain is extensive, with SSL7 shielding most of the lateral surface of the Cα3 domain. However, the SSL7 molecules are positioned such that they should allow binding to secretory IgA. The key IgA residues interacting with SSL7 are also bound by the leukocyte IgA receptor, FcαRI (CD89), thereby explaining how SSL7 potently inhibits IgA-dependent cellular effector functions mediated by FcαRI, such as phagocytosis, degranulation, and respiratory burst. Thus, the ability of S. aureus to subvert IgA-mediated immunity is likely to facilitate survival in mucosal environments such as the nasal passage and may contribute to systemic infections

Presence of Middle East respiratory syndrome coronavirus antibodies in Saudi Arabia : a nationwide, cross-sectional, serological study

BACKGROUND: Scientific evidence suggests that dromedary camels are the intermediary host for the Middle East respiratory syndrome coronavirus (MERS-CoV). However, the actual number of infections in people who have had contact with camels is unknown and most index patients cannot recall any such contact. We aimed to do a nationwide serosurvey in Saudi Arabia to establish the prevalence of MERS-CoV antibodies, both in the general population and in populations of individuals who have maximum exposure to camels. METHODS: In the cross-sectional serosurvey, we tested human serum samples obtained from healthy individuals older than 15 years who attended primary health-care centres or participated in a national burden-of-disease study in all 13 provinces of Saudi Arabia. Additionally, we tested serum samples from shepherds and abattoir workers with occupational exposure to camels. Samples were screened by recombinant ELISA and MERS-CoV seropositivity was confirmed by recombinant immunofluorescence and plaque reduction neutralisation tests. We used two-tailed Mann Whitney U exact tests, χ(2), and Fisher's exact tests to analyse the data. FINDINGS: Between Dec 1, 2012, and Dec 1, 2013, we obtained individual serum samples from 10 009 individuals. Anti-MERS-CoV antibodies were confirmed in 15 (0·15%; 95% CI 0·09-0·24) of 10 009 people in six of the 13 provinces. The mean age of seropositive individuals was significantly younger than that of patients with reported, laboratory-confirmed, primary Middle Eastern respiratory syndrome (43·5 years [SD 17·3] vs 53·8 years [17·5]; p=0·008). Men had a higher antibody prevalence than did women (11 [0·25%] of 4341 vs two [0·05%] of 4378; p=0·028) and antibody prevalence was significantly higher in central versus coastal provinces (14 [0·26%] of 5479 vs one [0·02%] of 4529; p=0·003). Compared with the general population, seroprevalence of MERS-CoV antibodies was significantly increased by 15 times in shepherds (two [2·3%] of 87, p=0·0004) and by 23 times in slaughterhouse workers (five [3·6%] of 140; p<0·0001). INTERPRETATION: Seroprevalence of MERS-CoV antibodies was significantly higher in camel-exposed individuals than in the general population. By simple multiplication, a projected 44 951 (95% CI 26 971-71 922) individuals older than 15 years might be seropositive for MERS-CoV in Saudi Arabia. These individuals might be the source of infection for patients with confirmed MERS who had no previous exposure to camels. FUNDING: European Union, German Centre for Infection Research, Federal Ministry of Education and Research, German Research Council, and Ministry of Health of Saudi Arabia