Neutrophils play a key role in the human immune response to Staphylococcus aureus infections. These professional phagocytes rapidly migrate to the site of infection to engulf bacteria and destroy them via specialized intracellular killing mechanisms. Here we describe a robust and relatively high-throughput flow cytometry assay to quantify phagocytosis of S. aureus by human neutrophils. We show that effective phagocytic uptake of S. aureus is greatly enhanced by opsonization, i.e. the tagging of microbial surfaces with plasma-derived host proteins like antibodies and complement. Our rapid assay to monitor phagocytosis can be used to study neutrophil deficiencies and bacterial evasion, but also provides a powerful tool to assess the opsonic capacity of antibodies, either in the context of natural immune responses or immune therapies.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surfaceexpressed b protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in b represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

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.

No abstract

Shigella is a major global pathogen and the etiological agent of shigellosis, a diarrheal disease that primarily affects low- and middle-income countries. Shigellosis is characterized by a complex, multistep pathogenesis during which bacteria use multiple invasion proteins to manipulate and invade the intestinal epithelium. Antibodies, especially against the O-antigen and some invasion proteins, play a protective role as titres against specific antigens inversely correlate with disease severity; however, the context of antibody action during pathogenesis remains to be elucidated, especially with Shigella being mostly an intracellular pathogen. In the absence of a correlate of protection, functional assays rebuilding salient moments of Shigella pathogenesis can improve our understanding of the role of protective antibodies in blocking infection and disease. In vitro assays are important tools to build correlates of protection. Only recently animal models to recapitulate human pathogenesis, often not in full, have been established. This review aims to discuss in vitro assays to evaluate the functionality of anti-Shigella antibodies in polyclonal sera in light of the multistep and multifaced Shigella infection process. Indeed, measurement of antibody level alone may limit the evaluation of full vaccine potential. Serum bactericidal assay (SBA), and other functional assays such as opsonophagocytic killing assays (OPKA), and adhesion/invasion inhibition assays (AIA), are instead physiologically relevant and may provide important information regarding the role played by these effector mechanisms in protective immunity. Ultimately, the review aims at providing scientists in the field with new points of view regarding the significance of functional assays of choice which may be more representative of immune-mediated protection mechanisms.

According to our preliminary results, prostaglandin antiglaucomatous analogs do not significantly enhance pigmentation in the superficial layers of the conjunctiva. The existence of the Tyrosinase enzyme in the superficial layers of the conjunctiva suggests that basal melanocytes may transfer their melanogenic apparatus to superficial epithelial cells.

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.