Infect Immun. 2014 Jul;82(7):2890-901.

SpyAD, a moonlighting protein of Group A Streptococcus contributing to bacterial division and host cell adhesion

Marilena Gallotta,a1 Giovanni Gancitano,a2 Giampiero Pietrocola,b Marirosa Mora,a Alfredo Pezzicoli,a Giovanna Tuscano,a Emiliano Chiarot,a Vincenzo Nardi Dei,a Annarita Taddei,c Simonetta Rindi,b Pietro Speziale,b Marco Soriani,a Guido Grandi,a3* Immaculada Margarita* and Giuliano Bensia


Novartis Vaccines and Diagnostics Srl, Siena, Italya; Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Pavia, Italyb; Centre for High Instruments, Electron Microscopy Section, University of Tuscia, Viterbo, Italyc

*Address correspondence to Immaculada Margarit, and Guido Grandi,

1Present address: Dynavax Technologies Corporation, Berkeley, CA 94710, USA

2Present address: Sebia srl, Florence, Italy

3Present address: Center for Integrative Biology, University of Trento, Trento, Italy



Group A Streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, indicative of an impaired capacity of the knock-out mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein).

PMID: 24778116



The Gram-positive pathogen Streptococcus pyogenes (group A Streptococcus, GAS) causes a broad range of human diseases including superficial pharyngitis and skin infections that can lead to suppurative sequelae, as well as invasive conditions like pneumonia, bacteremia, streptococcal toxic shock syndrome and necrotizing fasciitis (1-4).

A vaccine against GAS is not available yet, although several efforts have led to the discovery of putative vaccine candidates (5, 6). To achieve a highly selective identification of few protective GAS antigens, we recently applied a high throughput strategy, which combines parallel mass spectrometry analysis of peptides generated after protease treatment of live bacteria, analysis of immunogenic antigens by protein array, and quantification of antibody accessible antigens by flow-cytometry analysis. This allowed defining a three-protein formulation conferring consistent protection in mice against infection with multiple GAS serotypes (7, 8). Two out of three protective antigens, i.e. Streptolysin O and the chemokine protease SpyCEP, were well described for their role in GAS pathogenesis (9, 10), while the function of the third antigen Spy0269 was completely unknown and was the subject of the present study. Analysis of the publicly available GAS genomes in the NCBI database indicated that the Spy0269 gene was present in 20 out of 20 completely sequenced strains, with >93% identity along its 873 amino acids sequence. We also reported that Spy0269 was exposed on the surface of 18 out of 22 isolates analyzed by flow cytometry (FACS) using a specific mouse serum, and that the protein was highly recognized by 204 out of 239 sera from pharyngitis patients, indicating high surface expression during human infection (7, 11).

Using a combination of genetic, biochemical and cellular microbiology approaches, we have provided evidence that SPy0269, here renamed SpyAD (Streptococcus pyogenes Adhesion and Division protein) is a multifunctional protein that plays a role in GAS cell division and can mediate bacterial adhesion to epithelial host cells.


BE fig1

FIG 1 Analysis of SpyAD deletion mutant (DSpyAD) bacterial cultures. (A) Bacterial cultures of GAS 3348 wild-type strain, SpyAD gene deletion mutant (DSpyAD) and SpyAD complemented strains DSpyAD(pAMSpyAD) observed after 150 minutes of incubation. (B) Schematic representation of the sedimentation rate curves of the three strains, obtained by measuring the 620 nm optical densities in the upper part of the tubes at different time points. (C) Representative images of the three strains obtained by optical microscopy (high-contrast digital enhancement). (D) and (E) Representative images obtained by Scanning Electron Microscopy (SEM) of the GAS M1-3348 wild-type strain and of the DSpyAD gene deletion mutant respectively.


The striking long-chain phenotype observed when the SpyAD gene was deleted, depicted in Figure 1, as well as the evidence showing that GAS SpyAD is localized in the bacterial septum and that a recombinant version of the protein can interact with FtsZ, strongly suggested that SpyAD is involved in the process(es) leading to cell division. FtsZ has been reported as cytosolic in several bacterial species, but confocal results showing it can be stained by specific antibodies suggested that in the case of GAS this protein can be exposed on the bacterial surface. Overall, the obtained data reinforced the hypothesis that SpyAD could be involved in the bacterial cell division process, possibly modulating the GAS divisome machinery.

A second possible role of SpyAD as a novel GAS adhesin was evidenced by its capacity to bind to eukaryotic cells in vitro and to mediate bacterial adhesion to the nasal epithelium in vivo, as shown in Figure 2.


BE fig2

FIG 2 In vitro and in vivo cell adhesion of L. lactis expressing SpyAD. (A) Flow cytometry analysis of L. lactis expressing either full-length SpyAD (pAM-SpyAD) or the SpyAD/M1 chimera (pAM-SpyAD/M1) as compared to a strain carrying the vector alone (pAM). Filled and empty histograms indicate staining of bacteria with a serum raised against alum adjuvant alone and with a polyclonal mouse SpyAD antiserum respectively. (B) Bar graph showing in vitro adhesion of the three L. lactis strains to A549 human epithelial cells and specific inhibition of the binding by anti-SpyAD antibodies. The percentage of adhesion of L. lactis to A549 cells is reported on the y-axis. Sera dilutions tested for inhibition of cell binding are reported below the x-axis. Black, light grey and dark grey bars illustrate the results obtained when cells where incubated with the pAM, pAM–SpyAD and SpyAD/M1 L. lactis strains respectively (multiplicity of infection, MOI, was 20:1). Mean values and standard deviations (SD) from 6-8 experiments are represented. Statistical significance: **P < 0.01, ***P < 0.001. (C) In vivo adhesion of the three recombinant L. lactis strains to the nasal mucosa of CD1 mice. The strains used to infect mice intranasally (2-5 x 107 CFU/mouse) are indicated on the x-axis. The y-axis reports the number of recovered CFUs per million of bacteria inoculated by performing nasal washes 20 h after infection.


SpyAD could adhere to different epithelial cell lines and also to Human Brain Microvascular Endothelial cells (HBMEC, data not shown). The absence of strict cell type specificity was not unexpected, as GAS and other streptococci have a large pool of molecules which mediate host cell adhesion with various affinities (12, 13). A two-step mechanism has been proposed for GAS cell adhesion according to which an initial weak and reversible contact with host cells is followed by a second adhesion step mediated by different arrays of adhesins, which confer cell and tissue specificity (14). We hypothesize that SpyAD may interact with the host at a stage of infection in which a weak and low specific adhesion is required, possibly binding to cellular surface exposed proteins or the extracellular matrix (ECM). In vitro binding experiments to eukaryotic cell proteins actually indicated that SpyAD can bind to human collagen VI and keratin 1. Collagen VI is localized in vascular walls and in the interstitial space of the upper and lower airways, beneath basement membrane of the epithelium, and is a target for streptococcal adhesins, among which S. pyogenes M protein (15). Keratin 1 and its heterodimer type I partner, keratin 10, are major components of the cytoskeleton in suprabasal keratinocytes of stratified squamous epithelia (16). Keratin 1 is also present on the cell surface of endothelial cells (17). We can envisage that infections and other inflammatory conditions may significantly alter epithelial integrity, leading to partial or complete shedding of epithelial basal cells and lamina and unmasking of potential receptors for bacterial adhesion (18, 19). This scenario would well fit with the fact that GAS readily colonizes the nasal and the upper respiratory tract epithelia, tissues which frequently undergo tissue repair and remodeling.

The ability of some proteins to exert more than one biological function is well described both in eukaryotes and prokaryotes, and several “moonlighting” proteins have been identified in a wide range of human pathogens, including Group A Streptococcus. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, chaperonin 60, Hsp70 and peptidyl prolyl isomerase, are known bacterial moonlighting proteins playing a role in bacterial virulence (20-22).

In conclusion, based on the evidence reported in this study, we predict that induction of functional antibodies against SpyAD can impair the capacity of the pathogen to properly divide and to colonize specific host niches. This may contribute to the observed SpyAD-mediated highly protective immune response in mouse models of infection, which makes of this antigen an excellent candidate for a multicomponent broadly effective vaccine against Group A Streptococcus.



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