Increased Production of Outer Membrane Vesicles by Salmonella Interferes with Complement-Mediated Innate Immune Attack

Bacterial outer membrane vesicles (OMVs) enriched with bioactive proteins, toxins, and virulence factors play a critical role in host-pathogen and microbial interactions. The two-component system PhoP-PhoQ (PhoPQ) of Salmonella enterica orchestrates the remodeling of outer membrane lipopolysaccharide (LPS) molecules and concomitantly upregulates OMV production. In this study, we document a novel use of nanoparticle tracking analysis to determine bacterial OMV size and number. Among the PhoPQ-activated genes tested,; pagC; expression had the most significant effect on the upregulation of OMV production. We provide the first evidence that PhoPQ-mediated upregulation of OMV production contributes to bacterial survival by interfering with complement activation. OMVs protected bacteria in a dose-dependent manner, and bacteria were highly susceptible to complement-mediated killing in their absence. OMVs from bacteria expressing PagC bound to complement component C3b in a dose-dependent manner and inactivated it by recruiting complement inhibitor Factor H. As we also found that Factor H binds to PagC, we propose that PagC interferes with complement-mediated killing of Salmonella in the following two steps: first by engaging Factor H, and second, through the production of PagC-enriched OMVs that divert and inactivate the complement away from the bacteria. Since PhoPQ activation occurs intracellularly, the resultant increase in PagC expression and OMV production is suggested to contribute to the local and systemic spread of Salmonella released from dying host cells that supports the infection of new cells.; IMPORTANCE; Bacterial outer membrane vesicles (OMVs) mediate critical bacterium-bacterium and host-microbial interactions that influence pathogenesis through multiple mechanisms, including the elicitation of inflammatory responses, delivery of virulence factors, and enhancement of biofilm formation. As such, there is a growing interest in understanding the underlying mechanisms of OMV production. Recent studies have revealed that OMV biogenesis is a finely tuned physiological process that requires structural organization and selective sorting of outer membrane components into the vesicles. In Salmonella, outer membrane remodeling and OMV production are tightly regulated by its PhoPQ system. In this study, we demonstrate that PhoPQ-regulated OMV production plays a significant role in defense against host innate immune attack. PhoPQ-activated PagC expression recruits the complement inhibitor Factor H and degrades the active C3 component of complement. Our results provide valuable insight into the combination of tools and environmental signals that Salmonella employs to evade complement-mediated lysis, thereby suggesting a strong evolutionary adaptation of this facultative intracellular pathogen to protect itself during its extracellular stage in the host

Anti-SP0845^23–350 antibodies are functional as assessed by blood bactericidal assay.

<p>Pneumococcal cells (100–150 cfu) from strains TIGR4 (A), ATCC 6303 (B), ATCC 6314 (C) and D39 (D) were pretreated with either preimmune (PI, dark bar) or mouse anti-SP0845^23–350 hyperimmune (HI, grey bar) sera and incubated with peripheral blood from CBA/N mice at 37°C for 3 h with rotation. The surviving bacteria were enumerated by plating serial dilutions on TSA plates in duplicate. The data represents the mean ± SD values. The data was analyzed using Student's unpaired t test.</p

Purification and immunoblotting of SP0845.

<p>SP0845^23–350 was expressed in <i>E</i>. <i>coli</i> and purified using Ni-NTA affinity and DEAE sepharose anion-exchange chromatography. The purified recombinant protein was resolved on 12% SDS-PAG and stained with Coomassie brilliant blue R250 (A) or transferred to nitrocellulose membrane and visualized using anti-histidine affinity tag antibody followed by horseradish peroxidase conjugated goat anti-mouse Ig as secondary antibody and 3, 3' diaminobenzidine/ H₂O₂ as substrate (B). The molecular mass marker (in kDa) is shown on the left of the panels.</p

Identification of immunoreactive proteins from pneumococcal cell envelope fraction.

<p>The SB14 solubilized surface associated proteins of TIGR4 were resolved by 2D gel electrophoresis. The gel was either silver stained (A) or transferred to PVDF membrane, immunoblotted with polyclonal sera raised against whole heat-killed TIGR4 and developed using enhanced chemiluminescence reagent as substrate (B). The immunoreactive spots analyzed from the gel shown in panel A are numbered 1 through 8 (circled) and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118154#pone.0118154.t001" target="_blank">Table 1</a>. The molecular mass marker (in kDa) is shown to the right of panel A. The proteins were resolved by isoelectric focusing in the pH range 4 to 7 as marked in panel A.</p

SP0845 is accessible to antibodies.

<p>(A) Mid-logarithmic phase TIGR4 cells were incubated with mouse anti-SP0845^23–350 or mouse preimmune sera (negative control) followed by incubation with F(ab')₂ fragment phycoerythrin-conjugated goat anti-mouse IgG + IgM (H + L) antibody. The processed samples were observed under a fluorescence microscope. The fluorescent images obtained with anti-SP0845^23–350 and preimmune sera are in the upper left and right panels, respectively. The lower panels show the corresponding phase contrast images (magnification = 1000X). Surface expression of SP0845 was studied for pneumococcal strains TIGR4 (B), ATCC 6303 (C), ATCC 6314 (D), D39 (E) and R36A (F). Pneumococcal cells were incubated with either preimmune or anti-SP0845^23–350 sera for 1 hr followed by a FITC-conjugated F(ab')₂ fragment goat anti-mouse IgG + IgM (H + L) antibody as secondary antibody. The surface staining was detected by flow cytometry.</p

Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection <i>In Vivo</i>

<div><p><i>Streptococcus pneumoniae</i> is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of <i>S. pneumoniae</i> are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of <i>S. pneumoniae</i> TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of <i>sp0845</i> homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in <i>in vitro</i> grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.</p></div

SP0845-specific serum antibody endpoint titers.

<p>^<i>a</i> The SP0845-specific antibody endpoint titer was determined by ELISA after pooling serum from 12 mice immunized subcutaneously with recombinant SP0845. The endpoint titer was determined using two times the optical density obtained for the preimmune sera (diluted 1 in 100) as the cut off value.</p><p>SP0845-specific serum antibody endpoint titers.</p

SPD_0739 (homologue of SP0845) is involved in nucleoside import by pneumococci.

<p>(A) Wildtype (WT) D39, <i>spd_0739</i> deficient (KO), <i>spd_0739</i> deficient strain genetically complemented with <i>spd_0739</i> (GC) and <i>spd_0739</i> deficient strain transformed with pDC123_DS (vector control; VC) were propagated either in the absence or presence of 0.1 mM 5-fluorouridine (5FU). (B) Wildtype TIGR4 and its <i>sp0845</i> deficient derivative were propagated either in the absence or presence of 0.1 mM 5FU. (C) Wildtype D39 and a second independently generated <i>spd_0739</i> deficient mutant were grown either in the absence or presence of 0.1 mM 5FU. The absorbance of the pneumococcal cultures was recorded at 620 nm after 6 h. For panels A, B and C the statistical significance was determined using the Student's t test with the corresponding 'No 5FU' sample as the reference. (D) Wildtype D39 was grown in presence of 0.05 mM 5FU and the indicated solutes at a concentration of 0.5 mM. Six hours later the absorbance was recorded. Wildtype D39 in the absence of 5FU served as the positive control. Error bars represent mean ± SD. The data presented is a representative of three independent experiments each performed in duplicates. One-way ANOVA analysis was performed using D39 sample with 5FU in the absence of any competing solute as the reference.</p

Immunoreactive pneumococcal proteins identified in this study by mass spectrometry.

<p>^<i>a</i> For spot numbers 1 through 8 refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118154#pone.0118154.g001" target="_blank">Fig. 1</a>. Spot number 9 and 10 were identified from an independent experiment performed under similar experimental conditions.</p><p>^<i>b</i> Gene and protein annotations are according to the genome sequence of <i>S</i>. <i>pneumoniae</i> TIGR4 (GenBank accession no. AE005672). ORF, open reading frame.</p><p>^<i>c</i> Percentage of total protein sequence covered by the experimentally detected peptides.</p><p>^<i>d</i> Identification probability of the fragment match.</p><p>^<i>e</i> MASCOT scores greater than the cut-off value were considered statistically significant (<i>p</i> ≤ 0.05).</p><p>Immunoreactive pneumococcal proteins identified in this study by mass spectrometry.</p