Reactivating Immunity Primed by Acellular Pertussis Vaccines in the Absence of Circulating Antibodies: Enhanced Bacterial Control by TLR9 Rather Than TLR4 Agonist-Including Formulation.

Pertussis is still observed in many countries despite of high vaccine coverage. Acellular pertussis (aP) vaccination is widely implemented in many countries as primary series in infants and as boosters in school-entry/adolescents/adults (including pregnant women in some). One novel strategy to improve the reactivation of aP-vaccine primed immunity could be to include genetically- detoxified pertussis toxin and novel adjuvants in aP vaccine boosters. Their preclinical evaluation is not straightforward, as it requires mimicking the human situation where T and B memory cells may persist longer than vaccine-induced circulating antibodies. Toward this objective, we developed a novel murine model including two consecutive adoptive transfers of the memory cells induced by priming and boosting, respectively. Using this model, we assessed the capacity of three novel aP vaccine candidates including genetically-detoxified pertussis toxin, pertactin, filamentous hemagglutinin, and fimbriae adsorbed to aluminum hydroxide, supplemented-or not-with Toll-Like-Receptor 4 or 9 agonists (TLR4A, TLR9A), to reactivate aP vaccine-induced immune memory and protection, reflected by bacterial clearance. In the conventional murine immunization model, TLR4A- and TLR9A-containing aP formulations induced similar aP-specific IgG antibody responses and protection against bacterial lung colonization as current aP vaccines, despite IL-5 down-modulation by both TLR4A and TLR9A and IL-17 up-modulation by TLR4A. In the absence of serum antibodies at time of boosting or exposure, TLR4A- and TLR9A-containing formulations both enhanced vaccine antibody recall compared to current aP formulations. Unexpectedly, however, protection was only increased by the TLR9A-containing vaccine, through both earlier bacterial control and accelerated clearance. This suggests that TLR9A-containing aP vaccines may better reactivate aP vaccine-primed pertussis memory and enhance protection than current or TLR4A-adjuvanted aP vaccines.

Immunodominance in T cell responses elicited against different domains of detoxified pneumolysin PlyD1.

Detoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with Streptococcus pneumoniae in mouse models. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. BALB/c (H-2d) and C57BL/6 (H-2b) mice were vaccinated with Al(OH)3-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. Adjuvantation of PlyD1 enhanced the serological responses in both strains. In vitro restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. Overlap between H-2d and H-2b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. H-2d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2b epitopes were scattered over the protein. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development.

Antibodies to PcpA and PhtD protect mice against Streptococcus pneumoniae by a macrophage- and complement-dependent mechanism.

Currently marketed Streptococcus pneumoniae (Spn) vaccines, which contain polysaccharide capsular antigens from the most common Spn serotypes, have substantially reduced pneumococcal disease rates but have limited coverage. A trivalent pneumococcal protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad protein D (PhtD), and detoxified pneumolysin is being developed to provide broader, cross-serotype protection. Antibodies against detoxified pneumolysin protect against bacterial pneumonia by neutralizing Spn-produced pneumolysin, but how anti-PhtD and anti-PcpA antibodies protect against Spn has not been established. Here, we used a murine passive protection sepsis model to investigate the mechanism of protection by anti-PhtD and anti-PcpA antibodies. Depleting complement using cobra venom factor eliminated protection by anti-PhtD and anti-PcpA monoclonal antibodies (mAbs). Consistent with a requirement for complement, complement C3 deposition on Spn in vitro was enhanced by anti-PhtD and anti-PcpA mAbs and by sera from PhtD- and PcpA-immunized rabbits and humans. Moreover, in the presence of complement, anti-PhtD and anti-PcpA mAbs increased uptake of Spn by human granulocytes. Depleting neutrophils using anti-Ly6G mAbs, splenectomy, or a combination of both did not affect passive protection against Spn, whereas depleting macrophages using clodronate liposomes eliminated protection. These results suggest anti-PhtD and anti-PcpA antibodies induced by pneumococcal protein vaccines protect against Spn by a complement- and macrophage-dependent opsonophagocytosis.

A bivalent pneumococcal histidine triad protein D-choline-binding protein A vaccine elicits functional antibodies that passively protect mice from Streptococcus pneumoniae challenge.

Vaccines based on conserved pneumococcal proteins are being investigated because serotype coverage by pneumococcal polysaccharide and polysaccharide conjugate vaccines is incomplete and may eventually decrease due to serotype replacement. Here, we examined the functionality of human antibodies induced by a candidate bivalent choline-binding protein A- pneumococcal histidine triad protein D (PcpA-PhtD) vaccine. Pre- and post-immune sera from subjects who had been vaccinated with the PcpA-PhtD candidate vaccine were tested in an established passive protection model in which mice were challenged by intravenous injection with Streptococcus pneumoniae serotype 3 strain A66.1. Serum antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Bacterial surface binding by serum antibodies was determined by a flow cytometry-based assay. Sera from 20 subjects were selected based on low activity of pre-immune samples in the passive protection model. Bacterial surface binding correlated more strongly with anti-PcpA (0.87; p < 0.0001) than with anti-PhtD (0.71; p < 0.0001). The odds ratio for predicting survival in the passive protection assay was higher for the anti-PcpA concentration (470 [95% confidence interval (CI), 46.8 to >999.9]) than for the anti-PhtD concentration (3.4 [95% CI, 1.9 to 5.6]) or bacterial surface binding (9.4 [95% CI, 3.6 to 24.3]). Pooled post-immune serum also protected mice against a challenge with S. pneumoniae serotype 3 strain WU2. Both anti-PcpA and anti-PhtD antibodies induced by the bivalent candidate vaccine mediate protection against S. pneumoniae. The results also showed that the ELISA titer might be useful as a surrogate for estimating the functional activity of antibodies induced by pneumococcal protein vaccines.

PcpA Promotes Higher Levels of Infection and Modulates Recruitment of Myeloid-Derived Suppressor Cells during Pneumococcal Pneumonia.

We used two different infection models to investigate the kinetics of the PcpA-dependent pneumococcal disease in mice. In a bacteremic pneumonia model, we observed a PcpA-dependent increase in bacterial burden in the lungs, blood, liver, bronchoalveolar lavage, and spleens of mice at 24 h postinfection. This PcpA-dependent effect on bacterial burden appeared earlier (within 12 h) in the focal pneumonia model, which lacks bacteremia or sepsis. Histological changes show that the ability of pneumococci to make PcpA was associated with unresolved inflammation in both models of infection. Using our bacteremic pneumonia model we further investigated the effects of PcpA on recruitment of innate immune regulatory cells. The presence of PcpA was associated with increased IL-6 levels, suppressed production of TRAIL, and reduced infiltration of polymorphonuclear cells. The ability of pneumococci to make PcpA negatively modulated both the infiltration and apoptosis of macrophages and the recruitment of myeloid-derived suppressor-like cells. The latter have been shown to facilitate the clearance and control of bacterial pneumonia. Taken together, the ability to make PcpA was strongly associated with increased bacterial burden, inflammation, and negative regulation of innate immune cell recruitment to the lung tissue during bacteremic pneumonia.

Passive protection of mice against Streptococcus pneumoniae challenge by naturally occurring and vaccine-induced human anti-PhtD antibodies.

Currently marketed Streptococcus pneumoniae vaccines are based on polysaccharide capsular antigens from the most common strains. Pneumococcal histidine triad protein D (PhtD) is a conserved surface protein that is being evaluated as a candidate for a vaccine with improved serotype coverage. Here, we measured the functional activity of human anti-PhtD antibodies in a passive protection model wherein mice were challenged with a lethal dose of S. pneumoniae by intravenous injection. This functional activity was compared with anti-PhtD antibody concentrations measured by enzyme-linked immunosorbent assay (ELISA) to estimate the 50% protective dose (ED50). Anti-PhtD antibodies affinity purified from pooled normal human sera passively protected mice with an ED50 of 1679 ELISA units/ml (95% confidence interval, 1420-1946). Sera from subjects injected with aluminum-adjuvanted PhtD in a phase I trial had similar activity per unit of antibody (ED50 = 1331 ELISA units/ml [95% confidence interval, 762-2038]). Vaccine-induced activity in the passive protection model was blocked by pre-incubation with recombinant PhtD but not by a control S. pneumoniae antigen (LytB). These results show that human anti-PhtD antibodies, whether naturally acquired or induced by the PhtD candidate vaccine, are functional. This supports the development of the PhtD candidate as part of a broadly protective pneumococcal vaccine.

Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis.

OBJECTIVE: Due to the fact that current polysaccharide-based pneumococcal vaccines have limited serotype coverage, protein-based vaccine candidates have been sought for over a decade to replace or complement current vaccines. We previously reported that a trivalent Pneumococcal Protein recombinant Vaccine (PPrV), showed protection against pneumonia and sepsis in an infant murine model. Here we investigated immunological correlates of protection of PPrV in the same model. METHODS: C57BL/6J infant mice were intramuscularly vaccinated at age 1-3 weeks with 3 doses of PPrV, containing pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and detoxified pneumolysin mutant PlyD1. 3-4 weeks after last vaccination, serum and lung antibody levels to PPrV components were measured, and mice were intranasally challenged with a lethal dose of Streptococcus pneumoniae (Spn) serotype 6A. Lung Spn bacterial burden, number of neutrophils and alveolar macrophages, phagocytosed Spn by granulocytes, and levels of cytokines and chemokines were determined at 6, 12, 24, and 48h after challenge. RESULTS: PPrV vaccination conferred 83% protection against Spn challenge. Vaccinated mice had significantly elevated serum and lung antibody levels to three PPrV components. In the first stage of pathogenesis of Spn induced pneumonia (6-24h after challenge), vaccinated mice had lower Spn bacterial lung burdens and more phagocytosed Spn in the granulocytes. PPrV vaccination led to lower levels of pro-inflammatory cytokines IL-6, IL-1ß, and TFN-α, and other cytokines and chemokines (IL-12, IL-17, IFN-γ, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden. CONCLUSION: Trivalent PPrV vaccination results in increased serum and lung antibody levels to the vaccine components, a reduction in Spn induced lethality, enhanced early clearance of Spn in lungs due to more rapid and thorough phagocytosis of Spn by neutrophils, and correspondingly a reduction in lung inflammation and tissue damage.

Human antibodies to PhtD, PcpA, and Ply reduce adherence to human lung epithelial cells and murine nasopharyngeal colonization by Streptococcus pneumoniae.

Streptococcus pneumoniae adherence to human epithelial cells (HECs) is the first step in pathogenesis leading to infections. We sought to determine the role of human antibodies against S. pneumoniae protein vaccine candidates PhtD, PcpA, and Ply in preventing adherence to lung HECs in vitro and mouse nasopharyngeal (NP) colonization in vivo. Human anti-PhtD, -PcpA, and -Ply antibodies were purified and Fab fragments generated. Fabs were used to test inhibition of adherence of TIGR4 and nonencapsulated strain RX1 to A549 lung HECs. The roles of individual proteins in adherence were tested using isogenic mutants of strain TIGR4. Anti-PhtD, -PcpA, and -Ply human antibodies were assessed for their ability to inhibit NP colonization in vivo by passive transfer of human antibody in a murine model. Human antibodies generated against PhtD and PcpA caused a decrease in adherence to A549 cells (P < 0.05). Anti-PhtD but not anti-PcpA antibodies showed a protective role against mouse NP colonization. To our surprise, anti-Ply antibodies also caused a significant (P < 0.05) reduction in S. pneumoniae colonization. Our results support the potential of PhtD, PcpA, and Ply protein vaccine candidates as alternatives to conjugate vaccines to prevent non-serotype-specific S. pneumoniae colonization and invasive infection.

Elevated inflammatory markers combined with positive pneumococcal urinary antigen are a good predictor of pneumococcal community-acquired pneumonia in children.

BACKGROUND: Our objective was to evaluate procalcitonin (PCT) and C-reactive protein (CRP) as predictors of a pneumococcal etiology in community-acquired pneumonia (CAP) in hospitalized children. METHODS: Children requiring hospitalization for CAP were prospectively enrolled. The following indices were determined: antibodies against pneumococcal surface proteins (anti-PLY, pneumococcal histidine triad D, pneumococcal histidine triad E, LytB and pneumococcal choline-binding protein A), viral serology, nasopharyngeal cultures and polymerase chain reaction for 13 respiratory viruses, blood pneumococcal polymerase chain reaction, pneumococcal urinary antigen, PCT and CRP. Presumed pneumococcal CAP (P-CAP) was defined as a positive blood culture or polymerase chain reaction for Streptococcus pneumoniae or as a pneumococcal surface protein seroresponse (≥2-fold increase). RESULTS: Seventy-five patients were included from which 37 (49%) met the criteria of P-CAP. Elevated PCT and CRP values were strongly associated with P-CAP with odds ratios of 23 (95% confidence interval: 5-117) for PCT and 19 (95% confidence interval: 5-75) for CRP in multivariate analysis. The sensitivity was 94.4% for PCT (cutoff: 1.5 ng/mL) and 91.9% for CRP (cutoff: 100 mg/L). A value≤0.5 ng/mL of PCT ruled out P-CAP in >90% of cases (negative likelihood ratio: 0.08). Conversely, a PCT value≥1.5 ng/mL associated with a positive pneumococcal urinary antigen had a diagnostic probability for P-CAP of almost 80% (positive likelihood ratio: 4.59). CONCLUSIONS: PCT and CRP are reliable predictors of P-CAP. Low cutoff values of PCT allow identification of children at low risk of P-CAP. The association of elevated PCT or CRP with a positive pneumococcal urinary antigen is a strong predictor of P-CAP.

Safety and immunogenicity of a pneumococcal histidine triad protein D vaccine candidate in adults.

BACKGROUND: Pneumococcal vaccines based on conserved protein antigens have the potential to offer expanded protection against Streptococcus pneumoniae. OBJECTIVE: To explore safety and immunogenicity of a recombinant protein vaccine candidate against S. pneumoniae composed of adjuvanted pneumococcal histidine triad protein D (PhtD). METHODS: This phase I, exploratory, open-label, single-center clinical study enrolled adults (18-50 years). Participants in a pilot safety cohort received a single intramuscular injection of 6 µg. Following safety review, 3 dose cohorts were enrolled (6, 25, and 100 µg); participants received 2 injections administered approximately 30 days apart. Assignment of the second injection and successive dose cohorts were made after blinded safety reviews after each injection at each dose level. Safety endpoints included rates of solicited injection site and systemic reactions, unsolicited adverse events, serious adverse events, and safety laboratory tests. Immunogenicity endpoints included levels of anti-PhtD antibodies as measured by ELISA. RESULTS: Sixty-three participants were enrolled and received the pilot safety dose (n=3) or at least 1 dose of PhtD vaccine candidate at 6 µg (n=20), 25 µg (n=20), or 100 µg (n=20). No safety concerns were identified. No vaccine-related serious adverse event was reported. The most common solicited injection site reaction was pain and most common solicited systemic reactions were myalgia and headache; most reactions were mild and transient. Observed geometric mean concentrations (95% CI) were 200.99 ELISA units (148.46, 272.10), 352.07 (193.49, 640.63), and 699.15 (405.49, 1205.48) post-injection 1 in the 6, 25, and 100 µg dose cohorts, respectively, and 378.25 (275.56, 519.21), 837.32 (539.29, 1300.04), and 1568.62 (1082.92, 2272.16) post-injection 2. CONCLUSIONS: All dose levels were safe and immunogenic. The frequency of solicited reactions was highest at the 100 µg dose. Administration of a second injection significantly increased the levels of anti-PhtD antibodies (ClinicalTrials.gov registry no. NCT01444001).

Neutralizing antibodies elicited by a novel detoxified pneumolysin derivative, PlyD1, provide protection against both pneumococcal infection and lung injury.

Streptococcus pneumoniae pneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers and in vitro neutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.

Antibody response to Streptococcus pneumoniae proteins PhtD, LytB, PcpA, PhtE and Ply after nasopharyngeal colonization and acute otitis media in children.

We prospectively compared serum antibody levels of 5 Streptococcus pneumoniae (Spn) proteins: PcpA PhtD, PhtE Ply and LytB associated with nasopharyngeal (NP) colonization and acute otitis media (AOM) infection in a cohort of 6-30 mo old children. Antigen-specific antibody titers were determined by ELISA. A total of 731 visits among 168 children were studied. There were 301 Spn NP colonization episodes documented in 109 (65%) children and 42 Spn AOM episodes in 34 (20%) children. IgG antibody titers to the 5 proteins were significantly different among children over time (p < 0.001), with a rank order as follows: PcpA > PhtE = PhtD > Ply > LytB Characterization of IgG and IgM acute and convalescent serum antibody levels of Spn AOM infection showed the kinetics of the response differed among children, with the same rank order of antibody levels over time. Individual data showed that some children responded to AOM with an antibody increase to one or more of these Spn proteins but some children failed to respond. We conclude that antibody levels to Spn proteins PcpA PhtD, PhtE, Ply and LytB, all rise over time in children age 6 to 30 mo following natural exposure to Spn after NP colonization and AOM; however, there were significant differences in quantity of antibody elicited among these potential vaccine antigens.

Formulation, stability and immunogenicity of a trivalent pneumococcal protein vaccine formulated with aluminum salt adjuvants.

We investigated the immunogenicity, stability and adsorption properties of an experimental pneumococcal vaccine composed of three protein vaccine antigens; Pneumococcal histidine triad protein D, (PhtD), Pneumococcal choline-binding protein A (PcpA) and genetically detoxified pneumolysin D1 (PlyD1) formulated with aluminum salt adjuvants. Immunogenicity studies conducted in BALB/c mice showed that antibody responses to each antigen adjuvanted with aluminum hydroxide (AH) were significantly higher than when adjuvanted with aluminum phosphate (AP) or formulated without adjuvant. Lower microenvironment pH and decreased strength of antigen adsorption significantly improved the stability of antigens. The stability of PcpA and PlyD1 assessed by RP-HPLC correlated well with the immunogenicity of these antigens in mice and showed that pretreatment of the aluminum hydroxide adjuvant with phosphate ions improved their stability. Adjuvant dose-ranging studies showed that 28 µg Al/dose to be the concentration of adjuvant resulting in optimal immunogenicity of the trivalent vaccine formulation. Taken together, the results of theses studies suggest that the type of aluminum salt, strength of adsorption and microenvironment pH have a significant impact on the immunogenicity and chemical stability of an experimental vaccine composed of the three pneumococcal protein antigens, PhtD, PcpA, and PlyD1.

Structure-guided antigen engineering yields pneumolysin mutants suitable for vaccination against pneumococcal disease.

Pneumolysin (PLY) is a cholesterol-binding, pore-forming protein toxin. It is an important virulence factor of Streptococcus pneumoniae and a key vaccine target against pneumococcal disease. We report a systematic structure-driven approach that solves a long-standing problem for vaccine development in this field: detoxification of PLY with retention of its antigenic integrity. Using three conformational restraint techniques, we rationally designed variants of PLY that lack hemolytic activity and yet induce neutralizing antibodies against the wild-type toxin. These results represent a key milestone toward a broad-spectrum protein-based pneumococcal vaccine and illustrate the value of structural knowledge in formulating effective strategies for antigen optimization.

Vaccine-induced human antibodies to PspA augment complement C3 deposition on Streptococcus pneumoniae.

Pneumococcal surface protein (PspA) is a virulence factor expressed by all clinical isolates of Streptococcus pneumoniae. PspAs are variable in structure and have been grouped into clades and cross-reacting families based on sequence similarities and immunologic cross-reactivity. At least 98% of PspAs are found in PspA families 1 or 2. PspA has been shown to interfere with complement deposition on pneumococci, thus reducing opsonization and clearance of bacteria by the host immune system. Prior studies using pooled human sera have shown that PspA interferes with C3 deposition on a single strain of S. pneumoniae, WU2, and that mouse antibody to PspA can enhance the deposition of C3 on WU2. The present studies have demonstrated that these previous findings are representative of most normal human sera and each of seven different strains of S. pneumoniae. It was observed that PspAs of PspA families 1 and 2 could inhibit C3 deposition in the presence of immunoglobulin present in all but 3 of 22 normal human sera. These studies have also demonstrated that rabbit and human antibody to PspA can enhance the deposition of C3 on pneumococci expressing either family 1 or 2 PspAs and either capsular types 2, 3, or 11. A vaccine candidate that can elicit immunity that neutralizes or compensates for S. pneumoniae's ability to thwart host immunity would be of value.

Role of the novel OprD family of porins in nutrient uptake in Pseudomonas aeruginosa.

To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of Pseudomonas putida. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an opdK mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with opdK. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cis-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms.

Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide.

PsaA of Streptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+ transporter. Mutations in psaA cause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. A psaBC deletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather, psaA mutations indirectly affect this process through the disruption of Mn2+ transport. The deficiency in Mn2+ transport also causes hypersensitivity to oxidative stress from H2O2 and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+ and Mn2+ cations after a lag of about 15 h. Addition of Mn2+ alone or together with Fe2+ allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+ alone extended the 15-h lag phase to 25 h. Thus, while Fe2+ adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2 was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.