Biomedical subjects

Susan K Hollingshead

Publications and source records attributed to Susan K Hollingshead.

At least 19 recordsLinked to original sources

PubMed2006

Streptococcus pneumoniae sheds syndecan-1 ectodomains through ZmpC, a metalloproteinase virulence factor.

Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC.

Animals↗

PubMed2006

CCL5 modulates pneumococcal immunity and carriage.

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Recently, it has been shown that genetic polymorphisms can result in diminished expression of CCL5, which results in increased susceptibility to and progression of infectious diseases. We show that CCL5, together with Th cytokine mRNA expression, is temporally up-regulated during pneumococcal carriage. To determine the contribution of CCL5 to pneumococcal surface antigen A-specific humoral and cellular pneumococcal immunity, mice were treated with anti-CCL5 or control Abs before and during Streptococcus pneumoniae strain EF3030-challenge for the initiation of carriage. CCL5 blockade resulted in a decrease of CD4(+) and CD8(+) T cells as well as CD11b(+) cells in the spleen, cervical lymph node, lung, and nasopharyngeal associated lymphoid tissue during the recognition phase of the pneumococcal adaptive immune response. CCL5 blockade significantly reduced the Ag-specific IgG2a and IgG1 Abs in serum and IgA Ab levels in nasal washes. These decreases also corresponded to reductions in Ag-specific T cell (mucosal and systemic) responses. CCL5 inhibition resulted in decreasing the quantity of IL-4- and IFN-gamma-secreting CD4(+) T cells and increasing the number of Ag-specific IL-10-producing CD4(+) T cells; these changes combined also corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity.

Animals↗

PubMed2006

Pneumococcal surface protein A (PspA) family distribution among clinical isolates from adults over 50 years of age collected in seven countries.

The pneumococcal surface protein PspA, a cell-wall-associated surface protein, is a promising component for pneumococcal vaccines. In this study, the distribution of the PspA family was determined in a panel of invasive and clinically important pneumococcal isolates from adults over 50 years of age, collected between 1995 and 2002. One thousand eight hundred and forty-seven recent isolates from invasive pneumococcal disease were obtained from seven Western countries, together with clinical data. An ELISA-based serological method was standardized in order to determine the PspA family and clade distribution. Molecular tests were used when isolates were non-typable by ELISA (PspA family typing by PCR). Only 42 (2.3 %) isolates were non-typable by ELISA and PspA family typing by PCR was performed. Finally, 3 isolates were considered as non-pneumococcal and 1844 were classified as follows: 749 (40.6 %) were PspA family 1, 1078 (58.5 %) were PspA family 2, 13 (0.7 %) were PspA family 1 and 2 and 4 (0.2 %) remained non-typable. The cross-reactivity of antibodies to PspAs of different clades was confirmed. In conclusion, inclusion of PspA family 1 and family 2 in future pneumococcal vaccines would ensure broad coverage of pneumococcal strains infecting people over 50 years of age.

Aged↗

PubMed2006

Mn2+-dependent regulation of multiple genes in Streptococcus pneumoniae through PsaR and the resultant impact on virulence.

The concentration of Mn2+ is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in Streptococcus pneumoniae, was found to negatively affect the transcription of psaBCA, pcpA, rrgA, rrgB, rrgC, srtBCD, and rlrA in the presence of Mn2+. psaBCA encode an ABC-type transporter for Mn2+. pcpA, rrgA, rrgB, and rrgC encode several outer surface proteins. srtBCD encode a cluster of sortase enzymes, and rlrA encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn2+ concentrations in the wild-type strain and are elevated under both high and low Mn2+ concentrations in a psaR mutant strain. RlrA is an activator of rrgA, rrgB, rrgC, and srtBCD (D. Hava and A. Camilli, Mol. Microbiol. 45:1389-1406, 2002), suggesting that PsaR may indirectly control these genes through rlrA, while PsaR-dependent repression of psaBCA, pcpA, and rlrA transcription is direct. The impact of Mn2+-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of DeltapsaR, pcpA, and DeltapsaR DeltapcpA mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn2+ acting as a signal for the expression of virulence factors within different host sites.

ATP-Binding Cassette Transporters↗

PubMed2006

Validation of a multiplex pneumococcal serotyping assay with clinical samples.

We have recently developed a rapid pneumococcal serotyping method called "multibead assay" (J. Yu et al., J. Clin. Microbiol. 43:156-162, 2005) based on a multiplexed immunoassay for capsular polysaccharides in lysates of pneumococcal cultures. The multibead assay can identify 36 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/33C, 11A/11D/11F, 12A/12B/12F, 14, 15B/5C, 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F). More than 90% of the U.S. isolates express one of these serotypes (J. B. Robbins et al., J. Infect. Dis. 148:1136-1159, 1983). To validate the new assay, we examined 495 clinical isolates of pneumococci obtained in Brazil, Denmark, and Mexico. Pneumococci were serotyped by the Neufeld test in their countries of origin, and lysates of each strain were coded and mailed to the United States for the multibead assay at ambient temperature without any thermal protection. After breaking the code, 54 discrepancies (11% of samples) were noted, but 46 were due to nonreproducible technical problems or insufficient growth of the pneumococci. All of the isolates grew well for a second test, and therefore, the culture medium used for the multibead assay is adequate. The discrepancies persisted for eight isolates, involving the 6A, 11A, and 18C serotypes. Additional studies of the eight isolates showed that the discrepancies were due to differences in the reagents used in the multibead or Neufeld tests for these three serotypes. For instance, five isolates were typed as 6A with the Neufeld test but as nontypeable by the multibead assay. Selection of another new monoclonal antibody (Hyp6AG1) for the multibead assay resulted in all five discrepant isolates typing as 6A. This finding indicates the validity of the multibead assay and emphasizes the need to validate any new pneumococcal serotyping assay with a large number of clinical isolates from different locations. It also suggests the presence of serological subtypes among isolates expressing the 6A serotype.

Bacterial Capsules↗

PubMed2005

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage.

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] >> IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.

Adhesins, Bacterial↗

PubMed2005

Relative fitness of fluoroquinolone-resistant Streptococcus pneumoniae.

Fluoroquinolone resistance in Streptococcus pneumoniae is primarily mediated by point mutations in the quinolone resistance-determining regions of gyrA and parC. Antimicrobial resistance mutations in housekeeping genes often decrease fitness of microorganisms. To investigate the fitness of quinolone-resistant S. pneumoniae (QRSP), the relative growth efficiencies of 2 isogenic QRSP double mutants were compared with that of their fluoroquinolone-susceptible parent, EF3030, by using murine nasopharyngeal colonization and pneumonia models. Strains containing the GyrA: Ser81Phe, ParC: Ser79Phe double mutations, which are frequently seen in clinical QRSP, competed poorly with EF3030 in competitive colonization or competitive lung infections. However, they efficiently produced lung infection even in the absence of EF3030. The strain containing the GyrA: Ser81Phe, ParC: Ser79Tyr double mutations, which is seen more frequently in laboratory-derived QRSP than in clinical QRSP, demonstrated reduced nasal colonization in competitive or noncompetitive lung infections. However, the strain was equally able to cause competitive or noncompetitive lung infections as well as EF3030.

Animals↗

PubMed2004

Characterization of antibodies to PspA and PsaA in adults over 50 years of age with invasive pneumococcal disease.

We characterized antibody responses to two Streptococcus pneumoniae surface proteins, PspA and PsaA, in 14 adults over 50 years of age hospitalized with invasive pneumococcal disease (IPD), and in two groups of age-matched controls (18 patients with invasive disease due to other microorganisms and 35 patients hospitalized for non infectious conditions). All patients with IPD and all control subjects had detectable antibodies to both proteins on hospital admission. Three weeks later, the geometric mean concentrations of antibodies to PspA and PsaA in IPD patients were respectively 10 and 25 times higher than on admission. In contrast, acute and convalescent antibody levels were similar in control patients with invasive diseases due to other microorganisms.

Adhesins, Bacterial↗

PubMed2004

Typing of pneumococcal surface protein A (PspA) in Streptococcus pneumoniae isolated during epidemiological surveillance in Brazil: towards novel pneumococcal protein vaccines.

Pneumococcal protein vaccine based on pneumococcal surface protein A (PspA) is in development with the potential to offer broad range of protection against different strains. We have investigated the frequency of PspA family 1 (Fam1) and family 2 (Fam2) proteins among Streptococcus pneumoniae recovered from ongoing surveillance in Brazil. Fam1 and Fam2 were expressed in comparable rates among 366 isolates, with the potential coverage of 94.3%. PspA families were not associated to age group or source of isolates. However, considering the significant tendency of increasing prevalence of Fam2 associated to widespread dissemination of the genetically-related resistant strains, the monitoring of the PspA families derived from population-based data may be necessary in the context of vaccine development.

Adolescent↗

PubMed2004

Effects of PspA and antibodies to PspA on activation and deposition of complement on the pneumococcal surface.

Streptococcus pneumoniae infection is a frequent cause of pneumonia, otitis media, meningitis, and septicemia. Pneumococcal surface protein A (PspA) is an important virulence factor on the pathogen surface, and it is known to interfere with complement activation. In this study, flow cytometry was used to study the effects of PspA and antibodies to PspA on the deposition of complement C3 on the surface of a capsular type 3 strain, WU2, and its PspA- mutant, JY1119. Using naive mouse serum as a complement source, measurable deposition of C3 was observed within 4 min on PspA- pneumococci, and the amount of surface-bound C3 accumulated rapidly as the amount of serum was increased. In contrast, very little C3 was deposited on the PspA+ strain. In nonimmune mouse serum, the classical pathway was the dominant activation pathway triggered by PspA- pneumococci. Accordingly, EGTA blocked almost all of the complement activation. Moreover, a significant amount of C3 was still deposited on the PspA- strain when serum from factor B-deficient mice was used. This deposition was not observed on the PspA+ pneumococci, indicating that PspA may inhibit complement deposition via the classical pathway. Furthermore, under the conditions we tested, PspA also inhibited C3 deposition when the classical pathway was initiated by antibodies to capsular polysaccharide. Antibodies to PspA could overcome the anticomplementary effect of PspA, allowing for increased complement activation and C3 deposition onto PspA+ bacteria.

Animals↗

PubMed2004

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.

ATP-Binding Cassette Transporters↗

PubMed2004

PspA protects Streptococcus pneumoniae from killing by apolactoferrin, and antibody to PspA enhances killing of pneumococci by apolactoferrin [corrected].

Lactoferrin is an important component of innate immunity through its sequestration of iron, bactericidal activity, and immune modulatory activity. Apolactoferrin (ALF) is the iron-depleted form of lactoferrin and is bactericidal against pneumococci and several other species of bacteria. We observed that lactoferricin (LFN), an 11-amino-acid peptide from the N terminus of lactoferrin, is bactericidal for Streptococcus pneumoniae. Strains of S. pneumoniae varied in their susceptibility to ALF. Lactoferrin is bound to the pneumococcal surface by pneumococcal surface protein A (PspA). Using mutant PspA(-) pneumococci of four different strains, we observed that PspA offers significant protection against killing by ALF. Knockout mutations in genes for two other choline-binding proteins (PspC and PcpA) did not affect killing by ALF. PspA did not have to be attached to the bacterial surface to inhibit killing, because the soluble recombinant N-terminal half of PspA could prevent killing by both ALF and LFN. An 11-amino-acid fragment of PspA was also able to reduce the killing by LFN. Antibody to PspA enhanced killing by lactoferrin. These findings suggested that the binding of ALF to PspA probably blocks the active site(s) of ALF that is responsible for killing.

Amino Acid Sequence↗

PubMed2004

Evolutionary genetics of the capsular locus of serogroup 6 pneumococci.

The evolution of the capsular biosynthetic (cps) locus of serogroup 6 Streptococcus pneumoniae was investigated by analyzing sequence variation within three serotype-specific cps genes from 102 serotype 6A and 6B isolates. Sequence variation within these cps genes was related to the genetic relatedness of the isolates, determined by multilocus sequence typing, and to the inferred patterns of recent evolutionary descent, explored using the eBURST algorithm. The serotype-specific cps genes had a low percent G+C, and there was a low level of sequence diversity in this region among serotype 6A and 6B isolates. There was also little sequence divergence between these serotypes, suggesting a single introduction of an ancestral cps sequence, followed by slight divergence to create serotypes 6A and 6B. A minority of serotype 6B isolates had cps sequences (class 2 sequences) that were approximately 5% divergent from those of other serotype 6B isolates (class 1 sequences) and which may have arisen by a second, more recent introduction from a related but distinct source. Expression of a serotype 6A or 6B capsule correlated perfectly with a single nonsynonymous polymorphism within wciP, the rhamnosyl transferase gene. In addition to ample evidence of the horizontal transfer of the serotype 6A and 6B cps locus into unrelated lineages, there was evidence for relatively frequent changes from serotype 6A to 6B, and vice versa, among very closely related isolates and examples of recent recombinational events between class 1 and 2 cps serogroup 6 sequences.

Amino Acid Sequence↗

PubMed2003

Immunizations with pneumococcal surface protein A and pneumolysin are protective against pneumonia in a murine model of pulmonary infection with Streptococcus pneumoniae.

Intranasal infection of mice with certain strains of capsular group 19 Streptococcus pneumoniae can result in focal pneumonia in the absence of bacteremia. Using this model of murine pneumonia, we demonstrated that immunization with recombinant forms of either pneumococcal surface protein A (PspA) or PdB (a genetically detoxified derivative of pneumolysin) elicited significant protection against focal pulmonary infection. This may be the first demonstration that a proposed vaccine antigen can protect against pneumococcal pneumonia. The best protection was obtained by immunizing mice with a mixture of PspA and PdB, indicating that the protection elicited by these antigens can complement each other. This result is in agreement with previous studies that used pneumococcal sepsis and nasal colonization models and demonstrate that the best protein vaccines for prevention of infection may be those that include more than one protection-eliciting pneumococcal protein.

Administration, Intranasal↗

PubMed2003

Immunogenic protein contaminants in pneumococcal vaccines.

Currently available pneumococcal vaccines were examined for contamination by pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA). PspA could be detected in some (but not all) lots of 23-valent polysaccharide vaccine. Many lots of pneumococcal vaccines, including the heptavalent conjugate vaccine, were found to elicit small (but variable) antibody responses to PspA, PsaA, or both. The degree of contamination was highly variable, and this should be considered in pneumococcal vaccine evaluations or when capsular polysaccharide is used for pneumococcal antibody assays.

Adhesins, Bacterial↗

PubMed2003

Both family 1 and family 2 PspA proteins can inhibit complement deposition and confer virulence to a capsular serotype 3 strain of Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA), a virulence factor of Streptococcus pneumoniae, is exceptionally diverse, being classified into two major families which are over 50% divergent by sequence analysis. A family 1 PspA from strain WU2 was previously shown to impede the clearance of pneumococci from mouse blood and to interfere with complement deposition on the bacterial surface. To determine whether a family 2 PspA can perform the same role as family 1 PspA, the family 1 PspA (from strain WU2) was replaced with a family 2 PspA (from strain TIGR4) by molecular genetic methods to make an isogenic pair of strains expressing different PspA proteins. Surface binding of lactoferrin and interference with C3 deposition by the two types of PspA proteins were determined by flow cytometry, and virulence was assessed in a mouse bacteremia model. Although the family 2 PspA appeared to bind less human lactoferrin than did the family 1 PspA, both PspA proteins could interfere with complement deposition on the pneumococcal surface and could provide full virulence in the mouse infection model. A mutant form of the family 2 PspA with a deletion within the choline-binding region was also produced. Pneumococci with this mutant PspA failed to bind human lactoferrin even though the PspA was present on the pneumococcal surface. The mutant PspA only partially interfered with complement deposition and moderately attenuated virulence. These results suggest that family 1 and family 2 PspA proteins play similar roles in virulence and that surface accessibility of PspA is important for their function.

Amino Acid Sequence↗

PubMed2003

Regions of PspA/EF3296 best able to elicit protection against Streptococcus pneumoniae in a murine infection model.

Pneumococcal surface protein A (PspA) can elicit protection against Streptococcus pneumoniae in mouse infection models. PspA is classified by serology and amino acid sequence into two major families that are divided by sequence into five clades. The most variable portion of the molecule is the alpha-helical domain, which comprises the N-terminal half of PspA. Prior studies of a family 1 PspA protein observed that protective antibodies are reactive with epitopes in the alpha-helical domain and that most cross-protective epitopes mapped to the 108 most C-terminal amino acids of the alpha-helical region. In these studies, we have used six overlapping recombinant fragments of family 2, clade 3 PspA/EF3296 to map the protection-eliciting regions of its alpha-helical domain. The three fragments, which included the 104 most C-terminal amino acids of the alpha-helical domain (314 to 418), could each elicit protection against EF3296. A fragment comprising amino acids 75 to 305 failed to elicit significant protection. A fragment containing amino acids 1 to 115 elicited protection against EF3296 in BALB/c mice but not in CBA/N mice. All three fragments containing amino acids 314 to 418 were able to elicit cross-protection against pneumococci expressing PspA proteins of clades 2, 3, 4, and 5. Cross-protection elicited by these three fragments was easier to demonstrate in CBA/N mice than in BALB/c mice. The 1-to-115 fragment, however, elicited some cross-protection against clades 2 and 4 in BALB/c mice but not in CBA/N mice. These studies provide support for the importance of the C-terminal 104 and N-terminal 115 amino acids of the alpha-helical region of PspA in the elicitation of cross-protection.

Animals↗

PubMed2003

Effects of zinc deficiency and pneumococcal surface protein a immunization on zinc status and the risk of severe infection in mice.

Streptococcus pneumoniae is a major cause of illness and death in children in developing countries. In these children, zinc deficiency is associated with an increased risk of acute respiratory tract infections, which can be reduced by daily zinc administration. Severe infections decrease zinc levels in plasma and may thereby move individuals with preexisting low zinc stores into a vicious cycle of infection and unavailable zinc. Pneumococcal surface protein A (PspA) has emerged as a promising vaccine candidate, and immunization with this antigen protects animals from pneumococcal infection. In an animal experiment, we measured the effect of zinc depletion on the immune response to parenterally administrated PspA and assessed the effect of this PspA vaccination and zinc depletion on the severity of pneumococcal infection and on zinc status. Mice were kept on different diets for 5 weeks, immunized twice 14 days apart, and challenged intranasally with S. pneumoniae. Mice on the zinc-deficient diet showed substantially reduced immune responses to PspA, more extensive pneumococcal colonization in the nasal mucosa, more severe infections, and an increased risk of death. PspA immunization reduced the risk of severe disease, and the reduction in severity was reflected in substantially reduced zinc depletion from bones.

Animals↗