Amino Acid Changes in Elongation Factor Tu of Mycoplasma pneumoniae and Mycoplasma genitalium Influence Fibronectin Binding

Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu_Mp) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu_Mg), in spite of sharing 96% identity with EF-Tu_Mp, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu_Mp that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu_Mg. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu_Mp. Synthetic peptides corresponding to this region of EF-Tu_Mp (EF-Tu_Mp 340-358) blocked both recombinant EF-Tu_Mp and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu_Mg 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.Many pathogens express surface proteins that facilitate colonization and cellular invasion (12, 39, 44, 49, 55). The human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma genitalium, have genome sizes of 816,394 bp (20) and 580,070 bp (12), respectively, with the latter considered the smallest self-replicating biological cell (14, 38). These bacterial pathogens possess terminal tip-like structures comprised of specific membrane adhesins and adherence-related accessory proteins that mediate surface parasitism of target cells (5) and are essential for virulence (4). While adherence of virulent M. pneumoniae is mediated primarily by tip organelle-associated adhesins (10, 24), the absence of these proteins in hemadsorption-negative mutants (HA⁻ class II mutants) (17) still permits detectable adherence (18), suggesting the involvement of alternative mechanisms by which mycoplasmas bind to host cells.Recently, we showed that M. pneumoniae surface-associated elongation factor Tu (EF-Tu_Mp; MPN665) and the pyruvate dehydrogenase E1 beta subunit (MPN392) interact with fibronectin (Fn) (11). In addition, we demonstrated that HA⁻ class II mutants also bind Fn through EF-Tu (11). Fn is an abundantly available pathogen target (22) that exists in soluble form in blood fluids and plasma and in fibrillar form in the extracellular matrix (56). M. pneumoniae could readily access the extracellular matrix through virulence-related determinants following epithelial cell damage (29) and could directly bind to subepithelial tissue targets through EF-Tu interactions with Fn. Furthermore, these distinct pathogenic pathways may also contribute to the ability of M. pneumoniae to invade and to establish intracellular and perinuclear residence (9, 57).Detailed analyses of EF-Tu_Mp-Fn interactions revealed the critical role of the carboxyl region of EF-Tu (amino acids 192 to 219 and 314 to 394) in Fn recognition (3). Other mycoplasmas with tip organelles, such as Mycoplasma penetrans and Mycoplasma gallisepticum, have been reported to bind Fn through a 65-kDa protein (13) and the PlpA and Hlp3 proteins (34).Following our initial findings of EF-Tu_Mp-Fn interactions, surface-associated EF-Tu proteins from other microorganisms, including Lactobacillus johnsonii, Listeria monocytogenes, and Pseudomonas aeruginosa, were reported to bind mucin (16), fibrinogen (43), plasminogen, and factor H (32). Since EF-Tu is one of the most highly conserved proteins in mycoplasmas, it has been used to create an EF-Tu sequence-based mycoplasma phylogeny tree. This allows the classification of the human pathogens, M. genitalium and M. pneumoniae, along with M. gallisepticum, a poultry pathogen, in the same group (28). M. pneumoniae is an established pathogen of the respiratory tract (54) but has also been isolated from the urogenital tract (15). M. genitalium, an emerging sexually transmitted disease pathogen (27, 51), has also been associated with respiratory (6) and joint (50) pathologies. It has been suggested that the tissue-specific tropisms and pathogenic mechanisms of these two mycoplasmas are determined by genetic distinctions between them (19). Most of the open reading frames proposed for M. genitalium are present in M. pneumoniae. Overall, M. pneumoniae and M. genitalium share 67.4% average identity at the amino acid level, while conserved housekeeping proteins exhibit 70 to 97% identity (19). Among the latter proteins, EF-Tu displays a high sequence identity (96%).In this study, we compared EF-Tu-Fn binding between M. pneumoniae and M. genitalium and discovered biological and biochemical differences that facilitated the identification of key amino acids responsible for these interactions. Such distinctions provide evidence of unique colonization capabilities of these bacteria.     

Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the bla_KPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.Since the beginning of the last decade, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) isolates have been increasingly detected in the United States and subsequently in several regions worldwide (3, 4, 13, 17, 21). KPC enzymes efficiently hydrolyze all β-lactam molecules (1, 22), conferring various levels of resistance to all β-lactam compounds, including carbapenems (13). However, KPC-producing K. pneumoniae may appear susceptible to carbapenems, mainly meropenem (2, 13), by reference CLSI agar dilution or broth microdilution methods as well as by automated systems (6, 15, 17). Characteristically, it has been reported that automated systems may identify as many as 87% of KPC-KP isolates to be susceptible to meropenem (13). The detection of the susceptibility level of KPC-KP isolates to carbapenems has been shown to be difficult due to the phenotypic heterogeneity that they commonly exhibit (3, 10, 13). For instance, in agar diffusion methods such as disk diffusion or Etest, the heterogeneous growth to carbapenems of KPC-KP results in the appearance of scattered colonies within the inhibition zones (9, 13).These issues raise the need for cautious evaluation of susceptibility testing in KPC-KP isolates that are recovered in clinical laboratories. In our clinical laboratories, several KPC-KP isolates that appear susceptible by automated susceptibility assays or reference dilution assays contain heterogeneous subpopulations (D. Sofianou and K. Themeli-Digalaki, personal communications). It has been also shown that among Greek KPC-KP isolates, meropenem tends to exhibit lower MICs than imipenem or ertapenem (17, 20). In that respect, the aim of the present study was to characterize the heterogeneous mode of growth of apparently meropenem-susceptible KPC-KP clinical isolates by population analyses and bactericidal assays.     

Strategy To Create Chimeric Proteins Derived from Functional Adhesin Regions of Mycoplasma pneumoniae for Vaccine Development

The cell wall-less bacterium Mycoplasma pneumoniae is one of the most common agents of respiratory tract diseases in humans. Adhesin-mediated binding of the bacteria to host cells is a crucial step in colonization and subsequent pathogenesis. For the first time, we expressed 16 recombinant proteins covering almost the whole major adhesin P1 and the adherence-associated protein P30 to characterize these proteins immunologically and functionally. We describe a new in vitro assay using several human cell lines in combination with fluorescence-activated cell sorting analysis to screen antisera raised against the recombinant proteins quantitatively for adherence inhibition activity. The protein derived from the nearly C-terminal part of the P1 adhesin (amino acids [aa] 1288 to 1518) and the protein P30 (aa 17 to 274) especially showed prominent immunoreactivity with sera from M. pneumoniae-immunized guinea pigs as well as with M. pneumoniae-positive patient sera. We demonstrate that the same protein regions are involved in mediating cytadherence since antibodies against these adhesin regions decrease mycoplasma adhesion to human cells significantly. For further vaccine studies, we optimized the immunogenic and adherence-mediating properties of the antigen by combining both the P1 and the P30 regions in a novel chimeric protein. Antibodies against this protein show an increased reduction of M. pneumoniae adherence to human bronchial epithelial cells by 95%, which is comparable to results with polyspecific anti-M. pneumoniae animal serum. Our strategy results in a promising defined antigen candidate for reducing or even preventing M. pneumoniae colonization of the respiratory tract in future vaccination studies.Epidemiological studies confirm that between 5 and 10% of all community-acquired pneumonia cases, especially in children and young adults but also in elderly patients, are attributed to the cell wall-less bacterium Mycoplasma pneumoniae (49). Epidemic outbreaks in geographically close populations and the occurrence of extrapulmonary complications of the primary respiratory infections emphasize the significant impact of the agent on public health.Adhesion of M. pneumoniae to the host respiratory epithelium (cytadherence) is an essential first stage of infection and a precondition for successful colonization (19). Even though M. pneumoniae is one of the smallest and simplest microorganisms, M. pneumoniae cells exhibit a complex differentiated cell extension, the attachment organelle, that functions in different processes including cytadherence, cell division, and gliding (1, 32, 39). The analysis of mutants has resulted in the identification of an increasing number of proteins associated with cytadherence of M. pneumoniae (reviewed in reference 33), such as the transmembrane proteins P1 and P30, both densely clustered at the attachment organelle (31). The results of binding experiments and of the characterization of P1-deficient (avirulent) mutants established the role of the protein P1 as the main adhesin of the bacterium (18, 20, 39, 41). In addition, the P1 adhesin gene serves as an important marker for subtyping M. pneumoniae clinical isolates. Three subtypes (1 to 3) and three variants are discriminated on the basis of sequence differences in one or both of the repetitive elements, RepMP4 and RepMP2/RepMP3 (RepMP2/3), within this gene (7, 11, 30, 40, 45). The circulation of different subtypes and variants of M. pneumoniae in the human population is discussed as a possible reason for the epidemic outbreaks which occur every 3 to 7 years (36, 43).The protein P30 was first characterized by Dallo et al. (4). The C-terminal region of P30 is dominated by proline-rich repeating motifs similar to those found near the C terminus of P1 (4). In-frame deletions in these repeat regions resulted in a loss of cytadherence (34). Analysis of P30 mutants suggested an association of the protein with proper cell development, appropriate conformation of the adhesin P1 in the mycoplasma membrane, and receptor binding (16, 42).Besides their role in the attachment process, the adhesion proteins P1 and P30 act as potent immunogens that result in high titers of specific antibodies in serum of patients with acute M. pneumoniae infections (21, 24, 48). However, immunocompetent hosts with significant titers of anti-P1 antibodies are not protected from reinfection (28). Results of screening patient sera for adherence-inhibiting activity suggested that the P1 protein induces antibodies that are mostly not directed to cytadherence-mediating sites of the P1 protein (29).Considering the prominent role of adhesins in the interaction of M. pneumoniae and its host, the characterization of P1 and other adhesion-associated proteins of M. pneumoniae should identify regions that might serve as effective vaccine candidates. In the past, the investigation of mycoplasma adhesins was hampered by technical difficulties in protein expression. Unlike the universal genetic code, M. pneumoniae uses the TGA codon to incorporate tryptophan rather than as a stop codon (22, 44). To circumvent the problem, different strategies have been applied (14). The use of recombinant proteins covering defined adhesin regions turned out to be a suitable tool to identify protein regions with antigenic and adherence-mediating properties (3, 8, 46). However, these previous investigations were applied to only a few protein regions of P1.In this study, we present for the first time 15 recombinant proteins that provide almost complete coverage of the P1 adhesin of M. pneumoniae. Together with a recombinant protein derived from the adherence-associated protein P30, we characterized these adhesin regions by their serological reactions with sera both of immunized guinea pigs as model animals and of patients suffering from M. pneumoniae infections. The development of a novel adhesion inhibition assay enabled us to identify the functional attachment sites of M. pneumoniae adhesins. The consecutive determination of antigenic properties of distinct protein regions in humans and animals and their role in the attachment process provided the basis for a promising vaccine candidate characterized by a combination of immunogenic and adherence-mediating protein regions in a chimeric protein.     

The Effects of PspC on Complement-Mediated Immunity to Streptococcus pneumoniae Vary with Strain Background and Capsular Serotype

Streptococcus pneumoniae may evade complement activity by binding of factor H (FH), a negative regulator of the alternative pathway, to the surface protein PspC. However, existing data on the effects of FH binding to PspC on complement activity are conflicting, and there is also considerable allelic variation in PspC structure between S. pneumoniae strains that may influence PspC-dependent effects on complement. We have investigated interactions with complement for several S. pneumoniae strains in which the gene encoding PspC has been deleted. The degree of FH binding varied between strains and was entirely dependent on PspC for seven strains. Data obtained with TIGR4 strains expressing different capsular serotypes suggest that FH binding is affected by capsular serotype. Results of immunoblot analysis for C3 degradation products and iC3b deposition assays suggested that FH bound to PspC retained functional activity, but loss of PspC had strikingly varied effects on C3b/iC3b deposition on S. pneumoniae, with large increases on serotype 4, 6A, 6B, and 9V strains but only small increases or even decreases on serotype 2, 3, 17, and 23F strains. Repeating C3b/iC3b assays with TIGR4 strains expressing different capsular serotypes suggested that differences in the effect of PspC on C3b/iC3b deposition were largely independent of capsular serotype and depend on strain background. However, data obtained from infection in complement-deficient mice demonstrated that differences between strains in the effects of PspC on complement surprisingly did not influence the development of septicemia.Streptococcus pneumoniae is a common cause of invasive diseases such as pneumonia, meningitis, and septicemia even in immunocompetent subjects. One important component of host immunity to S. pneumoniae is the complement system, a series of approximately 30 serum and cell surface proteins organized into three enzyme cascades termed the classical, alternative, and mannan binding lectin (MBL) pathways (44). Infection experiments using complement-deficient mice have demonstrated that both the classical and alternative pathways are important for immunity to S. pneumoniae (2, 11, 15, 23, 43), and the high incidence of S. pneumoniae infections in patients with complement deficiencies confirms the relevance of complement for preventing disease in humans (3, 21). Both pathways lead to the formation of a C3 convertase that cleaves the central complement component C3, resulting in deposition of C3b on the surface of the pathogen that is further processed to iC3b. C3b and iC3b are opsonins, (44), and coating of S. pneumoniae with C3b/iC3b is vital for efficient phagocytosis of this organism by neutrophils (49). The importance of complement for immunity to S. pneumoniae is emphasized by the identification of several mechanisms by which the bacteria can inhibit complement activity. The choline binding surface protein PspA and the capsule prevent C3b/iC3b deposition on S. pneumoniae by mechanisms that remain unclear, whereas the secreted toxin pneumolysin seems to divert classical pathway activity away from the bacteria (1, 30, 32, 35, 39, 43, 48).PspC is another choline binding surface protein that is related to PspA and is important for virulence in models of nasopharyngeal colonization, septicemia, and pneumonia (16, 22, 33, 35, 38). PspC can bind to factor H (FH), a negative regulator of the alternative pathway (8, 13, 18). PspC and the closely related proteins encoded at the same genetic locus in different strains (termed SpsA, CbpA, PbcA, and Hic) could, therefore, prevent alternative pathway complement activity against S. pneumoniae by one of three potential mechanisms (7). First, FH may speed up the degradation of C3b by promoting the factor I-dependent cleavage of C3b bound to the bacterial surface to iC3b; second, FH causes the dissociation of factor B from the alternative pathway C3 convertase C3bBb, decreasing C3b deposition on the bacteria; and third, FH may also inhibit the formation on the bacterial surface of the C3 convertase C3bBb by preferentially binding C3b, thus preventing C3b binding to factor B. However, although the affinity of PspC for FH has been clearly demonstrated and the binding sites have been identified (6, 12, 13, 17, 18, 28, 38), the effects of this interaction on complement-mediated immunity to S. pneumoniae is relatively poorly defined. Loss of PspC can cause reduced iC3b (compatible with reduced processing of C3b to iC3b) (28) and increased C3b/iC3b (compatible with reduced C3 convertase activity) deposition on a capsular serotype 2 (ST2) strain D39 or an unencapsulated mutant derived from a capsular ST3 strain (19, 35). However, in contrast to Quin et al., Lu et al. and Li et al. found little effect on total C3 bound to a ΔpspC mutant derived from the D39 strain compared to the wild type unless this mutation was combined with mutation of pspA (25, 28, 35).The role of PspC on complement-mediated immunity in different strains could also be affected by the marked allelic variation in the structure of PspC. Ianelli et al. sequenced pspC from 43 different strains, and although the derived protein sequences had the same domain organization, each protein had a unique sequence that could be divided into 11 subgroups (17). The majority of PspC alleles contain a C-terminal cell wall choline binding motif (similar to PspA), but 17 of the allelic variants contain a cell wall anchor LPTXG motif instead. FH binding requires only the N-terminal 89 amino acids of PspC from the D39 strain and is dependent on a 12-amino-acid motif which is conserved between PspC alleles from different S. pneumoniae strains (28). However, this domain is not enough for full FH binding capacity and requires additional flanking amino acids that vary with allelic variation of PspC. Indeed, it is known that the ability of S. pneumoniae to bind to FH varies between strains (36). This variation in FH binding between strains seems to be independent of serotype but is increased on strains isolated from the blood or cerebrospinal fluid (CSF), under which conditions PspC expression is increased (29, 34). Overall, the existing data suggest that the effects of PspC on complement deposition on S. pneumoniae is not clear and could vary between strains. Furthermore, several other functions have been ascribed to PspC, including direct binding to C3 (which might counteract any FH-mediated reduction in C3b deposition) (4, 40) and aiding S. pneumoniae adhesion to host cells and translocation across epithelial layers (12, 13, 37, 38, 50), which could also affect virulence independent of any effect of PspC on complement-mediated immunity. Further clarification of the effects of PspC on opsonization of S. pneumoniae with C3b/iC3b is important for a better understanding of how PspC aids S. pneumoniae virulence. To address this question, in this study we have assessed the effects of loss of PspC on C3b/iC3b deposition on a range of S. pneumoniae strains representing different capsular STs.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

The Two Variants of the Streptococcus pneumoniae Pilus 1 RrgA Adhesin Retain the Same Function and Elicit Cross-Protection In Vivo

Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.Streptococcus pneumoniae is a main determinant of respiratory tract infections, such as otitis media, sinusitis, and community-acquired pneumonia, and is also responsible for invasive diseases, such as bacteremic pneumonia and meningitis (16, 17, 30, 43, 46, 50, 55). Nonetheless, pneumococci are normal components of the human commensal flora, asymptomatically colonizing the upper respiratory tracts of both children and healthy adults. Colonization is commonly followed by horizontal transmission of S. pneumoniae, leading to its spread within the community (4, 19, 33). Current glycoconjugate vaccines are efficacious against invasive disease caused by serotypes included in the vaccines; however, their potential to prevent carriage and related mucosal diseases, such as otitis media, is not optimal (10, 12, 23, 29, 34, 48). Furthermore, the partial geographic coverage and the phenomenon of serotype replacement associated with the introduction of the 7-valent conjugate vaccine limit to some extent its long-term effectiveness (11, 20, 31, 37). For these reasons, current research is focused on the identification of protein vaccine candidates able to elicit serotype-independent protection against S. pneumoniae infection. In this context, colonization could represent a critical point of intervention, and bacterial components involved in these mechanisms should be studied in order to determine their value as vaccine candidates.Adhesion of bacteria to the mucosa is considered an essential early step in the colonization process. The ability of S. pneumoniae to adhere to epithelial cells has been ascribed to a number of surface-exposed proteins, including PspC, PsaA, PsrP, PfbB, NanA, PavA, and pili (3, 21, 41, 42, 45, 49, 54).Pili were recently discovered in many Gram-positive pathogens, and although their biological function has not been fully elucidated, their presence has been mostly related to bacterial adhesion, biofilm formation, and translocation of epithelial barriers (1, 13, 35, 47). These structures are composed of subunits covalently linked by means of intermolecular isopeptide bonds (32, 36, 51-53). Furthermore, intramolecular isopeptide bonds have been found in most pilus subunits characterized to date (9, 26-28). These bonds may play a critical role in maintaining pilus integrity in the face of severe mechanical and chemical stress while bound to host cells and thus may provide a functional mode of stabilization for cell surface proteins involved in host pathogenesis.In S. pneumoniae, pili (pilus 1 and pilus 2) are encoded by two genomic islets (pilus islet 1 [PI-1] and PI-2) that are not present in all pneumococcal clinical isolates. A number of molecular epidemiological studies have highlighted the presence of PI-1 as a clonal property of S. pneumoniae isolates and have defined, based on sequence analysis, the classification of PI-1 into three major clades (2, 5, 7, 25, 38, 39). Mutants lacking PI-1 are impaired in adhesion to cultured epithelial cells in vitro and are less virulent in murine models of colonization, pneumonia, and bacteremia (6, 41). Interestingly, pilus 1 expression is known to increase host inflammatory responses that might disrupt the mucosal barrier and facilitate subsequent invasion by the bacteria (6).Pneumococcal pilus 1 is composed of three subunits (RrgA, RrgB, and RrgC); RrgB is the backbone component, and RrgA is the major ancillary protein, localized at the pilus tip and responsible for the adhesion properties of the pilus, whereas RrgC is the minor ancillary protein, likely located at the pilus base (21, 22, 41). In terms of sequence variability, RrgB is classified into three variants and RrgC is conserved, whereas RrgA exists in two major variants (clades I and II) (38). The recombinant form of RrgA clade I adheres in vitro to cultured A549 lung epithelial cells, as well as to purified extracellular matrix (ECM) components (collagen I, fibronectin, and laminin) (21, 41). In addition, RrgA, along with the other two pilus 1 components, is able to elicit protection from lethal challenge with the homologous strain in mouse models of active and passive immunization (18).In this work, we investigated whether the differences between the two variants had an effect on the biochemical characteristics, biological function, and immunological properties of the molecule. We found that (i) sequence variability was restricted to the “head” domain of RrgA, containing the putative adhesive motifs; (ii) the two RrgA variants were resistant to proteolytic cleavage, and this feature was dependent on the presence of intramolecular isopeptide bonds; (iii) the two variants were able to adhere to epithelial cells and ECM components at comparable levels, whereas a mutant (Asp444Ala) in the RGD tripeptide showed reduced binding; (iv) none of the individual fragments encompassing the N-terminal (NT), central (CP), and C-terminal (CT) portions of RrgA was able to maintain adhesive capacity; (v) antibodies against these fragments revealed the N terminus to be less accessible than the remaining portion of the molecule on the native pilus; and, finally, (vi) antibodies raised against each of the two RrgA variants were cross-reactive and cross-protective in murine passive-immunization studies.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Immune Responses to Recombinant Pneumococcal PsaA Antigen Delivered by a Live Attenuated Salmonella Vaccine

Streptococcus pneumoniae is a leading cause of morbidity and mortality among children worldwide and particularly in developing countries. In this study, we evaluated PsaA, a conserved antigen important for S. pneumoniae adhesion to and invasion into nasopharynx epithelia, for its ability to induce protective immunity against S. pneumoniae challenge when delivered by recombinant attenuated Salmonella vaccine (RASVs) strains. RASVs were engineered to synthesize PsaA peptides of various lengths. Vaccination with an RASV synthesizing full-length PsaA induced high titers of anti-PsaA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes, nasal washes, and lung homogenates) sites. BALB/c (haplotype H2^d) or C57BL/6 (haplotype H2^b) mice vaccinated either orally or intranasally exhibited a significant reduction in colonization of nasopharyngeal tissues after intranasal challenge with S. pneumoniae strains compared to controls, although protection was not observed with all challenge strains. None of the vaccine constructs provided protection against intraperitoneal challenge with S. pneumoniae strain WU2 (serotype 3). Immunization with RASVs synthesizing truncated PsaA generated lower titers of IgA and IgG and did not provide significant protection. Our results showed that RASVs synthesizing full-length PsaA can provide protection against nasal colonization by some S. pneumoniae strains. PsaA may be a useful addition to a multivalent vaccine, providing protection against pneumonia, otitis media, and other diseases caused by S. pneumoniae.Streptococcus pneumoniae is responsible for a number of serious diseases in humans, including pneumonia, meningitis, bacteremia, otitis media, and sinusitis (31). It is a major cause of childhood mortality, 90% of which occurs in developing countries. The current vaccines against pneumococcal infections include a 23-valent capsular polysaccharide vaccine for adults and a 7-valent conjugate vaccine licensed for children (75, 77). However, some nonvaccine serotypes have become prevalent in the face of continued use of polysaccharide vaccines (63, 79). Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations (28). There are also several concerns about the conjugate vaccines related to the cost and complexity of manufacture due to the different prevalent serotypes in different geographical areas. A meta-analysis showed that vaccination appears efficacious in reducing pneumococcal pneumonia in low-risk adults but not in high-risk groups (24). A more recent meta-analysis of 22 trials involving 101,507 participants found that the current 23-valent polysaccharide vaccine does not appear to be effective in preventing pneumonia, even in populations for which the vaccine is currently recommended (33, 52). There is a need to develop an improved and effective vaccine based on conserved antigens across all capsular serotypes to induce more effective and durable immune responses that could potentially protect against all clinically relevant pneumococcal capsular types and cover some high-risk groups who may not respond well to the current vaccine, while still keeping the cost low enough to be used in developing countries.Studies of S. pneumoniae protective antigens have identified several candidate proteins that may be useful as vaccine components and drug targets, including PsaA, PspA, PspC, autolysin, pneumolysin, several neuraminidase enzymes, PcsB, and SktP (25, 80, 81, 88).PsaA is a metal-binding lipoprotein with specificity for Mn²⁺ and Zn²⁺ (21, 41). psaA expression is upregulated during adherence to human lung epithelial cells and in blood or cerebrospinal fluid (20, 32, 61), and the protein plays a significant role in pneumococcal adherence and colonization. E-cadherin has been identified as the receptor for PsaA (1). These results indicate that PsaA is a critical factor in the first step for pneumococcal nasopharyngeal colonization and carriage. Mutations in psaA result in pleiotropic effects on a number of virulence functions in addition to adherence, including hypersensitivity to oxidative stress, a deficiency in Mn²⁺ transport and virulence (6, 14, 49, 57, 85). PsaA is a conserved antigen. It was present in all examined strains representing the 90 S. pneumoniae serogroups known at the time of the study, as well as other viridans streptococcal species (34, 54, 71). In addition, PsaA is immunogenic (9, 36), making it a desirable candidate for inclusion in a vaccine.The primary translation product of the psaA gene is a 309-amino-acid (aa) polypeptide that includes a 20-aa N-terminal leader sequence containing the prolipoprotein recognition sequence LXXC recognized by signal peptidase II, two (β/α)₄ domains, and an α-helical linker. Signal sequence cleavage results in a 290-aa mature protein anchored to the bacterial membrane via the resultant N-terminal Cys-linked lipid tail. The remainder of the protein is composed of the two (β/α)₄ domains linked by an α-helix, forming two lobes with a cleft where the metal-binding site is located (41, 62).Immunization with PsaA induced significant protection against S. pneumoniae colonization but only modest protection against invasive infection (8, 64, 81). Because PsaA and PspA have different functions in virulence, protection induced by these proteins may be additive. Indeed, promising results have been found for the combination of PsaA and PspA in the prevention of colonization and otitis media in animal models (3, 9, 58). Nasal immunization with six doses of lactic acid bacteria expressing psaA has been shown to induce anti-PsaA antibodies and to decrease colonization of the nasopharynx after intranasal challenge (59), although protection against intraperitoneal challenge was modest and not statistically significant. While these studies are promising, use of a more invasive vector may provide better stimulation of the immune system with fewer doses. Recombinant attenuated Salmonella vaccines (RASVs) can effectively colonize deep lymphoid tissues to induce long-lasting immune responses to delivered recombinant antigens as well as to vector antigens. In this work, we evaluated the utility of using a live attenuated Salmonella strain to deliver PsaA.     

Nested PCR-Linked Capillary Electrophoresis and Single-Strand Conformation Polymorphisms for Detection of Macrolide-Resistant Mycoplasma pneumoniae in Beijing,China

Mycoplasma pneumoniae is usually susceptible to macrolides, but macrolide-resistant strains have been found frequently in recent years. Mutations in domain V of the 23S rRNA gene of M. pneumoniae interfere with the binding of macrolides to rRNA and mediate macrolide resistance. In this study, we developed a rapid and inexpensive method that combines nested PCR (nPCR), single-strand conformation polymorphisms (SSCPs), and capillary electrophoresis (CE) to detect macrolide-resistant mutants directly from throat swabs. nPCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations, with results confirmed by sequencing. From January to December 2009, 665 throat swabs were collected in Beijing, China, yielding 110 samples that tested positive for M. pneumoniae by nPCR and serological testing. We randomly selected 64 positive throat swabs for CE-SSCP analysis. The A2063G mutation was found in 57 samples, and a coexisting T2611C mutation was identified in 1 sample. An A2063T mutation was identified in 1 sample. The total mutation rate was 91%. All mutant samples identified by nPCR-CE-SSCP were sequenced. The nPCR-CE-SSCP method could identify macrolide-resistant mutants directly from clinical samples. This is the first report of an nPCR-CE-SSCP assay for the detection of dominant mutations that confer macrolide resistance on M. pneumoniae. This approach would allow clinicians to choose appropriate therapy rapidly and could be used as a screening method for genetic mutations related to antibiotic resistance.Mycoplasma pneumoniae is a common cause of community-acquired respiratory tract infections, especially in children and young adults. Nearly 80% of the population shows evidence of exposure to M. pneumoniae by young adulthood, and epidemics occur every 3 to 7 years (5, 21, 31). For chemotherapy of M. pneumoniae infection in children, erythromycin (ERY), clarithromycin, and azithromycin (AZM) are considered to be first-choice agents. Because of the toxic effect on children, fluoroquinolones and tetracyclines are not recommended.M. pneumoniae is usually susceptible to macrolides; however, macrolide-resistant strains have been found frequently in recent years (16, 17, 22, 23, 24). The traditional method for assessing the susceptibility of M. pneumoniae to antibiotics depends on determination of the MICs of various macrolides after the isolation of M. pneumoniae strains. This method uses visual indicators of antibiotic effectiveness and is regarded as a reference method. However, it is time-consuming and labor-intensive; thus, it is less valuable in clinical practice than in research. Previous studies have shown that mutations in domain V of the 23S rRNA gene of M. pneumoniae that interfere with the binding of macrolides to rRNA can mediate M. pneumoniae resistance to certain macrolides (2, 8, 9, 19, 29, 32). Lucier et al. found that the A2063G mutation caused ERY resistance in M. pneumoniae strains (16). Okazaki et al. detected the same mutation in clinical samples resistant to ERY, while the A2064G mutation was also detected in resistant strains (22). The C2617A mutation was found to cause resistance to ERY, AZM, and telithromycin (35). Other mutations were also detected in resistant strains (4, 24). The correlation between mutations and macrolide resistance provides the possibility of direct detection of A2063G, A2064G, C2617A, or similar mutations with the goal of chemotherapy selection.A number of candidate methods have been used to detect mutations directly, including sequencing of PCR products, restriction fragment length polymorphism (RFLP) analysis, or other mutation detection methods, such as real-time PCR and high-resolution melt analysis (25, 33, 34). The single-strand conformation polymorphism (SSCP) technique is based on changes in the conformation of single-stranded DNA (ssDNA) that are induced by a single-base mutation. This can be detected by highly sensitive electrophoresis methods, such as capillary electrophoresis (CE). A strictly controlled SSCP testing procedure, coupled with CE as the separation tool, could reach nearly 100% sensitivity in mutant detection and could be more sensitive than sequencing (1, 6, 7, 10, 11, 13, 14, 26, 27, 28).In this study, we developed a nested-PCR (nPCR)-CE-SSCP method for rapid detection of antibiotic-resistant mutants of M. pneumoniae. Using this method, nPCR amplifies fragments of the M. pneumoniae 23S rRNA gene directly from the throat swabs of patients. The nPCR products are analyzed by SSCP, and single-stranded DNA is separated by CE. In this study, amplicons were sequenced, and the results were compared to those of CE-SSCP.     

Chlamydial and Periodontal Pathogens Induce Hepatic Inflammation and Fatty Acid Imbalance in Apolipoprotein E-Deficient Mice

Periodontitis and Chlamydia pneumoniae infection are independent risk factors for cardiovascular diseases. The aim of this study was to investigate the effect of C. pneumoniae and Aggregatibacter actinomycetemcomitans infection on hepatic inflammation and lipid homeostasis of apolipoprotein E-deficient mice. Mice were infected with viable C. pneumoniae intranasally three times for chronic infection or once for acute infection. Viable A. actinomycetemcomitans was administered 10 times intravenously alone or in concert with C. pneumoniae. Hepatic alterations were assessed by histochemistry, lipid quantification, and fatty acid profile analysis. The RNA expression levels and the presence of pathogens in the livers and lungs were detected by quantitative real-time PCR. Both pathogens were detected in the livers of the infected animals. Chronic C. pneumoniae infection induced marked changes in hepatic lipid homeostasis. A. actinomycetemcomitans infection resulted in inflammatory cell infiltration into the liver, accompanied by elevated hepatic RNA expression levels of inflammation-related genes and higher serum amyloid A and lipopolysaccharide concentrations. Our results indicate that proatherogenic pathogens infect the liver, causing proinflammatory alterations and lipid disturbances. This infection may maintain chronic systemic inflammation attributable to atherogenesis.Periodontitis, a chronic infection affecting the tooth-supporting tissues, and persistent Chlamydia pneumoniae infection, e.g., in the lungs and vasculature, are associated with an increased risk of cardiovascular diseases (CVD) (1, 14). A major periodontal pathogen, Aggregatibacter actinomycetemcomitans, and C. pneumoniae are gram-negative bacteria containing a potent proatherogenic outer membrane component, lipopolysaccharide (LPS). Seropositivity to these pathogens and their carriage are highly prevalent (15, 21, 38). Prospective studies have demonstrated that systemic exposure to A. actinomycetemcomitans is associated with a twofold risk for incident CVD (22). A recent survey showed that the periodontal pathogen burden, and especially the amount of A. actinomycetemcomitans, is associated with prevalent coronary heart disease (28). Similarly, reports have indicated an association between chronic C. pneumoniae infection and CVD and, further, a role of the total pathogen burden in the risk for coronary heart disease (2, 41).Many infections affecting the gastrointestinal tract, such as viral hepatitis and small intestinal bacterial overgrowth, may be associated with nonalcoholic fatty liver disease (NAFLD) (5, 39). However, the connection between infections by proatherogenic pathogens and liver damage is not well known. A recent report showed a significantly elevated seropositivity rate for C. pneumoniae among men with nonalcoholic steatohepatitis (4). In addition, C. pneumoniae replicates in mouse Kupffer cells and is able to induce a local proinflammatory state in the liver (20). Some epidemiological studies have reported an association between periodontitis and NAFLD (10, 25). In experimental animal studies, Escherichia coli LPS-induced periodontal inflammation generated NAFLD in a rat model (30). Moreover, in vitro studies have shown the capacity of various spirochetes to stimulate rat Kupffer cells to produce reactive oxygen species related to hepatic injury (18, 19).The aim of this study was to investigate the effect of acute and chronic C. pneumoniae infections, as well as A. actinomycetemcomitans infection, on the hepatic lipid and inflammation status of atherosclerosis-susceptible apolipoprotein E (apoE)-deficient mice. The degree and nature of hepatic lipid balance and inflammation were assessed by histochemistry, gene expression, total lipid quantification, and fatty acid profile analyses.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Screening of Streptococcus pneumoniae ABC Transporter Mutants Demonstrates that LivJHMGF,a Branched-Chain Amino Acid ABC Transporter,Is Necessary for Disease Pathogenesis

Bacterial ABC transporters are an important class of transmembrane transporters that have a wide variety of substrates and are important for the virulence of several bacterial pathogens, including Streptococcus pneumoniae. However, many S. pneumoniae ABC transporters have yet to be investigated for their role in virulence. Using insertional duplication mutagenesis mutants, we investigated the effects on virulence and in vitro growth of disruption of 9 S. pneumoniae ABC transporters. Several were partially attenuated in virulence compared to the wild-type parental strain in mouse models of infection. For one ABC transporter, required for full virulence and termed LivJHMGF due to its similarity to branched-chain amino acid (BCAA) transporters, a deletion mutant (ΔlivHMGF) was constructed to investigate its phenotype in more detail. When tested by competitive infection, the ΔlivHMGF strain had reduced virulence in models of both pneumonia and septicemia but was fully virulent when tested using noncompetitive experiments. The ΔlivHMGF strain had no detectable growth defect in defined or complete laboratory media. Recombinant LivJ, the substrate binding component of the LivJHMGF, was shown by both radioactive binding experiments and tryptophan fluorescence spectroscopy to specifically bind to leucine, isoleucine, and valine, confirming that the LivJHMGF substrates are BCAAs. These data demonstrate a previously unsuspected role for BCAA transport during infection for S. pneumoniae and provide more evidence that functioning ABC transporters are required for the full virulence of bacterial pathogens.Bacterial ABC transporters are an important class of transmembrane transporters that are involved in the import and export of a wide variety of substrates, including sugars, amino acids, peptides, polyamines, and cations (11, 12, 17, 39). A typical ABC transporter consists of four membrane-associated proteins consisting of two ATP-binding proteins (ATPases) and two membrane-spanning proteins (permeases) (11, 17). These may be fused in a variety of ways to form multidomain polypeptides, but typically permeases consist of six putative α-helical transmembrane segments that act as a channel through which substrates are transported across the membrane (11, 17). ABC transporters that import their substrate also contain a substrate-binding protein (SBP) that is present in the periplasm of gram-negative bacteria and most often as a lipoprotein bound to the outer surface of the membrane in gram-positive bacteria (17). These SBPs bind to the substrate before it is transferred across the cell membrane and therefore confer substrate specificity for the ABC transporter. Approximately 5% of the Escherichia coli and Bacillus subtilis genomes encode components of ABC transporters, highlighting the importance of ABC transporters for the physiology of both gram-positive and gram-negative bacteria (12, 30). ABC transporters are known to influence many cellular processes. including antibiotic resistance, nutrient acquisition, adhesion, protein secretion, environmental sensing, spore formation, conjugation, and growth under stress conditions (39). As a consequence, many ABC transporters have been shown by signature-tagged mutagenesis (STM) screens to be important for the virulence of a range of bacterial pathogens, including Yersinia spp., Staphylococcus aureus, and Streptococcus pneumoniae (9, 15, 22, 26), and these data have been supported by publications on the functions of individual ABC transporters (6, 7, 38, 45).The annotated genome sequence of the TIGR4 strain of the common gram-positive pathogen S. pneumoniae contains 73 ABC transporters (4, 13). Several ABC transporters required for substrate uptake have been described in some detail previously, and some of these are known to be important for full virulence, including the cation transporters PsaA, PiuA, PiaA, and PitA (6, 7, 27, 34) and the polyamine transporter PotABCD (44). Why disruption of these ABC transporter functions affect virulence is probably due to a variety of mechanisms. These include effects on micronutrient acquisition under stress conditions, such as reduced iron uptake after disruption of PiuA, PiaA, and PitA; increased sensitivity to oxidative stress due to loss of polyamine or manganese uptake by PotABCD or PsaA, respectively (25, 43, 44); and impaired adhesion to cell surfaces related to disruption of PsaA (2, 20). In addition, since intracellular levels of cations and other micronutrients can influence gene regulation (20), impaired uptake of micronutrients could affect bacterial adaptation to the host environment. SBP components of ABC transporters are attached to the external surface of the bacterial membrane, where they are exposed to interactions with the environment, and their sequences are usually highly conserved between different strains of the same bacterial species. As a consequence, SBPs have been investigated as potential protein vaccine candidates, and PiuA, PiaA, PsaA, and PotD have all been shown to be effective vaccines in animal models at preventing S. pneumoniae infection (12, 23).Given the importance of acquisition of various minerals and nutritional substrates for bacterial growth and virulence in vivo, some of the S. pneumoniae ABC transporters that have not yet been investigated are also likely to influence the pathogenesis of S. pneumoniae infections. Using mouse models of infection, we have therefore assessed the potential role during virulence of nine S. pneumoniae ABC transporters that have not, as far as we are aware, previously been investigated. Several ABC transporters were identified as required for full S. pneumoniae virulence in models of septicemia or pneumonia. The function of one of these ABC transporters, termed livJHMGF, which BLAST searches suggest is a member of the hydrophobic amino acid transporter subfamily and is likely to be a branched-chain amino acid (BCAA) transporter (40), was investigated in more detail.     

Toll-Like Receptor Stimulation Enhances Phagocytosis and Intracellular Killing of Nonencapsulated and Encapsulated Streptococcus pneumoniae by Murine Microglia

Toll-like receptors (TLRs) are crucial pattern recognition receptors in innate immunity that are expressed in microglia, the resident macrophages of the brain. TLR2, -4, and -9 are important in the responses against Streptococcus pneumoniae, the most common agent causing bacterial meningitis beyond the neonatal period. Murine microglial cultures were stimulated with agonists for TLR1/2 (Pam₃CSK₄), TLR4 (lipopolysaccharide), and TLR9 (CpG oligodeoxynucleotide) for 24 h and then exposed to either the encapsulated D39 (serotype 2) or the nonencapsulated R6 strain of S. pneumoniae. After stimulation, the levels of interleukin-6 and CCL5 (RANTES [regulated upon activation normal T-cell expressed and secreted]) were increased, confirming microglial activation. The TLR1/2, -4, and -9 agonist-stimulated microglia ingested significantly more bacteria than unstimulated cells (P < 0.05). The presence of cytochalasin D, an inhibitor of actin polymerizaton, blocked >90% of phagocytosis. Along with an increased phagocytic activity, the intracellular bacterial killing was also increased in TLR-stimulated cells compared to unstimulated cells. Together, our data suggest that microglial stimulation by these TLRs may increase the resistance of the brain against pneumococcal infections.Immunocompromised patients have a higher risk of developing bacterial infections in the central nervous system (CNS) (34, 37, 42). The list of the pathogens includes many organisms with low pathogenicity in the immunocompetent host (34, 37). Moreover, the distribution of the pathogens also differs from the immunocompetent host and depends on the nature of the immune defect. Patients with a decrease in B-lymphocyte function or with a loss of splenic function have an increased risk of meningitis caused by encapsulated bacteria, while patients with an impaired T-lymphocyte-macrophage system are more susceptible to CNS infections caused by intracellular pathogens (7, 42). One additional cause of this increased susceptibility to CNS infections probably is a decreased local immune defense (33).CNS infections not only are more frequent but also are associated with higher mortality rates and more severe long-term sequelae in immunocompromised than in immunocompetent individuals (9, 17, 34, 44). Polymicrobial infections, multiple organ system presentation, and the absence of typical clinical manifestations subsequent to the host''s diminished inflammatory response are challenging aspects in the management of these infections (34, 37, 42).The brain tissue shows a well-organized innate immune reaction in response to bacteria in the cerebrospinal fluid (CSF) (3, 21). Microglial cells, the resident phagocytes of the CNS, express Toll-like receptors (TLRs) that identify pathogen-associated molecular patterns (PAMPs) (41). The receptor-ligand interactions activate microglia to undergo morphological transformation as well as functional changes, such as the production of proinflammatory cytokines, chemokines, and reactive oxygen species, enhanced phagocytic activity, and antigen presentation (15, 39). This immune reaction cannot eliminate high amounts of pneumococci from the CSF but does prevent or minimize the invasion of these pathogens into the brain tissue, thereby limiting tissue destruction and neuronal injury.TLR2, -4, and -9 contribute to the recognition and response to Streptococcus pneumoniae in the CNS (31). A deficiency of TLR2, -4, or -9 or of the coreceptor CD14, which is necessary for TLR4 signaling increases the susceptibility of mice to S. pneumoniae (1, 11, 12, 40).Here, we hypothesized that activation of the innate immune response in microglia could increase the resistance of the brain tissue against CNS pneumococcal infections (14). This may be of particular interest in immunocompromised patients, whose outcome after S. pneumoniae meningitis is worse than that of immunocompetent individuals (9, 44). The aim of the present study was to investigate whether the stimulation of microglia by respective PAMPs can increase their ability to phagocytose and kill intracellular nonencapsulated and encapsulated S. pneumoniae strains, thereby protecting the brain during meningitis. Moreover, by using an encapsulated and a nonencapsulated pneumococcal strain, we assessed the protective effect of the capsule against phagocytosis by microglial cells.     

Streptococcus pneumoniae Autolysis Prevents Phagocytosis and Production of Phagocyte-Activating Cytokines

Streptococcus pneumoniae is a major pathogen in humans. The pathogenicity of this organism is related to its many virulence factors, the most important of which is the thick pneumococcal capsule that minimizes phagocytosis. Another virulence-associated trait is the tendency of this bacterium to undergo autolysis in stationary phase through activation of the cell wall-bound amidase LytA, which breaks down peptidoglycan. The exact function of autolysis in pneumococcal pathogenesis is, however, unclear. Here, we show the selective and specific inefficiency of wild-type S. pneumoniae for inducing production of phagocyte-activating cytokines in human peripheral blood mononuclear cells (PBMC). Indeed, clinical pneumococcal strains induced production of 30-fold less tumor necrosis factor (TNF), 15-fold less gamma interferon (IFN-γ), and only negligible amounts of interleukin-12 (IL-12) compared with other closely related Streptococcus species, whereas the levels of induction of IL-6, IL-8, and IL-10 production were similar. If pneumococcal LytA was inactivated by mutation or by culture in a medium containing excess choline, the pneumococci induced production of significantly more TNF, IFN-γ, and IL-12 in PBMC, whereas the production of IL-6, IL-8, and IL-10 was unaffected. Further, adding autolyzed pneumococci to intact bacteria inhibited production of TNF, IFN-γ, and IL-12 in a dose-dependent manner but did not inhibit production of IL-6, IL-8, and IL-10 in response to the intact bacteria. Fragments from autolyzed bacteria inhibited phagocytosis of intact bacteria and reduced the in vitro elimination of pneumococci from human blood. Our results suggest that fragments generated by autolysis of bacteria with reduced viability interfere with phagocyte-mediated elimination of live pneumococci.The pneumococcus Streptococcus pneumoniae is a leading cause of community-acquired pneumonia, meningitis, otitis media, and sinusitis and is a common cause of infection-related mortality in children and elderly people (28, 37).There is a large number of streptococcal species whose taxonomic classification is debated (14, 31). A number of streptococci, including alpha-hemolytic and nonhemolytic variants, constitute the viridans group, which can be further subdivided into the mitis, sanguinis, anginosus, salivarius, and mutans groups based on biochemical tests (14). Phenotypic and genetic tests consistently show that S. pneumoniae is closely related to and may be placed in the mitis subgroup (14, 30). Although the other members of the mitis group can cause sepsis and endocarditis (53), they are considerably less virulent than S. pneumoniae.Pneumococci are considered strictly extracellular pathogens, whose elimination depends on ingestion and killing by phagocytes (i.e., alveolar and tissue-resident macrophages and neutrophils recruited during the inflammatory process). Accordingly, an important determinant of pneumococcal pathogenicity is the thick, hydrophilic polysaccharide capsule, which impedes elimination by phagocytes in the absence of capsule-specific antibodies.The ability of phagocytes to kill microbes is augmented by the phagocyte-activating cytokines gamma interferon (IFN-γ) and tumor necrosis factor (TNF), which boost the bactericidal machinery and enhance killing and digestion of bacteria present within the phagosome (4, 39, 47). TNF is produced by monocytes/macrophages and activated T cells, while IFN-γ is produced by NK cells and T cells in response to interleukin-12 (IL-12) from macrophages. Thus, production of TNF, IFN-γ, and IL-12 is necessary for host defense against intracellular bacteria (8, 11, 21, 34, 48). More recently, these phagocyte-activating cytokines have also been shown to be essential for controlling extracellular gram-positive bacteria, including S. pneumoniae (36, 42, 50, 52, 54). Thus, a patient with an IL-12 deficiency was shown to suffer from recurrent episodes of pneumococcal infection (20). Phagocyte activation by TNF and/or IFN-γ might be required for decomposition of the thick, sturdy peptidoglycan (PG) layer of gram-positive bacteria after phagocytosis, while gram-negative bacteria may be more easily digested. Thus, human leukocytes produce more TNF, IFN-γ, and IL-12 when they are stimulated with gram-positive bacteria than when they are stimulated with gram-negative bacteria (23, 24).A peculiar property of S. pneumoniae is its tendency to undergo autolysis when it reaches the stationary phase of growth. This process is mediated by enzymes called autolysins (ALs), which, when activated, degrade cell wall PG. The major AL is an N-acetyl-muramyl-l-alanine amidase called LytA (27). Other pneumococcal ALs include LytB and LytC, which are believed to be involved mainly in modification of the cell wall during growth and division (16, 17). ALs are anchored to the cell wall via interactions with choline moieties on teichoic acid and lipoteichoic acid (LTA). Choline is necessary for pneumococcal growth, but culture in the presence of high concentrations of choline renders the bacteria incapable of undergoing autolysis (6, 19).Studies with mice have shown that S. pneumoniae with mutated LytA is less virulent than wild-type pneumococci (2, 7, 25). The reason for this is not clear, but two main hypotheses have been put forward. First, autolysis promotes the release of the intracellular toxin pneumolysin (Ply) (5, 33). Ply is an important determinant of virulence (3, 41) and interferes with several defense systems, including inhibition of ciliary beating (15), complement activation (38), and induction of intracellular oxygen radical production (33). Second, cell wall degradation products, such as soluble PG fragments and LTA released upon autolysis, have been suggested to augment the inflammatory response (9, 10, 44, 49).Here we examine a third possibility, that autolysis interferes with the generation of phagocyte-activating cytokines. We have previously shown that intact gram-positive bacteria provide a very efficient stimulus for IL-12 production by human monocytes, regardless of whether they are dead or alive (1, 23, 24), but that decomposed bacteria are inactive in this process and soluble components of the gram-positive cell wall, such as PG and LTA, even downregulate the production of IL-12 in response to intact bacteria in a dose-dependent manner (1). These observations led us to speculate that autolysis may promote virulence by generating bacterial cell wall fragments that block IL-12 production and thereby reduce IFN-γ production and phagocyte activation. Indeed, our data demonstrate that AL-mediated disintegration of pneumococci inhibits production of IFN-γ and also TNF in response to intact bacteria. Further, the presence of autolyzed bacteria reduced elimination of live pneumococci by blood cells in vitro.     

The Streptococcus pneumoniae Capsule Inhibits Complement Activity and Neutrophil Phagocytosis by Multiple Mechanisms

The Streptococcus pneumoniae capsule is vital for virulence and may inhibit complement activity and phagocytosis. However, there are only limited data on the mechanisms by which the capsule affects complement and the consequences for S. pneumoniae interactions with phagocytes. Using unencapsulated serotype 2 and 4 S. pneumoniae mutants, we have confirmed that the capsule has several effects on complement activity. The capsule impaired bacterial opsonization with C3b/iC3b by both the alternative and classical complement pathways and also inhibited conversion of C3b bound to the bacterial surface to iC3b. There was increased binding of the classical pathway mediators immunoglobulin G (IgG) and C-reactive protein (CRP) to unencapsulated S. pneumoniae, indicating that the capsule could inhibit classical pathway complement activity by masking antibody recognition of subcapsular antigens, as well as by inhibiting CRP binding. Cleavage of serum IgG by the enzyme IdeS reduced C3b/iC3b deposition on all of the strains, but there were still marked increases in C3b/iC3b deposition on unencapsulated TIGR4 and D39 strains compared to encapsulated strains, suggesting that the capsule inhibits both IgG-mediated and IgG-independent complement activity against S. pneumoniae. Unencapsulated strains were more susceptible to neutrophil phagocytosis after incubation in normal serum, normal serum treated with IdeS, complement-deficient serum, and complement-deficient serum treated with IdeS or in buffer alone, suggesting that the capsule inhibits phagocytosis mediated by Fcγ receptors, complement receptors, and nonopsonic receptors. Overall, these data show that the S. pneumoniae capsule affects multiple aspects of complement- and neutrophil-mediated immunity, resulting in a profound inhibition of opsonophagocytosis.The Gram-positive pathogen Streptococcus pneumoniae is one of the most common causes of pneumonia, septicemia, and meningitis in children and adults in both industrialized and developing parts of the world (10). This large burden of disease is compounded by the increased incidence of S. pneumoniae infections associated with HIV and by increasing antibiotic resistance among clinical isolates, and there is a strong need to understand the molecular pathogenesis of S. pneumoniae infections to assist the development of new therapeutic targets. Probably the most important virulence factor for S. pneumoniae is the extracellular capsule, a layer consisting of chains of monosaccharides that surrounds the bacteria. For S. pneumoniae strains, there are 91 antigenically distinct capsular serotypes, dictated by the order and type of the monosaccharide units within the polysaccharide chain and by different side branches (5, 27). The importance of the S. pneumoniae capsule for virulence is demonstrated by the facts that (i) all clinical isolates causing invasive disease are encapsulated; (ii) loss of the capsule by either genetic mutation or enzymatic degradation dramatically reduces S. pneumoniae virulence in animal models of infection (6, 28, 29, 43, 49); (iii) different capsular serotypes vary in the ability to cause invasive disease (9), and swapping capsular serotypes between strains affects virulence in animal models (21); and (iv) S. pneumoniae opaque-phase variants (which express a thicker capsule than transparent-phase variants) predominate during invasive infection (35). Furthermore, the capsule is the target for existing S. pneumoniae vaccines and widespread vaccination has led to the evolution of vaccine escape mutants expressing nonvaccine capsular serotypes, increasing the importance of a better understanding of how the capsule can affect virulence.One component of the immune system that is likely to be affected by the S. pneumoniae capsule is the complement system. Clinical and experimental evidence has shown the vital role of complement for host immunity to S. pneumoniae and that neutrophil phagocytosis of S. pneumoniae is largely dependent on complement activity (8, 15, 19, 22, 39, 52, 53). The complement system is organized into three enzyme cascades termed the classical, alternative, and mannan binding lectin (MBL) pathways (42). The classical complement pathway is activated by specific immunoglobulin G (IgG) and was generally considered an effector of the adaptive immune response, but recent data have demonstrated an important role for the classical pathway as part of the innate immune response to S. pneumoniae. S. pneumoniae cell wall phosphorylcholine (PC) is recognized by the serum proteins C-reactive protein (CRP) and serum amyloid P (SAP) (collectively termed pentraxins due to their structurally similarity) (40) and also by natural IgM (4). In addition, the cell surface lectin SIGN-R1 binds to the S. pneumoniae capsule (20). Recognition of S. pneumoniae by the pentraxins, natural IgM, and SIGN-R1 results in binding of the first component of the classical pathway, C1q, to the bacterial surface and complement activation. The MBL pathway is activated by binding of MBL to certain sugar residues found on the surface of pathogens. However, MBL binds poorly to S. pneumoniae and seems to have little effect on complement deposition on S. pneumoniae (8, 31), although MBL or other ficolins may directly opsonize microorganisms independent of complement activity. The alternative pathway is spontaneously activated unless the target cell is coated in sialic acid or complement-inhibitory proteins such as factor H (FH) (42) and is therefore a component of the innate immune response to S. pneumoniae. The alternative pathway probably also amplifies the amount of C3b/iC3b deposited on the bacterial surface once complement activation has been initiated by the classical or MBL pathway (8, 42). Each pathway leads to the formation of a C3 convertase that cleaves the central complement component C3, resulting in deposition of C3b on the surface of the pathogen that is further processed to iC3b. C3b and iC3b are opsonins mediating phagocytosis mainly through the complement receptor CR1 and CR3 receptor, respectively. As well as opsonizing bacteria, complement activation aids the inflammatory response through release of anaphylaxins such as C5a (42) and improves the adaptive immune response to S. pneumoniae through direct stimulation of B cells by the C3 breakdown product C3d (13).The external position of the capsule means it is ideally situated to modulate interactions between S. pneumoniae and host proteins and cells. Unencapsulated mutants have been shown to be more susceptible to phagocytosis, and there are limited data showing increased levels of complement deposition on their surface (1, 32, 47), but despite the importance of the capsule for S. pneumoniae virulence, there are few data on the mechanisms involved (32). Data obtained for other pathogens have shown a variety of mechanisms by which polysaccharide capsules can inhibit complement activity. The group B Streptococcus (GBS) and Neisseria meningitidis capsules contain sialic acid, which is thought to prevent alternative complement activity by creating a nonactivating surface and by binding to FH (23, 25, 46). Alternatively, the capsule may inhibit recognition of surface antigens by specific IgG, thereby preventing classical pathway activation or directly prevent binding of complement components to subcapsular targets of complement activity (37). In contrast, the capsule of Cryptococcus neoformans is a potent activator of alternative pathway activity and this is thought to aid immunity by depleting complement (50). For S. pneumoniae, whether the capsule prevents complement deposition indirectly through impairing recognition of the bacteria by IgG or has direct effects on bacterial interactions with non-IgG complement activators or other aspects of complement activity is not known. Given the importance of complement for neutrophil phagocytosis of S. pneumoniae (53), inhibition of opsonization with C3b/iC3b by the capsule could account for all of the effects of the capsule on phagocytosis. However, IgG bound to the bacterial surface and nonopsonic phagocytic molecules such as the mannose and scavenger receptors can mediate phagocytosis independently of complement, and these mechanisms of phagocytosis potentially could also be affected by the S. pneumoniae capsule. Indeed, recent data showing increased phagocytosis of an unopsonized, unencapsulated serotype 6B strain suggest that there can be a capsular effect on nonopsonic phagocytosis (44). Given the importance of the capsule for S. pneumoniae virulence and as a vaccine candidate, a more detailed understanding of the interactions of the capsule with complement and neutrophils would be beneficial.Using unencapsulated mutants from serotype 2 and 4 S. pneumoniae strains that are otherwise isogenic to the encapsulated parental strain, we have investigated the effect of the capsule on IgG-dependent and -independent complement deposition on the bacterial cell surface and on the binding of various complement mediators. We have also assessed the effects of the capsule on complement-dependent and complement-independent neutrophil phagocytosis.     

Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health problem in Central Asia, Southeast Asia, Africa, and Latin America (11, 52, 53). It was estimated that more than 21 million cases of typhoid fever and >200,000 deaths occurred in 2000 (10). The treatment of patients and management of asymptomatic carriers are becoming more difficult due to the worldwide emergence of multidrug-resistant (MDR) strains (2, 15, 29, 42, 43). Vaccination is considered the most promising strategy for the control of typhoid fever in developing countries (11, 19, 52, 53).Typhoid fever in children younger than 5 years old has often been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of less than optimal culture media (35, 46). Several studies have shown that the incidence of typhoid fever among children less than 5 years old is similar to that in school age children and young adults (14, 27, 34, 50, 51).The 3 licensed typhoid vaccines have limited efficacy, and none are suitable for young children under 5 years old. The use of heat-inactivated whole-cell vaccine was suspended in many countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were introduced in the late 1980s; both vaccines are well accepted and confer moderate protection (50 to 70%) in older children and adults. However, neither vaccine is licensed for routine immunization of infants (52).The Vi capsular polysaccharide is both an essential virulence factor and a protective antigen for S. Typhi (36, 38, 39). The concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is not suitable for routine immunization of infants and young children because of its age-related immunogenicity and T-cell independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae (12, 20), Vi covalently bound with protein conferred T-cell dependence and increased immunogenicity (48-50). To date, diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins (CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi polysaccharide in laboratory studies (16, 17, 32, 48-50; personal communications). An improved method was developed (24), utilizing adipic acid dihydrazide (ADH) as the linker and Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier. Clinical trials of Vi-rEPA conjugates conferred 89% protection in Vietnamese children 2 to 5 years old for 46 months (23, 26, 28). The level of serum IgG anti-Vi induced by Vi-rEPA conjugates was correlated with prevention of typhoid fever in these studies (7, 21-23, 26, 28).One limitation of using rEPA as the carrier protein is the lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by pharmaceutical companies in China and India were prepared (24, 48, 49). Modifications of conjugation procedures were made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility of stockpiling in disaster relief.Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule using alternating injections of polysaccharide and conjugate. Priming or boosting effects of polysaccharide on its conjugate vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no consistent conclusion about various types of polysaccharides studied (6, 9, 31, 40, 41). Here, we compared the immune response of Vi polysaccharide injected before or after the administration of Vi-DT with the responses of those receiving 2 injections of Vi-DT. We also investigated the dosage effect for the purpose of better formulation.     

Streptococcus pneumoniae Resistance to Complement-Mediated Immunity Is Dependent on the Capsular Serotype

Streptococcus pneumoniae strains vary considerably in the ability to cause invasive disease in humans, and this is partially associated with the capsular serotype. The S. pneumoniae capsule inhibits complement- and phagocyte-mediated immunity, and differences between serotypes in these effects on host immunity may cause some of the variation in virulence between strains. However, the considerable genetic differences between S. pneumoniae strains independent of the capsular serotype prevent an unambiguous assessment of the effects of the capsular serotype on immunity using clinical isolates. We have therefore used capsular serotype-switched TIGR4 mutant strains to investigate the effects of the capsular serotype on S. pneumoniae interactions with complement. Flow cytometry assays demonstrated large differences in C3b/iC3b deposition on opaque-phase variants of TIGR4(−)+4, +6A, +7F, and +23F strains even though the thicknesses of the capsule layers were similar. There was increased C3b/iC3b deposition on TIGR4(−)+6A and +23F strains compared to +7F and +4 strains, and these differences persisted even in serum depleted of immunoglobulin G. Neutrophil phagocytosis of the TIGR4(−)+6A and +23F strains was also increased, but only in the presence of complement, showing that the effects of the capsular serotype on C3b/iC3b deposition are functionally significant. In addition, the virulence of the TIGR4(−)+6A and +23F strains was reduced in a mouse model of sepsis. These data demonstrate that resistance to complement-mediated immunity can vary with the capsular serotype independently of antibody and of other genetic differences between strains. This might be one mechanism by which the capsular serotype can affect the relative invasiveness of different S. pneumoniae strains.The important Gram-positive pathogen Streptococcus pneumoniae has an extracellular polysaccharide capsule that inhibits complement activity, neutrophil phagocytosis, and bacterial killing by neutrophil extracellular traps (19, 23, 25, 26, 29, 31), as well as having major effects on bacterial interactions with the epithelium (8, 25, 26, 29, 31, 37). As a consequence, the capsule is essential for virulence (6, 38). Different strains of S. pneumoniae can express capsules with different structures, depending on the type of monosaccharide units and their bonds within the polysaccharide chain, the enzymes for the synthesis of which are encoded by genes within a specific locus in the genome (5, 27, 30). The different types of capsules are divided into 91 capsular serotypes. Although most S. pneumoniae strains can cause disease in humans, the ability to cause invasive infections (septicemia and meningitis) varies up to 60-fold between strains and is closely associated with the capsular serotype (4, 12). Some serotypes (e.g., 1, 4, 5, 7, and 14) are overrepresented among invasive disease isolates compared to the frequency of their isolation as nasopharyngeal commensals, while other capsular serotypes only rarely cause invasive disease despite being common nasopharyngeal commensals (4, 12, 15).The mechanisms causing capsular serotype-dependent variation in virulence are largely unknown but could reflect differences between the abilities of strains of different serotypes to inhibit host immune responses. Potentially, strains expressing capsular serotypes that strongly inhibit immunity could be more likely to establish invasive infection than strains with capsular serotypes that weakly inhibit host immunity, and this hypothesis is partially supported by existing experimental data. The virulence of different capsular serotypes varies markedly in mouse models of infection, but as there is only a weak relationship between virulence in mice and invasive potential in humans, the clinical relevance of these findings is unclear (1, 7, 9, 33). Because of the central role of complement and phagocytosis for systemic immunity to S. pneumoniae (11, 20, 45, 46), differences in the effects of different capsular serotypes on complement activity or phagocytosis are strong candidates for explaining why the serotype can affect virulence. Indeed, existing data show that resistance to complement activity and phagocytosis varies between strains with different capsular serotypes (18, 28, 46). However, in general, these studies have not controlled for strain phase variation or for noncapsular genetic variation between strains. S. pneumoniae has two main phase variants, opaque with an increased capsule thickness and transparent with a thinner capsule but increased expression of some surface proteins, such as PspC, that can affect complement activity (24, 31). Differences in phase variation between strains could therefore affect complement susceptibility. Furthermore, there is considerable genetic variation between S. pneumoniae strains independent of the capsular serotype. Only 60% of gene clusters are common to all S. pneumoniae strains, and the genome content differs by 8 to 10%, on average, between any two strains (10, 13, 16, 17). This genetic variation is partially linked to the capsular serotype (http://www.mlst.net/), and hence, the relationship between the capsular serotype and invasiveness could be due to noncapsular genetic variation rather than direct effects of the capsule.To overcome strain genetic variation confounding the assessment of capsular serotype interactions with the immune system, the capsular loci of one strain can be replaced with the capsular loci from another, creating otherwise isogenic strains expressing different capsular serotypes (29, 35, 43). Data obtained using capsular serotype-switched strains have shown that expression of capsular serotype 3 reduced complement deposition on a previously serotype 2 strain (2), increased the virulence in mice of an originally serotype 6B strain, and conversely decreased the virulence of a serotype 5 strain (22). Furthermore, a recent study demonstrated variations in resistance to neutrophil killing of unopsonized bacteria between capsular serotype-switched strains expressing different capsular serotypes and correlated reduced sensitivity to neutrophil killing with increased prevalence of that capsular serotype as a nasopharyngeal commensal (39). These studies have established the principle that the capsular serotype can affect the complement sensitivity, neutrophil killing, and virulence of S. pneumoniae independently of the strain background. However, as yet, there are only limited data on the effects of different capsular serotypes on complement-dependent immunity to S. pneumoniae and a more detailed assessment is required to help understand why a strain''s capsular serotype is linked to its invasive potential.We have used opaque- and transparent-phase variants of TIGR4 S. pneumoniae strains modified to express different capsular serotypes, two representative of relatively invasive capsular serotypes (4 and 7F) and two representative of less invasive serotypes (6A and 23F), to assess capsular serotype-dependent effects on immunity. We have investigated the effects of the capsular serotype on opsonization of S. pneumoniae with the complement-derived opsonins C3b and iC3b, as well as on neutrophil phagocytosis and virulence in a mouse model of septicemia.