A Live Recombinant Avirulent Oral Salmonella Vaccine Expressing Pneumococcal Surface Protein A Induces Protective Responses against Streptococcus pneumoniae

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Δcya Δcrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Δasd mutation was complemented by a plasmid vector possessing the asd⁺ gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd⁺ vector. After oral immunization, the recombinant Salmonella-PspA vaccine strain colonized the Peyer’s patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonella strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.     

Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies

The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 10⁷ CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.     

Can Results Obtained with Commercially Available MicroScan Microdilution Panels Serve as an Indicator of β-Lactamase Production among Escherichia coli and Klebsiella Isolates with Hidden Resistance to ExpandedSpectrum Cephalosporins and Aztreonam?

Among clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, there is an ever-increasing prevalence of β-lactamases that may confer resistance to newer β-lactam antibiotics that is not detectable by conventional procedures. Therefore, 75 isolates of these species producing well-characterized β-lactamases were studied using two MicroScan conventional microdilution panels, Gram Negative Urine MIC 7 (NU7) and Gram Negative MIC Plus 2 (N+2), to determine if results could be utilized to provide an accurate indication of β-lactamase production in the absence of frank resistance to expanded-spectrum cephalosporins and aztreonam. The enzymes studied included Bush groups 1 (AmpC), 2b (TEM-1, TEM-2, and SHV-1), 2be (extended spectrum β-lactamases [ESBLs] and K1), and 2br, alone and in various combinations. In tests with E. coli and K. pneumoniae and the NU7 panel, cefpodoxime MICs of ≥2 μg/ml were obtained only for isolates producing ESBLs or AmpC β-lactamases. Cefoxitin MICs of >16 μg/ml were obtained for all strains producing AmpC β-lactamase and only 1 of 33 strains producing ESBLs. For the N+2 panel, ceftazidime MICs of ≥4 μg/ml correctly identified 90% of ESBL producers and 100% of AmpC producers among isolates of E. coli and K. pneumoniae. Cefotetan MICs of ≥ 8 μg/ml were obtained for seven of eight producers of AmpC β-lactamase and no ESBL producers. For tests performed with either panel and isolates of K. oxytoca, MICs of ceftazidime, cefotaxime, and ceftizoxime were elevated for strains producing ESBLs, while ceftriaxone and aztreonam MICs separated low-level K1 from high-level K1 producers within this species. These results suggest that microdilution panels can be used by clinical laboratories as an indicator of certain β-lactamases that may produce hidden but clinically significant resistance among isolates of E. coli, K. pneumoniae, and K. oxytoca. Although it may not always be possible to differentiate between strains that produce ESBLs and those that produce AmpC, this differentiation is not critical since therapeutic options for patients infected with such organisms are similarly limited.     

Surface Antigen Exposure by Bismuth Dimercaprol Suppression of Klebsiella pneumoniae Capsular Polysaccharide

The bacterial capsule is an important virulence determinant in animal and plant disease. Bacterial capsule and slime can be inhibited by bismuth compounds, especially when complexed with lipophilic thiol chelators. Bismuth dimercaprol (BisBAL) at 1 ppm of Bi³⁺ repressed Klebsiella pneumoniae capsule expression in defined medium by nearly 90%, which exposed subsurface structures. The phagocytic index for BisBAL-treated bacteria increased from <10 to 360 bacteria per 100 neutrophils in the presence of complement and anticapsular or anti-O antigen antiserum. BisBAL treatment also enhanced the reactivity of monoclonal antibodies (MAbs) specific for the O1-antigen lipopolysaccharide (LPS) or the LPS core in a dose-dependent manner as indicated by the results of enzyme-linked immunosorbent assays. When anti-O1 MAb was used, the reactivity increased significantly for fully encapsulated O1:K1 or O1:K2 cells but not for O1:K⁻ cells. Deposition of C3b also increased significantly for BisBAL-treated O1:K1 or O1:K2 cells but not for O1:K⁻ cells. Survival of a serum-sensitive strain was <0.1% when nonimmune human serum absorbed with O1:K1 cells was used and 107% when BisBAL-treated cells were used for absorption. Outer membrane proteins were also more accessible on the surface of K. pneumoniae after BisBAL treatment. Thus, at subinhibitory levels, BisBAL inhibited capsule expression, which promoted phagocytosis, enhanced the reactivity of specific antibodies for LPS O antigen, LPS core epitopes, or outer-membrane proteins, and enhanced complement interaction with encapsulated K. pneumoniae. By unmasking bacterial surface structures and enhancing the immune system reactivity to bacteria, bismuth thiols may prove useful as adjuncts for vaccination.     

Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1β (IL-1β) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1β secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1β secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1β secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1β secretion through the NLRP3 inflammasome.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

A sopB deletion mutation enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines

SopB is a virulence factor of Salmonella encoded by SPI-5. Salmonella sopB deletion mutants are impaired in their ability to cause local inflammatory responses and fluid secretion into the intestinal lumen and also can enhance the immunogenicity of a vectored antigen. In this study, we evaluated the effects on immunogenicity and the efficacy of a sopB deletion mutation on two Salmonella enterica serovar Typhimurium vaccine strains with different attenuating mutations expressing a highly antigenic α-helical region of the Streptococcus pneumoniae surface protein PspA from an Asd⁺-balanced lethal plasmid. After oral administration to mice, the two pairs of strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens. The levels of antigen-specific serum immunoglobulin G (IgG) and mucosal IgA were higher in mice immunized with sopB mutants. Enzyme-linked immunospot assay results indicated that the spleen cells from mice immunized with a sopB mutant showed higher interleukin-4 and gamma interferon secretion levels than did the mice immunized with the isogenic sopB⁺ strain. The sopB mutants also induced higher numbers of CD4⁺ CD44^hi CD62L^hi and CD8⁺ CD44^hi CD62L^hi central memory T cells. Eight weeks after primary oral immunization, mice were challenged with 100 50% lethal doses of virulent S. pneumoniae WU2. Immunization with either of the sopB mutant strains led to increased levels of protection compared to that with the isogenic sopB⁺ parent. Together, these results demonstrate that the deletion of sopB leads to an overall enhancement of the immunogenicity and efficacy of recombinant attenuated Salmonella vaccine strains.     

A k2A-positive Klebsiella pneumoniae causes liver and brain abscess in a Saint Kitt's man

Klebsiella pneumoniae isolated in community-acquired pneumonia is increasingly found in primary pyogenic liver abscesses. The presence of magA in K. pneumoniae has been implicated in hypermucoviscosity and virulence of liver abscess isolates. The K2 serotype has also been strongly associated with hypervirulence. We report the isolation of non-magA, K2 K. pneumoniae strain from a liver abscess of a Saint Kitt''s man who survived the invasive syndrome.     

Relative importance of T-cell subsets in monocytotropic ehrlichiosis: a novel effector mechanism involved in Ehrlichia-induced immunopathology in murine ehrlichiosis

Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4⁺ Th1 cells, the expansion of tumor necrosis factor alpha (TNF-α)-producing CD8⁺ T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-α. Here, we investigated the role of CD4⁺ and CD8⁺ T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (10³ and 10⁵ bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4⁺ T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8⁺ T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8⁺ T cells abrogated TNF-α and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4⁺ T-cell numbers, and increased the frequency of Ehrlichia-specific CD4⁺ Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4⁺ T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4⁺ and CD8⁺ T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8⁺ T cells mediate Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4⁺ T-cell apoptosis.     

The Potassium Transporter Trk and External Potassium Modulate Salmonella enterica Protein Secretion and Virulence

Potassium (K⁺) is the most abundant intracellular cation and is essential for many physiological functions of all living organisms; however, its role in the pathogenesis of human pathogens is not well understood. In this study, we characterized the functions of the bacterial Trk K⁺ transport system and external K⁺ in the pathogenesis of Salmonella enterica, a major food-borne bacterial pathogen. Here we report that Trk is important for Salmonella to invade and grow inside epithelial cells. It is also necessary for the full virulence of Salmonella in an animal infection model. Analysis of proteins of Salmonella indicated that Trk is involved in the expression and secretion of effector proteins of the type III secretion system (TTSS) encoded by Salmonella pathogenicity island 1 (SPI1) that were previously shown to be necessary for Salmonella invasion. In addition to the role of the Trk transporter in the pathogenesis of Salmonella, we discovered that external K⁺ modulates the pathogenic properties of Salmonella by increasing the expression and secretion of effector proteins of the SPI1-encoded TTSS and by enhancing epithelial cell invasion. Our studies demonstrated that K⁺ is actively involved in the pathogenesis of Salmonella and indicated that Salmonella may take advantage of the high K⁺ content inside host cells and in the intestinal fluid during diarrhea to become more virulent.     

Structure and gene cluster of the capsular polysaccharide of multidrug resistant carbapenemase OXA-48-producing Klebsiella pneumoniae strain KPB536 of the genetic line ST147

The Gram-negative opportunistic pathogen Klebsiella pneumoniae is a significant cause of community-acquired and healthcare-associated infections for which multidrug resistance is a concern worldwide. A major virulence determinant of K. pneumoniae is a polysaccharide capsule (CPS) which forms a barrier around the bacterial cell wall, providing protection from environmental pressures and immune responses of eukaryotic organisms. More than 70 chemical capsule structures of serologically typeable K. pneumoniae strains are known. However, there are little data on the CPS structure and cps gene cluster organization of clinical multidrug resistant K. pneumoniae strains.Our investigation of multidrug resistant carbapenemase OXA-48-producing K. pneumoniae strain KPB536 identified a capsular type that was structurally similar to K. pneumoniae K10 but different from any K. pneumoniae CPS reported so far. The content and organization of the cps gene cluster in K. pneumoniae KPB536 also was determined. The catalytic functions of glycosyltransferases coded by the cps_KPB536 gene cluster were assigned by comparison with those responsible for the synthesis of glycoside linkages in the CPSs of K. pneumoniae types K10 and K61.     

Oral Immunization with a Salmonella typhimurium Vaccine Vector Expressing Recombinant Enterotoxigenic Escherichia coli K99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5⁺ ETEC infections, a balanced lethal asd⁺ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd⁺. Introduction of this plasmid into an attenuated Salmonella typhimurium Δaro Δasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.     

Risk factor analysis of invasive liver abscess caused by the K1 serotype <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The increasing prevalence of Klebsiella pneumoniae liver abscess in Asian countries is attributable to virulent strains of the K1 serotype. We investigated the risk factors for the K1 serotype K. pneumoniae liver abscess. A case-control study was performed using the database of a nationwide study of liver abscess in Korea. Multivariate logistic regression analysis was performed for 78 cases of the K1 serotype K. pneumoniae liver abscess and 81 controls with non-Klebsiella. Diabetes mellitus was the significant risk factor (OR 2.13; 95% CI 1.026 ~ 4.428; P = 0.042) for the K1 serotype K. pneumoniae liver abscess. Biliary disorders had a strong negative association (OR 0.18; 95% CI 0.078 ~ 0.410; P < 0.001). This study suggests that diabetes mellitus is a more significant risk factor for the K1 serotype K. pneumoniae liver abscess than for the non-Klebsiella liver abscess. Contributing members of the Korean Study Group for Liver Abscess are listed in the Acknowledgments.     

Aerobactin Mediates Virulence and Accounts for Increased Siderophore Production under Iron-Limiting Conditions by Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae

Hypervirulent (hypermucoviscous) Klebsiella pneumoniae (hvKP) strains are an emerging variant of “classical” K. pneumoniae (cKP) that cause organ and life-threatening infection in healthy individuals. An understanding of hvKP-specific virulence mechanisms that enabled evolution from cKP is limited. Observations by our group and previously published molecular epidemiologic data led us to hypothesize that hvKP strains produced more siderophores than cKP strains and that this trait enhanced hvKP virulence. Quantitative analysis of 12 hvKP strains in iron-poor minimal medium or human ascites fluid showed a significant and distinguishing 6- to 10-fold increase in siderophore production compared to that for 14 cKP strains. Surprisingly, high-pressure liquid chromatography (HPLC)-mass spectrometry and characterization of the hvKP strains hvKP1, A1142, and A1365 and their isogenic aerobactin-deficient (ΔiucA) derivatives established that aerobactin accounted for the overwhelming majority of increased siderophore production and that this was not due to gene copy number. Further, aerobactin was the primary factor in conditioned medium that enhanced the growth/survival of hvKP1 in human ascites fluid. Importantly the ex vivo growth/survival of hvKP1 ΔiucA was significantly less than that of hvKP1 in human ascites fluid, and the survival of outbred CD1 mice challenged subcutaneously or intraperitoneally with hvKP1 was significantly less than that of mice challenged with hvKP1 ΔiucA. The lowest subcutaneous and intraperitoneal challenge inocula of 3 × 10² and 3.2 × 10¹ CFU, respectively, resulted in 100% mortality, demonstrating the virulence of hvKP1 and its ability to cause infection at a low dose. These data strongly support that aerobactin accounts for increased siderophore production in hvKP compared to cKP (a potential defining trait) and is an important virulence factor.     

Listeria monocytogenes desensitizes immune cells to subsequent Ca2+ signaling via listeriolysin O-induced depletion of intracellular Ca2+ stores

Listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, is a prototype of the cholesterol-dependent cytolysins (CDCs) secreted by several pathogenic and nonpathogenic gram-positive bacteria. In addition to mediating the escape of the bacterium into the cytosol, this toxin is generally believed to be a central player in host-pathogen interactions during L. monocytogenes infection. LLO triggers the influx of Ca²⁺ into host cells as well as the release of Ca²⁺ from intracellular stores. Thus, many of the cellular responses induced by LLO are related to calcium signaling. Interestingly, in this study, we report that prolonged exposure to LLO desensitizes cells to Ca²⁺ mobilization upon subsequent stimulations with LLO. Cells preexposed to LLO-positive L. monocytogenes but not to the LLO-deficient Δhly mutant were found to be highly refractory to Ca²⁺ induction in response to receptor-mediated stimulation. Such cells also exhibited diminished Ca²⁺ signals in response to stimulation with LLO and thapsigargin. The presented results suggest that this phenomenon is due to the depletion of intracellular Ca²⁺ stores. The ability of LLO to desensitize immune cells provides a significant hint about the possible role played by CDCs in the evasion of the immune system by bacterial pathogens.     

Evidence for clonal dissemination of the serotype K1 Klebsiella pneumoniae strain causing invasive liver abscesses in Korea

Seventy-three liver abscess isolates of serotype K1 Klebsiella pneumoniae from a nationwide collection in Korea were genotypically characterized using pulsed-field gel electrophoresis and multilocus sequence typing. We found that serotype K1 K. pneumoniae strains that caused liver abscesses in Korea were genotypically related and that most were sequence type 23.     

Streptococcus pyogenes Serotype M1 Encodes Multiple Pathways for Entry into Human Epithelial Cells

The ability of a serotype M1 strain of Streptococcus pyogenes to efficiently invade A549 human lung epithelial cells was previously shown to be dependent on bacterial exposure to human or bovine serum proteins or synthetic peptides containing the sequence RGD. In this study, stimulation by invasion agonists was determined to be dependent on expression of the streptococcal cell surface protein, M1. Fetal bovine serum (FBS), fibronectin (Fn), the extracellular matrix protein laminin (Lm), and RGD-containing peptides were tested for their abilities to promote epithelial cell invasion and adherence by isogenic M1⁺ and M1⁻ strains of S. pyogenes. In the absence of an agonist, invasion and adherence were comparable for the two bacterial strains. FBS, Fn, and Lm stimulated invasion of the M1⁺ strain as much as 70-fold but failed to significantly affect invasion by the M1⁻ mutant. Adherence of the wild-type strain was stimulated by these same agonists. Epithelial cell adherence by the M1⁻ strain, however, was unaffected by the presence of Fn or Lm. Several RGD-containing peptides were found to promote invasion independently of M1 expression. Binding of ¹²⁵I-Fn was reduced 88% by the M1⁻ mutation and Fn was found to bind purified M1 protein, suggesting that Fn mediates invasion by direct binding to M1. To determine if host integrins might be involved in internalization of streptococci, several anti-integrin monoclonal antibodies (MAbs) were tested for their abilities to inhibit invasion. Antibody directed against integrin β1 inhibited FBS-, Fn-, and Lm-mediated invasion but did not abrogate RGD-peptide-stimulated invasion. MAb directed against the epithelial cell Fn receptor, integrin α5β1, inhibited Fn and FBS-mediated invasion but did not specifically inhibit Lm-mediated invasion. These results indicate that S. pyogenes has evolved multiple mechanisms for invasion of eukaryotic cells, at least two of which involve interactions between M1 protein, host integrins, and integrin ligands.     

Expression of Mucin-Type Glycoprotein K88 Receptors Strongly Correlates with Piglet Susceptibility to K88+ Enterotoxigenic Escherichia coli, but Adhesion of This Bacterium to Brush Borders Does Not

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88⁺ ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab⁺ or K88ac⁺ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab⁺ and K88ac⁺ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88⁺ ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88⁺ ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab⁺ and K88ac⁺ E. coli or a correlate for expression for such a receptor.     

Group B Streptococcus and Streptococcus suis Capsular Polysaccharides Induce Chemokine Production by Dendritic Cells via Toll-Like Receptor 2- and MyD88-Dependent and -Independent Pathways

Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1β (IL-1β), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.     

Analysis of Host Cells Associated with the Spv-Mediated Increased Intracellular Growth Rate of Salmonella typhimurium in Mice

The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv⁺ or Spv⁻ S. typhimurium strains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv⁺ versus Spv⁻ S. typhimurium was not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv⁺ and Spv⁻ salmonellae. To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv⁺ and Spv⁻ S. typhimurium strains. InvA⁻ Spv⁺ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA⁻ Spv⁻ salmonellae were only slightly attenuated relative to InvA⁺ Spv⁻ salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv⁻ S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.