Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters.

Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an approximately 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram-positive bacterium.

Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis.

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.

Deletion of purE attenuates Brucella melitensis infection in mice.

We previously showed that a purE mutant (delta purE201) of Brucella melitensis 16M is attenuated for growth in cultured human monocytes (E. S. Drazek, H. H. Houng, R. M. Crawford, T. L. Hadfield, D. L. Hoover, and R. L. Warren, Infect. Immun. 63:3297-3301, 1995). To determine if this strain is attenuated in animals, we compared the growth of the delta purE201 mutant with that of strain 16M in BALB/c mice. The number of bacteria in the spleen and spleen weight peaked for both strains between 1 and 2 weeks postinfection (p.i.), though the number of delta purE201 cells was significantly less than the number of 16M cells recovered from the spleens of infected mice. During the next 6 weeks, delta purE201 was essentially eliminated from infected mice (three of five mice sterile; < 100 CFU in two of live mice at 8 weeks p.i.), whereas bacteria persisted at a high level in the spleens of 16M-infected mice (about 106 CFU per spleen). The number of bacteria in the livers and lungs of mice infected with either strain paralleled those in the spleen. Mice infected with 16M had a strong inflammatory response, developing dramatic and prolonged splenomegaly (five to eight times normal spleen weight) and producing serum interleukin-6. In contrast, mice infected with delta purE201 developed only mild, transient splenomegaly at 1 week p.i. and produced no interleukin-6 in their serum. We further characterized the host response to infection by measuring changes in immune spleen cell populations by flow cytometry. CD4- and CD8-positive lymphocytes declined by I week in both experimental groups, while MAC-1-positive cells increased. T-cell subpopulations remained low or declined further, and MAC-1 cells increased to three times normal levels during 8 weeks of infection with 16M but returned to normal by 4 weeks after infection with delta purE201. These results document infectivity and attenuation of delta purE201 and suggest that it should be further evaluated as a potential vaccine.

A noncovalent complex vaccine prepared with detoxified Escherichia coli J5 (Rc chemotype) lipopolysaccharide and Neisseria meningitidis Group B outer membrane protein produces protective antibodies against gram-negative bacteremia.

Earlier studies showed that purified IgG from sera of rabbits immunized with a boiled Escherichia coli J5 (Rc chemotype) whole cell vaccine protected neutropenic rats against gram-negative bacterial sepsis. In the present study, de-O-acylated J5 lipopolysaccharide (J5 DLPS) as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (GBOMP) elicited anti-J5 LPS antibodies in rabbits. IgG prepared from immune rabbit sera protected neutropenic rats against lethal challenge with Pseudomonas aeruginosa 12:4:4 (Fisher Devlin immunotype 6). Sixteen of 26 rats treated with the postimmune serum IgG were protected compared with none of 20 rats treated with the control rabbit serum IgG (P < .001). In vitro binding studies showed binding of anti-J5 IgG to several gram-negative bacteria. These results indicate that a subunit vaccine made of J5 DLPS as a noncovalent complex with GBOMP may protect against gram-negative bacteremia.

The importance of a lipopolysaccharide-initiated, cytokine-mediated host defense mechanism in mice against extraintestinally invasive Escherichia coli.

Extraintestinally invasive Escherichia coli (EC) that possess both a complete LPS and K1 capsule evade both complement-mediated bacteriolysis and neutrophil-mediated killing. Since C3H/HeJ mice that are hyporesponsive to LPS were uniquely susceptible to lethal infection with EC of this phenotype, we speculated there was an LPS-initiated host defense mechanism against this pathogenic phenotype. The LPS-normoresponsive C3H/HeN as well as the C3H/HeJ mice cleared these EC from the circulation within 4 h of intravenous administration. Whereas electron micrographs of the liver demonstrated these EC undergoing degeneration within the phagolysosomes of of both macrophages and Kupffer cells of C3H/HeN mice, these EC replicated within these cells of the C3H/HeJ mice. Restoration of anti-EC activity of C3H/HeJ mice occurred with activation of Kupffer cells and peritoneal macrophages in vivo with BCG and in vitro with IFN-gamma, but not with LPS. Pretreatment of C3H/HeJ mice with a combination of recombinant murine IL-1 and TNF-alpha also restored the killing of K1(+)-EC but did not enhance the killing of a K1(-)-EC mutant. These data are consistent with the hypothesis that (a) there is no intrinsic inability of C3H/HeJ phagocytes to kill EC, but (b) an LPS-initiated, cytokine-mediated host defense mechanism is required for such killing. These studies emphasize the importance of bacterial surface characteristics in the interaction with specific host defenses.

Antibody and cytokine responses in a mouse pulmonary model of Shigella flexneri serotype 2a infection.

A murine pulmonary model was used to study the mucosal immune response to Shigella flexneri serotype 2a infection. Inoculation of BALB/cJ mice with shigellae via the intranasal route resulted in bacterial invasion of bronchial and alveolar epithelia with concomitant development of acute suppurative bronchiolitis and subsequent development of lethal pneumonia. The pathology of pulmonary lesions resembled the colitis that characterizes shigellosis in humans and primates. Significant protection against a lethal dose of S. flexneri 2a was observed in mice previously infected with two sublethal doses of the homologous strain. Immunity against lethal challenge was associated with decreased bacterial invasion of the mucosal epithelium. Over the course of two sublethal challenges, which constituted primary and secondary immunizations, mice developed pulmonary and serum immunoglobulin G and A antibody recognizing both lipopolysaccharide and invasion plasmid antigens IpaB and IpaC. Immune mice and naive control mice differed in lung lavage cytokine levels following lethal challenge. Immune mice developed significantly elevated levels of pulmonary gamma interferon within 6 h of challenge, while naive control mice developed elevated levels of this cytokine later during the initial 24-h period. Both groups had elevated levels of gamma interferon during the 24- to 48-h period of infection. Both groups also had elevated levels of tumor necrosis factor alpha within 6 h of challenge, but the control mice had significantly higher levels at the 48- and 72-h time points. Elevated levels of interleukin-4 were observed only in immunized mice. This cytokine appeared within 24 h and receded between 48 and 72 h. Fluorescence-activated cell sorter analysis of lung parenchymal cells showed that both groups experienced an initial influx of monocytes, but the proportion of this cell type began to recede in immunized mice after 48 h of infection, while peak levels were maintained in the control animals. These studies suggest that elements of local B lymphocyte activity, as well as Th1 and Th2 lymphocyte activity, may contribute to the survival of immune mice after intranasal challenge with shigellae.

A murine monoclonal antibody defines a unique epitope shared by Klebsiella lipopolysaccharides.

A hybridoma secreting a monoclonal antibody (MAb) directed against Klebsiella lipopolysaccharide (LPS) was derived from spleen cells of mice immunized a smooth, nonencapsulated Klebsiella strain (Friedländer 201; serogroup O1). The MAb, called V/9-5 (immunoglobulin G2a), cross-reacted with LPS preparations produced from reference strains for the Klebsiella O serogroups O1, O2ab, O2ac, O3, O4, O5, and O12. Furthermore, the MAb reacted with LPSs from serogroup reference strains O6/O8, O9, and O11, which are regarded as being identical to O1, O2, and O4, respectively. When testing the supernatant of clinically isolated Klebsiella strains by means of an inhibition enzyme-linked immunosorbent assay, we found that 86 (92.4%) of 93 Klebsiella pneumoniae subsp. pneumoniae isolates and 24 (96.0%) of 25 K. oxytoca isolates harbored the cross-reactive epitope. By contrast, two laboratory strains of K. pneumoniae subsp. rhinoscleromatis did not react with MAb V/9-5. The MAb proved to be specific for the genus Klebsiella, since it did not react with any of a total of 73 strains belonging to other gram-negative bacterial genera. In conjunction with other LPS-specific MAbs, MAb V/9-5 might become a useful reagent for rapid identification of klebsiellae in clinical specimens. Furthermore, the epitope recognized by MAb V/9-5 might serve as a target epitope for the production of human MAbs for immunotherapeutic purposes.

Instability of expression of lipooligosaccharides and their epitopes in Neisseria gonorrhoeae.

We assessed variation in the expression of lipooligosaccharide (LOS) components and their epitopes within populations of a strain of Neisseria gonorrhoeae by using the monoclonal antibodies (MAbs) O6B4 and 3F11 and immunoenzymatic, immuno-colloidal gold electron microscopic, and sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedures. Wild-type organisms varied in binding of both MAbs. We used the intensity of immunoenzymatic colony blot color to distinguish four binding variants for each MAb: red (R), pink (P), and colorless (nonreactive [N]) and an N back to R (N-R) revertant. R to P to R and R to N to R variation occurred at frequencies of 0.2% and 0.02%, respectively. The electrophoretic LOS profiles and MAb immunoblot patterns of the R, P, and N-R variants were the same as those of the wild type. LOSs of the N variants, in contrast, were of lower Mr, bound neither 3F11 nor O6B4 MAb, and contained as their major component the 3.6-kilodalton LOS that bears the L8LOS epitope of N. meningitidis. Results of immunoelectron microscopic studies were consistent with LOS binding patterns. Large number of colloidal gold particles were deposited about both R and P variants, distally from R organisms, but proximally from P organisms. N variant organisms, like their LOS, bound neither of the MAbs. N-R variant organisms were like the wild type in that they showed much variation in the amounts of MAb they bound.

Stability of expression of Neisseria gonorrhoeae lipooligosaccharides.

We compared multiple lipooligosaccharide (LOS) extracts from individual strains of Neisseria gonorrhoeae. Each of the extracts was prepared from single mass cultures grown on solid media under similar conditions but separated by time. We found only subtle variations in the number, electrophoretic mobility, and concentration of components of the LOSs from individual strains. We found no variation in the expression of a 3.6-kilodalton LOS component that carries the L8 LOS epitope. A significant variation in the 3-deoxy-D-manno-octulosonic acid content was found among different extracts from the same strain, but this variation appeared to be unrelated to the other LOS characteristics studied.

Heterogeneity of molecular size and antigenic expression within lipooligosaccharides of individual strains of Neisseria gonorrhoeae and Neisseria meningitidis.

We determined the Mr of neisserial lipooligosaccharides (LOS) by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis, minimal loading concentrations, and Salmonella isogenic rough mutant LOS as Mr standards. Salmonella LOS were resolved into three components. The migration distance of each component was linearly related to its theoretical Mr (r = 0.99). Neisserial LOS also contained multiple components whose calculated Mr ranged from 3,200 to 7,100. The relative abundance of components and their MrS varied greatly among strains. Meningococcal LOS were composed almost exclusively of two closely migrating components; gonococcal LOS were more heterogeneous. LOS from a gonococcus selected for resistance to a Pseudomonas pyocin contained only a single component that was different from and of intermediate Mr among the three components of the parent strain. A monoclonal antibody directed against the meningococcal L8 LOS epitope was used to determine whether heterogeneity of antigen expression reflected Mr heterogeneity. Single components of the L8 meningococcal LOS and of the LOS of 3 of 19 gonococcal strains bound the monoclonal antibody. Gonococcal LOS components that expressed the L8 epitope were of a similar Mr (4,800). Smaller components of these same LOS did not express the epitope.