Virulence plasmid-borne spvB and spvC genes can replace the 90-kilobase plasmid in conferring virulence to Salmonella enterica serovar Typhimurium in subcutaneously inoculated mice.

In a mouse model of systemic infection, the spv genes carried on the Salmonella enterica serovar Typhimurium virulence plasmid increase the replication rate of salmonellae in host cells of the reticuloendothelial system, most likely within macrophages. A nonpolar deletion in the spvB gene greatly decreased virulence but could not be complemented by spvB alone. However, a low-copy-number plasmid expressing spvBC from a constitutive lacUV5 promoter did complement the spvB deletion. By examining a series of spv mutations and cloned spv sequences, we deduced that spvB and spvC could be sufficient to confer plasmid-mediated virulence to S. enterica serovar Typhimurium. The spvBC-bearing plasmid was capable of replacing all of the spv genes, as well as the entire virulence plasmid, of serovar Typhimurium for causing systemic infection in BALB/c mice after subcutaneous, but not oral, inoculation. A point mutation in the spvBC plasmid preventing translation but not transcription of spvC eliminated the ability of the plasmid to confer virulence. Therefore, it appears that both spvB and spvC encode the principal effector factors for Spv- and plasmid-mediated virulence of serovar Typhimurium.

Pathogenesis of infection by clinical and environmental strains of Vibrio vulnificus in iron-dextran-treated mice.

Vibrio vulnificus is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico. In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality. V. vulnificus-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage. In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease. All six V. vulnificus strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains. At lethal doses, all strains caused s. c. skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 10(8) CFU/g, especially in perivascular regions. These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses. In non-iron dextran-treated mice, strains required 10(5)-fold-higher inocula to cause an identical disease process as with iron dextran treatment. These results demonstrate that s.c. inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by V. vulnificus.

Construction and phenotypic evaluation of a Vibrio vulnificus vvpE mutant for elastolytic protease.

Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V. vulnificus infections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the elastase were assessed by constructing vvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.

Constitutively expressed phoP inhibits mouse-virulence of Salmonella typhimurium in an Spv-dependent manner.

In Salmonella typhimurium, the transcription of several virulence genes including spvB is regulated by the PhoP/PhoQ regulatory system. To further examine the relationship between the PhoP/PhoQ and Spv systems for virulence in mice, we examined a non-polar phoP mutation combined with different virulence plasmid genotypes for effects on virulence of S. typhimurium in the mouse model. PhoP-/Spv+ and PhoP-/Spv- mutants were not detectably recovered from the spleens of subcutaneously or orally inoculated mice. The phoP gene constitutively expressed from the lacZ promoter of a low copy number vector (phoP(C)) only partially complemented the non-polar phoP mutation for mouse-virulence in both the Spv+ and Spv- backgrounds; both PhoP(C) strains exhibited virulence equal only to a PhoP+/Spv- strain. Interestingly, in a PhoP+ background, the phoP(C) gene reduced splenic infection of the Spv+ but not Spv- salmonellae after subcutaneous or oral inoculation compared with the PhoP+ parents. Additionally, the phoP(C) gene in an Spv+ background reduced the net growth of salmonellae in macrophages in vitro; phoP(C) in an Spv- background was without effect. These data suggest that the constitutive expression of the phoP gene attenuates the virulence of S. typhimurium in mice in an Spv-dependent manner.

Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice.

Following oral inoculation of BALB/c mice, Salmonella abortusovis strain SS44 was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes compared with S. typhimurium strain SL1344, whereas splenic infections were equivalent between the two serovars. SS44 was cured of its virulence plasmid or subjected to mutagenesis of the spv genes, and the Spv(-) derivatives were tested for virulence in mice. Plasmid-cured S. abortusovis SU40 retained virulence in BALB/c mice when inoculated intraperitoneally. On the other hand, mice infected orally with SU40 had greatly reduced splenic infection compared to those infected with wild-type SS44. Similar results were obtained after Tn5 insertion mutagenesis of the spvR gene or deletion of the spvABCD locus. These results suggest that in the gut-associated lymphoid tissues S. abortusovis may replicate less than S. typhimurium and that the S. abortusovis virulence plasmid primarily affects systemic infection after oral inoculation but not after intraperitoneal administration in the mouse model.

Growth of Staphylococcus aureus in Diprivan and Intralipid: implications on the pathogenesis of infections.

BACKGROUND: The incidence and severity of infections are increased when Intralipid or Diprivan are administered to patients. Intralipid promotes infection, presumably by inhibiting the reticuloendothelial system, thereby suppressing the host's constitutive immunity, whereas Diprivan supposedly promotes infection by supporting bacterial growth and increasing the inoculating dose. This study considers whether bacterial replication alone in Intralipid and Diprivan adequately explains the increased risk of infection associated with these agents or whether other factors might also be involved. METHODS: Staphylococcus aureus was cultured in 10% Intralipid or Diprivan at clinically relevant conditions or in Intralipid containing 0.005% (w/v) sodium EDTA, a current additive, to measure growth. To determine whether Intralipid affected infection, New Zealand white rabbits were injected intravenously with S. aureus with or without Intralipid. Twenty-four hours later, bacteria in lung, liver, spleen, and kidney tissues were enumerated. RESULTS: S. aureus failed to grow in Diprivan or Intralipid containing 0.005% EDTA. Whereas S. aureus did replicate in plain Intralipid, growth was delayed until the bacteria conditioned the media. Once initiated, growth was slow at clinically relevant temperatures. The administration of Intralipid to rabbits significantly increased the recovery of staphylococci from the kidneys, P < 0.001, relative to the other tissues 24 h after an intravenous inoculation with S. aureus, compared with rabbits receiving S. aureus with no Intralipid. CONCLUSIONS: These results suggest that Diprivan, and possibly Intralipid, represent poor media for the growth of S. aureus and may promote infection through mechanisms other than increased inoculum size.

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.

Systemic infection of mice by wild-type but not Spv- Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha.

The spv genes of the virulence plasmid of Salmonella typhimurium and other nontyphoidal serovars of S. enterica are involved in systemic infection by increasing the replication rate of the bacteria in host tissues beyond the intestines. We considered the possibility that the Spv virulence function is to evade suppression by the host response to infection. To examine this possibility, gamma interferon (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) were neutralized in BALB/c mice by intraperitoneal administration of monoclonal antibodies. Neutralization of IFN-gamma and/or TNF-alpha resulted in increased splenic infection with wild-type salmonellae after oral inoculation; however, Spv- salmonellae were defective at increasing splenic infection in cytokine-depleted mice. The use of a temperature-sensitive marker plasmid, pHSG422, indicated that neutralization of IFN-gamma caused less killing of wild-type S. typhimurium, while neutralization of TNF-alpha resulted in an increased in vivo replication rate for wild-type salmonellae. These results demonstrate that the Spv virulence function is not to evade suppression of bacterial infection normally mediated by IFN-gamma or TNF-alpha.

Epidural catheter reconnection. Safe and unsafe practice.

BACKGROUND: An in vitro model of epidural catheter contamination was used to determine if disconnected catheters can be safely reconnected. METHODS: Epidural catheters were filled with brain-heart infusion (BHI) broth or preservative-free saline containing 5 micrograms/ml fentanyl. Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus (1.10(5) colony-forming units) was injected into the initial 1.1 +/- 0.24 inch (2.75 +/- 0.60 cm) of the catheters. To study the effect of bacteria settling in a vertically oriented catheter on the advancement of bacteria along the catheter, bacteria were incubated with catheters in the vertical and the horizontal positions. To determine if bacteria are swept further into a catheter when fluid in it is displaced, catheters were inclined 30 degrees and the fluid in them was allowed to drain from the distal end to various extents. Bacteria were incubated with the catheter held horizontally. After incubation, the catheters were serially sectioned, and the resulting segments were eluted with buffered saline-containing gelatin (BSG), which was collected on BHI agar plates for colony counts. This determined if a segment of the catheter remained internally sterile distal to the point of disconnection. Effectiveness of decontaminating the exterior of the catheter was also tested as follows: Catheters (n = 10) were first immersed in BSG containing 1.10(5) S, aureus, immediately immersed in betadine for 2 min, exposed to air for 3 min, cut with a sterile instrument, and reconnected to a sterile screw cap catheter connector. Reconnected catheters were perfused with 10 ml BSG for 1 hr. Collected perfusate (100 microliters) was removed for direct colony count; the remaining perfusate was mixed with an equal volume of BHI and incubated overnight. A 100 microliters aliquot of BHI-BSG mixture was sampled the next day. No bacteria were cultured from either the perfusate or BHI-BSG mixture. RESULTS: Eight hours after contamination, as long as the fluid in the catheter was static, no bacteria were detected more than 13 inches (32.5 cm) from the contaminated end of catheters filled with BHI and no more than 8 inches (20 cm) from the end of those filled with fentanyl solution. This finding was not affected by incubation of the catheter in the vertical position. Fluid displacement less than 8 inches (20 cm) had no effect on dissemination, but when fluid was displaced 13 inches (32.5 cm), bacteria were found at the end of the catheter, 35 inches (87.5 cm) away. No bacteria were recovered from the perfusate of reconnected catheters after the catheters were cleaned with betadine and cut with a sterile instrument. CONCLUSIONS: There may be an area distal to the disconnected end of an epidural catheter where its interior remains sterile for at least 8 hr. Such an area exists only when the fluid in the catheter remains static. Furthermore, the exterior of the catheter can be adequately cleaned to prevent bacteria from entering the catheter when reconnected at that point.

Use of incompatible plasmids to control expression of antigen by Salmonella typhimurium and analysis of immunogenicity in mice.

Salmonella spp. have been investigated as live vaccine vectors because they are heat stable and can elicit humoral, cellular, and secretory immune responses. However, the expression of some foreign antigens is toxic to bacterial vectors. We therefore studied an approach for the controlled expression of antigen in Salmonella typhimurium wherein the antigen is not expressed in vitro but is expressed in vivo. A model antigen, beta-galactosidase, was expressed from the trc promoter on one plasmid, while repression was achieved by Lacl expressed in trans from a second plasmid. The second repressor plasmid was incompatible with the expression plasmid encoding beta-galactosidase. Loss by segregation of the repressor plasmid in vitro correlated with increased expression of beta-galactosidase. Oral inoculation of mice with salmonellae containing both plasmids induced serum IgG but not nasal, salivary, or biliary IgA antibody to beta-galactosidase. Serum IgG as well as biliary IgA anti-S. typhimurium antibody, but not salivary or nasal IgA, were also detected. This salmonella vector system for the controlled expression of recombinant antigens may be of value for inducing systemic but not mucosal immunity to antigens that are toxic to bacterial vectors.

Molecular analysis of spv virulence genes of the Salmonella virulence plasmids.

Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models. This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory. A common nomenclature has been devised for the five genes, i.e. spv for salmonella plasmid virulence. The first gene, spvR, encodes a positive activator for the following four genes, spvABCD. DNA sequence analysis of the spv genes from Salmonella typhimurium, Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences. The spv genes are induced at stationary phase and in carbon-poor media, and optimal expression is dependent on the katF locus. The virulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

The Salmonella typhimurium virulence plasmid increases the growth rate of salmonellae in mice.

The virulence plasmids of Salmonella typhimurium and other invasive Salmonella serovars have long been associated with the ability of these bacteria to cause systemic infection beyond the intestines in orally inoculated animals. Genetic analysis of virulence genes on the high-molecular-weight plasmids has revealed that no more than five genes spanning a 6.2-kb region are sufficient to replace the entire plasmid for conferring virulence. However, the exact virulence function(s) encoded by these genes has not been elucidated. In this report, we measured the possible effect of the virulence plasmid on the growth rate of S. typhimurium in mice by two complementary procedures. The first procedure used segregation of a temperature-sensitive plasmid in vivo to provide a measure of bacterial divisions and the number of recovered marker plasmid-containing salmonellae as a measure of killing. In the second procedure, aroA deletions were transduced into virulence plasmid-containing and plasmid-cured S. typhimurium. Since AroA- salmonellae are inhibited for growth in vivo, if the virulence plasmid affected only growth rate, no difference in the recoveries of the paired AroA- strains would be seen. Virulence plasmid-containing S. typhimurium segregated the marker plasmid more rapidly than did the virulence plasmid-cured strain, and AroA- derivatives of both strains were recovered equally from mice. Therefore, the S. typhimurium virulence plasmid increased growth rate but had no detectable effect on killing or bacterial movement into deep tissues. To examine whether the plasmid accomplished this function by affecting the intracellular/extracellular location of bacteria, orally infected mice were injected with gentamicin to kill the extracellular bacteria. Wild-type and plasmid-cured S. typhimurium strains were equally resistant to gentamicin in vivo and hence most likely located intracellularly to equal degrees. When wild-type and plasmid-cured S. typhimurium strains were sequestered within peritoneal chambers in mice, the resulting extracellular growth was equal. Therefore, the virulence plasmid increases the growth rate of S. typhimurium in mice, probably within mouse cells.

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid.

The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.

The Salmonella typhimurium virulence plasmid encodes a positive regulator of a plasmid-encoded virulence gene.

The 90-kb virulence plasmid of Salmonella typhimurium is necessary for invasion beyond the Peyer's patches to the mesenteric lymph nodes and spleens of orally inoculated mice. Two Tn5 insertions located on the left side of a previously identified 14-kb virulence region (P. A. Gulig and R. Curtiss III, Infect. Immun. 58:3262-3271, 1988) and mapping 272 bp from each other exhibited opposite effects on splenic infection of mice after oral inoculation. spvR23::Tn5 decreased splenic infection by 1,000-fold, whereas a spv-14::Tn5 mutant outcompeted wild-type S. typhimurium for splenic infection by 27-fold in mice fed mixtures of mutated and wild-type S. typhimurium. spvR23::Tn5 was complemented by a virulence plasmid subclone with an insert sequence encoding only an 891-bp open reading frame specifying a 33,000-molecular-weight protein. The amino acid sequence of this open reading frame had significant homology to members of the LysR family of positive regulatory proteins; thus, the gene was named spvR (salmonella plasmid virulence). To examine the possible regulatory effects of spvR on other virulence genes, we constructed a lacZ operon fusion in a downstream virulence gene, spvB. When spvR subcloned behind the lac promoter was provided on a separate plasmid in trans to the spvB-lacZ operon fusion, transcription of spvB increased 15-fold. spv-14::Tn5, which conferred a competitive advantage to S. typhimurium, increased the expression of a spvR-lacZ operon fusion in cis. spvR is therefore a positive regulator of spvB and an essential virulence gene of S. typhimurium. As opposed to having spvR subcloned behind the lac promoter, the wild-type spvR gene present on the virulence plasmid did not function to positively regulate spvB-lacZ in trans when salmonellae were grown to the log phase in L broth, suggesting that this regulatory system is activated in vivo during infection.

Genetic and DNA sequence analysis of the Salmonella typhimurium virulence plasmid gene encoding the 28,000-molecular-weight protein.

We have confirmed that the 28,000-molecular-weight (28K) protein encoded by the virA gene of the 90-kilobase Salmonella typhimurium virulence plasmid is a virulence factor. It was previously shown that a Tn5 insertion, vir-22::Tn5, located in the virulence plasmid greatly attenuated virulence for mice and inhibited the production of a 28K protein (P.A. Gulig and R. Curtiss III, Infect. Immun. 56:3262-3271, 1988). Plasmid pYA426 fully complemented vir-22::Tn5 to virulence by increasing splenic infection after oral inoculation and encoded the 28K protein. To identify the virulence gene(s) of pYA426 mutated by vir-22::Tn5, we constructed nested deletions in pYA426 and examined deletion derivatives for their abilities to complement vir-22::Tn5. Only derivatives still producing the 28K protein complemented vir-22::Tn5. Furthermore, the smallest complementing derivative encoded only the 28K protein, as determined by DNA sequence analysis. Therefore, the 28K protein is sufficient for complementation of the attenuating mutation vir-22::Tn5 and must be the virulence factor inhibited by the insertion. We determined the nucleotide sequence of the 1.2-kilobase BamHI-EcoRI fragment encoding the 28K protein and identified the structural gene, virA. A 723-base-pair open reading frame which encodes a peptide with a molecular weight of 27,572 was found.

Virulence plasmids of Salmonella typhimurium and other salmonellae.

Related high molecular weight plasmids of several serotypes and species of Salmonella have been associated with virulence in a variety of animal models of infection. The primary virulence plasmid phenotype is in the ability of salmonellae to spread beyond the initial site of infection, the intestines. The mechanism of this plasmid-mediated invasive infection has not been identified, but may be a complex interaction in the host-pathogen relationship. A common region of the salmonella plasmids has been associated with virulence, and specific virulence genes and their products are now being identified; however, much is yet to be accomplished in this field. The combined analysis of pathogenesis and genetics associated with the salmonella virulence plasmids may identify new systems of bacterial virulence and the genetic basis for this virulence.

Hybridization studies with a DNA probe derived from the virulence region of the 60 Mdal plasmid of Salmonella typhimurium.

Plasmid DNA of 68 strains of Salmonella that belonged to 18 serovars and exhibited 48 different plasmid profiles was examined for hybridization with a 32P-labelled DNA probe which consisted of a 3750 base pairs (bp) HindIII-HindIII fragment derived from the virulence region of the 60 megadalton (Mdal) plasmid of Salmonella typhimurium. The 32 Mdal plasmid of S. cholerae-suis, the 50 Mdal plasmid of S. dublin, the 36 Mdal plasmid of S. enteritidis, the 60 Mdal plasmid of S. gallinarum, the 60 Mdal plasmid of S. pullorum, and the 60 Mdal plasmid of S. typhimurium, plasmids that have been associated with virulence, all hybridized with the probe. Digestion of plasmid DNA of these strains with PvuII and hybridization with the probe revealed that the plasmids of strains of all six serovars contained fragments of approximately 2520 and 1520 bp that hybridized with the probe. Similarly, hybridization with BglI digests of DNA of the virulence-associated plasmids of strains of these six serovars showed that all six plasmids contained a fragment of approximately 3690 bp that hybridized with the probe. No other plasmids of these strains nor any plasmids of 12 other Salmonella serovars hybridized with the probe. Chromosomal DNA did not hybridize with the probe. The 60 Mdal plasmids of S. gallinarum and S. pullorum showed similar digestion patterns with restriction endonucleases BglI, BglII and PvuII.

Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b.

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.

Selective delivery of antigens by recombinant bacteria.

The means to attenuate Salmonella and to endow such avirulent strains with the ability to express colonization and virulence antigens from other pathogens has achieved considerable progress during the past several years. One can therefore begin to design and construct strains with specificity to a given animal host and to express in a defined way specific colonization and virulence antigens in a manner to stimulate long-lasting immunity to the Salmonella and to the pathogen supplying the genetic information for the colonization and virulence antigens. Since most pathogens colonize on or invade through mucosal surfaces, the use of recombinant bivalent Salmonella vaccine strains to stimulate a mucosal immune response would induce the development of a first line of defense against a diversity of pathogens. Mucosal immunity should therefore reduce contagious spread of many pathogens since the dose to overcome the mucosal immune barrier would be increased to result in a diminished likelihood of infection. The fact that the recombinant Salmonella vaccine strains also induce humoral and cellular immune responses justifies their use for induction of long-lasting immunity. Although considerable progress has been made in targeting antigens to the GALT by use of avirulent Salmonella, a similar strategy for delivery of antigens to the BALT has yet to be discovered and developed. In addition to constituting a system for induction of immunity against a diversity of pathogens, the recombinant avirulent Salmonella system should provide a means to explore parameters of the mucosal immune response. This would include investigation of the location and duration of memory, the age dependence of induction of mucosal immunity, and the means for the possible induction of oral tolerance with regard to either the mucosal or humoral response to an antigen expressed by the recombinant Salmonella. It is also possible to contemplate using the avirulent Salmonella to target expression of various modulators of the immune system such as interleukin-2 and interferon-gamma to the GALT and thus further enhance the immune response. Lastly, one can introduce into avirulent Salmonella strains genes for putative colonization antigens in order to investigate whether induction of an immune response against the putative colonization antigen does or does not interfere with infection. This system, therefore, permits another means to analyze the relative importance of various bacterial surface attributes in conferring pathogenicity to the microbe.