Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium.

Salmonella typhimurium-infected macrophages were examined by electron microscopy to determine whether intracellular survival of S. typhimurium is associated with failure of bacteria containing phagosomes to fuse with secondary lysosomes. S. typhimurium 14028 actively inhibited phagosome-lysosome fusion and appeared to preferentially divide within unfused phagocytic vesicles. In comparison with Escherichia coli, S. typhimurium inhibited phagosome-lysosome fusion in peritoneal macrophages, J774 macrophages, and bone marrow-derived macrophages from both BALB/c (itys) and SWR/J (ityr) mice. The mechanism responsible for Salmonella inhibition of phagosome-lysosome fusion is unknown but requires viable salmonellae, is not blocked by opsonization with fresh normal mouse serum, and is not due to lipopolysaccharide. Inhibition of phagosome-lysosome fusion may play a critical role in survival of salmonellae within macrophages and in virulence.     

Inhibition of IFN-gamma-stimulated proinflammatory cytokines by vasoactive intestinal peptide (VIP) correlates with increased survival of Salmonella enterica serovar typhimurium phoP in murine macrophages.

Vasoactive intestinal peptide (VIP)is a novel Th2 cytokine that has been shown previously to rescue rats and mice from the lethal effect of bacterial lipopolysaccharide (LPS). We report that VIP inhibited production of the proinflammatory cytokines, tumor necrosis factor-alpha(TNF-alpha)and interleukin-1beta (IL-1beta), at the mRNA level and that the inhibitory effect of VIP was maintained when macrophages were cocultured with an immunostimulatory concentration of interferon-gamma (IFN-gamma)(100 U/ml). The concentration of VIP that had optimal inhibitory effect was (1010) M. Furthermore, VIP prevented macrophage killing of a phoP mutant of Salmonella enterica serovar typhimurium, which is usually attenuated for virulence as a result of its inability to survive inside macrophages. However, although the effect of VIP on inducible nitric oxide synthase (iNOS) was less clear, N-monoethyl arginine (NEMA)(an iNOS inhibitor)did not rescue S. typhimurium from IFN- gamma-induced death, in accordance with previous reports that suggest that iNOS is not an important Salmonella killing pathway in macrophages within the first 24 h. VIP is a potent inhibitor of inflammatory pathways that lead to significant pathologic conditions. However, it increases survival of the normally avirulent phoP mutant and is able to inhibit IFN-gamma-stimulated killing of wild-type S. typhimurium in murine macrophages. Thus, VIP inhibits the proinflammatory type 1 response, thus favoring Salmonella survival.     

Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium.

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.     

Study of the role of the htrB gene in Salmonella typhimurium virulence.

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.     

Interaction of Nocardia asteroides with Rabbit Alveolar Macrophages: Association of Virulence, Viability, Ultrastructural Damage, and Phagosome-Lysosome Fusion

In vitro-maintained rabbit alveolar macrophages were infected with three strains of Nocardia asteroides. It was found that N. asteroides GUH-2 was resistant to macrophage killing, while N. asteroides 14759 was intermediate in resistance, and N. asteroides 10905 had little resistance to killing by macrophages. These observations correlated well with the data on relative virulence previously determined in mice. To establish the intracellular events leading to these differences, we determined the occurrence of phagosome-lysosome fusion in infected macrophages by both electron and fluorescent microscopic methods. It was found that the virulent strain GUH-2 inhibited phagosome-lysosome fusion; the intermediately virulent strain, 14759, partially inhibited fusion; and the less-virulent strain, 10905, was unable to inhibit fusion. In addition, electron microscopy of infected macrophages demonstrated that cells of the virulent strain, GUH-2, were not damaged, and only some of the cells of the intermediately virulent strain, 14759, were damaged, while most of the cells of the less virulent strain, 10905, exhibited considerable cellular destruction. These data indicated a direct correlation between the virulence of these organisms and their resistance to killing by alveolar macrophages, their lack of macrophage-induced ultrastructural damage, and their ability to inhibit phagosome-lysosome fusion. Thus, it appears that inhibition of phagosome-lysosome fusion in alveolar macrophages may be one of the mechanisms of pathogenicity of virulent strains of N. asteroides.     

Innate resistance of mice to Salmonella typhi infection.

The basis for the natural resistance of mice to Salmonella typhi was examined. In contrast to Salmonella typhimurium, the virulence of S. typhi for mice was independent of the mouse strain and was not affected by inactivation of murine macrophages with silica. However, mice were more susceptible to S. typhi when given iron alone or iron and an iron chelator. The results suggest that the failure of S. typhi to undergo net growth in murine tissues reflects an inability of the bacterium to multiply rather than rapid killing by resident macrophages.     

The full expression of the ity phenotype in ityr mice requires C3 activation by Salmonella lipopolysaccharide.

Our previous study has shown that the rapid and sufficient activation of complement by Salmonella lipopolysaccharide occurs in genetically resistant (Ityr) A/J mice. To assess whether the level of complement activation by a virulent strain of Salmonella typhimurium regulates the level of murine natural resistance, we compared levels of serum complement activation by S. typhimurium and kinetics of serum-opsonized S. typhimurium grown in macrophages using several strains of resistant (Ityr) and susceptible (Itys) mice. Itys macrophages killed intracellular S. typhimurium to the same extent as did Ityr macrophages when the pathogen was opsonized with Ityr serum. Opsonization of S. typhimurium with Itys serum reduced intracellular killing activity in Ityr macrophages to the same level as seen with Itys macrophages. Incubation of S. typhimurium with 25% Mg2+ EGTA (5 mm MgCl2-3 mm ethylene glycol-bis (beta-aminotheyl either)-N,N,N',N'-tetraacetic acid)-chelated Ityr serum resulted in higher levels of C3 deposition onto the surface of this bacteria, C3b generation and also C3 consumption, compared with that with Mg2+ EGTA-chelated Itys serum. Opsonization of S. typhimurium with A/J serum prior to infection increased early resistance in Itys mice. Infection with a virulent strain of S. typhimurium induced the expression of interleukin-10 (IL-10) mRNA at higher levels in C57BL/6 mice than in A/J mice. However, opsonization of S. typhimurium with A/J serum decreased bacterial growth in the spleen of C57BL/6 mice to the same level as observed for A/J mice in association with decreased expression levels of IL-10 mRNA. Moreover, administration of anti-C3 antibodies reduced the resistance of A/J mice in association with a decrease in serum levels of C3. These results indicate that the high level of complement activation via the alternative pathway in Ityr serum by a virulent strain of S. typhimurium reduces the virulence of this pathogen, which may contribute to the full expression of Ity phenotype in Ityr mice.     

A possible mechanism for host-specific pathogenesis ofSalmonellaserovars

We have identified the complement receptors on human and murine macrophages involved in the recognition ofSalmonellaserovars, and investigated their relevance to the intracellular survival.S. typhiwas capable of surviving within human monocyte-derived macrophages, whereasS. typhimuriumwas not. Conversely,S. typhimurium, but notS. typhi, resisted intracellular killing by murine macrophages, demonstrating that the intracellular survival ofSalmonellaserovars is host-dependent. In the presence of serum opsonin, human monocyte-derived macrophages recognizedS. typhiandS. typhimuriumvia complement receptor type 1 (CR1) and type 3 (CR3), respectively. In contrast, murine macrophages recognizedS. typhiandS. typhimuriumvia CR3 and CR1, respectively. These findings demonstrate that the intracellular fate ofSalmonellaserovars following phagocytosis may depend on the type of complement receptors involved in their recognition, in that CR1-mediated recognition is closely correlated to subsequent intracellular survival. The Tn5 insertion mutant ofS. typhimuriumwhich lacks the ability to interact with CR1 was sensitive to intracellular killing by murine macrophagesin vitro, and was much less virulent to micein vivo, confirming the relevance of CR1-mediated bacterial recognition to the pathogenicity ofS. typhimuriumfor mice. These results suggest that selective recognition ofSalmonellaserovars through CR1 may lead to their subsequent intracellular survival, and is responsible for the host-specific pathogenesis ofSalmonellaserovars.     

Capacity of recombinant gamma interferon to activate macrophages for Salmonella-killing activity.

The ability of recombinant gamma interferon (rIFN-gamma) to activate macrophages for Salmonella-killing activity was kinetically examined in relation to phagosome-lysosome fusion and H2O2 generation. Resident peritoneal macrophages of BALB/c mice incubated with 10(2) to 10(3) U of rIFN-gamma per ml for 12 h exhibited enhanced bactericidal activity against Salmonella typhimurium, although H2O2 generation was unaltered. In contrast, macrophages incubated with equal doses of rIFN-gamma for 48 h showed both an enhanced Salmonella-killing activity and an increased generation of H2O2. To evaluate Salmonella-killing activities of macrophages, intracellular bacteria were assayed at 0, 2, and 8 h after infection. During the initial 2 h of infection, 12-h-activated macrophages, as well as the unstimulated control macrophages, showed a decline in bacterial population at the same rate. Over the next 6 h of infection, however, the number of viable bacteria in activated macrophages remained unchanged, whereas the number of bacteria in control macrophages significantly (P less than 0.05) increased. Similar results were obtained in 48-h-activated macrophages. On the other hand, macrophages incubated with 10 to 10(3) U of rIFN-gamma exhibited enhanced fusion of lysosomes to Salmonella-containing phagosomes in both the 12-h- and 48-h-stimulated stages. Moreover, when 48-h-activated macrophages were incubated concomitantly with superoxide dismutase and catalase, Salmonella-killing activity was not affected. These results indicate that rIFN-gamma per se is able to activate peritoneal macrophages to induce Salmonella-killing activity and suggest that increased phagosome-lysosome fusion followed by an oxygen-independent killing mechanism is primarily responsible for the enhanced Salmonella-killing activity in rIFN-gamma-activated macrophages.     

Activation of macrophages by lymphokines: enhancement of phagosome-lysosome fusion and killing of Coccidioides immitis.

Previously, it was shown that arthroconidia of Coccidioides immitis appear to inhibit phagosome-lysosome fusion and survive within normal mouse peritoneal macrophages. However, when these macrophages are exposed to antigen-stimulated T lymphocytes from immune mice, activation occurs, leading to enhanced phagosome-lysosome fusion and killing of C. immitis. Results indicate that the activation of macrophages can be effected after incubation with soluble lymphocyte product(s) (lymphokines). The activation of macrophages results if the macrophages are exposed to the lymphokine before, but not after, infection. The results indicate that the lymphocyte population responsible for the elaboration of the lymphokine is phenotypically Lyt1+2- and that activation of macrophages by the lymphokine can occur across H-2 histocompatibility barriers.     

Effect of a lysolecithin analogue on nonspecific resistance to infection of mice

The effect of racemic 1-octadecyl-2-methoxy-sn-glycero-3 phosphorylcholine (ET-18-OCH3) on the nonspecific resistance of mice to infection with Salmonella typhimurium was investigated. Two S. typhimurium strains with different virulence were studied and no effect was observed in either case at concentrations of ET-18-OCH3 up to 100 micrograms/mouse. However, a concentration of 500 micrograms/mouse caused decreased resistance to S. typhimurium, correlating with a depression of carbon clearance. Treatment of macrophages with ET-18-OCH3 in vitro inhibited phagosome-lysosome fusion, but had no effect on zymosan-induced luminol-dependent chemiluminescence. The relationship between the adjuvant and nonspecific anti-infectious activity of ET-18-OCH3 and other compounds is discussed.     

Plasmid-associated virulence of Salmonella typhimurium.

We investigated the role of the 100-kilobase (kb) plasmid of Salmonella typhimurium in the virulence of this organism for mice. Three strains, LT2-Z, SR-11, and SL1344, which possessed 100-kb plasmids with identical restriction enzyme digestion profiles, were cured of their respective 100-kb plasmids after Tnmini-tet was used to label plasmids. Curing wild-type virulent strains SR-11 and SL1344 raised peroral 50% lethal doses from 3 x 10(5) and 6 x 10(4) CFU, respectively, to greater than 10(8) CFU. Both wild-type strains had intraperitoneal 50% lethal doses of less than 50 CFU, whereas the intraperitoneal 50% lethal doses for cured SR-11 and SL1344 were less than 50 and 400 CFU, respectively. Reintroduction of the Tnmini-tet-labeled, 100-kb plasmid restored wild-type virulence. Invasion from Peyer's patches to mesenteric lymph nodes and spleens after peroral inoculation was the stage of pathogenesis most affected by curing S. typhimurium of the 100-kb plasmid. Wild-type S. typhimurium replicated in spleens of mice inoculated intravenously to a greater extent than did plasmid-cured derivatives. Wild-type and cured strains equally adhered to and invaded Henle-407, HEp-2, and CHO cells; furthermore, the presence of the 100-kb plasmid was not necessary for replication of S. typhimurium within CHO cells. The 100-kb plasmid had no effect on phagocytosis and killing of S. typhimurium by murine peritoneal macrophages in vitro and in vivo. Similarly, wild-type and plasmid-cured strains were resistant to killing by 90% normal human, rabbit, and guinea pig sera. All wild-type and plasmid-cured S. typhimurium strains possessed complete lipopolysaccharide, as determined by silver staining solubilized cells in sodium dodecyl sulfate-polyacrylamide gels. We have confirmed the role of the 100-kb plasmid of S. typhimurium in virulence, primarily in invasion to mesenteric lymph nodes and spleens after peroral inoculation of mice. Involvement of the 100-kb plasmid in infection of mesenteric lymph nodes and spleens suggests a role for the plasmid in the complex interaction of S. typhimurium with cells of the reticuloendothelial system.     

Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection.

The sigma factor RpoS mediates the stationary-phase expression of a large group of genes, including those involved in resistance to a variety of environmental stresses, such as starvation, oxidation, and low pH. In addition, RpoS has been shown to regulate Salmonella virulence. In Salmonella typhimurium, RpoS controls the expression of the Salmonella plasmid virulence (spv) genes, which are required for systemic infection. However, the mechanism by which RpoS affects the pathogenicity of Salmonella remains incompletely defined. In this study, we focused on the ability of rpoS to affect the early stages of the infection process of S. typhimurium. An rpoS mutant of S. typhimurium exhibited wild-type abilities to attach to and invade Int-407 cells and J774 macrophage-like cells. In addition, rpoS did not affect the intracellular survival of S. typhimurium in either J774 macrophage-like cells or rat bone marrow-derived macrophages. However, the rpoS mutant demonstrated a decreased ability to colonize murine Peyer's patches after oral inoculation than its wild-type virulent parent strain showed. In addition, virulence plasmid-cured derivatives of the rpoS mutant were recovered in lower numbers from murine Peyer's patches than were plasmid-cured derivatives of the isogenic wild-type S. typhimurium. This indicates that RpoS regulation of chromosomally encoded genes is important for colonization of the gut-associated lymphoid tissue (GALT) by S. typhimurium. Microscopic analysis of histological sections taken from Peyer's patches after peroral infection of mice showed that, unlike its wild-type virulent parent strain, the isogenic rpoS mutant did not destroy the follicle-associated epithelium of the GALT. Furthermore, the rpoS mutant demonstrated a decreased ability to adhere to histological sections of murine Peyer's patches than its wild-type parent showed. Our data provide evidence for a role of RpoS in the interaction of Salmonella with cells of the GALT, specifically the Peyer's patches. This implicates the involvement of rpoS in the initial stages of systemic infection by Salmonella as opposed to infection leading to gastroenteritis.     

Salmonella typhimurium mutants lacking flagella or motility remain virulent in BALB/c mice

Nonmotile flagellated (mot) and nonflagellated (fla) mutants of Salmonella typhimurium LT-2 were isolated from a collection of mutants with random Tn10-insertion mutations. Both classes of mutants were resistant to infection by the flagellotropic bacteriophage chi. The nonflagellated (fla::Tn10) mutants did not react with H antigen-specific antisera and did not possess flagella when examined by electron microscopy, and sheared-cell extracts were devoid of flagellin. The nonmotile (mot::Tn10) mutants reacted with H-specific antisera and expressed paralyzed flagella that were indistinguishable from wild-type flagella. The Tn10 insertions in strain LT-2 were mapped to loci in regions II (flh and mot) and III (fli) of the flagellar genes, and the mutations were transduced into the mouse-virulent S. typhimurium strains SR-11 and SL1344. Lack of motility reduced the ability of S. typhimurium to invade Henle cells in vitro, yet the virulence in mice of the nonmotile mutants of SR-11 and SL1344 was unaffected by the inactivity or loss of flagella. Wild-type SR-11 had a 50% lethal dose (LD50) in BALB/c mice following oral (p.o.) challenge of 2.4 x 10(4) CFU. The p.o. LD50 of the SR-11 fli-8007::Tn10 mutant was 4.5 x 10(4) CFU. The mot-8008::Tn10 mutation in SR-11 conferred paralyzed flagella and increased the p.o. LD50 in mice to 2.2 x 10(5) CFU, but this was not statistically significant. A similar increase in the p.o. LD50 was observed when the SL1344 mot-8008::Tn10 mutant was tested in mice. Wild-type SR-11 and the isogenic nonflagellated and nonmotile mutants were equally virulent in mice challenged via intraperitoneal injection.     

Invasion and replication of Salmonella typhimurium in animal cells.

A total of 81 avirulent Tn10 insertion mutants of Salmonella typhimurium have previously been described. These mutants were selected for the inability to survive in murine macrophages. We have characterized the abilities of the most avirulent of these mutants to adhere to, invade, and replicate in both macrophages and nonphagocytic epithelial cells. The results suggest that most mutants contain a defect that is specific to survival within professional phagocytes. These mutants invaded and replicated normally within nonphagocytic human colon adenocarcinoma cells (Caco-2) but did not survive in the macrophage cell line J774. One mutant invaded both macrophages and epithelial cells much less efficiently than the parental strain. The defect associated with this mutant appears to be a result of decreased adherence to animal cells.     

Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection.

The role of macrophages in the innate immunity of (CBA/N female X DBA/2N male)F1 female mice to Salmonella typhimurium was assessed with silica, an agent which has been reported to selectively inactivate macrophages. Silica, administered intravenously to mice, markedly decreased the phagocytic capacity of splenic macrophages but had no effect on splenic responsiveness to the B-cell mitogen lipopolysaccharidide or the T-cell mitogen phytohemagglutinin, nor did it affect the frequency of surface immunoglobulin-positive cells (B cells). Silica given to mice 1 day before intraperitoneal challenge decreased the 50% lethal dose of S. typhimurium 100-fold. The incidence of survival of mice given silica up to 14 days before infection with a sublethal dose of organisms was also decreased. This susceptibility could also be demonstrated when silica was given 10 days, but not 20 days, after S. typhimurium infection. Poly-2-vinylpyridine-N-oxide, a lysosomal stabilizing agent, abrogated the silica effect. Deaths among silica-treated mice followed uncontrolled multiplication of the organism in the spleen. These results provide direct evidence that macrophages play an essential role in natural immunity to murine typhoid and demonstrate the efficacy of silica as a tool to analyze macrophage function.     

Salmonella typhimurium loci involved in survival within macrophages.

A set of Tn10 mutants of Salmonella typhimurium which have a diminished capacity to survive in murine macrophages and decreased virulence in mice has been described previously. In this study, we characterized 30 of these mutants and determined map locations of Tn10 insertions for 23 of these strains. In addition, short fragments of transposon-flanking DNA were cloned, and the nucleotide sequence was determined for 23 mutants. Seven mutants carried transposon insertions in known genes, representing six loci: htrA, prc, purD, fliD, nagA, and smpB. The possible roles of these genes in Salmonella virulence are discussed. One insertion was found to be in an unknown gene which shared homology with the open reading frames Bv' and Bv located in the pin inversion system of Shigella boydii. In one mutant, Tn10 was found to be inserted in a gene with significant homology to adhE of Escherichia coli and Clostridium acetobutylicum. The map location and degree of homology indicate that the Salmonella gene encodes a related, but different, dehydrogenase. In 14 of the mutants analyzed, Tn10 was inserted into genes which had no significant homologies to entries in the DNA and protein data bases. In conclusion, 16 insertions define loci, termed ims for impaired macrophage survival, which have not yet been described in S. typhimurium but have been shown previously to be necessary for full virulence in mice. Although most ims loci are distributed randomly throughout the genome, a cluster was found between 75 and 78 min on the Salmonella chromosome.     

Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.     

Flagella help Salmonella typhimurium survive within murine macrophages.

In this study, we evaluated how flagella enhance the pathogenicity of Salmonella typhimurium in strain C57BL/6J mice. When mice were infected orally with flagellated or nonflagellated S. typhimurium, equivalent numbers of bacteria colonized the gastrointestinal tracts of the animals, but the number of flagellated organisms increased faster once colonization began in the spleens and livers. To evaluate this differential rate of Salmonella growth, the rate of blood clearance, and the kinetics of net multiplication of salmonellae in splenic tissue after intravenous challenge, the two groups of mice were compared. We found that clearance of bacteria from the blood was the same for flagellated or nonflagellated strains. However, the number of flagellated bacteria in the spleen increased logarithmically until the death of the animals, whereas the number of nonflagellated salmonellae increased only slightly. In contrast, both flagellated and nonflagellated strains grew exponentially in the spleens of mice pretreated with silica, a macrophage toxic agent. In an in vitro macrophage assay, flagellated salmonellae survived longer than nonflagellated organisms. These results indicate that flagella either protect S. typhimurium from the intracellular killing mechanisms of murine macrophages or that flagella enhance the ability of S. typhimurium to multiply within murine macrophages.     

Susceptibility of lipopolysaccharide-responsive and -hyporesponsive ItyS Mice to infection with rough mutants of Salmonella typhimurium.

The R5 (chemotype Rb) but not the R10 (chemotype Rd) mutant of murine pathogen Salmonella typhimurium 395MS was extremely virulent in intraperitoneal infections of C57BL/10ScCr mice carrying the ityS and lpsD alleles. C57BL/6J (ityS lpsN) and C3H/HeJ (ityR lpsD) mice showed a much higher resistance to the R5 mutant. Further studies were performed with peritoneal macrophages in vitro in order to elucidate susceptibility in lipopolysaccharide (LPS)-hyporesponsive mice carrying ItyS. The intracellular killing capacity of the ItyS LpsD macrophages was lower than that of the ItyS LpsN macrophages for the R5 mutant and may partly explain the increased susceptibility of the ItyS LpsD mice. The deep rough mutant, R10, was rapidly killed intracellularly by the ItyS LpsD macrophages. Processing of the bacteria in macrophages that had phagocytosed R5 or R10 bacteria was followed for up to 18 days by endotoxin measurements (limulus assay) and immunostaining, with monoclonal antibodies to various parts of the LPS molecule being used. Only 0.1% or less of the macrophage-associated bacteria remained alive after 48 h of incubation, and none were alive on day 7. Although immunostaining showed that LPS was present in both the LpsD and LpsN macrophages during the whole incubation period of 18 days, endotoxin activity in the LpsD macrophages on day 7 was lower than that in the LpsN macrophages, indicating that qualitative modifications of the chemical composition or physical state of the LPS molecule occurred. The interleukin-6 response in the ItyS LpsD macrophages was delayed and of shorter duration compared with that in the ItyS LpsN macrophages. The results suggest that the difference between the LPS-hyporesponsive and -responsive ItyS mice in susceptibility to infection with the R5 mutant was due to the lower activation state of the LpsD macrophages during infection, leading to a lower intracellular bactericidal systems of the macrophages. A rapid killing of the bacterium should restrict the infection and may partly compensate for a diminished inflammatory response. The persistence of LPS within the cells is discussed.