Multiple Fimbrial Adhesins Are Required for Full Virulence of Salmonella typhimurium in Mice

Adhesion is an important initial step during bacterial colonization of the intestinal mucosa. However, mutations in the Salmonella typhimurium fimbrial operons lpf, pef, or fim only moderately alter mouse virulence. The respective adhesins may thus play only a minor role during infection or S. typhimurium may encode alternative virulence factors that can functionally compensate for their loss. To address this question, we constructed mutations in all four known fimbrial operons of S. typhimurium: fim, lpf, pef, and agf. A mutation in the agfB gene resulted in a threefold increase in the oral 50% lethal dose (LD₅₀) of S. typhimurium for mice. In contrast, an S. typhimurium strain carrying mutations in all four fimbrial operons (quadruple mutant) had a 26-fold increased oral LD₅₀. The quadruple mutant, but not the agfB mutant, was recovered in reduced numbers from murine fecal pellets, suggesting that a reduced ability to colonize the intestinal lumen contributed to its attenuation. These data are evidence for a synergistic action of fimbrial operons during colonization of the mouse intestine and the development of murine typhoid fever.     

The Salmonella typhimurium AhpC Polypeptide Is Not Essential for Virulence in BALB/c Mice but Is Recognized as an Antigen during Infection

The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium. We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046–4048, 1997). We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not. An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed by S. typhimurium during infection of BALB/c mice and constitutes a target for the immune system.     

The Ferric Enterobactin Transporter Fep Is Required for Persistent Salmonella enterica Serovar Typhimurium Infection

Most bacterial pathogens require iron to grow and colonize host tissues. The Gram-negative bacterium Salmonella enterica serovar Typhimurium causes a natural systemic infection of mice that models acute and chronic human typhoid fever. S. Typhimurium resides in tissues within cells of the monocyte lineage, which limit pathogen access to iron, a mechanism of nutritional immunity. The primary ferric iron import system encoded by Salmonella is the siderophore ABC transporter FepBDGC. The Fep system has a known role in acute infection, but it is unclear whether ferric iron uptake or the ferric iron binding siderophores enterobactin and salmochelin are required for persistent infection. We defined the role of the Fep iron transporter and siderophores in the replication of Salmonella in macrophages and in mice that develop acute followed by persistent infections. Replication of wild-type and iron transporter mutant Salmonella strains was quantified in cultured macrophages, fecal pellets, and host tissues in mixed- and single-infection experiments. We show that deletion of fepB attenuated Salmonella replication and colonization within macrophages and mice. Additionally, the genes required to produce and transport enterobactin and salmochelin across the outer membrane receptors, fepA and iroN, are needed for colonization of all tissues examined. However, salmochelin appears to be more important than enterobactin in the colonization of the spleen and liver, both sites of dissemination. Thus, the FepBDGC ferric iron transporter and the siderophores enterobactin and salmochelin are required by Salmonella to evade nutritional immunity in macrophages and cause persistent infection in mice.     

Stability of erp Loci during Borrelia burgdorferi Infection: Recombination Is Not Required for Chronic Infection of Immunocompetent Mice

Borrelia burgdorferi can persistently infect mammals despite their production of antibodies directed against bacterial proteins, including the Erp lipoproteins. We sequenced erp loci of bacteria reisolated from laboratory mice after 1 year of infection and found them to be identical to those of the inoculant bacteria. We conclude that recombination of erp genes is not essential for chronic mammalian infection.     

Glucose 6-Phosphate Dehydrogenase Is Required for Salmonella typhimurium Virulence and Resistance to Reactive Oxygen and Nitrogen Intermediates

Salmonella typhimurium zwf mutants lacking glucose 6-phosphate dehydrogenase (G6PD) activity have increased susceptibility to reactive oxygen and nitrogen intermediates as well as attenuated virulence in mice. Abrogation of the phagocyte respiratory burst oxidase during experimental infection with zwf mutant Salmonella causes a prompt restoration of virulence, while inhibition of inducible nitric oxide synthase results in delayed lethality. These observations suggest that G6PD-dependent bacterial antioxidant defenses play an important pathogenic role during early salmonellosis and additionally may help to antagonize NO-dependent antimicrobial mechanisms later in the course of infection.     

Effect of Delayed-Type Hypersensitivity Reaction and Transferred Lymphokine on the Resistance of Mice to Salmonella typhimurium Infection

Immune mice which exhibited a delayed-type hypersensitivity reaction to bovine serum albumin after bovine serum albumin immunization and stimulation and normal mice that had been transferred with a lymphokine-rich fraction from the supernatant of concanavalin A-stimulated spleen cell cultures demonstrated resistance to Salmonella infection.     

Salmonella typhimurium Infection in Mice Induces Nitric Oxide-Mediated Immunosuppression through a Natural Killer Cell-Dependent Pathway

Splenocytes isolated from C57BL/6J female mice 3 to 7 days after inoculation with an attenuated strain of Salmonella typhimurium produced high levels of nitric oxide (39 to 77 μM) and gamma interferon (IFN-γ). Additionally, spleen cell cultures from Salmonella-inoculated mice were markedly suppressed in their ability to generate an in vitro plaque-forming cell (PFC) response to sheep erythrocytes. Depletion of natural killer (NK) cells from the immune splenocyte population markedly reduced nitric oxide production, prevented suppression of PFC responses, and completely abrogated IFN-γ release. Treatment of NK cell-depleted immune cells with IFN-γ restored nitric oxide production to levels comparable to those of intact immune cells and also restored the immunosuppression. These results suggest that NK cells regulate the induction of nitric oxide-mediated immunosuppression following infection with S. typhimurium through the production of IFN-γ.     

Oral Immunization of Mice with Killed Salmonella typhimurium Vaccine

A study was undertaken to assess the efficacy of oral, parenteral, and intraperitoneal immunization methods of administering killed Salmonella typhimurium vaccine to mice and to evaluate the effectiveness of single and multiple doses of the vaccine containing varied numbers of the killed bacteria. A further objective of this study was to evaluate the effect of adding substances to the vaccine to which have been ascribed "adjuvant" properties. The protection was estimated by isolation of bacteria from the spleen and feces after oral challenge of the mice with live S. typhimurium. The results showed that one or more doses of 10(10) organisms given orally led to significant protection. This rate of protection increased proportionately with the number of doses up to 10 doses, which offered 100% protection. Streptomycin, when added to multiple doses of 10(9) or more organisms given orally, increased the degree of protection, but beryllium sulfate and pertussis vaccine did not. Although multiple doses afforded similar systemic protection by all three routes of immunization, oral immunization yielded significantly greater local protection than that observed after subcutaneous or intraperitoneal immunization.     

Neither the Bvg− Phase nor the vrg6 Locus of Bordetella pertussis Is Required for Respiratory Infection in Mice

In Bordetella species, the BvgAS sensory transduction system mediates an alteration between the Bvg⁺ phase, characterized by expression of adhesins and toxins, and the Bvg⁻ phase, characterized by the expression of motility and coregulated phenotypes in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. Since there is no known environmental or animal reservoir for B. pertussis, the causative agent of whooping cough, it has been assumed that this phenotypic alteration must occur within the human host during infection. Consistent with this hypothesis was the observation that a B. pertussis mutant, SK6, containing a TnphoA insertion mutation in a Bvg-repressed gene (vrg6) was defective for tracheal and lung colonization in a mouse model of respiratory infection (D. T. Beattie, R. Shahin, and J. Mekalanos, Infect. Immun. 60:571–577, 1992). This result was inconsistent, however, with the observation that a Bvg⁺ phase-locked B. bronchiseptica mutant was indistinguishable from the wild type in its ability to establish a persistent respiratory infection in rabbits and rats (P. A. Cotter and J. F. Miller, Infect. Immun. 62:3381–3390, 1994; B. J. Akerley, P. A. Cotter, and J. F. Miller, Cell 80:611–620, 1995). To directly address the role of Bvg-mediated signal transduction in B. pertussis pathogenesis, we constructed Bvg⁺ and Bvg⁻ phase-locked mutants and compared them with the wild type for their ability to colonize the respiratory tracts of mice. Our results show that the Bvg⁺ phase of B. pertussis is necessary and sufficient for respiratory infection. By constructing a strain with a deletion in the bvgR regulatory locus, we also show that ectopic expression of Bvg⁻ phase phenotypes decreases the efficiency of colonization, underscoring the importance of Bvg-mediated repression of gene expression in vivo. Finally, we show that the virulence defect present in strain SK6 cannot be attributed to the vrg6 mutation. These data contradict an in vivo role for the Bvg⁻ phase of B. pertussis.     

Enzyme Activities of the Livers of Mice Infected with Salmonella typhimurium

The activities of the mouse liver enzymes tryptophan oxygenase, tyrosine aminotransferase, and pyruvate kinase were measured after infection with three dose levels of Salmonella typhimurium strain SR-11. Infection occurred in all groups as evidenced by an increase in bacterial numbers and by death of the animals. The activities of the enzymes increased in all groups during the course of the infection. The results obtained during infection are compared with those obtained after endotoxin injection.     

Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages

Salmonella enterica serovar Typhimurium is able to resist antimicrobial peptide killing by induction of the PhoP-PhoQ and PmrA-PmrB two-component systems and the lipopolysaccharide (LPS) modifications they mediate. Murine cathelin-related antimicrobial peptide (CRAMP) has been reported to inhibit S. Typhimurium growth in vitro and in vivo. We hypothesize that infection of human monocyte-derived macrophages (MDMs) with Salmonella enterica serovar Typhi and S. Typhimurium will induce human cathelicidin antimicrobial peptide (CAMP) production, and exposure to LL-37 (processed, active form of CAMP/hCAP18) will lead to upregulation of PmrAB-mediated LPS modifications and increased survival in vivo. Unlike in mouse macrophages, in which CRAMP is upregulated during infection, camp gene expression was not induced in human MDMs infected with S. Typhi or S. Typhimurium. Upon infection, intracellular levels of ΔphoPQ, ΔpmrAB, and PhoP^cS. Typhi decreased over time but were not further inhibited by the vitamin D₃-induced increase in camp expression. MDMs infected with wild-type (WT) S. Typhi or S. Typhimurium released similar levels of proinflammatory cytokines; however, the LPS modification mutant strains dramatically differed in MDM-elicited cytokine levels. Overall, these findings indicate that camp is not induced during Salmonella infection of MDMs nor is key to Salmonella intracellular clearance. However, the cytokine responses from MDMs infected with WT or LPS modification mutant strains differ significantly, indicating a role for LPS modifications in altering the host inflammatory response. Our findings also suggest that S. Typhi and S. Typhimurium elicit different proinflammatory responses from MDMs, despite being capable of adding similar modifications to their LPS structures.     

Endogenous Interleukin-12 Is Not Required for Resolution of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection in Mice

A Th1 immune response involving gamma interferon (IFN-gamma) production is required to eliminate Chlamydophila abortus infections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12(-/-) and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12(-/-) mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12(-/-) mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-gamma. The low level of IFN-gamma was essential for survival of IL-12(-/-) infected mice. Both WT and IL-12(-/-) mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-gamma and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge with C. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12(-/-) mice. The lack of IL-12 resulted in few infiltrating CD4(+) T cells in the liver relative to the number in WT mice, although the number of CD8(+) T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.     

Priming of T-Cell Responses in Mice by Porins of Salmonella typhimurium

An imbalance in signals delivered to T cells via T-cell receptor and accessory molecules can lead to anergy, apoptosis, or both. In the present study we have demonstrated that Salmonella typhimurium infection in mice leads to a progressive loss of CD4⁺ T helper (Th) cell population, abnormal T-cell death by apoptosis and loss of accessory molecules (B7 and intracellular adhesion molecule-1) on macrophages. Quantification of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-γ (IFN-γ) secretion revealed a Th2-type of response in lymphocytes isolated from spleen. However, preimmunization of mice with porins resulted in an increased CD4⁺ Th cell population and accessory molecules on the surface of macrophages. Quantification of cytokines revealed a Th1-type of response. We conclude that preimmunization of mice with porins provides a microenvironment in which a well-balanced accessory molecule and cytokine network is established, which results in the prevention of cell death by apoptosis.     

Susceptibility to Salmonella typhimurium Infection and Effectiveness of Vaccination in Mice Deficient in the Tumor Necrosis Factor Alpha p55 Receptor

Mice defective in the ability to produce the tumor necrosis factor alpha p55 receptor (TNFαp55R) were orally challenged with a number of Salmonella typhimurium HWSH derivatives that differ in virulence. In comparison to TNFαp55R^+/+ mice, TNFαp55R^−/− mice succumbed earlier to challenge with wild-type S. typhimurium HWSH and S. typhimurium HWSH purE. In contrast, TNFαp55R^−/− mice were able to control an S. typhimurium HWSH aroA challenge, although greater numbers of Salmonella organisms were present in the tissues for a longer time period than was observed with TNFαp55R^+/+ mice. Vaccination of normal and TNFαp55R knockout animals with S. typhimurium HWSH aroA showed that TNFαp55R^−/− mice, unlike TNFαp55R^+/+ mice, were not protected against a virulent S. typhimurium HWSH challenge. Splenocytes from TNFαp55R^−/− mice exhibited a reduced ability to proliferate in the presence of S. typhimurium antigen compared to TNFαp55R^+/+ mice. Thus, TNFαp55R is essential for controlling Salmonella growth in tissues and for recall of immunity in murine salmonellosis.     

A Study of Lymphocytotoxic Antibodies in Multiparous Women Having Had At Least Four Pregnancies

The study of anti-HL-A immunization was carried out on 26 sera taken from multiparous women who had had at least 4 pregnancies and who had never been transfused. In 20 of these cases, it was possible to define the HL-A phenotype of the husband and wife. As many well-defined sera were detected in multiparous women as in primiparous women. However, persistence of antibodies from one pregnancy to another can make the sera of the multiparous women more polyspecific than the sera of the primiparous women. One cross-reaction was described between the W14, HL-A5 and W5 antigens, and the cross-reaction between the HL-A7 and HL-A8 antigens was confirmed.     

Identification of Salmonella typhimurium Genes Required for Colonization of the Chicken Alimentary Tract and for Virulence in Newly Hatched Chicks

From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described.Poultry are a major source of Salmonella food poisoning for humans. More than 2,000 serotypes have been identified, mainly as a result of human food poisoning, the vast majority of which produce little or no systemic disease in healthy adult animals. Salmonellae are usually associated with food poisoning by virtue of their ability to colonize the alimentary tracts of livestock, particularly poultry. This results in considerable contamination of carcasses at slaughter, with entry of salmonellae into human food. A small number of serotypes typically produce typhoid-like diseases in adults of a restricted number of host species. These include Salmonella typhi and S. paratyphi in humans, S. cholerae-suis in pigs, S. dublin in cattle, and S. gallinarum in birds. Although transmission of these serotypes is primarily fecal-oral, they do not colonize the alimentary tract extensively in their respective host species and are not excreted in the feces in large numbers, except in the advanced stages of systemic disease. Thus, carcass contamination by most of these serotypes is infrequent, and they rarely enter the human food chain. S. typhimurium and S. enteritidis are exceptional in being able to colonize the alimentary tracts of poultry and cause typhoid in mice.While infection of adult poultry leads to limited excretion of Salmonella in the feces, infection of newly hatched chicks, which have a relatively simple gut flora, results in rapid multiplication and extensive excretion. This can lead to rapid spread of a Salmonella strain through a flock as the housing and water and feeding systems become contaminated. Present methods for control are either inadequate or create additional problems. Improvements in hygienic measures are costly, and antibiotic therapy or prophylaxis can result in the production and spread of antibiotic-resistant strains (43). Vaccination with live, attenuated Salmonella vaccines is becoming more attractive and acceptable (11). Colonization of young chickens by Salmonella can be reduced by oral inoculation with preparations of gut flora derived from adult chickens. This has been termed competitive exclusion and provides the young chicken with an established gut flora early in life (28). The level of resistance obtained is, nevertheless, less than that of an adult bird that has been vaccinated.In the chicken alimentary tract, ingested foodstuff reaches the crop, which acts as a storage organ where little, if any, digestion occurs. The crop is lined with squamous epithelium which is normally colonized by lactobacilli, and, as a result of fermentation, this organ has a pH of 4 to 5. Further along, the proventriculus secretes HCl and is immediately followed by the gizzard, which is a muscular organ in which food is ground. Distal to the small intestine, the ceca branch off from the ileum as two closed tubes, just before it opens into the cloaca. Facultative anaerobic bacteria such as lactobacilli, streptococci, and Escherichia coli are found in moderately high densities throughout the gut, whereas obligate anaerobes are found in high numbers only in the cecum. Upon colonization of the alimentary tract, the highest viable counts of Salmonella are obtained from the cecum, cloaca, and ileum and, to a lesser extent, from the crop (3). The higher counts obtained from the more distal regions of the gut may be the result of the lower flow rate (42), and the reabsorption of fluids from the intestinal contents may also contribute to this.Very few data regarding the mechanism whereby Salmonella colonize the alimentary tracts of chickens are available. Potentially, it could include the ability to multiply in the intestinal contents, growth in or migration through the mucus layer, and attachment to and invasion of the epithelium. Some Salmonella serotypes colonize more efficiently than others (3, 44); thus, S. montevideo persists in the guts of chickens and is consequently excreted in the feces for longer than S. typhimurium. Serotypes that produce typhoid-like disease in poultry, such as S. gallinarum and S. pullorum, or other animals, such as S. cholerae-suis, colonize the alimentary tracts of chickens very poorly in the absence of clinical disease.Physical attachment has been suggested as a mechanism for colonization (45), and there is some evidence that fimbriae promote colonization of the murine intestine by S. typhimurium (47, 48) and of the chicken intestine by S. infantis (3) and S. enteritidis (49). Barrow et al. (3) also found that a nonmotile mutant of S. infantis did not colonize as well as the wild type, whereas nonmotility made no difference to colonization by S. typhimurium phage type 14. One interpretation of this apparent anomaly was that the nonspecific chemical method used for mutagenesis may have generated additional mutations affecting colonization. However, the possibility that different serotypes use alternative mechanisms for colonization also needs to be considered.To improve our understanding of colonization of the chicken alimentary tract by Salmonella, we sought to identify some of the microbial determinants required. We report here the isolation of transposon mutants of S. typhimurium F98 which showed reduced excretion in the feces following experimental oral inoculation and have identified the sites of transposon insertion. The role of the identified genes in colonization was confirmed by transducing the transposon mutations to a fresh S. typhimurium F98 genetic background with a derivative of bacteriophage P22 and by the construction of defined mutations. Many of the identified genes were also found to be required for virulence of S. typhimurium F98 in newly hatched chicks.