Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Sustained Protection in Mice Immunized with Fractional Doses of Salmonella Enteritidis Core and O Polysaccharide-Flagellin Glycoconjugates

  • Raphael Simon ,

    rsimon@medicine.umaryland.edu

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Jin Y. Wang,

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Mary A. Boyd,

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Mohan E. Tulapurkar,

    Affiliations Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Division of Pulmonary and Critical Care, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Girish Ramachandran,

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Sharon M. Tennant,

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Marcela Pasetti,

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • James E. Galen,

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯
  • Myron M. Levine

    Affiliations Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, United States of America

    ⨯

Abstract

Non-typhoidal Salmonella (NTS) serovars S. Enteritidis and S. Typhimurium are a major cause of invasive bacterial disease (e.g., bacteremia, meningitis) in infants and young children in sub-Saharan Africa and also occasionally cause invasive disease in highly susceptible hosts (young infants, the elderly, and immunocompromised subjects) in industrialized countries. No licensed vaccines exist against human NTS infections. NTS core and O polysaccharide (COPS) and FliC (Phase 1 flagellin subunits) each constitute protective antigens in murine models. S. Enteritidis COPS conjugated to FliC represents a promising vaccine approach that elicits binding and opsonophagocytic antibodies and protects mice against lethal challenge with virulent S. Enteritidis. We examined the protective efficacy of fractional dosages of S. Enteritidis COPS:FliC conjugate vaccines in mice, and also established that protection can be passively transferred to naïve mice by administering sera from mice immunized with conjugate. Mice were immunized with three doses of either 10 µg, 2.5 µg (full dose), 0.25 µg, or 0.025 µg S. Enteritidis COPS:FliC conjugate at 28 day intervals. Antibody titers to COPS and FliC measured by ELISA fell consonant with progressively smaller vaccine dosage levels; anti-FliC IgG responses remained robust at fractional dosages for which anti-COPS serum IgG titers were decreased. Nevertheless, >90% protection against intraperitoneal challenge was observed in mice immunized with fractional dosages of conjugate that elicited diminished titers to both FliC and COPS. Passive transfer of immune sera from mice immunized with the highest dose of COPS:FliC to naïve mice was also protective, demonstrating the role of antibodies in mediating protection. These results provide important insights regarding the potency of Salmonella glycoconjugate vaccines that use flagellin as a carrier protein.

Citation: Simon R, Wang JY, Boyd MA, Tulapurkar ME, Ramachandran G, Tennant SM, et al. (2013) Sustained Protection in Mice Immunized with Fractional Doses of Salmonella Enteritidis Core and O Polysaccharide-Flagellin Glycoconjugates. PLoS ONE 8(5): e64680. https://doi.org/10.1371/journal.pone.0064680

Editor: Dipshikha Chakravortty, Indian Institute of Science, India

Received: February 12, 2013; Accepted: April 17, 2013; Published: May 31, 2013

Copyright: © 2013 Simon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by Middle Atlantic RCE Program, National Institute of Allergy and Infectious Diseases/National Institutes of Health 2 U54 AI057168. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Non-typhoidal Salmonella (NTS) infections are a global problem, with distinct regional clinico-epidemiological differences. In industrialized countries, NTS are common causes of bacterial gastroenteritis and occasionally cause invasive disease (meningitis, septicemia, bacteremia, etc.) in susceptible groups such as young infants, the elderly and immunocompromised subjects [1]. In sub-Saharan Africa, invasive salmonellosis caused by multiple antibiotic-resistant NTS strains are among the most common causes of invasive bacterial disease in infants and young children, with a case fatality rate between 15–30% [2]. Importantly, two serovars, S. Typhimurium (and monophasic variants) and S. Enteritidis, cause 80–95% of invasive disease in sub-Saharan Africa [1], [2], making the concept of control by vaccination epidemiologically feasible.

Salmonella lipopolysaccharide (LPS) and flagellin (the structural protein subunit of polymeric flagella filaments) are protective antigens in animal models [3], [4]. The conserved core and serogroup-specific O polysaccharide (COPS) constitute the polysaccharide portion of LPS. Unconjugated NTS COPS is a poor immunogen that does not elicit immunologic memory in animal models [4], [5] and unconjugated bacterial polysaccharides, including capsular polysaccharides, are also, in general, weakly immunogenic in human infants [6]. In contrast, conjugation of Salmonella COPS with proteins has been shown to improve anti-polysaccharide humoral responses and to induce protection in mice [4], [5], [7].

We reported previously that S. Enteritidis COPS:FliC conjugates were immunogenic and protective in mice against virulent S. Enteritidis strain R11 (originally isolated from the blood of a Malian child) and the antibodies in post-vaccination sera manifested opsonophagocytic activity [4]. We report herein that S. Enteritidis COPS:FliC conjugates protect even when administered in fractional dosages that elicit diminished anti-FliC and COPS antibody responses (compared to the 2.5 µg “full dose” that was reported previously [4]), and that passive transfer of serum from conjugate-immunized mice protects naïve mice against otherwise lethal S. Enteritidis challenge.

Materials and Methods

Ethics Statement

All animal experiments carried out in this work were approved by the University of Maryland Baltimore Office of Animal Welfare Assurance (OAWA), under approved Animal Use Protocol 0909007.

Bacterial Strains

Characteristics and growth conditions of wild-type S. Enteritidis R11 and attenuated derivative CVD 1941 (ΔguaBA ΔclpP) have been previously described [4].

Purification of LPS, COPS and Flagellin, and Synthesis of COPS:FliC Conjugates

Purification of LPS, COPS and FliC from CVD 1941 and their characterization were performed as described [4]. Direct conjugation between COPS and FliC monomers was accomplished at a 1∶1 ratio of polysaccharide to protein, using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP, Research Organics, OH) [8]. Unreacted protein and polysaccharide were removed by size-exclusion chromatography with Superdex 200 (GE/Amersham, NJ) and anion exchange Q membrane chromatography (Sartorius, Germany) [4].

Mice

Female outbred CD-1 mice (8–10 week old) were purchased from Charles River Laboratories (Wilmington, MA). Animal protocols were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee.

Immunization and Challenge

Mice were injected intramuscularly (IM) in the right hind limb at 0, 28 and 56 days with either 10 µg (a 4-fold dose), 2.5 µg (full dose), 0.25 µg (1/10th dose) or 0.025 µg (1/100th dose) of polysaccharide conjugated to FliC in 50 µl of sterile PBS. Sera were obtained before vaccination and at day 77. Mice were challenged intraperitoneally (IP) on day 84, with 1×106 CFU of S. Enteritidis R11 (IP LD50 = 2.2×105). To assess the protective effect of passively transferred antibodies, naïve mice were injected intravenously (IV) through the tail vein with 100 µl of PBS (negative controls) or with pooled sera from mice immunized with PBS (normal serum negative controls) or with 10 µg of COPS:FliC (immune serum) diluted with PBS to 434 ELISA Units (EU) of anti-LPS IgG and 500,000 EU anti-FliC IgG per dose. Mice were infected IP 2–3 hours later with 5×105 CFU of S. Enteritidis R11. Mice were monitored for 21 days after challenge, recording overall health, weight loss and mortality. Moribund mice exhibiting signs including lethargy, non-responsiveness and ≥20% weight loss were euthanized and recorded as dead.

Serum Antibody Analysis

IgG levels against LPS or FliC were measured by ELISA, and end-point titers reported as ELISA units (EU)/ml, as previously described [4]. Seropositivity was defined as a titer four-fold above the Geometric Mean Titer (GMT) of sera from mice immunized with PBS.

Statistical Analysis

Serological responses of groups of mice were compared by Mann-Whitney rank sum test and mortality incidences by Fisher’s exact test (FET), using Sigma-Stat software package; p≤0.05 was considered significant.

Results

Humoral Response to Full and Fractional Dosages of COPS:FliC Conjugate

Higher geometric mean titers (GMT) and seropositive levels against both antigens were seen as a function of immunization with increasing amounts of COPS:FliC. Maximal titers (GMT = 2,000,000–6,000,000 EU/ml) and minimal animal-to-animal variation (100% seropositive) for anti-FliC IgG were achieved at dosages ≥0.25 µg (Fig. 1A). Mice immunized with 0.025 µg COPS:FliC (a 1/100th fractional dose), also exhibited elevated anti-FliC IgG compared to controls, but the titers were lower (GMT = 1,449 EU/ml) and in some mice no anti-FliC IgG antibody responses could be detected (only 75% of mice were seropositive post-immunization). Anti-LPS IgG titers were generally lower and with higher animal-to-animal variability compared to anti-FliC IgG titers (Figure 1). Immunization with 10 µg (4-fold dose) or 2.5 µg (a full dose) of COPS:FliC elicited anti-LPS IgG GMTs of 885 EU/ml and 308 EU/ml, respectively, whereas immunization with 0.25 µg (1/10th fractional dose) or 0.025 µg (1/100th fractional dose) resulted in GMT’s of <80 EU/ml. Anti-LPS IgG was detected in 92% of the mice immunized with the 10 µg dosage level and in 83% of mice given a 2.5 µg dose. However, less than 60% of animals became seropositive for anti-LPS IgG after immunization with 0.25 µg or 0.025 µg of COPS:FliC.

thumbnail
Download:
Figure 1. Serum titers and seropositive levels in mice receiving varying doses of COPS:FliC.

Mice were immunized with PBS or three doses of the indicated amount of COPS:FliC. Serum was obtained 21 days after the third dose, and levels of specific antibody in individual mice (grey diamonds), and the geometric mean titer (black rectangles) and percent seropositive (white rectangles) within groups were determined by ELISA for A. FliC, or B. LPS. Statistical significance (P values) for COPS:FliC immunized groups relative to PBS controls assessed by Mann-Whitney rank sum test are indicated.

https://doi.org/10.1371/journal.pone.0064680.g001

Protection after Immunization

IP challenge with wild type strain R11 caused 100% mortality in controls (Table 1). Mice actively immunized with three doses of COPS:FliC were significantly protected at all dosage levels tested (≥90% vaccine efficacy).

thumbnail
Download:
Table 1. Efficacy of different doses of Salmonella Enteritidis COPS:FliC conjugate in protecting mice from lethal challenge with wild-type S. Enteritidis R11a.

https://doi.org/10.1371/journal.pone.0064680.t001

Protection by Passive Immunization

Challenge with R11 caused >80% mortality in mice to whom normal serum or PBS was passively administered (Table 2). In contrast, mice to whom immune sera from COPS:FliC conjugate-immunized mice was passively transferred were significantly protected from fatal S. Enteritidis challenge, as <15% mortality was observed (p = 0.005 versus normal serum controls).

thumbnail
Download:
Table 2. Efficacy of passive immunization into naïve mice with sera from mice immunized with COPS:FliC in protecting mice from lethal challenge with wild-type S. Enteritidis R11a.

https://doi.org/10.1371/journal.pone.0064680.t002

Discussion

Vaccine strategies focused towards generating antibodies against Salmonella Typhi capsular polysaccharide are effective in preventing typhoid fever in humans. Unconjugated Vi polysaccharide vaccines are licensed for use in adults and older children and provide ∼55–60% protection for up to three years [9], before antibody levels plummet. A Vi-conjugate vaccine consisting of Vi linked to recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa was immunogenic in Vietnamese children, pre-school children, toddlers and infants [10], [11], [12]. In a large-scale, randomized, controlled field trial in pre-school children in Vietnam, the Vi-rEPA conjugate exhibited 89% efficacy over 46 months of follow-up [10], [13]. Following the pioneering path blazed by Vi-rEPA conjugate in preventing typhoid fever, COPS-carrier protein glycoconjugate vaccines are now being pursued as a strategy to prevent paratyphoid A fever and invasive NTS infections [14], [15], [16].

Parenteral conjugate vaccines evoke primarily humoral systemic immune responses. Although Salmonella are intracellular pathogens, they are vulnerable to antibodies while they are extracellular during bacteremic dissemination [17]. Various critical threshold levels of serum IgG to Vi have been proposed as a correlate of the protection elicited by Vi-based vaccines [9], [18], [19]. Nevertheless, the antibody mediators and mechanistic correlates of protection in humans against NTS are as yet undefined. Accumulating evidence indicates that anti-Salmonella antibodies function through two main (and measurable) mechanisms, direct serum bactericidal activity (SBA) via the C9 complement membrane attack complex, and opsonophagocytosis of bacteria into phagocytes.

The importance of SBA to NTS is unclear as isolates from blood of certain serovars are resistant to complement-mediated lysis through the expression of long-chain OPS and the protein encoded by the resistance to complement killing (rck) gene [20], [21]. Nevertheless, both complement-resistant and susceptible S. Typhimurium strains are similarly susceptible to opsonophagocytic uptake and killing by oxidative burst [4], [22], [23]. We reported opsonophagocytic activity in sera from mice immunized with COPS:FliC [4]. That passively transferred cell-free immune sera from mice immunized with the highest dose of COPS:FliC (10 µg) recapitulated the protection seen with active immunization indicates that protection is likely mediated by systemic antibodies.

Using the homologous FliC as the carrier protein for conjugation to COPS offers several advantages. Antibody titers to the FliC carrier protein were generally higher than those to COPS. Protection at lower vaccine dosage levels could be due to high antibody levels to FliC, or to modest antibody responses to both antigens if the biological activities of anti-COPS and anti-FliC work synergistically. If in future clinical trials Salmonella COPS:FliC glycoconjugates prove to be protective vaccines in humans, these possibilities should be considered in attempting to identify antibody titer cut-offs that constitute a threshold for protective immunity.

Immune responses directed at the COPS hapten and to a carrier protein representing a protective antigen of the homologous pathogen could function synergistically to limit immune escape. The possible selection of Vi-negative S. Typhi strains has been raised as constituting a potential theoretical consequence if Vi-based vaccines were to become widely used in populations in endemic areas [24], [25]. In some endemic areas, S. Typhi putatively lacking Vi have in rare instances been isolated from the blood [26], [27]. However, it is presumed that susceptibility to complement exerts selective pressure similarly for both S. Typhi Vi and NTS long-chain OPS expression [20], [28]. Monophasic variants of S. Typhimurium lacking phase 2 flagellin FljB have been reported [29]. Isolates presumed devoid of flagella (i.e., non-motile, H- strains) apparently derived from S. Typhimurium parents occur but are rare [29]. S. Typhimurium mutants deficient in flagellin are pathogenic after oral infection in mice, however expression of flagella is documented as an important virulence determinant that contributes to cell invasion and inflammation in vitro [30]. Most circulating S. Typhimurium, S. Enteritidis and other serovar NTS strains associated with invasive disease are expected to be vulnerable to both anti-FliC and anti-OPS antibodies. The primary results reported herein demonstrate the efficacy of small fractional doses of S. Enteritidis COPS:FliC conjugate and document that passively transferred antibodies confer protection. These observations provide further impetus for pursuing this conjugate vaccine strategy to control invasive NTS disease in young children in sub-Saharan Africa.

Author Contributions

Conceived and designed the experiments: RS JG ST ML MP MB. Performed the experiments: RS JW MT GR. Analyzed the data: RS. Wrote the paper: RS ML MP ST JG MB GR MT JW.

References

  1. 1. Jones TF, Ingram LA, Cieslak PR, Vugia DJ, Tobin-D'Angelo M, et al. (2008) Salmonellosis outcomes differ substantially by serotype. J Infect Dis 198: 109–114.
  2. 2. Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA (2012) Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa. The Lancet 379: 2489–2499.
  3. 3. Simon R, Tennant SM, Galen JE, Levine MM (2011) Mouse models to assess the efficacy of non-typhoidal Salmonella vaccines: Revisiting the role of host innate susceptibility and routes of challenge. Vaccine 29: 5094–106.
  4. 4. Simon R, Tennant SM, Wang JY, Schmidlein PJ, Lees A, et al. (2011) Salmonella enterica serovar Enteritidis core O polysaccharide conjugated to H:g,m flagellin as a candidate vaccine for protection against invasive infection with S. Enteritidis. Infect Immun 79: 4240–4249.
  5. 5. Watson DC, Robbins JB, Szu SC (1992) Protection of mice against Salmonella Typhimurium with an O-specific polysaccharide-protein conjugate vaccine. Infect Immun 60: 4679–4686.
  6. 6. Pollard AJ, Perrett KP, Beverley PC (2009) Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines. Nat Rev Immunol 9: 213–220.
  7. 7. Svenson SB, Nurminen M, Lindberg AA (1979) Artificial Salmonella vaccines: O-antigenic oligosaccharide-protein conjugates induce protection against infection with Salmonella Typhimurium. Infect Immun 25: 863–872.
  8. 8. Lees A, Nelson BL, Mond JJ (1996) Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents. Vaccine 14: 190–198.
  9. 9. Klugman KP, Koornhof HJ, Robbins JB, Le Cam NN (1996) Immunogenicity, efficacy and serological correlate of protection of Salmonella Typhi Vi capsular polysaccharide vaccine three years after immunization. Vaccine 14: 435–438.
  10. 10. Lin FY, Ho VA, Khiem HB, Trach DD, Bay PV, et al. (2001) The efficacy of a Salmonella Typhi Vi conjugate vaccine in two-to-five-year-old children. N Engl J Med 344: 1263–1269.
  11. 11. Canh DG, Lin FY, Thiem VD, Trach DD, Trong ND, et al. (2004) Effect of dosage on immunogenicity of a Vi conjugate vaccine injected twice into 2- to 5-year-old Vietnamese children. Infect Immun 72: 6586–6588.
  12. 12. Thiem VD, Lin FY, Canh do G, Son NH, Anh DD, et al. (2011) The Vi conjugate typhoid vaccine is safe, elicits protective levels of IgG anti-Vi, and is compatible with routine infant vaccines. Clin Vaccine Immunol 18: 730–735.
  13. 13. Mai NL, Phan VB, Vo AH, Tran CT, Lin FY, et al. (2003) Persistent efficacy of Vi conjugate vaccine against typhoid fever in young children. N Engl J Med 349: 1390–1391.
  14. 14. Simon R, Levine MM (2012) Glycoconjugate vaccine strategies for protection against invasive Salmonella infections. Human Vaccines and Immunotherapeutics 8: 494–498.
  15. 15. Martin LB (2012) Vaccines for typhoid fever and other salmonelloses. Curr Opin Infect Dis 25: 489–499.
  16. 16. Konadu EY, Lin FY, Ho VA, Thuy NT, Van Bay P, et al. (2000) Phase 1 and phase 2 studies of Salmonella enterica serovar Paratyphi A O-specific polysaccharide-tetanus toxoid conjugates in adults, teenagers, and 2- to 4-year-old children in Vietnam. Infect Immun 68: 1529–1534.
  17. 17. MacLennan CA, Gondwe EN, Msefula CL, Kingsley RA, Thomson NR, et al. (2008) The neglected role of antibody in protection against bacteremia caused by nontyphoidal strains of Salmonella in African children. J Clin Invest 118: 1553–1562.
  18. 18. Acosta CJ, Hong-Hui Y, Ning W, Qion G, Qun D, et al. (2005) Efficacy of a locally produced, Chinese Vi polysaccharide typhoid fever vaccine during six years of follow-up. Vaccine 23: 5618–5623.
  19. 19. Klugman KP, Gilbertson IT, Koornhof HJ, Robbins JB, Schneerson R, et al. (1987) Protective activity of Vi capsular polysaccharide vaccine against typhoid fever. Lancet 2: 1165–1169.
  20. 20. Grossman N, Schmetz MA, Foulds J, Klima EN, Jimenez-Lucho VE, et al. (1987) Lipopolysaccharide size and distribution determine serum resistance in Salmonella Montevideo. J Bacteriol 169: 856–863.
  21. 21. Roantree RJ, Rantz LA (1960) A study of the relationship of the normal bactericidal activity of human serum to bacterial infection. J Clin Invest 39: 72–81.
  22. 22. Gondwe EN, Molyneux ME, Goodall M, Graham SM, Mastroeni P, et al. (2010) Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans. Proc Natl Acad Sci U S A 107: 3070–3075.
  23. 23. Tennant SM, Wang JY, Galen JE, Simon R, Pasetti MF, et al. (2011) Engineering and preclinical evaluation of attenuated nontyphoidal Salmonella strains serving as live oral vaccines and as reagent strains. Infect Immun 79: 4175–4185.
  24. 24. Arya SC (1999) Efficacy of Vi polysaccharide vaccine against Salmonella Typhi. Vaccine 17: 1015–1016.
  25. 25. Arya SC (1997) Efficacy of Salmonella Typhi Vi capsular polysaccharide vaccine in South Africa. Vaccine 15: 244.
  26. 26. Baker S, Sarwar Y, Aziz H, Haque A, Ali A, et al. (2005) Detection of Vi-negative Salmonella enterica serovar Typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan. J Clin Microbiol 43: 4418–4425.
  27. 27. Mehta G, Arya SC (2002) Capsular Vi polysaccharide antigen in Salmonella enterica serovar Typhi isolates. J Clin Microbiol 40: 1127–1128.
  28. 28. Robbins JD, Robbins JB (1984) Reexamination of the protective role of the capsular polysaccharide (Vi antigen) of Salmonella Typhi. J Infect Dis 150: 436–449.
  29. 29. Levy H, Diallo S, Tennant SM, Livio S, Sow SO, et al. (2008) PCR method to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella isolates from the blood of patients with clinical enteric fever. J Clin Microbiol 46: 1861–1866.
  30. 30. Schmitt CK, Ikeda JS, Darnell SC, Watson PR, Bispham J, et al. (2001) Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect Immun 69: 5619–5625.