Attenuation of Salmonella enterica Serovar Typhimurium by Altering Biological Functions of Murein Lipoprotein and Lipopolysaccharide
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感染与免疫杂志 2005年第12期
Departments of Microbiology and Immunology Pathology, University of Texas Medical Branch, Galveston, Texas 77555-1070
ABSTRACT
We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.
TEXT
Braun/murein lipoprotein (Lpp) and lipopolysaccharide (LPS) synergize to produce proinflammatory cytokines in primary macrophages and LPS-nonresponsive mice (10). We recently demonstrated the presence of two functional copies of the lpp gene (lppA and lppB) on the chromosome of Salmonella enterica serovar Typhimurium 14028. When compared to wild-type (WT) S. enterica serovar Typhimurium, the lppAB double-knockout (DKO) mutant was found to be highly attenuated in both in vitro and in vivo models (7).
To understand the relative contributions of lppA and lppB genes in Salmonella virulence, we generated lppA and lppB single-knockout (SKO) mutants (2, 7). Although the invasive potentials of the lppA and lppB SKO mutants for human colonic epithelial cells (T84) were similar but significantly higher than that of the lppAB DKO mutant, which was minimally invasive (2, 7), LppA appeared more potent than LppB in vivo (2). Subsequently, we constructed an lppA lppB msbB triple-knockout (TKO) mutant of S. enterica serovar Typhimurium (2) to minimize LPS-induced cellular reactions in the host. The msbB (multicopy repressor of the htrB [high-temperature requirement]) gene encodes an enzyme required for attaching myristic acid to the lipid A moiety of LPS, thereby enhancing LPS's biological activity (1, 8). Although highly attenuated, the lppA lppB msbB TKO mutant did not survive the host's hostile environment (2).
In this study, we developed and characterized lppA msbB and lppB msbB DKO mutants and expected that such mutants would be highly attenuated but able to survive inside the host. Our future goal is to test and compare such newly developed mutants with other known Salmonella mutants (e.g., phoP/Q and aroA) as candidates for vaccine development and examine their ability to deliver heterologous antigens. Further, we will also use genomic and proteomic approaches to understand Lpp's role in S. enterica serovar Typhimurium virulence and host responses.
Construction and in vitro characterization of lppA msbB and lppB msbB DKO mutants. The strategy for constructing lppA msbB and lppB msbB DKO targeted mutants was similar to that used to develop lppA and lppB SKO mutants as we recently described using a pDMS197 suicide vector and an Escherichia coli S17-1pir strain (7). Subsequently, the recombinant plasmids from the E. coli S17-1pir strain were delivered to an S enterica serovar Typhimurium YS1 strain (msbB deficient) (3). The transconjugants were selected on MSB agar plates containing the appropriate antibiotics and sucrose (4). We confirmed the identity of lppA msbB and lppB msbB DKO mutants by PCR and Southern and Western blot analyses (7). The relevant features of the cultures used were described in our recent publications (2, 7).
The invasive ability of lppA msbB and lppB msbB DKO mutants, as determined by a gentamicin protection assay, was 1,000-fold lower in T84 cells, a finding similar to that with the lppAB DKO mutant (P 0.003 by the Student's t test; data not shown) (2, 7) when compared to findings with the WT S. enterica serovar Typhimurium. The levels of invasion of T84 cells by the msbB (P = 0.03), as well as by the lppA or lppB, SKO mutant (P = 0.004) were reduced 10- and 100-fold, respectively, compared to invasion by WT S. enterica serovar Typhimurium (data not shown). We also showed that these effects on invasion of lppAB and msbB genes could be complemented (7).
Similarly, motility of the lppA msbB and lppB msbB DKO mutants (data not shown) was significantly less (50%; P = 0.04) than that of WT S. enterica serovar Typhimurium, and the lppAB DKO mutant was nonmotile (2, 7). Interestingly, the msbB SKO mutant was highly motile, even more so than WT S. enterica serovar Typhimurium (by 69%; P = 0.02), while the lppA and lppB SKO mutants exhibited motility 34% less (P = 0.05) than that of the WT bacterium. The number of flagella could have been affected in the msbB SKO mutant, causing its increased motility (9). Although the lppA msbB and lppB msbB DKO mutants were more motile than the lppAB DKO mutant (P = 0.008), they were not highly motile like the msbB SKO mutant, indicating a predominant effect of Lpp on bacterial motility. The decreased invasion rate was attributed to neither a lesser binding of the mutants (lppA msbB and lppB msbB) to the host cells nor a motility defect (7; data not shown).
Tumor necrosis factor alpha (TNF-) levels, detected in the supernatants of RAW264.7 murine macrophages infected with the heat-killed lppA msbB or lppB msbB DKO mutant (2, 7), were reduced by 60 to 70% compared to the level induced by the WT S. enterica serovar Typhimurium (P = 0.003), as determined by enzyme-linked immunosorbent assay (7) (Fig. 1A). TNF- levels with the msbB SKO mutant were reduced by 35%, which was similar to levels in macrophages infected with the lppA and lppB SKO mutants compared to those in WT bacterium-infected macrophages (P = 0.02) (Fig. 1A). Similarly, T84 cells infected with live lppA msbB and lppB msbB DKO mutants produced significantly lower interleukin-8 (IL-8) levels compared to levels in WT S. enterica serovar Typhimurium-infected cells (Fig. 1B). On the other hand, T84 cells infected with either the msbB, lppA, and lppB SKO mutants or the WT bacterium produced similar amounts of IL-8 (2, 6). IL-8 production was not invasion related, since similar levels were detected when T84 cells were incubated with either live or heat-killed lppA msbB or lppB msbB DKO mutants.
In vivo characterization of lppA msbB and lppB msbB DKO mutants. We chose the lppB msbB DKO mutant for immunization studies because the LppA SKO mutant was more potent (i) in inducing lethality in mice at higher doses and (ii) in producing more cytokines in vivo compared to the LppB SKO mutant (2). However, for other in vivo studies, we used both lppA msbB and lppB msbB DKO mutants.
Mice (8-week-old female C57BL6 mice; 20 per group) were inoculated intraperitoneally with 1 x 104 CFU of the WT, lppA msbB, or lppB msbB DKO mutants of S. enterica serovar Typhimurium. Similarly, mice were infected with lppA, lppB, and msbB SKO mutants. One group was inoculated with phosphate-buffered saline (PBS) as a control. Five mice from each group were bled and sacrificed at days 1, 3, 4, and 7 postinfection, and their livers and spleens were examined for bacterial survival and multiplication (2). Sections from these organs were fixed in 10% buffered formalin and processed for hematoxylin-and-eosin staining. Histopathological examination of tissue sections was performed using a four- and five-tiered (for spleen and liver, respectively) grading system (2). Serum levels of cytokines/chemokines (TNF-, gamma interferon [IFN-], and KC [equivalent of human IL-8]) were assayed with a Cytometric bead array kit (2).
In another experiment, nine groups of mice were used, one of which was a PBS-injected group as a noninfected control. The remaining eight groups of mice were infected with either one of four different doses (1 x 103, 1 x 104, 1 x 105, and 1 x 106 CFU) of WT bacteria or its lppB msbB mutant by the intraperitoneal route (5). After 8 weeks, all surviving mice in the lppB msbB mutant-immunized group were challenged with the highest dose of WT S. enterica serovar Typhimurium (1 x 106 CFU). The PBS control group of mice was also challenged with the highest dose of WT S. enterica serovar Typhimurium after 8 weeks. Deaths during the next month in all groups were recorded.
All of the mice inoculated with WT S. enterica serovar Typhimurium died at the dose level of 1 x 103 CFU. The group infected with even a 3-log-higher dose of lppB msbB DKO mutant had no deaths (data not shown). On the other hand, mice immunized with 1 x 103, 1 x 104, 1 x 105, and 1 x106 CFU of the lppB msbB DKO mutant were protected (in a dose-dependent manner [10, 40, 90, and 100%]) against challenge with a lethal dose of WT S. enterica serovar Typhimurium (1 x 106 CFU). The nonimmunized group of mice, which were instead given PBS, died within 3 days after challenge with WT S. enterica serovar Typhimurium.
Quantitation of bacteria in livers and spleens of mice infected with lppA msbB and lppB msbB DKO mutants indicated these strains could survive and replicate under in vivo conditions, unlike the lppA lppB msbB TKO mutant (Fig. 2). As shown by the number of bacteria at day 1 postinfection, more WT bacteria invaded the liver and spleen (2.7 to 3.7 logs) than did either the lppA msbB or lppB msbB DKO mutant bacteria (1.9 to 2 logs), indicating attenuation in the invasive ability of these mutants (Fig. 2).
TNF- production, compared to that of the noninfected control, increased in the sera of mice infected with the WT bacterium for 4 days postinoculation (Fig. 3A). Serum TNF- levels were reduced (94 to 96%) in mice infected with lppA msbB and lppB msbB DKO mutants and were similar to those in lppA and lppB SKO mutants on day 4 postinfection, compared to that of mice infected with the WT S. enterica serovar Typhimurium. Although the TNF- levels induced with the msbB SKO mutant were 82% lower, compared to animals infected with the WT bacterium on day 4 after infection, similar levels of this cytokine (444 versus 694 pg/ml) were noted in WT versus msbB SKO mutant-infected mice after 3 days.
Similarly, IFN- levels peaked on day 3 postinfection in mice infected with the WT S. enterica serovar Typhimurium, but the lppA msbB and lppB msbB DKO mutants were only able to induce a minimum level of this cytokine (Fig. 3B). While IFN- levels decreased by 41% in mice infected with the WT bacterium on day 3 versus day 4, they increased to 3 times greater levels (from 80 to 231 pg/ml) on day 4 compared to day 3 postinfection with the msbB SKO mutant.
As shown in Fig. 3C, KC induction was highly reduced in mice infected with the lppA msbB and lppB msbB DKO mutants (98%), compared to WT Salmonella. A marginal increase in the KC level was noted in the sera of mice infected with the msbB SKO mutant over 4 days; however, these levels were much lower than those in mice infected with the WT bacterium. Overall these data indicated a greater contribution of Lpp in inducing these cytokines/chemokines, compared to MsbB, and that the Lpp/MsbB DKO mutants produced minimal host responses. These data were further confirmed by histopathological examination of tissue sections from mice infected with the lppA msbB and lppB msbB DKO mutants (score of 1+ versus 4+ for the WT bacterium for liver and 1+ versus 3+ for spleen). We observed similar histopathological changes with the lppAB DKO mutant (2).
Collectively, our data indicated that Salmonella strains with deletions in msbB and either of the lpp genes were highly attenuated. Immunological characterization of such mutants is crucial to determine their suitability as live attenuated vaccines against salmonellosis or to deliver heterologous antigens and compare them with existing mutants for efficacy.
ACKNOWLEDGMENTS
This work was supported by a grant from the Advanced Technology Program of the Texas Higher Education Coordinating Board that was awarded to A.K.C. A.A.F. is a recipient of the McLaughlin Postdoctoral Fellowship, which supported part of this research. C.L.G. is an NSF predoctoral fellow.
We thank Mardelle Susman for editing the manuscript.
REFERENCES
1. Clementz, T., Z. Zhou, and C. R. Raetz. 1997. Function of the Escherichia coli msbB gene, a multicopy suppressor of htrB knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate incorporation by HtrB. J. Biol. Chem. 272:10353-10360.
2. Fadl, A. A., J. Sha, G. R. Klimpel, J. P. Olano, D. W. Niesel, and A. K. Chopra. 2005. Murein lipoprotein is a critical outer membrane component involved in Salmonella enterica serovar Typhimurium systemic infection. Infect. Immun. 73:1081-1096.
3. Low, K. B., M. Ittensohn, T. Le, J. Platt, S. Sodi, M. Amoss, O. Ash, E. Carmichael, A. Chakraborty, J. Fischer, S. L. Lin, X. Luo, S. I. Miller, L. Zheng, I. King, J. M. Pawelek, and D. Bermudes. 1999. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat. Biotechnol. 17:37-41.
4. Murray, S. R., D. Bermudes, K. S. de Felipe, and K. B. Low. 2001. Extragenic suppressors of growth defects in msbB Salmonella. J. Bacteriol. 183:5554-5561.
5. Reed, L., and H. Muench. 1938. A simple method of estimating fifty per cent end-points. Am. J. Hygiene 27:493-497.
6. Schuerer-Maly, C. C., L. Eckmann, M. F. Kagnoff, M. T. Falco, and F. E. Maly. 1994. Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide. Immunology 81:85-91.
7. Sha, J., A. A. Fadl, G. R. Klimpel, D. W. Niesel, V. L. Popov, and A. K. Chopra. 2004. The two murein lipoproteins of Salmonella enterica serovar Typhimurium contribute to the virulence of the organism. Infect. Immun. 72:3987-4003.
8. Somerville, J. E., Jr., L. Cassiano, B. Bainbridge, M. D. Cunningham, and R. P. Darveau. 1996. A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide. J. Clin. Investig. 97:359-365.
9. Sunshine, M. G., B. W. Gibson, J. J. Engstrom, W. A. Nichols, B. D. Jones, and M. A. Apicella. 1997. Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization. J. Bacteriol. 179:5521-5533.
10. Zhang, H., J. W. Peterson, D. W. Niesel, and G. R. Klimpel. 1997. Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production. J. Immunol. 159:4868-4878.(A. A. Fadl, J. Sha, G. R.)
ABSTRACT
We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.
TEXT
Braun/murein lipoprotein (Lpp) and lipopolysaccharide (LPS) synergize to produce proinflammatory cytokines in primary macrophages and LPS-nonresponsive mice (10). We recently demonstrated the presence of two functional copies of the lpp gene (lppA and lppB) on the chromosome of Salmonella enterica serovar Typhimurium 14028. When compared to wild-type (WT) S. enterica serovar Typhimurium, the lppAB double-knockout (DKO) mutant was found to be highly attenuated in both in vitro and in vivo models (7).
To understand the relative contributions of lppA and lppB genes in Salmonella virulence, we generated lppA and lppB single-knockout (SKO) mutants (2, 7). Although the invasive potentials of the lppA and lppB SKO mutants for human colonic epithelial cells (T84) were similar but significantly higher than that of the lppAB DKO mutant, which was minimally invasive (2, 7), LppA appeared more potent than LppB in vivo (2). Subsequently, we constructed an lppA lppB msbB triple-knockout (TKO) mutant of S. enterica serovar Typhimurium (2) to minimize LPS-induced cellular reactions in the host. The msbB (multicopy repressor of the htrB [high-temperature requirement]) gene encodes an enzyme required for attaching myristic acid to the lipid A moiety of LPS, thereby enhancing LPS's biological activity (1, 8). Although highly attenuated, the lppA lppB msbB TKO mutant did not survive the host's hostile environment (2).
In this study, we developed and characterized lppA msbB and lppB msbB DKO mutants and expected that such mutants would be highly attenuated but able to survive inside the host. Our future goal is to test and compare such newly developed mutants with other known Salmonella mutants (e.g., phoP/Q and aroA) as candidates for vaccine development and examine their ability to deliver heterologous antigens. Further, we will also use genomic and proteomic approaches to understand Lpp's role in S. enterica serovar Typhimurium virulence and host responses.
Construction and in vitro characterization of lppA msbB and lppB msbB DKO mutants. The strategy for constructing lppA msbB and lppB msbB DKO targeted mutants was similar to that used to develop lppA and lppB SKO mutants as we recently described using a pDMS197 suicide vector and an Escherichia coli S17-1pir strain (7). Subsequently, the recombinant plasmids from the E. coli S17-1pir strain were delivered to an S enterica serovar Typhimurium YS1 strain (msbB deficient) (3). The transconjugants were selected on MSB agar plates containing the appropriate antibiotics and sucrose (4). We confirmed the identity of lppA msbB and lppB msbB DKO mutants by PCR and Southern and Western blot analyses (7). The relevant features of the cultures used were described in our recent publications (2, 7).
The invasive ability of lppA msbB and lppB msbB DKO mutants, as determined by a gentamicin protection assay, was 1,000-fold lower in T84 cells, a finding similar to that with the lppAB DKO mutant (P 0.003 by the Student's t test; data not shown) (2, 7) when compared to findings with the WT S. enterica serovar Typhimurium. The levels of invasion of T84 cells by the msbB (P = 0.03), as well as by the lppA or lppB, SKO mutant (P = 0.004) were reduced 10- and 100-fold, respectively, compared to invasion by WT S. enterica serovar Typhimurium (data not shown). We also showed that these effects on invasion of lppAB and msbB genes could be complemented (7).
Similarly, motility of the lppA msbB and lppB msbB DKO mutants (data not shown) was significantly less (50%; P = 0.04) than that of WT S. enterica serovar Typhimurium, and the lppAB DKO mutant was nonmotile (2, 7). Interestingly, the msbB SKO mutant was highly motile, even more so than WT S. enterica serovar Typhimurium (by 69%; P = 0.02), while the lppA and lppB SKO mutants exhibited motility 34% less (P = 0.05) than that of the WT bacterium. The number of flagella could have been affected in the msbB SKO mutant, causing its increased motility (9). Although the lppA msbB and lppB msbB DKO mutants were more motile than the lppAB DKO mutant (P = 0.008), they were not highly motile like the msbB SKO mutant, indicating a predominant effect of Lpp on bacterial motility. The decreased invasion rate was attributed to neither a lesser binding of the mutants (lppA msbB and lppB msbB) to the host cells nor a motility defect (7; data not shown).
Tumor necrosis factor alpha (TNF-) levels, detected in the supernatants of RAW264.7 murine macrophages infected with the heat-killed lppA msbB or lppB msbB DKO mutant (2, 7), were reduced by 60 to 70% compared to the level induced by the WT S. enterica serovar Typhimurium (P = 0.003), as determined by enzyme-linked immunosorbent assay (7) (Fig. 1A). TNF- levels with the msbB SKO mutant were reduced by 35%, which was similar to levels in macrophages infected with the lppA and lppB SKO mutants compared to those in WT bacterium-infected macrophages (P = 0.02) (Fig. 1A). Similarly, T84 cells infected with live lppA msbB and lppB msbB DKO mutants produced significantly lower interleukin-8 (IL-8) levels compared to levels in WT S. enterica serovar Typhimurium-infected cells (Fig. 1B). On the other hand, T84 cells infected with either the msbB, lppA, and lppB SKO mutants or the WT bacterium produced similar amounts of IL-8 (2, 6). IL-8 production was not invasion related, since similar levels were detected when T84 cells were incubated with either live or heat-killed lppA msbB or lppB msbB DKO mutants.
In vivo characterization of lppA msbB and lppB msbB DKO mutants. We chose the lppB msbB DKO mutant for immunization studies because the LppA SKO mutant was more potent (i) in inducing lethality in mice at higher doses and (ii) in producing more cytokines in vivo compared to the LppB SKO mutant (2). However, for other in vivo studies, we used both lppA msbB and lppB msbB DKO mutants.
Mice (8-week-old female C57BL6 mice; 20 per group) were inoculated intraperitoneally with 1 x 104 CFU of the WT, lppA msbB, or lppB msbB DKO mutants of S. enterica serovar Typhimurium. Similarly, mice were infected with lppA, lppB, and msbB SKO mutants. One group was inoculated with phosphate-buffered saline (PBS) as a control. Five mice from each group were bled and sacrificed at days 1, 3, 4, and 7 postinfection, and their livers and spleens were examined for bacterial survival and multiplication (2). Sections from these organs were fixed in 10% buffered formalin and processed for hematoxylin-and-eosin staining. Histopathological examination of tissue sections was performed using a four- and five-tiered (for spleen and liver, respectively) grading system (2). Serum levels of cytokines/chemokines (TNF-, gamma interferon [IFN-], and KC [equivalent of human IL-8]) were assayed with a Cytometric bead array kit (2).
In another experiment, nine groups of mice were used, one of which was a PBS-injected group as a noninfected control. The remaining eight groups of mice were infected with either one of four different doses (1 x 103, 1 x 104, 1 x 105, and 1 x 106 CFU) of WT bacteria or its lppB msbB mutant by the intraperitoneal route (5). After 8 weeks, all surviving mice in the lppB msbB mutant-immunized group were challenged with the highest dose of WT S. enterica serovar Typhimurium (1 x 106 CFU). The PBS control group of mice was also challenged with the highest dose of WT S. enterica serovar Typhimurium after 8 weeks. Deaths during the next month in all groups were recorded.
All of the mice inoculated with WT S. enterica serovar Typhimurium died at the dose level of 1 x 103 CFU. The group infected with even a 3-log-higher dose of lppB msbB DKO mutant had no deaths (data not shown). On the other hand, mice immunized with 1 x 103, 1 x 104, 1 x 105, and 1 x106 CFU of the lppB msbB DKO mutant were protected (in a dose-dependent manner [10, 40, 90, and 100%]) against challenge with a lethal dose of WT S. enterica serovar Typhimurium (1 x 106 CFU). The nonimmunized group of mice, which were instead given PBS, died within 3 days after challenge with WT S. enterica serovar Typhimurium.
Quantitation of bacteria in livers and spleens of mice infected with lppA msbB and lppB msbB DKO mutants indicated these strains could survive and replicate under in vivo conditions, unlike the lppA lppB msbB TKO mutant (Fig. 2). As shown by the number of bacteria at day 1 postinfection, more WT bacteria invaded the liver and spleen (2.7 to 3.7 logs) than did either the lppA msbB or lppB msbB DKO mutant bacteria (1.9 to 2 logs), indicating attenuation in the invasive ability of these mutants (Fig. 2).
TNF- production, compared to that of the noninfected control, increased in the sera of mice infected with the WT bacterium for 4 days postinoculation (Fig. 3A). Serum TNF- levels were reduced (94 to 96%) in mice infected with lppA msbB and lppB msbB DKO mutants and were similar to those in lppA and lppB SKO mutants on day 4 postinfection, compared to that of mice infected with the WT S. enterica serovar Typhimurium. Although the TNF- levels induced with the msbB SKO mutant were 82% lower, compared to animals infected with the WT bacterium on day 4 after infection, similar levels of this cytokine (444 versus 694 pg/ml) were noted in WT versus msbB SKO mutant-infected mice after 3 days.
Similarly, IFN- levels peaked on day 3 postinfection in mice infected with the WT S. enterica serovar Typhimurium, but the lppA msbB and lppB msbB DKO mutants were only able to induce a minimum level of this cytokine (Fig. 3B). While IFN- levels decreased by 41% in mice infected with the WT bacterium on day 3 versus day 4, they increased to 3 times greater levels (from 80 to 231 pg/ml) on day 4 compared to day 3 postinfection with the msbB SKO mutant.
As shown in Fig. 3C, KC induction was highly reduced in mice infected with the lppA msbB and lppB msbB DKO mutants (98%), compared to WT Salmonella. A marginal increase in the KC level was noted in the sera of mice infected with the msbB SKO mutant over 4 days; however, these levels were much lower than those in mice infected with the WT bacterium. Overall these data indicated a greater contribution of Lpp in inducing these cytokines/chemokines, compared to MsbB, and that the Lpp/MsbB DKO mutants produced minimal host responses. These data were further confirmed by histopathological examination of tissue sections from mice infected with the lppA msbB and lppB msbB DKO mutants (score of 1+ versus 4+ for the WT bacterium for liver and 1+ versus 3+ for spleen). We observed similar histopathological changes with the lppAB DKO mutant (2).
Collectively, our data indicated that Salmonella strains with deletions in msbB and either of the lpp genes were highly attenuated. Immunological characterization of such mutants is crucial to determine their suitability as live attenuated vaccines against salmonellosis or to deliver heterologous antigens and compare them with existing mutants for efficacy.
ACKNOWLEDGMENTS
This work was supported by a grant from the Advanced Technology Program of the Texas Higher Education Coordinating Board that was awarded to A.K.C. A.A.F. is a recipient of the McLaughlin Postdoctoral Fellowship, which supported part of this research. C.L.G. is an NSF predoctoral fellow.
We thank Mardelle Susman for editing the manuscript.
REFERENCES
1. Clementz, T., Z. Zhou, and C. R. Raetz. 1997. Function of the Escherichia coli msbB gene, a multicopy suppressor of htrB knockouts, in the acylation of lipid A. Acylation by MsbB follows laurate incorporation by HtrB. J. Biol. Chem. 272:10353-10360.
2. Fadl, A. A., J. Sha, G. R. Klimpel, J. P. Olano, D. W. Niesel, and A. K. Chopra. 2005. Murein lipoprotein is a critical outer membrane component involved in Salmonella enterica serovar Typhimurium systemic infection. Infect. Immun. 73:1081-1096.
3. Low, K. B., M. Ittensohn, T. Le, J. Platt, S. Sodi, M. Amoss, O. Ash, E. Carmichael, A. Chakraborty, J. Fischer, S. L. Lin, X. Luo, S. I. Miller, L. Zheng, I. King, J. M. Pawelek, and D. Bermudes. 1999. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat. Biotechnol. 17:37-41.
4. Murray, S. R., D. Bermudes, K. S. de Felipe, and K. B. Low. 2001. Extragenic suppressors of growth defects in msbB Salmonella. J. Bacteriol. 183:5554-5561.
5. Reed, L., and H. Muench. 1938. A simple method of estimating fifty per cent end-points. Am. J. Hygiene 27:493-497.
6. Schuerer-Maly, C. C., L. Eckmann, M. F. Kagnoff, M. T. Falco, and F. E. Maly. 1994. Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide. Immunology 81:85-91.
7. Sha, J., A. A. Fadl, G. R. Klimpel, D. W. Niesel, V. L. Popov, and A. K. Chopra. 2004. The two murein lipoproteins of Salmonella enterica serovar Typhimurium contribute to the virulence of the organism. Infect. Immun. 72:3987-4003.
8. Somerville, J. E., Jr., L. Cassiano, B. Bainbridge, M. D. Cunningham, and R. P. Darveau. 1996. A novel Escherichia coli lipid A mutant that produces an antiinflammatory lipopolysaccharide. J. Clin. Investig. 97:359-365.
9. Sunshine, M. G., B. W. Gibson, J. J. Engstrom, W. A. Nichols, B. D. Jones, and M. A. Apicella. 1997. Mutation of the htrB gene in a virulent Salmonella typhimurium strain by intergeneric transduction: strain construction and phenotypic characterization. J. Bacteriol. 179:5521-5533.
10. Zhang, H., J. W. Peterson, D. W. Niesel, and G. R. Klimpel. 1997. Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production. J. Immunol. 159:4868-4878.(A. A. Fadl, J. Sha, G. R.)