IAI FigSearch
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, Y. R.
Right arrow Articles by Fierer, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, Y. R.
Right arrow Articles by Fierer, J.
Infection and Immunity, April 2006, p. 2498-2502, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2498-2502.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mutation of Phosphotransacetylase but Not Isocitrate Lyase Reduces the Virulence of Salmonella enterica Serovar Typhimurium in Mice

Yang Re Kim,1,{dagger} Shaun R. Brinsmade,2 Zheng Yang,1 Jorge Escalante-Semerena,2 and Joshua Fierer1,3*

VA Healthcare, San Diego, California,1 Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin and,2 Department of Medicine, University of California at San Diego School of Medicine, San Diego, California3

Received 21 June 2005/ Returned for modification 21 July 2005/ Accepted 10 January 2006


   ABSTRACT
Top
 Abstract
Text
 References
 
A phosphotransacetylase (pta) mutant of Salmonella enterica serovar Typhimurium was attenuated in mice but survived normally in macrophages. Complementation of the pta mutation in trans restored virulence. An isocitrate lyase (aceA) mutant was virulent, so the inability to use acetate as a sole carbon source does not explain the phenotype.


   TEXT
Top
 Abstract
 Text
References
 
Isocitrate lyase (Icl) catalyzes the first step in the glyoxylate shunt that enables many bacteria, including Salmonella enterica, to use acetate as both a carbon and an energy source (22). Munoz-Elias and McKinney recently reported that icl mutants of Mycobacterium tuberculosis are killed by mouse macrophages and are avirulent in mice (16). To determine whether isocitrate lyase (aceA) plays an analogous role in S. enterica pathogenesis, we made mutants of S. enterica serovar Typhimurium 14028s. The mutant cannot utilize acetate or propionate as a sole carbon source (Table 1). As controls, we inactivated the pta gene, which encodes phosphotransacetylase (Pta), the enzyme responsible for the interconversion of acetyl coenzyme A (CoA) and acetyl phosphate (1); the pta strain cannot grow in high acetate concentrations (~50 mM) (11, 21). The Pta enzyme also catalyzes the interconversion of propionyl-P and propionyl-CoA during the anaerobic degradation of L-threonine and during the catabolism of odd-chain fatty acids (7). Pta activity is needed to recapture propionate excreted by S. enterica during growth on 1,2-propanediol (17). In S. enterica expression of the 1,2-propanediol utilization (pdu) genes has been linked by in vivo expression technology studies to reduced fitness during infection (4). As a control for the inability to utilize propionate, we mutated prpB, which encodes the 2-methylisocitrate lyase that catalyzes the conversion of 2-methylisocitrate to pyruvate and succinic acid, a necessary step in the metabolism of propionic acid (6, 8). We were also prompted by the recently established connection between short-chain fatty acid catabolism and sirtuin (19) to investigate the role of cobB (sirtuin protein deacetylase [20]) in virulence.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Strains used

 
There was no significant difference in the growth of aceA, prpB, and cobB mutants in LB broth at 37°C compared to strain 14028s, except that the cob/pta and pta mutants had slightly increased lag times. All strains reached the same optical density by 8 h (Fig. 1). However, the growth of the cobB/pta and pta mutants was significantly reduced in LB broth under anaerobic conditions (10 ml of autoclaved broth injected into sterile Vacutainer tubes with methylene blue as an Eh indicator), and they reached only half the density of strain 14028s after 8 h. This did not occur in anaerobic Trypticase soy broth (TSB), which has added glucose, suggesting that pta is required for anaerobic growth on amino acids (7).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Growth curves for strain 14028s and for five mutants grown aerobically and anaerobically in LB and TSB at 37°C. Anaerobic conditions were achieved by boiling the media to drive off dissolved air and injecting the hot broth into sterile, stoppered 16-by-125-mm vacuum tubes (Vacutainer; BD). The anaerobic growth of pta mutant in LB broth was slower and still did not reach the level of the control strain even after 24 h.

 
To test for virulence in mice, bacteria were grown in overnight at 37°C in TSB, washed twice in PBS, and resuspended in sterile saline. aceA and prpB mutants were fully virulent in genetically susceptible BALB/cJ that have a mutant Nramp1 (18) (Fig. 2A and B). In order to determine whether the aceA mutant was attenuated in immune mice, as was reported by McKinney et al. for M. tuberculosis isocitrate lyase 1 gene mutants (15), we infected congenic, resistant BALB/c.Nramp1 mice that express the wild-type Nramp1 gene from DBA/2 mice (18). We extended the experiment to 21 days after infection, well into the acquired immunity phase of the infection. Neither the prpB nor the aceA mutants were attenuated in these resistant mice, as determined by enumerating the CFU in livers and spleens (Fig. 3). We concluded that, unlike M. tuberculosis (15), isocitrate lyase is not required for salmonellae to survive in immune mice and that the ability to utilize acetate or propionate as an energy source is not required for S. enterica virulence. This finding is in agreement with the recent report of Fang et al. that an aceA mutant of serovar Typhimurium was not attenuated when injected intraperitoneally into C3H/HeN mice (5). However, these authors found that prolonged survival of an aroA mutant of strain 14028s in the mesenteric lymph nodes of 129sv mice depended on aroA, although the effect was only apparent in these nodes and after 4 weeks (5).


Figure 2
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Survival curves for BALB/c mice infected intraperitoneally with ~30 CFU of either strain 14028s or one of the mutants. The survival curves for the prbB (A) and aceA (B) mutants did not differ significantly from the control organism, strain 14028s (eight mice/group). (C) Mice (n = 15 to 17) infected with the cob/pta or pta mutant survived significantly longer than the mice infected with strain 14028s (P < 0.01). There was no significant difference in the survival of mice infected with the cob/pta double mutant and the pta mutant (P > 0.2).

 

Figure 3
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3. Isocitrate lyase (aceA) and prpB are not required for virulence in genetically resistant BALB/c.D2 mice. Mice were infected intraperitoneally with 2 x 104 14028s organisms or one of the mutants. Six mice in each group were sacrificed on days 5 (not shown), 14, and 21 after infection. There were no significant differences between CFU counts in either the spleens or the livers of mice infected with the two strains at any of these time points.

 
Although the aceA mutant was virulent in both genetically resistant and susceptible mice, one of our control strains, the cobB/pta mutant, was attenuated in BALB/c mice (Fig. 2C). We then used P22 transduction to construct strains carrying each one of those mutations. The cobB mutant was fully virulent (not shown), but the pta mutant was attenuated to the same extent as the double mutant (Fig. 2C). To establish the relationship between lethality and bacterial growth in vivo, we infected BALB/c mice with the pta mutant (95 CFU) and strain 14028s (65 CFU). Three days after infection there were 1,000-fold more viable 14028s than pta mutant organisms (5.25 ± 0.58 versus 2.18 ± 0.44), and all of the mice infected with strain 14028s were dead by day 4. Mice infected with the pta mutant survived until day 6 after infection but by then they had >106 CFU/spleen, all of which were still resistant to antibiotics and unable to grow on acetate. These results show that although the pta mutant grew more slowly in BALB/c mice than 14028s, it ultimately killed them. Thus, the pta mutant was partially attenuated in these genetically susceptible mice.

We coinfected BALB/c.D2 mice intraperitoneally with equal numbers of the pta mutant and strain 14028s, and after only 1 day there were more 14028s organisms in the spleens, although the difference in livers was not statistically significant. At all later time points there were only 2 to 6% as many pta as 14028s in spleens and livers, and the differences were highly significant (Fig. 4). The pta mutant was similarly impaired in BALB/c.D2 mice when the inoculum was grown to mid-log phase and if mice were infected intravenously (not shown). To confirm that the pta mutation was responsible for the attenuation of the mutant, we complemented the mutation in trans with the plasmid pPTA15 (Table 1), which restored the ability to grow on acetate and to grow in anaerobic LB broth. The complemented strain was also virulent in BALB/c.D2 mice; the competitive index (CI) for the complemented strain was nearly 0 (Fig. 5).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		FIG. 4. The pta mutant is attenuated in BALB/c.D2 mice. A comparison of the CFU of pta mutant and 14028s recovered from spleens and livers 1, 3, 5, 10, and 21 days after infection is shown. There were six mice in each group, and the geometric means ± the standard errors of the mean are shown. All differences between the groups were highly significant (P < 0.01), except on day 1 after infection, when the statistical significance differences were P = 0.05 in the spleen and P = 0.5 in the liver.

 

Figure 5
View larger version (8K):
[in this window]
[in a new window]
  		FIG. 5. Restoration of phosphotransacetylase function in trans restores the virulence of pta mutant. The CI was calculated for either the pta mutant or the mutant complemented in trans (ptacomp) compared to strain 14028s. BALB/c.D2 mice were infected intraperitoneally with 4 x 104 14028s organisms, and 10 days later the animals were sacrificed. Each point is the CI from an individual mouse (log transformed), and the bars show the geometric mean. A CI of 0 means that the two strains were recovered in the same ratio as they were injected. A CI of –2 means that 100-fold more 14028s organisms were recovered. The ratio is nearly –2 for the pta mutant but nearly 0 when pta was complemented in trans. We obtained similar results on days 5 and 20 after infection.

 
Since S. enterica is a facultative intracellular pathogen that grows inside macrophages, we determined the effect of the pta mutation on survival inside periodate-elicited peritoneal macrophages. We opsonized bacteria with 20% normal human serum and coinfected adherent macrophages for 30 min with the 14028 and pta mutant strains. After 0, 4, and 18 h of incubation in Dulbecco modified Eagle medium with 20 µg of gentamicin/ml, we lysed the macrophages to determine the surviving CFU. We did not find a difference in survival between the 14028s strain and the pta mutant (data not shown). This confirms the recent report of Kim and Falkow (10) that a pta mutant is not more susceptible to macrophage killing.

Lawhon et al. did not find that S. enterica pta mutants are attenuated in mice (12), so we expected the pta mutant to be a negative control in these experiments. We cannot be sure why our results differ from theirs, but they used a double ackA-pta mutant and they tested virulence only in BALB/c mice by determining the oral 50% lethal dose (12). Since we found that the pta strain killed BALB/c mice, although at a slower rate, it is possible that Lawhon et al. overlooked the attenuation of the mutant in an oral infection model, in which there tends to be greater variation in time to death within a group. It is also possible that the ackA (acetate kinase) mutation affected virulence in some way. AckA is the enzyme that phosphorylates short-chain fatty acids (i.e., acetate and propionate), yielding acyl~P, which is in turn converted to the CoA derivative by Pta. During growth on acetogenic substrates (e.g., glucose), pta mutants have no acetyl~P, whereas ack mutants accumulate acetyl~P (25).

We can only speculate on why the pta mutation attenuates serovar Typhimurium infections in mice. Attenuation is not due only to their inability to use acetate for energy, since the aceA mutant was virulent. It is possible that Pta has some other function in Salmonella and that is currently under investigation. It has also been claimed that acetyl~P can act as a P donor and thus play a role in signal transduction (23, 25). Acetyl~P can phosphorylate OmpR, leading to the repression of flagellum synthesis in Escherichia coli (14) and pta mutants are hyperflagellated. However, McCleary determined that the kinetics of phosphorylation of PhoB by acetyl~P made it unlikely that acetyl~P served that function in vivo (13). There are many two-component regulators in S. enterica, and we cannot exclude that acetyl~P plays a physiological role in activating one or more of them in S. enterica. Indeed, Chamnongpol and Groisman showed that acetyl~P can donate a phosphate to a mutant PhoP protein that functions in the absence of the sensor PhoQ and that activity requires Pta (3). They also found that the native PhoP protein could be phosphorylated by acetyl~P, although less efficiently. The exact mechanism of attenuation in the pta mutant remains to be determined.


   ACKNOWLEDGMENTS
 
We thank Bruce Zwilling for breeding pairs of the BALB/c.D2 Nramp1 congenic mice and Sharon Okamoto for technical assistance.

This work was supported by NIH grants R01 AI47884 (J.F.) and R01 GM40313 (J.E.-S.).


   FOOTNOTES
 
* Corresponding author. Mailing address: VA Healthcare San Diego, 3350 La Jolla Village Dr., San Diego, CA 92161. Phone: (858) 552-7446. Fax: (858) 552-4398. E-mail: jfierer{at}ucsd.edu. Back

Editor: F. C. Fang

{dagger} Present address: Kangman St. Mary’s Hospital, Seoul, South Korea. Back


   REFERENCES
Top
 Abstract
 Text
 References
 
1. Brown, T. D., M. C. Jones-Mortimer, and H. L. Kornberg. 1977. The enzymatic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J. Gen. Microbiol. 102:327-336.[Medline]
2. Castilho, B. A., P. Olfson, and M. J. Casadaban. 1984. Plasmid insertion mutagenesis and Iac gene fusions with mini-Mu bacteriophage transposons. J. Bacteriol. 158:488-495.[Abstract/Free Full Text]
3. Chamnongpol, S., and E. A. Groisman. 2000. Acetyl phosphate-dependent activation of a mutant PhoP response regulator that functions independently of its cognate sensor kinase. J. Mol. Biol. 300:291-305.[CrossRef][Medline]
4. Conner, C. P., D. M. Heithoff, S. M. Julio, R. L. Sinsheimer, and M. J. Mahan. 1998. Differential patterns of acquired virulence genes distinguish Salmonella strains. Proc. Natl. Acad. Sci. USA 95:4641-4645.[Abstract/Free Full Text]
5. Fang, F. C., S. J. Libby, M. E. Castor, and A. M. Fung. 2005. Isocitrate lyase (AceA) is required for Salmonella persistence but not for acute lethal infection in mice. Infect. Immun. 73:2547-2549.[Abstract/Free Full Text]
6. Grimek, T. L., H. Holden, I. Rayment, and J. C. Escalante-Semerena. 2003. Residues C123 and D58 of the 2-methylisocitrate lyase (PrpB) enzyme of Salmonella enterica are essential for catalysis. J. Bacteriol. 185:4837-4843.[Abstract/Free Full Text]
7. Hesslinger, C., S. A. Fairhurst, and G. Sawers. 1998. Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate. Mol. Microbiol. 27:477-492.[CrossRef][Medline]
8. Horswill, A. R., and J. C. Escalante-Semerena. 2001. In vitro conversion of propionate to pyruvate by salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate. Biochemistry 40:4703-4713.[CrossRef][Medline]
9. Horwitz, A. H., C. Morandi, and G. Wilcox. 1980. Deoxiribonucleic acid sequence of araBAD promoter mutants of Escherichia coli. J. Bacteriol. 142:659-667.[Abstract/Free Full Text]
10. Kim, C. C., and S. Falkow. 2004. Delineation of upstream signaling events in the salmonella pathogenicity island 2 transcriptional activation pathway. J. Bacteriol. 186:4694-4704.[Abstract/Free Full Text]
11. LaRossa, R. A., T. K. Van Dyk, and D. R. Smulski. 1990. A need for metabolic insulation: lessons from sulfonylurea genetics, p. 109-122. In Z. Barak, D. M. Chipman, and J. V. Schloss (ed.), Biosynthesis of branched chain amino acids. Weinheim and Balaban, Rehovot, Israel.
12. Lawhon, S. D., R. Maurer, M. Suyemoto, and C. Altier. 2002. Intestinal short-chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA. Mol. Microbiol. 46:1451-1464.[CrossRef][Medline]
13. McCleary, W. R. 1996. The activation of PhoB by acetylphosphate. Mol. Microbiol. 20:1155-1163.[CrossRef][Medline]
14. McCleary, W. R., J. B. Stock, and A. J. Ninfa. 1993. Is acetyl phosphate a global signal in Escherichia coli? J. Bacteriol. 175:2793-2798.[Free Full Text]
15. McKinney, J. D., K. Honer zu Bentrup, E. J. Munoz-Elias, A. Miczak, B. Chen, W.-T. Chan, D. Swenson, J. C. Sacchettini, W. R. Jacobs, and D. G. Russell. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735-738.[CrossRef][Medline]
16. Muñoz-Elias, E. J. and J. D. McKinney. 2005. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11:638-645.[CrossRef][Medline]
17. Palacios, S., V. J. Starai, and J. C. Escalante-Semerena. 2003. Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol. J. Bacteriol. 185:2802-2810.[Abstract/Free Full Text]
18. Potter, M., A. D. O'Brien, E. Skamene, P. Gros, A. Forget, P. A. L. Kongshavn, and J. S. Wax. 1983. A BALB/c congenic strain of mice that carries a genetic locus (Ity) controlling resistance to intracellular parasites. Infect. Immun. 40:1234-1235.[Abstract/Free Full Text]
19. Starai, V. J., I. Celic, R. N. Cole, J. D. Boeke, and J. C. Escalante-Semerena. 2002. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298:2390-2392.[Abstract/Free Full Text]
20. Tsang, A. W., A. R. Horswill, and J. C. Escalante-Semerena. 1998. Studies of regulation of expression of the propionate (prpBCDE) operon provide insights into how Salmonella typhimurium LT2 integrates its 1,2-propanediol and propionate catabolic pathways. J. Bacteriol. 180:6511-6518.[Abstract/Free Full Text]
21. Van Dyk, T. K., and R. A. LaRossa. 1987. Involvement of ack-pta operon products in {alpha}-ketobutyrate metabolism by Salmonella typhimurium. Mol. Gen. Genet. 207:435-440.[CrossRef][Medline]
22. Walsh, K., and D. E. Koshland, Jr. 1984. Determination of flux through the branch point of two metabolic cycles: the tricarboxylic acid cycle and the glyoxylate shunt. J. Biol. Chem. 259:9646-9654.[Abstract/Free Full Text]
23. Wanner, B. L. 1995. Signal transduction and cross regulation in the Escherichia coli phosphate regulon by PhoR, CreC, and acetyl phosphate, p. 203-221. In J. A. Hoch and T. J. Silhavy (ed.), Two-component signal transduction. American Society for Microbiology, Washington, D.C.
24. Way, J. C., M. A. Davis, D. Morisato, D. E. Roberts, and N. Kleckner. 1984. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32:369-379.[CrossRef][Medline]
25. Wolfe, A. J. 2005. The acetate switch. Microbiol. Mol. Biol. Rev. 69:12-50.[Abstract/Free Full Text]

Infection and Immunity, April 2006, p. 2498-2502, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2498-2502.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, Y. R.
Right arrow Articles by Fierer, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, Y. R.
Right arrow Articles by Fierer, J.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals