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Proc Natl Acad Sci U S A. May 28, 2002; 99(11): 7675–7680.
Published online May 21, 2002. doi:  10.1073/pnas.112210499
PMCID: PMC124319
Microbiology

Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp.

Abstract

The importance of inorganic polyphosphate (poly P) and poly P kinase (PPK), the enzyme principally responsible for its synthesis, has been established previously for stationary-phase survival of Escherichia coli and virulence in Pseudomonas aeruginosa. The gene (ppk) that encodes PPK is highly conserved among many bacterial pathogens, including Shigella and Salmonella spp. In view of the phylogenetic similarity of the enteropathogens and the frequency with which virulence factors are expressed in stationary phase, the ppk gene of pathogenic Shigella flexneri, Salmonella enterica serovar Dublin, and Salmonella enterica serovar typhimurium have been cloned and deleted. In some of these mutants lacking ppk, the phenotypes included features indicative of decreased virulence such as: (i) growth defects, (ii) defective responses to stress and starvation, (iii) loss of viability, (iv) polymyxin sensitivity, (v) intolerance to acid and heat, and (vi) diminished invasiveness in epithelial cells. Thus PPK may prove, as it has with P. aeruginosa, to be an attractive target for antibiotics, with low toxicity because PPK is not found in higher eukaryotes.

Inorganic polyphosphate (poly P) is a chain of tens or many hundreds of phosphate residues linked by “high-energy” phosphoanhydride bonds. Poly P is ubiquitous, having been found in every cell examined (1), and performs varied functions depending on the cell and circumstances (2). Escherichia coli mutants lacking polyphosphate kinase (PPK), the enzyme responsible for the synthesis of poly P from ATP, are deficient in responses to stresses and stringencies and fail to survive in stationary phase (3, 4). The gene ppk that encodes PPK is highly conserved in Gram-negative bacteria (5), including some 20 pathogens. Mutation of ppk in six enteropathogens rendered them impaired in motility on a semisolid agar surface (6), indicative of a loss in ability to invade and establish systemic infections in host cells. A ppk mutant of Pseudomonas aeruginosa was also defective in quorum sensing and the dependent virulence factors, elastase and rhamnolipid; the mutant was also deficient in biofilm formation and was not lethal in a burned-mouse pathogenesis model (7). Vibrio cholerae ppk mutants also show defects in growth, motility, and surface attachment, features linked to virulence (8).

Poly P is involved in the expression in E. coli of RpoS (9), the sigma factor responsible for activation of more than 50 genes required for survival during starvation, UV radiation, oxidative damage, and osmotic stress (10, 11). In addition to a decrease in long-term survival in the stationary phase, increased sensitivities to oxidative, osmotic, and heat stresses, and defects in adaptive growth in minimal media are among the phenotypic features exhibited by the ppk mutant (3); these can all be linked to a decreased expression of the rpoS gene (9). On the basis of all these factors, it would seem that poly P is likely needed for the virulence of Shigella and Salmonella spp.

Shigella flexneri, a facultative intracellular pathogen, is the etiological agent of bacillary dysentery. The capacity of this bacterium to enter human epithelial cells depends on secreted proteins encoded by a regulon of virulence genes. Expression of the genes is controlled by multiple environmental signals (12). The ability of S. flexneri in stationary phase to survive for several hours at pH 2.5 likely accounts for the low infective dose in shigellosis. The acid resistance depends on expression of rpoS; a deletion mutant is highly acid sensitive (13). Interdependence with rpoS expression in E. coli (9) can be added to the similarity of S. flexneri in the expressions of several invasion operons (e.g., viz, ipa, mxi, and spa) that are maximal in stationary-phase cultures (13, 14). Evidence that infective S. flexneri is in a stationary, nondividing state and that expression of stationary-phase-specific genes is essential for survival and virulence provides support for a role for poly P in its pathogenesis.

Salmonellae are Gram-negative facultative anaerobes and, when acquired by the ingestion of contaminated food or water, can cause a range of diseases depending on the serovar and host (1517). Salmonella enterica serovar typhimurium (S. typhimurium) causes inflammatory diarrhea in calves and also elicits a systemic disease in mice. It shows a broad host range, infecting cattle, pigs, sheep, horses, poultry, and rodents (18); similar diseases caused by Salmonella enterica serovar Dublin (S. dublin) are found principally in cattle.

In salmonellae, as in S. flexneri, evidence exists for the regulation of virulence by RpoS. S. typhimurium harbors a large (80–100 kDa) plasmid (19), the absence of which results in attenuation or loss of virulence. The spvABCD (salmonella plasmid virulence) operon found on this plasmid greatly enhances the ability of the bacteria to proliferate in extraintestinal tissues and thus is required for the induction of systemic disease in mice (20).

To investigate the role of poly P in the virulence of Shigella and Salmonella spp., null mutants of ppk were prepared and their phenotypes, with particular relation to virulence factors, were examined.

Materials and Methods

Reagents.

ATP, creatine kinase, DNase I, and RNase IIIa were from Roche Molecular Biochemicals. Creatine phosphate, 3-(N-morpholino)propanesulfonic acid (Mops), kanamycin, ampicillin, tetracycline, amino acids, and BSA were from Sigma. [γ-32P]ATP, Hybond-N+ nylon membranes, and carrier-free 32Pi were from Amersham Pharmacia. Polyethyleneimine-cellulose F TLC plates were from Merck. Restriction enzymes were from New England Biolabs. The DIG (digoxigenin) DNA labeling kit was from Boehringer Mannheim, and [32P]poly P was as described (21).

Strains, Plasmids, and Phages.

Strains, plasmids, and phages are listed in Table Table1.1. Plasmids were introduced into E. coli by transformation and into S. typhimurium strains by electrotransformation with a Bio-Rad Gene Pulser.

Table 1
Strains, phage, and plasmids

DNA Manipulations and Analysis.

DNA manipulations and analysis were as described by Sambrook et al. (23).

Preparation of Part of ppk Gene of S. typhimurium WRAY.

Partial sequence of S. typhimurium ppk was obtained by a BLAST search of the TIGR (The Institute for Genomic Research) genome sequence database by using the E. coli ppk sequence. This sequence was amplified by PCR with two synthetic primers (SalI forward primer, 5′-CCGTGAATAAAACGGAGTATAGGTAG-3′; SalI reverse primer, 5′-AAAATGTCATCCAGGCAG-3′); genomic DNA of S. typhimurium was the template.

Genomic Library Construction and Screening of S. typhimurium WRAY.

Wild-type (WT) DNA was partially digested with Sau3AI. The fragments were ligated into BamHI-digested λ DASHII and packaged by using Gigapack II Gold (Stratagene). The library was screened by using the labeled PCR fragment (partial ppk gene, 659 bp) to obtain clones that carry the entire ppk gene. From the positive clones, a 5.7-kb XbaI–KpnI fragment was identified that contained the whole ppk gene. This fragment was cloned into pBluescript II KS (+) (Stratagene) that had been digested with XbaI and KpnI, yielding the plasmid pKS7. The 5.7-kb fragment in pKS7 was sequenced.

Construction of ppk-Deletion Mutant of S. typhimurium.

A 2.072-kb PCR fragment containing the ppk coding sequence was generated with pKS7 as the template and the primers 5′-CGTGAATAAAAACGGAGTAT-3′ and 5′-ATGAAAGCTGTTTGAGCCG-3′. This fragment was ligated into pBluescript II KS (+) that had been digested with SmaI to construct the plasmid pKS10. It was further digested with ApaI and EcoRI to remove the SalI site in the vector. The resulting 5.1-kb fragment was treated with T4 DNA polymerase to create blunt ends, and the ends were then self-ligated. The plasmid pKS10-1 thus prepared was digested with SalI to remove a 800-bp fragment from the midportion of the ppk gene to obtain a 4.3-kb fragment. It was ligated to a kanamycin-resistance gene cassette contained within a SalI restriction fragment of pUC4K. The resulting plasmid, pKS10-6, was digested with KpnI and XbaI; the fragment was blunt-ended by using T4 DNA polymerase and cloned into pKNG101, yielding pKSS1. The plasmid pKSS1 was introduced into the WT S. typhimurium FIRN from E. coli S17-1 λ pir by conjugal transfer. Cointegrant conjugants containing no plasmid and representing a single homologous recombination were isolated by plating on LB agar plates supplemented with nalidixic acid (50 μg/ml) and carbenicillin (60 μg/ml), and the genotype was confirmed by PCR. The strains were subjected to sucrose selection; clones with a double-homologous recombination event (streptomycin-sensitive and kanamycin-resistant) were identified and further tested by PCR and assay of PPK.

Construction of S. enterica Serovar Dublin Δppk Δppx::kan Mutants.

P1vir lysate of E. coli strain CF5802 was used to transduce the Δppk Δppx::kan mutation into E. coli Hfr strain KL16. Conjugation was performed between KL16 Δppk Δppx::kan (donor) and S. typhimurium strain SL7519 [F] mutL111::Tn10 (recipient). A P22 lysate prepared from the resultant SL7519 Δppk Δppx::kan strain was then used to transduce the mutation into S. dublin SVA47. Three independent S. dublin Δppk Δppx::kan mutants were isolated and verified by PCR.

Construction of S. flexneri Δppk Δppx::kan Mutants.

P1vir lysate of E. coli strain CF5802 was used to transduce the Δppk Δppx::kan mutation into WT S. flexneri strain 2a.

Biochemical Assays.

PPK and exopolyphosphatase (PPX) activities were assayed as described (24, 25). Poly P levels were determined by the nonradioactive method (26).

Survival.

Survival of stationary-phase cells.

See legend to Fig. Fig.33.

Figure 3
Long-term survival. WT and mutants of S. typhimurium (A) and S. dublin (B) were tested. Long-term survival in LB was assayed as described (3).

Heat-shock survival.

See legend to Fig. Fig.44.

Figure 4
Heat-shock survival. WT and mutants of Salmonella spp. and S. flexneri were grown overnight (≈16 h) in LB. The stationary-phase cells were washed and diluted in 0.9% NaCl to a cell density of about 5 × 103 per ml. Samples (2 ml) ...

Acid tolerance.

Stationary-phase cells grown overnight (≈20 h) in LB were washed in saline, resuspended in acidified LB (pH 3.0 for S. flexneri and 3.3 for Salmonella spp.), and incubated aerobically at 37°C; cultures were diluted and plated on LB to measure viability.

Polymyxin B resistance.

See legend to Fig. Fig.55 for details.

Figure 5
Polymyxin B resistance. In ppk mutant of S. typhimurium, resistance is restored after complementation with PPK. For induction of PPK, cells were grown to an OD540 of 0.8 in LB to which 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) was added ...

Surface attachment.

As described (8), 96-well assay plates of polyvinyl chloride (Falcon 3911 Microtester III flexible) were from Becton Dickinson.

Gentamycin-protection assays.

See legend of Fig. Fig.66.

Figure 6
Invasion and growth in epithelial cells and survival in macrophages. The gentamycin-protection assays were as described (28). (B) WT and mutant of S. typhimurium were grown overnight in LB to stationary phase. The cells were diluted in PBS and opsonized ...

Nucleotide Sequence Accession Number.

The GenBank accession number for the sequence of S. typhimurium WRAY reported in this study is AF085682.

Results

PPK, PPX, and poly P in WT and Mutant Pathogens.

Levels of PPK, PPX, and poly P in the enteric pathogens S. flexneri, S. typhimurium, and S. dublin were similar to those observed for E. coli (4) (Table (Table2).2). PPK of the pathogens was localized, as in E. coli, in the membrane fraction but at 2 to 3 times the level. PPX downstream in the ppk operon resembled E. coli PPK in levels and membrane localization. The null ppk mutants of all these enteric strains exhibited barely detectable levels of PPK, PPX, and poly P (Table (Table2).2).

Table 2
PPK, PPX, and poly P in cell lysates

Growth in Rich Media.

Of the several enterics, only the S. flexneri ppk mutants show a defect in growth and short-term loss of survival (Fig. (Fig.1).1). By contrast, the Salmonella spp. ppk mutants showed relatively little loss of viability even after 2 days of incubation, but as with E. coli (3), suffered profound losses in survival after many days of incubation (see below).

Figure 1
Growth studies. WT and mutant S. flexneri were grown aerobically at 37°C in LB medium. Samples were analyzed for growth (OD540) and viability (cfu/ml).

In LB medium, S. flexneri grew to an OD540 of 4 and a cell density of 4 × 109 colony-forming units (cfu)/ml. Unlike WT, the ppk mutant, after a normal rate of growth for 3 h, declined in the next 2 h to an OD540 of 0.3 and a cell density of 8 × 106 cfu/ml. Thereafter, the mutant cultures recovered to near WT levels by about 16 h. The increase in mutant cell density after the abrupt drop coincided with appearance of a small-colony phenotype; these variants represented about 60% of the number of typical large WT colonies present in overnight (≈16-h) cultures.

The ppk mutant of S. flexneri also differed from WT; when the cells were grown aerobically in nutrient broth (Difco) the growth rate was significantly slower up to 6 h, and a lower cell density was reached after 16 h of growth. Remarkably, mutants grown in either LB or nutrient broth aggregated and settled out within a few minutes, unlike WT, which remained uniformly dispersed in the growth medium (data not shown).

Growth in Minimal Media.

PPK mutants of Salmonella spp. could be adapted to grow in Mops-buffered minimal medium (27), but the ppk mutant of S. flexneri grew poorly. Supplementation of the medium with amino acids restored the growth of the mutant to WT levels (data not shown).

Growth After Nutrient Downshift.

S. typhimurium WT grown in LB medium and diluted (1:100) into Mops-buffered minimal medium suffered a lag of about 4 h before the start of exponential growth with a generation time of 76 min. After a similar lag, the ppk mutant grew more slowly, with a generation time of about 120 min (Fig. (Fig.22A). WT growth was restored when the mutant was complemented with a plasmid containing ppk. S. dublin WT subjected to a similar nutrient downshift grew with no lag and a doubling time of 60 min; the ppk mutant grew slowly to about 3 h before the onset of WT exponential growth (Fig. (Fig.22B).

Figure 2
Growth after downshift. (A) S. typhimurium WT complemented with vector, and mutant with either vector or vector containing ppk gene, were grown aerobically at 37°C in LB for about 20 h. The cultures were then diluted (1:100) into prewarmed (37°C) ...

Effect of pH on Growth.

Many bacteria endure transient encounters with very low or high pH, well outside the growth range (13, 14). The Enterobacteriaceae must cope with low-pH stress during their passage through the stomach to the intestine (29, 30). Growth of WT and the ppk mutant were compared in LB medium supplemented with 0.4% glucose and adjusted to pH values ranging from 3.0 to 8.0. S. flexneri WT cells grew well across a broad pH range of 5 to 8 and even at pH 4.3 after a prolonged lag period; the ppk mutant also grew well from pH 5 to 8 but failed to grow at all at pH 4.3; neither WT nor mutant cells could grow at pH 3.3 (data not shown).

The WT and mutant of S. typhimurium did not differ in growth at pH values between 5.0 and 8; the density of the WT was about 2-fold higher after 7 h of growth between pH 5.0 and 7.0. Both WT and mutant did not grow at pH 3.0; growth of the WT was better than the mutant at pH 4.0; WT reached a density of 0.8 after 7 h compared with 0.3 for the mutant. The growth rate of both WT and mutant S. dublin, like S. typhimurium, did not differ at pH values between 5.0 and 8.0; but the density of the WT was about 3-fold higher after 7 h at pH 5.0–7.0. Both WT and mutant did not grow at pH 3.0. Growth of the WT and the mutant at pH 4.0 commenced after a 4-h lag, but no significant difference occurred in the growth curves (data not shown).

Virulence Factors.

Acid tolerance.

Stationary-phase cells grown in rich medium were exposed to pH 3.0 (for 1 h at 37°C). Survival of WT S. flexneri was reduced by 104-fold, but was still 50-fold better than that of the mutant (Table (Table3).3). Unlike S. flexneri, less difference occurred in acid tolerance in Salmonella spp. between WT and mutants. The S. typhimurium mutant showed a 3-fold lesser acid tolerance than WT; the S. dublin mutant showed a 6-fold lower acid tolerance (Table (Table3).3).

Table 3
Survival of S. flexneri and Salmonella spp. in acidic medium

Long-term survival.

In LB medium, survival of the S. typhimurium mutant declined to about 0.001% after 8 days of incubation and still further after 10 days, whereas the WT remained at about 6% of the initial value (Fig. (Fig.3).3). At 10 days, the colony size of the mutant was like that of the WT, but in the next 4 days the culture grew back to about 1% of the initial number with two-thirds of the colonies being variants of small size. When stationary-phase cultures of the WT and mutant were present at a 1:1 ratio, the survival of the mutant increased to about 20% of the initial value until 8 days; thereafter, viability of both WT and mutant in the mixed culture dropped drastically to about 0.001% (data not shown).

The S. dublin mutant showed only a modest loss of viability to about 3% after 7 days of incubation, whereas the WT remained at about 20% of the initial value. When stationary-phase cultures of the WT and mutant were present at a 1:1 ratio, the viability of the mutant decreased rapidly to about 0.2% of the initial value after 3 days of incubation compared with 20% for the WT. Thereafter, the mutant population decreased another 10-fold after 9 days, whereas the WT remained near the same level (data not shown).

Heat resistance.

On entry into stationary phase, the E. coli WT develops a tolerance to heat at 55–57°C (3). Stationary-phase WT S. flexneri exposed to 55°C for 3 min retained 70% of their viability compared with less than 0.5% for the mutant (Fig. (Fig.44A). S. typhimurium WT survival after 2 min was 30% compared with less than 4% for the mutant (Fig. (Fig.44B). The heat resistance of S. dublin was relatively greater than that of S. typhimurium; little loss of viability occurred after 4 min and, even after 10 min, 9% of the WT survived compared with 2% for the mutant (Fig. (Fig.44C).

Polymyxin B resistance.

Survival of mutant S. typhimurium in the stationary phase in polymyxin B at 10 μg/ml was 65% compared with 100% for WT; at 20 μg/ml only 15% of the mutant survived compared with 65% for WT (N.N.R. and A.K., unpublished data). In a repetition of this experiment with a wider range of polymyxin B levels, the resistance of the mutant was again significantly less than that of the WT and could be restored to WT levels by complementation with ppk (Fig. (Fig.5).5). Similar results were obtained with S. dublin (data not shown).

Motility and surface attachment.

The mutants of Salmonella spp. were impaired in their swimming motility as observed with E. coli and other bacteria (6). WT S. flexneri is nonmotile. With regard to adherence to an abiotic surface, predictive of a capacity to form biofilms, the Salmonella spp. mutants showed a 20–35% decrease relative to WT in their adherence to polystyrene (data not shown).

Invasion and growth in epithelial cells and survival in macrophages.

The S. typhimurium mutant was only half as invasive in HEp-2 epithelial cells measured at 2 h and was still at half the WT level after 24 h (Fig. (Fig.66A), as measured by a gentamycin-protection assay. Survival in the macrophage RAW 264.7 was unaffected for the WT, but the mutant declined progressively when assayed after 4 and 24 h (Fig. (Fig.66B).

Discussion

The purpose of this study was to confirm and extend the hypothesis that poly P and PPK, the enzyme that makes it, are needed for virulence in the important enteric pathogens S. flexneri, S. typhimurium, and S. dublin. The hypothesis was based on several facts: (i) poly P participates in activation of stationary-phase responses and starvation in E. coli and so is essential for its survival (2, 3); (ii) virulence factors of many pathogens are expressed in stationary phase (1, 4, 31); (iii) the ppk gene is highly conserved among these and many other pathogens (2, 5); and (iv) virulence in mice of P. aeruginosa (7) and virulence factors of Neisseria meningitidis (32), V. cholerae (8), and Helicobacter pylori (C.D.F., C.-M. Tzeng, and A.K., unpublished results) all depend on the intact ppk gene. An additional incentive in this study was to widen the range of comparisons and relationships in the enzymology, metabolism, genetics, and physiology of poly P.

Organization of the ppk operon and the levels and membrane location of PPK were similar in the three enteric pathogens and resembled those of E. coli (Table (Table2).2). Although knockout of ppk reduced PPK and poly P to levels below detection, it should be noted that another PPK activity (PPK2) has been discovered in ppk mutants of P. aeruginosa that was not detected by the standard assays for PPK; this pathway is likely responsible for significant accumulation of poly P in the ppk mutants of this organism (H. Zhang and A.K., unpublished results).

Of the three enteric pathogens, only S. flexneri showed a strong defect in the extent of growth in both rich and minimal media (Fig. (Fig.1),1), which was accompanied by a profound loss in viability in a few hours. By contrast, the Salmonella spp. showed only a modest diminution of growth rate after a nutrient downshift (Fig. (Fig.2)2) and lost viability only after many days (Fig. (Fig.3).3). With the loss of viability, the large WT colony type was succeeded by the emergence of a small-colony variant, much as had been observed with E. coli (3). When mutant and WT Salmonella spp. were cultured together in a 1:1 ratio, both the WT and mutant S. typhimurium lost viability, whereas under similar conditions the S. dublin WT was spared. The basis for these results remains to be studied.

The phenotypes of the enteric pathogens include defects in several factors (Table (Table4),4), some of which have been related to virulence. These virulence factors include diminished capacity to withstand low pH or elevated temperature or polymyxin B, impairment in motility, attachment to an abiotic surface, and invasiveness in eukaryotic cells in culture. On the basis of these in vitro criteria of virulence, tests of the mutants in an animal host are now clearly indicated.

Table 4
Defects in PPK mutants

Cloning of the ppk gene of the three enteric pathogens makes their overexpression feasible, as well as making their PPKs available for structural and functional studies. These should complement the comparative studies of the PPKs of E. coli, P. aeruginosa, V. cholerae, and H. pylori, which have revealed striking differences in their kinetics and specificities (5).

PPK is attractive as a target for antibiotics, because the absence of any similar enzyme in higher eukaryotic species makes toxicity less likely. Large-scale screening for inhibitors of E. coli and P. aeruginosa PPKs have produced candidates, unique among known kinases, and active at low concentration (S. Lee, ICOS Corp., Bothell, WA, personal communication). Such compounds may prove useful not only as drugs but also as reagents for studies in which prompt inhibition of PPK can be achieved and for which conditional mutants are not yet available.

Acknowledgments

We thank the undergraduate students Amit Prasad and Ted Su for excellent technical assistance and Drs. B. A. D. Stocker and A. T. Maurelli for some strains and phages. The PAN Facility at Stanford University provided primer synthesis and nucleotide sequencing. We also thank Leroy Bertsch for critical reading of the manuscript. This work was supported by a grant from the National Institute of General Medical Sciences, National Institutes of Health.

Abbreviations

poly P
inorganic polyphosphate
PPK
polyphosphate kinase
PPX
exopolyphosphatase
S. typhimurium
Salmonella enterica serovar typhimurium
S. dublin
S. enterica serovar Dublin
WT
wild type
cfu
colony-forming unit

Footnotes

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF085682).

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