| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, MD 20814-4799, USA
2 Mucosal Immunology Laboratory, Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Charlestown, MA 02129, USA
3 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA
Correspondence
Anthony T. Maurelli
amaurelli{at}usuhs.mil
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Present address: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, NY 10021, USA.
Present address: Universitätsklinikum Erlangen, Fredrich-Alexander-Universität, Loschgestr. 15, 91054 Erlangen, Germany.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The presence of virulence genes in ‘pathogenicity islands’ reflects this form of gene acquisition (Lee, 1996). Thus, horizontal gene transfer can radically transform an organism from a benign resident of a eukaryotic host into a pathogen capable of colonizing a new niche within the host and, incidentally, causing harm to the host.
A complement to the process of bacterial pathogens' evolution by gene acquisition is the progressive adaptation of the bacteria to the new niche by mutation and selection for improved fitness. This process is called pathoadaptive evolution (Sokurenko et al., 1999). An important element of this process is the selection of ‘black holes’ in pathogen genomes, that is, the inactivation or loss of genes that are incompatible with, and even antagonistic to, the new pathogenic lifestyle (Maurelli, 2007). These incompatible genes, which we define as antivirulence loci (AVL), are present in the genome of the non-pathogenic ancestor but absent or inactive in the pathogen because expression of AVL in the pathogen is detrimental to expression of some virulence phenotype. Thus, deletion or mutational inactivation of AVL is selected for in the pathogen.
Bacteria of the genus Shigella are the causative agents of bacillary dysentery in humans and primates. In the early stage of the disease, Shigella produce several enterotoxins that are believed to facilitate their passage from the small intestine to the colon (Fasano et al., 1997). Once in the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, and spread to adjacent cells. Intracellular bacterial growth and tissue destruction lead to an intense inflammatory response with migration of polymorphonuclear neutrophils (PMNs) to the site of infection (Sansonetti et al., 1999).
We previously demonstrated the contribution of ‘black holes’ to the evolution of Shigella from the non-pathogenic gut commensal Escherichia coli (Maurelli et al., 1998). The cadA gene, encoding lysine decarboxylase in E. coli K-12, was shown to be part of a large region uniformly altered in all Shigella spp., and its expression is incompatible with several phenotypes of Shigella virulence (Day et al., 2001; McCormick et al., 1999). These observations support the concept of bacterial pathogen evolution by selection for increased fitness through gene inactivation or deletion.
The genetic similarities between Shigella and E. coli are sufficient to justify grouping them in the same genus. Shigella spp. may even be considered clones, or subgroups, of E. coli (Pupo et al., 2000). However, there are several well-known metabolic differences that distinguish the organisms and aid in their clinical differentiation. Among these are auxotrophic requirements of Shigella that are not found among isolates of E. coli. Most clinical isolates of Shigella fail to grow on minimal medium. Of these auxotrophic isolates, 98 % grow on minimal medium supplemented with methionine, tryptophan and nicotinic acid (Ahmed et al., 1988). Many strains of Shigella require only nicotinic acid supplementation for growth on minimal medium. This requirement is due to mutations in nadA and/or nadB in Sh. flexneri (Gemski et al., 1971; Mantis & Sansonetti, 1996)
The L-aspartate-dihydroxyacetone phosphate pathway is used by E. coli K-12 for de novo synthesis of NAD (Fig. 1). L-Aspartate oxidase, encoded by nadB, forms a multi-enzyme complex with quinolinate synthetase A, the product of nadA. This enzyme complex catalyses the oxidation of L-aspartate to iminoaspartate, which is then condensed with dihydroxyacetone phosphate (DHAP) to form quinolinate (QUIN). Quinolinate phosphoribosyltransferase, the product of nadC, converts QUIN to nicotinic acid mononucleotide, which enters the pathway for NAD synthesis. In the absence of functional nadA and/or nadB, QUIN is not made and no nicotinic acid mononucleotide is available to synthesize NAD. However, exogenous nicotinic acid can be converted to nicotinic acid mononucleotide by the action of nicotinate phosphoribosyltransferase, the product of pncB (Fig. 1). This pathway bypasses the need for nadA and nadB and synthesis of QUIN and is the basis of the nicotinic acid requirement for Shigella growth on minimal medium (Gemski et al., 1971).
|
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) used throughout this study. Other strains of Shigella used were: 2457T (Sh. flexneri 2a) (Formal et al., 1958) and its avirulent, plasmid-cured derivative strain BS103 (Maurelli et al., 1984); BS504 (Sh. dysenteriae 1), BS512 (Sh. boydii), and BS514 (Sh. sonnei). BS176 is a spontaneous virulence-plasmid-cured derivative of M90T (Sansonetti et al., 1986). Overnight cultures for virulence assays were inoculated from single pigmented colonies growing on tryptic soy broth (TSB, Bacto) plates with 1.5 % agar and 0.025 % Congo red (CR; Sigma) at 37 °C (Maurelli et al., 1984) and grown in Luria–Bertani (LB) medium at 37 °C. The wild-type strain of Salmonella typhimurium employed was 
3306 (Gulig & Curtiss, 1987). ATM266 is an EIEC strain (Harris et al., 1982). E2348/69 is a strain of enteropathogenic E. coli (EPEC) obtained from Gail Hecht (University of Illinois at Chicago, USA). E. coli K-12 MC4100 was the source of DNA for PCR reactions to amplify the nadA and nadB loci. Ye37 is a Yersinia enterocolitica isolate WA obtained from Peter Feng (FDA, College Park, MD, USA). BS813 is the nadA+ nadB+ transformant of M90T. For plating efficiency determination of M90T, M9 glucose minimal medium supplemented with methionine (10 µg ml–1) and tryptophan (20 µg ml–1) was used. When required, 1 mM QUIN (Fluka), or 10 µg ml–1 nicotinic acid (Sigma) was added.
Molecular techniques.
The primers used for amplification of the nadA and nadB loci were as follows: nadAPF 5'-CAAGCAACTCTATGTCGGTGG-3' and nadAM 5'-TGGCAAGGCCAATACACAGC-3' allowed amplification of the entire nadA locus and its promoter, while nadBPF 5'-CAAAGGGTTAGAGTGTCTCG-3' and nadBM 5'-CGTGGGCCAGACCAGAACTATTCC-3' allowed amplification of the entire nadB locus and its promoter, using Taq DNA polymerase (Qiagen).
For cloning, we used the commercially available plasmids pGEM-T (Promega) and pBluescript II-SK (Stratagene). Transformants were obtained by electroporation of competent cells from cold water treatment and screened for ampicillin resistance. Ampicillin-resistant clones were then confirmed by PCR and their phenotype was monitored by determining plating efficiency on minimal medium containing the appropriate supplements.
Invasion assays.
Invasion assays were performed as described previously (Sandlin et al., 1996) with some alterations. Bacteria were grown overnight at 37 °C then diluted 1 : 50 into 5.0 ml LB and grown with agitation at 37 °C to OD600 
0.6. Standardized culture volumes were removed, diluted, titered, and used to infect semi-confluent monolayers of mammalian cells at 37 °C in Dulbecco's modified essential medium (DMEM) with or without the indicated concentrations of QUIN, nicotinic acid or nicotinic acid mononucleotide (Sigma). The input inoculum was 1x108 c.f.u. in a volume of 1 ml. After 30 min, infected monolayers were washed extensively with 1xPBS to remove both the QUIN and extracellular bacteria and incubated for an additional 90 min in DMEM with 50 µg gentamicin ml–1. Monolayers were subsequently washed with PBS and lysed in the presence of 0.5 % Triton X-100. Released intracellular bacteria were plated on TSB-CR agar and colonies were counted to determine the percentage invasion compared to the input titre. The same protocol was used to test the effect of QUIN on intracellular growth of bacteria except that the lysis step was performed after 0.5, 1.5, 2.5, 3.5, 4.5 and 5.5 h.
Alternatively, instead of lysing the infected cells, monolayers were fixed with methanol and stained with Giemsa 45, 90, 135 or 180 min after final gentamicin treatment, and examined by light microscopy for evidence of bacterial invasion. At least 200 infected cells were counted. The frequency of protrusion formation, indicating intracellular spreading capacity of the bacteria, was determined as the percentage of cells with internalized bacteria that displayed cytoplasmic protrusions.
Plaque assay.
Plaque assays were performed essentially as described previously (Oaks et al., 1985) with some alterations. Bacterial cultures were grown overnight, diluted 1 : 50 into 5.0 ml fresh LB, and grown with agitation at 37 °C to OD600 0.6. Standardized culture volumes were removed, diluted, titered, and used to infect confluent monolayers of HeLa cells for 2 h at 37 °C in a 6.0 % CO2 atmosphere. The input inoculum was 1x104 c.f.u. in a volume of 100 µl. Agarose overlays with or without the indicated concentrations of QUIN were then applied to infected monolayers and allowed to incubate at 37 °C in a 6 % CO2 atmosphere for an additional 72 h. Neutral red staining allowed for enumeration of plaques to determine the plaque-forming efficiency compared to the input titre. Plaque diameter was calculated as the average of 10 plaques.
PMN transepithelial migration assay.
PMN migration was assessed as previously described (McCormick et al., 1998). For assessment of Sh. flexneri-induced PMN migration in the presence of QUIN, 25 µl washed bacterial suspension (6x108 bacteria) was added directly to the basolateral surface (upper compartment) of inverted T84 cell monolayers for 90 min at 37 °C. Next, the T84 cell monolayers were washed to remove non-adherent bacteria and QUIN (5 or 100 µM) was added to both the apical and basolateral reservoirs for 30 min. PMNs isolated from volunteers were subsequently added to the basolateral surface (1x106 per monolayer) and assessed for PMN transmigration in the continued presence of QUIN. Transmigration was quantified by assaying for myeloperoxidase, the PMN azurophilic marker, and is expressed as cell equivalents as described previously (McCormick et al., 1998; Parkos et al., 1991). A 1 µM solution of N-formylmethionyl leucyl phenylalanine (fMLP; Sigma), a potent PMN chemoattractant, was used as a control.
Protein extraction and preparation.
To study the amount of expressed proteins, 20 ml of exponential cultures of M90T grown with or without 0.4 mM QUIN were standardized using OD600 measurement and harvested by centrifugation (13 000 g, 5 min). Supernatants were filtered through a 0.45 µm pore size filter (Millipore) and secreted proteins were precipitated by the addition of trichloroacetic acid (10 % final concentration), followed by acetone washing. The final pellet of precipitated proteins was resuspended in 200 µl sample buffer. Whole-cell lysates were generated by washing the pellets once with 20 ml ice-cold, sterile 1xPBS, followed by centrifugation of 1 ml aliquots and suspension in 500 µl Laemmli sample buffer (Bio-Rad).
To study the effect of QUIN on CR-induced secretion via the type III secretion system (T3SS), whole-cell fractions and supernatants of CR-treated Sh. flexneri were prepared as described previously (Bahrani et al., 1997). Briefly, 40 ml samples from exponential cultures of M90T were harvested by centrifugation (13000 g, 5 min). Each pellet was resuspended in 2 ml sterile PBS and split into two 1 ml aliquots incubated with 30 µg CR ml–1 for 1 h at 37 °C, with or without 0.4 mM QUIN. After the incubation period, the samples were centrifuged again (13 000 g, 5 min). Whole-cell lysates and precipitated proteins from the supernatants were prepared as described above.
To study the effect of QUIN on the low-calcium-induced T3SS of Y. enterocolitica we used the same protocol with the exception that CR was replaced by 5 mM EGTA to chelate cations from the medium.
SDS-PAGE and immunoblotting.
All samples were boiled and 10 µl of each were loaded on 10 % acrylamide SDS-PAGE gels as described by Laemmli (1970). After electrophoresis, proteins were transferred to a nitrocellulose membrane. Immunoblotting procedures were carried out with mouse monoclonal antibodies 2F1 and 2G2, which recognize IpaB and IpaC, respectively (Mills et al., 1988). Horseradish peroxidase-labelled sheep anti-mouse IgG (Amersham Bioscience) was used as secondary antibody and visualized by enhanced chemiluminescence with the visualizer Western blot detection kit (Upstate) in a LAS-3000 system using Image reader LAS 3000 v2.1 software (Fuji.). The images obtained were analysed using Image Gauge v4.22 software (Fuji) allowing quantification of the signals observed. For the study of the effect of QUIN on the calcium-deprivation-induced T3SS of Y. enterocolitica, SDS-PAGE gels were stained with Imperial protein stain (Pierce).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 0.5 mM QUIN almost completely inhibited invasion of HeLa cells by M90T, whereas 1 mM nicotinic acid or nicotinic acid mononucleotide had no adverse effect on invasion. Addition of 1 mM QUIN caused no visible changes in HeLa cell morphology and up to 2.5 mM QUIN caused no change in pH of the tissue culture medium.
|
).
|
). The formation of cytoplasmic protrusions extending from infected cells and containing one or more bacteria at the end is a reliable measure of intracellular motility. To examine the effect of QUIN on protrusion formation in Shigella-infected cells, bacteria were first allowed to invade semi-confluent HeLa monolayers and then 400 µM QUIN was added and the monolayers incubated at 37 °C. At various time points, the monolayers were fixed, stained and examined for protrusions emanating from infected cells. Fig. 3(a) shows that QUIN-treated monolayers displayed the same frequency of protrusion formation from Shigella-infected cells as untreated monolayers. Thus, QUIN does not inhibit the actin-based intracellular motility of Shigella.
|
). Since QUIN blocked Shigella invasion it was added to infected monolayers only after an initial period in order to allow the bacteria to invade. Fig. 3(b)
shows that QUIN impaired plaque formation in a dose-dependent manner and at much lower concentrations than were required to block invasion. The mean plaque diameter formed by M90T in the cell monolayer in the absence of QUIN was 1.53 mm. Fig. 3(c) shows that the mean plaque size decreased with increasing concentrations of QUIN. This effect of QUIN on plaque size was accompanied by a marked effect on plaque shape, with QUIN causing the formation of faint and irregularly shaped plaques compared to the large, well-defined plaques of the control (Fig. 3d). Therefore, the major post-invasion effect of QUIN is a strong inhibition of the ability of the bacteria to spread from cell to cell, although it had no effect on the intracellular motility of Shigella.
QUIN inhibits Shigella-induced transepithelial migration of PMNs
A model system for the ability of Shigella to induce the severe inflammatory response typical of dysentery is the induction of PMN transepithelial migration across a monolayer of polarized T84 cells (McCormick et al., 1998). We tested QUIN for its effect on the ability of Shigella to induce transepithelial migration of PMNs after invasion of a monolayer of polarized epithelial cells. Since QUIN inhibited invasion of M90T into polarized T84 cells (Table 2), the assay was modified to allow the bacteria to invade the polarized T84 cells for 90 min before different concentrations of QUIN were added to the monolayers. Fig. 4 demonstrates that QUIN has a potent inhibitory effect on the ability of Shigella to induce transepithelial migration of PMNs.
|
|
QUIN is a specific inhibitor of Shigella virulence
We tested another Sh. flexneri serotype as well as representative strains of Sh. dysenteriae, Sh. boydii, Sh. sonnei and EIEC to determine whether our observations on the inhibition of virulence by QUIN could be extended to the four species of Shigella as well as to the closely related EIEC. As shown in Fig. 5, 400 µM QUIN largely inhibited invasion of mammalian cells by all these strains.
|
), the same concentration of QUIN had no effect on invasion of T84 cells by Sal. typhimurium strain 3306 (Table 2).
While Shigella induction of PMN transepithelial migration was significantly inhibited by as little as 5 µM QUIN (Fig. 4), Sal. typhimurium still induced PMN migration in the presence of 100 µM QUIN (Table 2). We also tested E2348/69, an EPEC strain that induces PMN transepithelial migration in this model system. It was still able to induce PMN transepithelial migration in the presence of 100 µM QUIN (Table 2). These data strongly suggest that the antivirulence properties of QUIN observed in this assay are limited to Shigella spp.
Effect of QUIN on intracellular growth and virulence factor expression in Shigella
One possible explanation for the observations that QUIN inhibited cell-to-cell spread and plaque formation, yet did not inhibit intracellular motility of the bacteria is that QUIN inhibits intracellular growth or survival of Shigella. However, when we measured the effect of QUIN on growth of Shigella after invasion of HeLa cells, we found that intracellular growth of the bacteria was not inhibited by 400 µM QUIN (data not shown).
Since Shigella can utilize QUIN for growth on minimal medium (data not shown) by converting QUIN to nicotinic acid mononucleotide by the action of the nadC gene product (Fig. 1), we can assume that QUIN can enter the bacterial cell. Therefore, we wished to determine whether the antivirulence effects result from direct repression of Shigella virulence gene expression by QUIN. We used Western blot analysis of the IpaB and IpaC virulence proteins expressed during exponential culture of Sh. flexneri M90T with or without QUIN to address this question. The amount of protein detected in the pellet (non-secreted proteins) plus the amount of protein TCA-precipitated from the supernatant (secreted proteins) were compared for the two conditions. We found less than a twofold reduction in the amount of IpaB and IpaC expressed in the presence of QUIN compared to the amount expressed during growth without QUIN (1.68±0.12- and 1.55±1.03-fold reduction for IpaB and IpaC, respectively). By comparison with the well-studied growth temperature regulation of Shigella virulence, low growth temperature (30 °C) causes 60-fold reduced expression of ipaB as compared to expression at 37 °C (Andrews et al., 1991; Hromockyj & Maurelli, 1989; McCormick et al., 1998). Therefore, it is not likely that the inhibitory effect of QUIN on Shigella virulence is due to QUIN interaction at the level of virulence gene transcription/translation.
Effect of QUIN on secretion of invasion effectors via the Shigella type III secretion system
Invasion, intracellular and intercellular spread, and the ability to induce PMN transepithelial migration all require the secretion of Ipa proteins from Shigella. Mutations in the T3SS, which mediates secretion of these invasion effectors, abolish these virulence phenotypes (McCormick et al., 1998; Menard et al., 1993; Schuch et al., 1999). We therefore used Western blot analysis of M90T culture supernatants to determine whether QUIN could alter the secretion of Ipa proteins via the T3SS. When wild-type Sh. flexneri are incubated with CR, secretion of Ipa proteins through the T3SS is induced (Parsot et al., 1995). Fig. 6 shows that incubation of M90T with 400 µM QUIN strongly impaired the secretion of IpaB and C that is normally observed when bacteria are incubated with CR. Quantitative analysis showed that the secretion of IpaB and C in the presence of QUIN was reduced 81.9 % and 82.8 %, respectively.
|
). Coomassie staining of SDS-PAGE-analysed protein extracts obtained from Yersinia cultures showed that addition of QUIN to the growth medium did not affect the amount of virulence effectors detected in the supernatant of a calcium-deprived culture (data not shown), which suggests that the effects of QUIN on T3SS-induction are specific to Shigella spp.
nadA+ nadB+ transformants of Sh. flexneri 5a are attenuated in virulence
We wanted to study the effect of reconstructing the NAD pathway in Shigella. The wild-type (functional) nadA and nadB genes with their native promoters from E. coli K-12 were cloned together in pBluescript and transformed into Sh. flexneri 5a strain M90T in order to determine whether expression of these putative AVL in the wild-type Shigella would attenuate virulence. The nadA+ nadB+ transformant of M90T, BS813, was able to grow on minimal medium in the absence of nicotinic acid, confirming the functional activity of the transformed genes. Invasion assays with BS813 in HeLa cells showed a 100-fold reduction of invasion capacity compared to M90T (0.0023±0.0003 % vs 0.21±0.03 %, respectively). In fact, BS813 gave results similar to BS176, the non-invasive plasmid-cured derivative of M90T (i.e. 0.0019±0.0001 %), and to M90T in the presence of 0.5 mM QUIN (Table 1). BS813 was also unable to form plaques in the plaque assay on HeLa cells, even at very high input concentrations (plaque-forming efficiency <10–8). PMN transmigration experiments could not be performed with BS813 due to the assay's dependence on efficient invasion of Shigella into T84 cells. However, invasion and plaque assay results are sufficient to confirm the antivirulence nature of nadA and nadB when functional alleles of these genes are expressed in Shigella.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Mutations in nadA and nadB are responsible for this auxotrophy in Sh. flexneri 2a (Gemski et al., 1971) and our analysis of these loci in other strains of Shigella and EIEC strongly suggested them as good AVL candidates for Shigella (A.-L. Prunier and others, unpublished).
Several intermediates of the NAD synthesis pathway were tested for their capacity to interfere with the invasive phenotype of Shigella and only QUIN was shown to have an effect. QUIN inhibited three essential phenotypes of Shigella virulence: bacterial invasion, cell-to-cell spread and induction of PMN transepithelial migration. Moreover, we found that QUIN strongly inhibited CR-induced T3SS secretion of the Shigella virulence factors IpaB and IpaC, which are key to the entry of the bacteria into eukaryotic cells and provide a pore for the delivery of other effectors (Enninga et al., 2005; Mounier et al., 1997).
Interference with Shigella-induced actin polymerization could explain how QUIN blocks the entry step. However, any interference of this type must be limited to the early invasion stage, as we observed no gross alteration of the bacteria's ability to polymerize actin in the cytoplasm of the host cell after invasion. When bacteria were allowed to invade cells before addition of QUIN, they produced normal numbers and lengths of cytoplasmic protrusions that contained bacteria. This observation demonstrated the ability of intracellular bacteria to polymerize actin normally in the presence of QUIN.
The plaque assay showed that QUIN also blocked a late stage in Shigella infection, cell-to-cell spread. Since intracellular bacteria were motile in the presence of QUIN and appeared capable of producing cytoplasmic extensions that could ‘invade’ adjacent cells, the inhibitory effect of QUIN on cell-to-cell spread is not on actin-based motility per se but on some subsequent step in the spreading process. QUIN could be blocking the actual ‘invasion’ of the bacteria-containing protrusion into the adjacent cell. Alternatively, QUIN could be blocking the ability of the bacteria to lyse the protrusion and escape into the cytoplasm of the adjacent cell.
The failure to spread to adjacent cells is consistent with the inhibition of another virulence phenotype by QUIN, the induction of PMN transepithelial migration. Previous observations in our laboratory have shown that an icsA mutant of Sh. flexneri is severely impaired in its ability to induce transepithelial migration of PMNs (Fernandez et al., 2001). This mutant is unable to catalyse polymerization of actin in the host cytoplasm and, as such, is unable to spread intercellularly. Therefore, induction of PMN transepithelial migration is dependent not only on invasive ability and expression of a functional T3SS (McCormick et al., 1999; Zurawski et al., 2006), but also on the ability to spread to adjacent cells.
While QUIN displays a remarkable ability to inhibit invasion of L2 cells by all four species of Shigella (Fig. 5), its inhibitory properties appear to be restricted to Shigella spp. Sal. typhimurium is also an invasive pathogen of the intestinal epithelium and is closely related to E. coli and Shigella. Both Shigella and Sal. typhimurium invade a variety of cell types in tissue culture, while their respective in vivo targets are different, and they both require the activities of T3SS secretory pathways. Yet QUIN dramatically inhibits Shigella invasion of tissue culture cells whereas it has no effect on invasion by Sal. typhimurium.
Analogous results were observed in the PMN transepithelial migration assay where QUIN blocked PMN signalling by Shigella but had no effect on signalling by Sal. typhimurium. Similarly, a strain of EPEC was insensitive to QUIN and induced PMN transepithelial migration in the presence of 100 µM QUIN. It should be noted that, unlike Shigella, Sal. typhimurium has a completely functional pathway for de novo NAD synthesis and synthesizes QUIN (Penfound & Foster, 1996). The EPEC strain used in this study is a nicotinic acid prototroph and, presumably, also has a functional pathway for de novo NAD synthesis. The model of pathoadaptive evolution by inactivation of AVL predicts that since these pathogens can synthesize QUIN, virulence of Sal. typhimurium and EPEC should be insensitive to inhibition by QUIN. Our results confirm this prediction.
The precise mechanism by which QUIN acts to inhibit Shigella virulence is still unknown. It is possible that QUIN acts directly on the mammalian cell to inhibit Shigella invasion. However, pretreatment of HeLa cell monolayers with 0.4 mM QUIN for up to 18 h prior to infection with M90T had no effect on bacterial invasion of the monolayer cells (data not shown). In addition, pretreatment of the bacteria with QUIN before addition to the cell monolayer was ineffective at blocking bacterial invasion. In fact, inhibition of invasion was only observed when QUIN was present during incubation of the bacteria with the cell monolayer. QUIN did not affect growth of Sh. flexneri in HeLa cells, and it did not significantly impair the expression of IpaB and IpaC virulence factors. It is possible that a bacterial or host target for the action of QUIN only becomes exposed once the bacterium comes in contact with the eukaryotic cell and is subsequently inhibited by QUIN.
QUIN shows a strong inhibitory effect on the induction of T3SS by CR, as shown by reduced levels of secreted IpaB and IpaC when the compound is added during the secretion assay (Fig. 6). The fact that all the Shigella virulence properties monitored in this study (invasion, cell-to-cell spread, and induction of PMN migration) are inhibited by QUIN is consistent with this last observation, since all of these phenotypes require a functional T3SS. As the mediator of T3SS induction by CR has yet to be identified in Shigella, we cannot deduce from this observation the precise mechanism by which QUIN exercises its effect. However, this result is consistent with the hypothesis that a bacterial target for the action of QUIN (probably a T3SS component) only becomes exposed once the virulence properties of the bacterium are induced, either by growth at 37 °C and addition of CR, or by contact with the eukaryotic cell. Furthermore, the QUIN target is specific to Shigella, since we saw no evidence in this work for the impairment of T3SS function in EPEC, Sal. typhimurium or Y. enterocolitica.
We have proposed two main criteria for the identification of AVL in pathogens. First, the AVL must be present and expressed in closely related or ancestral species occupying the non-virulent ancestral niche but absent from (or mutated in) pathogenic clones living in host tissues. Second, expression of the AVL by the pathogen in host tissues must attenuate virulence (Maurelli, 2007). Therefore, in order to prove that nadA/B fulfil the definition of AVL, we restored the functionality of nadA/B in Shigella. We observed a consistent inability of the resulting strain, BS813, to invade tissue culture cells. These results, combined with the results of virulence assays that exhibit a strong inhibitory effect of QUIN on Shigella virulence, demonstrate powerful pathoadaptive selection against the expression of the nadA and nadB genes in Shigella spp. and show that they are AVL for Shigella.
In summary, we have identified a new set of AVL for Shigella spp., and our results support the concept of bacterial pathogen evolution via inactivation of AVL. The results also demonstrate the potential of genomic and phenotypic screening for differences between bacterial pathogens and related commensals as a powerful tool for discovery of AVL. Nordfelth et al. (2005) recently screened a 9400 compound library to identify small molecules that specifically inhibited the T3SS of Y. pseudotuberculosis. In our study, we used a totally different approach and also identified a small molecule that specifically blocks T3SS of Shigella. Thus, identification of AVL can provide a powerful tool to discover new compounds that specifically inhibit bacterial virulence and therefore could be used in the treatment of infectious diseases. Recognition of pathoadaptive mutation as a process of bacterial pathogen evolution and application of the sort of converse comparative genomics we describe in this report (i.e. identification of genes and/or phenotypes present in a non-pathogenic ancestor and absent in the bacterial pathogen) should enable investigators to identify AVL in other pathogens. This information will provide new avenues for investigation of areas ranging from pathogenic mechanisms to the identification of novel antimicrobial agents.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: I. R. Henderson
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Andrews, G. P., Hromockyj, A. E., Coker, C. & Maurelli, A. T. (1991). Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens. Infect Immun 59, 1997–2005.
Bahrani, F. K., Sansonetti, P. J. & Parsot, C. (1997). Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation. Infect Immun 65, 4005–4010.[Abstract]
Bernardini, M. L., Mounier, J., d'Hauteville, H., Coquis-Rondon, M. & Sansonetti, P. J. (1989). Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A 86, 3867–3871.
Day, W. A., Jr, Fernandez, R. E. & Maurelli, A. T. (2001). Pathoadaptive mutations that enhance virulence: genetic organization of the cadA regions of Shigella spp. Infect Immun 69, 7471–7480.
Enninga, J., Mounier, J., Sansonetti, P. & Tran Van, N. G. (2005). Secretion of type III effectors into host cells in real time. Nat Methods 2, 959–965.[CrossRef][Medline]
Fasano, A., Noriega, F. R., Liao, F. M., Wang, W. & Levine, M. M. (1997). Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut 40, 505–511.
Fernandez, I. M., Silva, M., Schuch, R., Walker, W. A., Siber, A. M., Maurelli, A. T. & McCormick, B. A. (2001). Cadaverine prevents the escape of Shigella flexneri from the phagolysosome: a connection between bacterial dissemination and neutrophil transepithelial signaling. J Infect Dis 184, 743–753.[CrossRef][Medline]
Flachmann, R., Kunz, N., Seifert, J., Gutlich, M., Wientjes, F. J., Laufer, A. & Gassen, H. G. (1988). Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB. Eur J Biochem 175, 221–228.[Medline]
Formal, S. B., Dammin, G. J., Labrec, E. H. & Schneider, H. (1958). Experimental Shigella infections: characteristics of a fatal infection produced in guinea pigs. J Bacteriol 75, 604–610.
Gemski, P., Formal, S. B. & Baron, L. S. (1971). Identification of two widely separated loci conferring nicotinic acid dependence on wild-type Shigella flexneri 2a. Infect Immun 3, 500–503.
Groisman, E. A. & Ochman, H. (1996). Pathogenicity islands: bacterial evolution in quantum leaps. Cell 87, 791–794.[CrossRef][Medline]
Gulig, P. A. & Curtiss, R., III (1987). Plasmid-associated virulence of Salmonella typhimurium. Infect Immun 55, 2891–2901.
Harris, J. R., Wachsmuth, I. K., Davis, B. R. & Cohen, M. L. (1982). High-molecular-weight plasmid correlates with Escherichia coli enteroinvasiveness. Infect Immun 37, 1295–1298.
Hromockyj, A. E. & Maurelli, A. T. (1989). Identification of Shigella invasion genes by isolation of temperature-regulated inv : lacZ operon fusions. Infect Immun 57, 2963–2970.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Lee, C. A. (1996). Pathogenicity islands and the evolution of bacterial pathogens. Infect Agents Dis 5, 1–7.[Medline]
Mantis, N. J. & Sansonetti, P. J. (1996). The nadB gene of Salmonella typhimurium complements the nicotinic acid auxotrophy of Shigella flexneri. Mol Gen Genet 252, 626–629.[Medline]
Maurelli, A. T. (2007). Black holes, antivirulence genes, and gene inactivation in the evolution of bacterial pathogens. FEMS Microbiol Lett 267, 1–8.[CrossRef][Medline]
Maurelli, A. T., Blackmon, B. & Curtiss, R., III (1984). Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun 43, 397–401.
Maurelli, A. T., Fernandez, R. E., Bloch, C. A., Rode, C. K. & Fasano, A. (1998). ‘Black holes’ and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proc Natl Acad Sci U S A 95, 3943–3948.
McCormick, B. A., Siber, A. M. & Maurelli, A. T. (1998). Requirement of the Shigella flexneri virulence plasmid in the ability to induce trafficking of neutrophils across polarized monolayers of the intestinal epithelium. Infect Immun 66, 4237–4243.
McCormick, B. A., Fernandez, M. I., Siber, A. M. & Maurelli, A. T. (1999). Inhibition of Shigella flexneri-induced transepithelial migration of polymorphonuclear leucocytes by cadaverine. Cell Microbiol 1, 143–155.[CrossRef][Medline]
Menard, R., Sansonetti, P. J. & Parsot, C. (1993). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899–5906.
Mills, J. A., Buysse, J. M. & Oaks, E. V. (1988). Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies. Infect Immun 56, 2933–2941.
Mounier, J., Bahrani, F. K. & Sansonetti, P. J. (1997). Secretion of Shigella flexneri Ipa invasins on contact with epithelial cells and subsequent entry of the bacterium into cells are growth stage dependent. Infect Immun 65, 774–782.[Abstract]
Nordfelth, R., Kauppi, A. M., Norberg, H. A., Wolf-Watz, H. & Elofsson, M. (2005). Small-molecule inhibitors specifically targeting type III secretion. Infect Immun 73, 3104–3114.
Oaks, E. V., Wingfield, M. E. & Formal, S. B. (1985). Plaque formation by virulent Shigella flexneri. Infect Immun 48, 124–129.
Parkos, C. A., Delp, C., Arnaout, M. A. & Madara, J. L. (1991). Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction. J Clin Invest 88, 1605–1612.[Medline]
Parsot, C., Menard, R., Gounon, P. & Sansonetti, P. J. (1995). Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol Microbiol 16, 291–300.[Medline]
Penfound, T. & Foster, J. W. (1996). Biosynthesis and recycling of NAD. In Escherichia coli and Salmonella: Cellular and Molecular Biology, pp. 721–730. Edited by F. C. Neidhart and others. Washington, DC: American Society for Microbiology.
Pupo, G. M., Lan, R. & Reeves, P. R. (2000). Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc Natl Acad Sci U S A 97, 10567–10572.
Sandlin, R. C., Goldberg, M. B. & Maurelli, A. T. (1996). Effect of O side-chain length and composition on the virulence of Shigella flexneri 2a. Mol Microbiol 22, 63–73.[CrossRef][Medline]
Sansonetti, P. J., Kopecko, D. J. & Formal, S. B. (1982). Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun 35, 852–860.
Sansonetti, P. J., Ryter, A., Clerc, P., Maurelli, A. T. & Mounier, J. (1986). Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun 51, 461–469.
Sansonetti, P. J., Tran, V. N. & Egile, C. (1999). Rupture of the intestinal epithelial barrier and mucosal invasion by Shigella flexneri. Clin Infect Dis 28, 466–475.[Medline]
Schuch, R., Sandlin, R. C. & Maurelli, A. T. (1999). A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination. Mol Microbiol 34, 675–689.[CrossRef][Medline]
Sokurenko, E. V., Hasty, D. L. & Dykhuizen, D. E. (1999). Pathoadaptive mutations: gene loss and variation in bacterial pathogens. Trends Microbiol 7, 191–195.[CrossRef][Medline]
Straley, S. C., Plano, G. V., Skrzypek, E., Haddix, P. L. & Fields, K. A. (1993). Regulation by Ca2+ in the Yersinia low-Ca2+ response. Mol Microbiol 8, 1005–1010.[CrossRef][Medline]
Zurawski, D. V., Mitsuhata, C., Mumy, K. L., McCormick, B. A. & Maurelli, A. T. (2006). OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of virulence. Infect Immun 74, 5964–5976.
Received 12 February 2007;
revised 4 April 2007;
accepted 5 April 2007.
This article has been cited by other articles:
|
|
 |
|
  G. N. Schroeder and H. Hilbi Molecular Pathogenesis of Shigella spp.: Controlling Host Cell Signaling, Invasion, and Death by Type III Secretion Clin. Microbiol. Rev., January 1, 2008; 21(1): 134 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-L. Prunier, R. Schuch, R. E. Fernandez, and A. T. Maurelli Genetic Structure of the nadA and nadB Antivirulence Loci in Shigella spp. J. Bacteriol., September 1, 2007; 189(17): 6482 - 6486. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
