The fate of not sensing phosphate

Phosphate, in terms of cellular content, is the fifth most important element. Its homeostasis is controlled by the two-component system (TCS) PhoBR. The pstSCAB-phoU operon, encoding the phosphate specific transport system (Pst), is among the genes regulated by PhoBR. It mediates phosphate acquisition and is required for virulence in pathogenic bacteria. Recently, we demonstrated in uropathogenic Escherichia coli (UPEC) that inactivation of the Pst system negatively regulates expression of type 1 fimbriae and alters the ability to colonize the mouse urinary tract.

Phosphate Homeostasis

Phosphate homeostasis is controlled by the two component system (TCS) PhoBR, where PhoR encodes the inner-membrane histidine kinase and PhoB, the cytoplasmic response regulator. In phosphate starvation conditions, i.e., when the extracellular concentration falls below 4 µM, PhoBR activates genes belonging to the Pho regulon, which encodes genes involved in phosphate acquisition and metabolism, such as the Pst system, an ABC transporter (Hsieh and Wanner, Curr Opin Microbiol 2010). In addition to its role in phosphate transport, the Pst system also represses PhoBR, as its inactivation results in constitutive expression of PhoBR regulated genes, regardless of environmental phosphate availability. Thereby, inactivation of the Pst system mimics phosphate starvation conditions. Moreover, the Pst system is also required for virulence (Crepin et al., Vet Micro 2011; Lamarche et al., FEMS Micro Rev 2007). However, the mechanisms connecting the Pst system, the Pho regulon (PhoBR) and virulence are not well defined.

Mechanisms Connecting PhoBR TCS and Virulence in UPEC

Urinary tract infections (UTIs) affect, annually, millions of persons worldwide. Each year, in the US, the associated cost (medical visits and treatments) for UTIs is estimated at 2.5 billion dollars (Hannan et al., FEMS Micro Rev 2012). Uropathogenic Escherichia coli (UPEC) strains are responsible for up to 85% of community acquired UTIs (Griebling, Urologic Diseases in America 2007). Despite the availability of effective antibiotic therapies, episodes of resistance, recurrence and persistence are common.

Recently, we showed that the pst mutant of the UPEC strain CFT073 is less able to colonize the mouse urinary tract, which is directly linked to the decreased expression of type 1 fimbriae (pili), a key virulence factor in UPEC strains. These fimbriae are found at the bacterial cell surface and they mediate, notably, adhesion to host cells. In UPEC strains, they are expressed in the bladder and mediated adhesion to urothelial cells, allowing the colonization of the bladder. In addition to mediating adhesion to bladder cells, type 1 fimbriae also promote its invasion and the formation of intracellular bacterial communities (IBC) (Hannan et al., FEMS Micro Rev 2012). Not surprisingly, the pst mutant is impaired in its invasion capacity. As formation of IBCs allows the persistence of UPEC strains in the host, the virulence defect of the pst mutant could be attributed to the cumulative effects of the defect in colonization and invasion of bladder cells, leading to the inability to persist in the host.

In our study, we observed that the decreased expression of type 1 fimbriae, in the pst mutant, was concomitant with the altered expression of the regulators fimB, ipuA and ipbA. However, as ipuA and ipbA were described in a strain devoid of fimB and fimE (Bryan et al., Infect Immun 2006), the major regulators of type 1 fimbriae, we hypothesized that the decreased expression of fim, in the pst mutant, is mainly attributed to fimB, while ipuA and ipbA may have an accessory role.

We also identified that one of the mechanisms by which the Pho regulon alters expression of type 1 fimbriae seems to be through the alarmone ppGpp, a second messenger produced by nutritional starvation involved in global physiological adaptation. Indeed, we observed that the pst mutant produces 3-fold less ppGpp than the wild-type strain, which is in agreement with what was previously observed in an E. coli K-12 strain (Spira et al., J Bacteriol 1995; Spira and Yagil, Mol Gen Genet 1998). As production of ppGpp activates expression of fimB, which mediates expression of type 1 fimbriae (Aberg et al., Mol Microbiol 2006), we hypothesized that the decrease expression of fimB and then, the type 1 fimbriae, is connected to the altered production of ppGpp in the pst mutant.

A basal level of cellular ppGpp is essential for proper Pho regulon expression. Furthermore, physiological induction of the Pho regulon, when the extracellular concentration of P_i falls below 4 µM, also induces synthesis of ppGpp (Spira et al., J Bacteriol 1995; Spira and Yagil, Mol Gen Genet 1998; Bougdour and Gottesman, Proc Natl Acad Sci U S A 2007). Its synthesis relies on the GDP pyrophosphokinase/GTP pyrophosphokinase RelA, while it is degraded by the guanosine 3′-diphosphate 5′-triphosphate 3′-diphosphatase SpoT. Furthermore, SpoT can also induce synthesis of ppGpp through its synthase activity. Indeed, phosphate starvation conditions activate ppGpp synthesis through SpoT (Spira and Yagil, Mol Gen Genet 1998; Bougdour and Gottesman, Proc Natl Acad Sci U S A 2007). As our results show that constitutive activation of the Pho regulon, through inactivation of the pst system, represses production of ppGpp, it suggests that constitutive activation of PhoBR has a deleterious effect on bacterial adaptation to starvation. Interestingly, spoT is not differentially expressed in the pst mutant, suggesting that the Pho regulon, PhoB, may regulate, directly or indirectly, SpoT at a post-transcriptional level. Thereby, inactivation of the PST system could inhibit the synthase or activate the hydrolase activity of SpoT. Further experiments are required to elucidate regulatory mechanisms connecting the Pho regulon and production of ppGpp, and its role in type 1 fimbriae expression.

Role of the PhoBR Two-Component System (TCS)

In tested conditions, i.e., LB broth, human urine and in vivo (infected bladder), type 1 fimbriae were downregulated in the pst mutant. As for constitutive activation of PhoBR, through inactivation of pst, physiological activation of PhoBR, through phosphate starvation, had the same effect on fim expression. Interestingly, bladder and urine are phosphate-replete conditions, i.e., phoBR and the Pho regulon are not induced in these environments. Thereby, strains found in the bladder produce type 1 fimbriae and cause UTI. In this way, during UTI, it would be possible to modulate the expression level of phoBR in order to decrease the production of type 1 fimbriae and potentially decrease colonization of the urinary tract. Indeed, as TCS are absent from humans and other animals, they are choice targets for therapeutic strategies. Due to increased use of antibiotics, pathogenic strains are increasingly resistant. However, small chemical molecule libraries have been developed and have shown an inhibitory effect on these regulatory systems (Stephenson and Hoch, Curr Med Chem 2004). Furthermore, some small molecules have been shown to inhibit the expression or the activity of virulence factors (Cegelski et al., Nat Rev Microbiol 2008). In this manner, small molecules activating the Pho regulon, through inactivation of the Pst system or by direct activation of PhoBR, could be considered as therapeutic agents.

Role of Other TCS in Environmental Regulation of Adherence Factors

TCS are widely present in bacteria and regulate gene expression or protein function by responding to various environmental signals. They are thus important for bacterial adaptation and virulence. In our study, we determined that the constitutive activation of the TCRS PhoBR is detrimental to the bacteria. Indeed, constitutive activation of PhoBR inhibits expression of the type 1 fimbriae and consequently, decreased the colonization of the mouse urinary tract.

It has previously been demonstrated that several TCS are able to regulate the expression of adherence and motility factors such as fimbriae, curli and flagella. Indeed, the QseBC TCS, which responds to quorum sensing, is involved in type 1 fimbriae, curli and flagella expression. Recently, it has been shown in the UPEC strain UTI89 that deletion of qseC, encoding the histidine kinase, resulted in a reduction of type 1 fimbriae and flagella expression/production as well as curli expression (Kostakioti et al., Mol Microbiol 2009; Kostakioti et al., Infect Immun 2012). The authors demonstrated that in the absence of qseC, the response regulator QseB remained constitutively active and led to the downregulation of type 1 fimbriae, curli and flagella. As with the deregulation of phoBR, constitutive activation of qse leads to a preferentially OFF orientation of the fimS promoter, the phase variable promoter of type 1 fimbriae, leading to a decreased expression of type 1 fimbriae genes (Kostakioti et al., Mol Microbiol 2009). Moreover, in enterohemorrhagic E. coli, decreased flagella production is the consequence of the direct binding of QseB to the promoter of flhDC, the master regulators of flagella (Clarke and Sperandio, Mol Microbiol 2005).

If constitutive activation of both PhoB and QseB downregulates expression of type 1 fimbriae, deletion or inactivation of other TCRSs may also inhibit expression of fimbriae or flagella. This is especially the case for BarA-UvrY, CpxAR and RcsCDB TCRSs, where CpxAR and RcsCDB respond to stresses affecting the bacterial envelope whereas the stimulus to BarA-UvrY remains to be determined. In a UPEC strain, deletion of the response regulator uvrY resulted in decreased invasion of uroepithelial cells, while in the avian pathogenic E. coli (APEC) strain χ7122, deletion of barA and uvrY decreased adhesion and invasion of chicken embryo fibroblasts. These defects are related to the decreased production of type 1 and P fimbriae (Herren et al., Infect Immun 2006; Palaniyandi et al., PLoS One 2012). Similarly to barA-uvrY, deletion of cpxR led to an assembly defect of P fimbriae in a UPEC strain. Indeed, it has been shown that CpxR binds to the pap promoter and favors the ON orientation of this promoter (Hung et al., EMBO J 2001). As well, deletion of rcsB decreased expression of fimB, increased expression of fimE and lead to the repression of type 1 fimbriae.

Several TCSs in E. coli are thus implicated in the regulation of adhesion and motility. Contrary to the response regulators UvrY, CpxR and RcsB, which activate expression of fimbriae genes, PhoB and QseB repress it. However, with the exception of CpxR, it remains unclear if this regulation occurs by a direct binding of these regulators at promoter sites, or indirectly by positively regulating other genes implicated in direct regulation of fimbrial gene expression.

Concluding Remarks

In conclusion, downregulation of type 1 fimbriae was identified as a key factor connecting the Pst system, the TCS PhoBR and virulence. Indeed, low production of type 1 fimbriae is directly linked to the attenuated virulence of the pst mutant. These fimbriae are repressed both in vitro and in vivo in the pst mutant. We also began to dissect the regulatory pathway linking the Pst system, the PhoBR TCRS and expression of type 1 fimbriae. Indeed, the decreased expression of type 1 fimbriae is linked to the differential expression of their regulators, which seems to rely, at least in part, on production of ppGpp. The specific molecular mechanisms connecting the Pho regulon (the Pst system) and the type 1 fimbriae remain to be determined. Elucidation of mechanisms controlling type 1 fimbriae through activation of the Pho regulon and regulation by other TCS may open new avenues for therapeutics or prophylactics against urinary tract infections.

Crépin S, Houle S, Charbonneau MÈ, Mourez M, Harel J, Dozois CM. Decreased expression of type 1 fimbriae by a pst mutant of uropathogenic Escherichia coli reduces urinary tract infection. Infect Immun. 2012;80:2802–15. doi: 10.1128/IAI.00162-12.