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Summary Acinetobacter baumannii has emerged as a leading nosocomial pathogen, infecting a wide range of anatomic sites including the respiratory tract and the bloodstream. In addition to being multi‐drug resistant, little is known about the molecular basis of A. baumannii pathogenesis. To better understand A. baumannii virulence, a combination of a transposon‐sequencing (TraDIS) screen and the neutropenic mouse model of bacteremia was used to identify the full set of fitness genes required during bloodstream infection. The lytic transglycosylase MltB was identified as a critical fitness factor. MltB cleaves the MurNAc‐GlcNAc bond of peptidoglycan, which leads to cell wall remodeling. Here we show that MltB is part of a complex network connecting resistance to stresses, membrane homeostasis, biogenesis of pili and in vivo fitness. Indeed, inactivation of mltB not only impaired resistance to serum complement, cationic antimicrobial peptides and oxygen species, but also altered the cell envelope integrity, activated the envelope stress response, drastically reduced the number of pili at the cell surface and finally, significantly decreased colonization of both the bloodstream and the respiratory tract.

The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC . This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.

Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon. We showed that a pst mutation results in virulence attenuation in extraintestinal pathogenic Escherichia coli (ExPEC) strains. Our results indicate that the bacterial cell surface of the pst mutants is altered. In this study, we show that pst mutants of ExPEC strains display an increased sensitivity to different cationic antimicrobial peptides and vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is significantly less abundant in pst mutants. Among differentially expressed genes in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to lipid A, forming the 1-diphosphate species, was found to be downregulated. Our results strongly suggest that the Pho regulon is involved in lipid A modifications, which could contribute to bacterial surface perturbations. Since the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A modification mechanism could be widely distributed among gram-negative bacterial species.

The pstSCAB-phoU operon encodes the phosphate-specific transport system (Pst). Loss of Pst constitutively activates the Pho regulon and decreases bacterial virulence. However, specific mechanisms underlying decreased bacterial virulence through inactivation of Pst are poorly understood. In uropathogenic Escherichia coli (UPEC) strain CFT073, inactivation of pst decreased urinary tract colonization in CBA/J mice. The pst mutant was deficient in production of type 1 fimbriae and showed decreased expression of the fimA structural gene which correlated with differential expression of the fimB, fimE, ipuA, and ipbA genes, encoding recombinases, mediating inversion of the fim promoter. The role of fim downregulation in attenuation of the pst mutant was confirmed using a fim phase-locked-on derivative, which demonstrated a significant gain in virulence. In addition, the pst mutant was less able to invade human bladder epithelial cells. Since type 1 fimbriae contribute to UPEC virulence by promoting colonization and invasion of bladder cells, the reduced bladder colonization by the pst mutant is predominantly attributed to downregulation of these fimbriae. Elucidation of mechanisms mediating the control of type 1 fimbriae through activation of the Pho regulon in UPEC may open new avenues for therapeutics or prophylactics against urinary tract infections.

The pstSCAB-phoU operon encodes the phosphate-specific transport system (Pst). Loss of Pst constitutively activates the Pho regulon and decreases bacterial virulence. However, specific mechanisms underlying decreased bacterial virulence through inactivation of Pst are poorly understood. In uropathogenic Escherichia coli (UPEC) strain CFT073, inactivation of pst decreased urinary tract colonization in CBA/J mice. The pst mutant was deficient in production of type 1 fimbriae and showed decreased expression of the fimA structural gene which correlated with differential expression of the fimB , fimE , ipuA , and ipbA genes, encoding recombinases, mediating inversion of the fim promoter. The role of fim downregulation in attenuation of the pst mutant was confirmed using a fim phase-locked-on derivative, which demonstrated a significant gain in virulence. In addition, the pst mutant was less able to invade human bladder epithelial cells. Since type 1 fimbriae contribute to UPEC virulence by promoting colonization and invasion of bladder cells, the reduced bladder colonization by the pst mutant is predominantly attributed to downregulation of these fimbriae. Elucidation of mechanisms mediating the control of type 1 fimbriae through activation of the Pho regulon in UPEC may open new avenues for therapeutics or prophylactics against urinary tract infections.

Extra-intestinal pathogenic E. coli cause urinary tract infections (UTIs), newborn meningitis, abdominal sepsis and septicemia. UTIs affect millions of women annually, and result in significant health care costs and morbidity worldwide. Uropathogenic E. coli (UPEC) is the predominant urinary tract pathogen, causing up to 85% of UTIs. Despite appropriate therapy, recurrent episode of UTI are common and bacterial strains are increasingly more resistant to many currently used antimicrobial agents. The pstSCAB-phoU operon encodes the phosphate specific transport system (Pst) and belongs to the Pho regulon, which is regulated by the two-component regulatory system (TCRS) PhoBR. Inactivation of the Pst system in E. coli and other bacteria leads to constitutive activation of the Pho regulon, perturbations in cellular adaptation, and decreased virulence. The role of the Pst system in uropathogenic E. coli (UPEC) was assessed by deleting the pstSCA genes in UPEC strain CFT073. In competitive (co-challenge) and single-strain infections, the pst mutant was attenuated for colonization of both the bladder and kidneys of CBA/J mice and was impaired for production of Type 1 fimbriae. Type 1 fimbriae are essential for UPEC virulence and their phase-variable expression is positively and negatively regulated by FimB and FimE, respectively. In vitro, in LB broth and human urine, repression of the fim structural gene fimA in the pst mutant correlated with increased orientation of the fim promoter in the OFF-position. In vivo, down-regulation of fimA in CFT073 Δpst correlated with the up-regulation of fimE. To confirm the specific role of repression of fim expression by the pst mutant during UTI, fim phase locked-ON pst derivatives of the pst mutant and WT CFT073 strains were constructed. Compared to the pst mutant, the fim phase locked-ON pst derivative demonstrated a significant gain in colonization of the bladder, that was similar to that of CFT073 WT and CFT073 fim locked-ON strains. As Type 1 fimbriae are important for UPEC virulence, by promoting adhesion, our results suggest that the reduced bladder colonization by the pst mutant during UTI is predominantly attributed to down-regulation of these fimbriae. Since the Pho regulon is controlled by the TCRS PhoBR, molecules inducing the expression of the Pho regulon through inactivation of Pst or activation of PhoBR could potentially impair UPEC virulence by inhibiting colonization and the infection cycle, which is dependent on expression of type 1 fimbrial adhesins.

During the course of infection, bacteria must coordinately regulate gene expression in response to environmental stimuli. The phosphate (Pho) regulon is controlled by the two component-regulatory system PhoBR. PhoBR is activated during starvation and regulates genes involved in phosphate homeostasis. Several studies have highlighted the importance of the Pho regulon in bacterial pathogenesis, showing how induction of PhoBR, in addition to regulating genes participating in phosphate metabolism, leads to modulation of many cellular processes. The pleiotropic effects of Pho regulon activation include attenuated virulence and alteration of many virulence traits, including adhesion to host cells and resistance to cationic antimicrobial peptides, acidity and oxidative stresses. This review provides an overview of the relationship between the Pho regulon and virulence in Escherichia coli and illustrates that, in addition to regulating phosphate homeostasis, the Pho regulon plays a key role in regulating stress responses and virulence.

Bacterial pathogens regulate virulence factor gene expression coordinately in response to environmental stimuli, including nutrient starvation. The phosphate (Pho) regulon plays a key role in phosphate homeostasis. It is controlled by the PhoR/PhoB two-component regulatory system. PhoR is an integral membrane signaling histidine kinase that, through an interaction with the ABC-type phosphate-specific transport (Pst) system and a protein called PhoU, somehow senses environmental inorganic phosphate (P(i)) levels. Under conditions of P(i) limitation (or in the absence of a Pst component or PhoU), PhoR activates its partner response regulator PhoB by phosphorylation, which, in turn, up- or down-regulates target genes. Single-cell profiling of PhoB activation has shown recently that Pho regulon gene expression exhibits a stochastic, "all-or-none" behavior. Recent studies have also shown that the Pho regulon plays a role in the virulence of several bacteria. Here, we present a comprehensive overview of the role of the Pho regulon in bacterial virulence. The Pho regulon is clearly not a simple regulatory circuit for controlling phosphate homeostasis; it is part of a complex network important for both bacterial virulence and stress response.

Background Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain χ7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. Results Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain χ7122. Conclusion Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence.