Search

Acinetobacter baumannii infects a wide range of anatomic sites including the respiratory tract and bloodstream. Despite its clinical importance, little is known about the molecular basis of A. baumannii pathogenesis. We previously identified the UDP-N-acetyl-D-galactosaminuronic acid (UDP-GalNAcA) biosynthesis genes, gna-gne2, as being critical for survival in vivo. Herein, we demonstrate that Gna-Gne2 are part of a complex network connecting in vivo fitness, cell envelope homeostasis, and resistance to antibiotics. The Δgna-gne2 mutant exhibits a severe fitness defect during bloodstream infection. Capsule production is abolished in the mutant strain, which is concomitant with its inability to survive in human serum. In addition, the Δgna-gne2 mutant was more susceptible to vancomycin and unable to grow on MacConkey plates, indicating an alteration in cell envelope integrity. Analysis of lipid A by mass spectrometry showed that the hexa- and hepta-acylated species were affected in the gna-gne2 mutant. Finally, the Δgna-gne2 mutant was more susceptible to several classes of antibiotics. Together, this study demonstrates the importance of UDP-GalNAcA in the pathobiology of A. baumannii. By interrupting its biosynthesis, we showed that this molecule plays a critical role in capsule biosynthesis and maintaining the cell envelope homeostasis.

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.

The phagosomal transporter (Pht) family of the major facilitator superfamily (MFS) is encoded by phylogenetically related intracellular gammaproteobacteria, including the opportunistic pathogen Legionella pneumophila . The location of the pht genes between the putative thymidine kinase ( tdk ) and phosphopentomutase ( deoB ) genes suggested that the phtC and phtD loci contribute to thymidine salvage in L. pneumophila . Indeed, a phtC + allele in trans restored pyrimidine uptake to an Escherichia coli mutant that lacked all known nucleoside transporters, whereas a phtD + allele did not. The results of phenotypic analyses of L. pneumophila strains lacking phtC or phtD strongly indicate that L. pneumophila requires PhtC and PhtD function under conditions where sustained dTMP synthesis is compromised. First, in broth cultures that mimicked thymidine limitation or starvation, L. pneumophila exhibited a marked requirement for PhtC function. Conversely, mutation of phtD conferred a survival advantage. Second, in medium that lacked thymidine, multicopy phtC + or phtD + alleles enhanced the survival of L. pneumophila thymidylate synthase ( thyA )-deficient strains, which cannot synthesize dTMP endogenously. Third, under conditions in which transport of the pyrimidine nucleoside analog 5-fluorodeoxyuridine (FUdR) would inhibit growth, PhtC and PhtD conferred a growth advantage to L. pneumophila thyA + strains. Finally, when cultured in macrophages, L. pneumophila required the phtC-phtD locus to replicate. Accordingly, we propose that PhtC and PhtD contribute to protect L. pneumophila from dTMP starvation during its intracellular life cycle.

A. baumannii is a significant cause of bacterial bloodstream infection in humans. Since multiple antibiotic resistance is becoming more common among strains of A. baumannii, there is an urgent need to develop novel tools to treat infections caused by this dangerous pathogen. To develop knowledge-guided treatment approaches for A. baumannii, a thorough understanding of the mechanism by which this pathogen causes bloodstream infection is required. Here, using a mouse model of infection, we report the identification of A. baumannii genes that are critical for the ability of this pathogen to cause bloodstream infections. This study lays the foundation for future research on A. baumannii genes that can be targeted to develop novel therapeutics against this emerging human pathogen., Acinetobacter baumannii is emerging as a leading global multiple-antibiotic-resistant nosocomial pathogen. The identity of genes essential for pathogenesis in a mammalian host remains largely unknown. Using transposon-directed insertion-site sequencing (TraDIS), we identified A. baumannii genes involved in bacterial survival in a leukopenic mouse model of bloodstream infection. Mice were inoculated with a pooled transposon mutant library derived from 109,000 mutants, and TraDIS was used to map transposon insertion sites in the genomes of bacteria in the inoculum and of bacteria recovered from mouse spleens. Unique transposon insertion sites were mapped and used to calculate a fitness factor for every insertion site based on its relative abundance in the inoculum and postinfection libraries. Eighty-nine transposon insertion mutants that were underrepresented after experimental infection in mice compared to their presence in the inocula were delineated as candidates for further evaluation. Genetically defined mutants lacking feoB (ferrous iron import), ddc (d-ala-d-ala-carboxypeptidase), and pntB (pyridine nucleotide transhydrogenase subunit) exhibited a fitness defect during systemic infection resulting from bacteremia. In vitro, these mutants, as well as a fepA (ferric enterobactin receptor) mutant, are defective in survival in human serum and within macrophages and are hypersensitive to killing by antimicrobial peptides compared to the survival of the parental strain under these conditions. Our data demonstrate that FepA is involved in the uptake of exogenous enterobactin in A. baumannii. Genetic complementation rescues the phenotypes of mutants in assays that emulate conditions encountered during infection. In summary, we have determined novel A. baumannii fitness genes involved in the pathogenesis of mammalian infection. IMPORTANCE A. baumannii is a significant cause of bacterial bloodstream infection in humans. Since multiple antibiotic resistance is becoming more common among strains of A. baumannii, there is an urgent need to develop novel tools to treat infections caused by this dangerous pathogen. To develop knowledge-guided treatment approaches for A. baumannii, a thorough understanding of the mechanism by which this pathogen causes bloodstream infection is required. Here, using a mouse model of infection, we report the identification of A. baumannii genes that are critical for the ability of this pathogen to cause bloodstream infections. This study lays the foundation for future research on A. baumannii genes that can be targeted to develop novel therapeutics against this emerging human pathogen.

Genetic complementation in many bacteria is commonly achieved by reintroducing functional copies of the mutated or deleted genes on a recombinant plasmid. Chromosomal integration systems using the Tn 7 transposon have the advantage of providing a stable single-copy integration that does not require selective pressure. Previous Tn 7 systems have been developed, although none have been shown to work effectively in a variety of enterobacteria. We have developed several mini-Tn 7 and transposase vectors to provide a more versatile system. Transposition of Tn 7 at the chromosomal att Tn 7 site was achieved by a classical conjugation approach, wherein the donor strain harbored the mini-Tn 7 vector and the recipient strain possessed the transposase vector. This approach was efficient for five different pathogenic enterobacterial species. Thus, this system provides a useful tool for single-copy complementation at an episomal site for research in bacterial genetics and microbial pathogenesis. Furthermore, these vectors could also be used for the introduction of foreign genes for use in biotechnology applications, vaccine development, or gene expression and gene fusion constructs.

Summary The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a β-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of β-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a β-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.

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.

The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a β-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of β-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a β-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.

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.

Expression of genes of the locus of enterocyte effacement (LEE) is essential for adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelial cells. Gut factors that may modulate LEE gene expression may therefore influence the outcome of the infection. Because nitric oxide (NO) is a critical effector of the intestinal immune response that may induce transcriptional regulation in enterobacteria, we investigated its influence on LEE expression in EHEC O157:H7. We demonstrate that NO inhibits the expression of genes belonging to LEE1, LEE4, and LEE5 operons, and that the NO sensor nitrite-sensitive repressor (NsrR) is a positive regulator of these operons by interacting directly with the RNA polymerase complex. In the presence of NO, NsrR detaches from the LEE1/4/5 promoter regions and does not activate transcription. In parallel, two regulators of the acid resistance pathway, GadE and GadX, are induced by NO through an indirect NsrR-dependent mechanism. In this context, we show that the NO-dependent LEE1 down-regulation is due to absence of NsrR-mediated activation and to the repressor effect of GadX. Moreover, the inhibition of expression of LEE4 and LEE5 by NO is due to loss of NsrR-mediated activation, to LEE1 down-regulation and to GadE up-regulation. Lastly, we establish that chemical or cellular sources of NO inhibit the adherence of EHEC to human intestinal epithelial cells. These results highlight the critical effect of NsrR in the regulation of the LEE pathogenicity island and the potential role of NO in the limitation of colonization by EHEC., Author Summary Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are food-borne pathogens for humans causing bloody diarrhea and, especially in children under five years old, kidney damages leading to death in 5% of cases. Antibiotics are contra-indicated because they are suspected to increase the severity of the disease. Therefore, it is crucial to develop alternative preventive or therapeutic strategies to fight EHEC infection. To reach this goal, a deeper knowledge of host-pathogen interaction is required. A critical step in EHEC infection is the adhesion of bacterial cells to intestinal epithelial cells. In response to the bacterial infection, the host triggers an immune response directed against the pathogen. The current study shows that a main effector of this immune response, nitric oxide (NO), dramatically reduces the capacity of EHEC to adhere to intestinal epithelial cells. We have investigated the molecular mechanisms involved and identified a NO-sensor regulator that controls the expression of the genes required for EHEC adhesion. This finding underlines that NO could be a potential protective factor limiting the development of EHEC-induced diseases and provides a new avenue of investigation for the development of therapeutic strategies against infections with O157:H7 bacteria.