铜绿假单胞菌phoQ基因敲除对氨基糖苷类抗生素耐药性的影响

目的 分析氨基糖苷类抗生素敏感或耐药铜绿假单胞菌菌株二元信号系统PhoQ/PhoP编码基因序列并确定该系统与耐约性关系.方法 采用PCR获得铜绿假单胞菌菌株全长phoQ和phoP基因片段,T-A克隆后测序.构建phoQ和phoP基因原核表达系统,Ni-NTA亲和层析法提纯目的 重组表达产物rPhoQ和rPhoP,皮内注射免疫法制备兔抗血清,免疫双扩散法测定抗血清效价.采用Red重组系统敲除氨基糖苷类抗生素耐药铜绿假单胞菌菌株phoQ基因,采用PCR、测序和Western blot对phoQ突变株进行鉴定.采用试管稀释法测定各铜绿假单胞菌野生株和突变株对4种氨基糖苷类抗生素的最低抑菌浓度(MIC).结果 与GenBank中相关序列比较,所克隆的phoP和phoQ基因核苷酸和氨基酸相似性分别为98.7%～99.6%和98.7～100%、98.4%～99.8%和99.1%～100%.采用pET-42a和E. coli BL21DE3系统成功地表达了rPhoQ和rPhoP.rPhoQ和rPhoP兔抗血清免疫双扩效价分别为1:4和1:8抗血清.经PCR、测序和Western blot鉴定,两株phoQ突变株phoQ基因及产物均缺失.上述phoQ突变株对4种氨基糖苷类抗生素的MIC值分别为其野生株的1/512～1/2048.结论 PhoQ/PhoP是序列保守的铜绿假单胞菌二元信号转导系统,该系统介导细菌对氨基精苷类抗生素的耐药性.     

铜绿假单胞菌phoQ基因敲除对氨基糖苷类抗生素耐药性的影响

目的 分析氨基糖苷类抗生素敏感或耐药铜绿假单胞菌菌株二元信号系统PhoQ/PhoP编码基因序列并确定该系统与耐约性关系.方法 采用PCR获得铜绿假单胞菌菌株全长phoQ和phoP基因片段,T-A克隆后测序.构建phoQ和phoP基因原核表达系统,Ni-NTA亲和层析法提纯目的 重组表达产物rPhoQ和rPhoP,皮内注射免疫法制备兔抗血清,免疫双扩散法测定抗血清效价.采用Red重组系统敲除氨基糖苷类抗生素耐药铜绿假单胞菌菌株phoQ基因,采用PCR、测序和Western blot对phoQ突变株进行鉴定.采用试管稀释法测定各铜绿假单胞菌野生株和突变株对4种氨基糖苷类抗生素的最低抑菌浓度(MIC).结果 与GenBank中相关序列比较,所克隆的phoP和phoQ基因核苷酸和氨基酸相似性分别为98.7%～99.6%和98.7～100%、98.4%～99.8%和99.1%～100%.采用pET-42a和E. coli BL21DE3系统成功地表达了rPhoQ和rPhoP.rPhoQ和rPhoP兔抗血清免疫双扩效价分别为1:4和1:8抗血清.经PCR、测序和Western blot鉴定,两株phoQ突变株phoQ基因及产物均缺失.上述phoQ突变株对4种氨基糖苷类抗生素的MIC值分别为其野生株的1/512～1/2048.结论 PhoQ/PhoP是序列保守的铜绿假单胞菌二元信号转导系统,该系统介导细菌对氨基精苷类抗生素的耐药性.     

PhoP/Q regulated genes inSalmonella typhi: identification of melittin sensitive mutants

Many of the genes (pags (phoPactivatedgenes) andprgs (phoPrepressedgenes) ) regulated by the PhoP and PhoQ proteins (PhoP/Q) are necessary for survival ofSalmonella typhimuriumin murine macrophages and pathogenesis in mice. Although a great deal is known about theS. typhimuriumphoP/Qregulon, little has been done with the human specific pathogenS. typhi, prompting us to investigateS. typhiphoP/Qregulated genes. IsogenicphoP12(null) andphoP24(constitutive) strains were constructed inS. typhiTy2 andS. typhimuriumC5 strains. Comparison of whole cell proteins from these strains by SDS-PAGE showed differences in both the number and molecular mass of PhoP/Q regulated proteins. This suggested thatS. typhiandS. typhimuriummay have different PhoP/Q regulated proteins and/or that their regulation may be different. A genetic procedure was developed to isolate mutations in PhoP/Q regulated genes. This involved random MudJ transposon mutagenesis of aphoP12mutant, creatinglacZ-gene fusions, and screening for Lac⁺ or Lac^- colonies. A mobilizable plasmid carrying thephoP24mutant gene was conjugated into these insertion mutants. Those that changed from Lac^- to Lac⁺ were inferred to bepag::MudJ insertions and those that changed from Lac⁺ to Lac^- were inferred to beprg::MudJ insertions. Five mutants with PhoP/Q regulated MudJ fusions were found by this scheme. The mutations were termedpqa(PhoPQ activated) andpqr(PhoPQ repressed) to distinguish them from other PhoP/Q regulated genes. Thepqa/pqr::MudJ mutations were transduced intoS. typhiphoP⁺ andphoP24strains by Vi-I phage transduction. Characterization of the mutants (Southern blot analysis, β-galactosidase activity on indicator plates and in liquid cultures) strongly suggested that their MudJ insertion mutations were in five different genes. Further characterization involved determining cationic peptide sensitivity and mouse virulence. Two mutants were found to be sensitive to the antimicrobial peptide melittin.     

The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis

At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis.     

The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis

The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37 degrees C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.     

Broad-spectrum antimicrobial activity of human intestinal defensin 5.

Defensins are antibiotic peptides expressed in human and animal myeloid and epithelial cells. Due to the limited availability of natural peptides, the properties of human epithelial defensins have not been studied. We assayed the microbicidal activity of recombinant human intestinal defensin 5 (rHD-5) in the presence of salt (O to 150 mM NaCl) with varied pH (pH 5.5 to pH 8.5) and trypsin (25 and 250 microg/ml). rHD-5 exhibits microbicidal activity against Listeria monocytogenes, Escherichia coli, and Candida albicans. In contrast to cryptdins, the mouse intestinal defensins, rHD-5 is active against both mouse-virulent wild-type Salmonella typhimurium and its isogenic, mouse-avirulent phoP mutant. In the presence of salt, rHD-5 activity was reduced, and at 100 mM NaCl, activity against S. typhimurium was abolished. However, at all salt concentrations tested, rHD-5 remained bactericidal to L. monocytogenes. Activity against L. monocytogenes was not pH dependent but was diminished at pH 5.5 against wild-type S. typhimurium. This acid-induced resistance may have been mediated by the virulence gene regulator phoP, since the phoP mutant was equally sensitive at pH 5.5 and pH 7.4. In the presence of trypsin, rHD-5 was partially cleaved, but even then, rHD-5 at 100 microg/ml decreased the number of CFU of wild-type S. typhimurium by more than 99%. The persistence of microbicidal activity of rHD-5 under these conditions supports the notion that naturally occurring human intestinal defensin is an effective arm of mucosal host defense.     

Cationic defensins arise from charge-neutralized propeptides: a mechanism for avoiding leukocyte autocytotoxicity?

Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mammalian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic granules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil-specific rabbit defensin cDNAs that code for preproprotein precursors to the mature defensins NP-3a, NP-4, and NP-5. The preprodefensins NP-3a, NP-4, and NP-5, like the previously characterized preprodefensins, lack consensus sequences for N-linked glycosylation, suggesting that defensins are targeted to lysosome-like granules by a mechanism not dependent on the mannose-6-phosphate receptor. Analysis of all seven known myeloid prodefensins revealed a structure wherein an anionic propiece neutralizes the cationicity of the mature peptide. Because defensins apparently require cationic epitopes for cell membrane permeabilization and cytotoxicity, charge neutralization of mature peptides by their anionic propieces may prevent autocytotoxicity during defensin synthesis and processing.     

Cryptdins: antimicrobial defensins of the murine small intestine.

Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria. Recent studies by Ouellette and associates (A. J. Ouellette, R. M. Greco, M. James, D. Frederick, J. Naftilan, and J. T. Fallon, J. Cell Biol. 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported. We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells. Both peptides contained 35 amino acid residues, including the characteristic defensin "signature" of six invariantly conserved cysteines. Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J. Cell Biol. 108:1687-1695, 1989). Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro. Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages. Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S. typhimurium 14028S. Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties. The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L. monocytogenes and Salmonella species.     

Infection with an avirulent phoP mutant of Neisseria meningitidis confers broad cross-reactive immunity

Successful vaccines against serogroup A and C meningococcal strains have been developed, but current serogroup B vaccines provide protection against only a limited range of strains. The ideal meningococcal vaccine would provide cross-reactive immunity against the variety of strains that may be encountered in any community, but it is unclear whether the meningococcus possesses immune targets that have the necessary level of cross-reactivity. We have generated a phoP mutant of the meningococcus by allele exchange. PhoP is a component of a two-component regulatory system which in other bacteria is an important regulator of virulence gene expression. Inactivation of the PhoP-PhoQ system in Salmonella leads to avirulence, and phoP mutants have been shown to confer protection against virulent challenge. These mutants have been examined as potential live attenuated vaccines. We here show that a phoP mutant of the meningococcus is avirulent in a mouse model of infection. Moreover, infection of mice with the phoP mutant stimulated a bactericidal immune response that not only killed the infecting strain but also showed cross-reactive bactericidal activity against a range of strains with different serogroup, serotype, and serosubtyping antigens. Sera from the mutant-infected mice contained immunoglobulin G that bound to the surface of a range of meningococcal strains and mediated opsonophagocytosis of meningococci by human phagocytic cells. The meningococcal phoP mutant is thus a candidate live, attenuated vaccine strain and may also be used to identify cross-reactive protective antigens in the meningococcus.     

Immunogenicity against human papillomavirus type 16 virus-like particles is strongly enhanced by the PhoPc phenotype in Salmonella enterica serovar Typhimurium

Recombinant Salmonella strains have been widely used to deliver heterologous antigens and induce immune responses in vaccinated animals and humans. It remains to be established, however, how these bacteria mount an immune response; this has prevented the rational design of vaccines. Here we report for the first time that a particular genetic program, PhoPc, is necessary for recombinant Salmonella strains to induce an antibody response to a heterologous antigen, the human papillomaviruses type 16 (HPV16) virus-like particle (VLP). The PhoPc phenotype results from a point mutation in phoQ, the gene encoding the sensor component of a two-component regulatory system (PhoP-PhoQ) that controls the expression of a number of virulence factors in Salmonellae. To demonstrate that immunogenicity of the viral antigen expressed by the bacterial vector was dependent on the PhoPc phenotype, we have expressed the phoQ mutant gene (phoQ24) in two differently attenuated Salmonella enterica serovar Typhimurium strains. Our data show extrachromosomal phoQ24 to be dominant over the chromosomal copy of the phoQ gene, conferring the PhoPc phenotype on the recipient strains. In addition, activation of PhoPQ-regulated genes by the plasmid-encoded PhoQ24 did not alter bacterial survival and conferred immunogenicity to the HPV16 VLP expressed in the two S. enterica serovar Typhimurium backgrounds, inducing the production of HPV-specific antibodies in mice. This strongly suggests that at least one of the PhoP-regulated genes is necessary for mounting an efficient antibody response to HPV16 VLP. This finding sets the stage for further development of a Salmonella-based vaccine against HPV infection and cervical cancer.     

Two-component bacterial regulation systems: Targets of a search for new antibacterial drugs

A review of the literature presents data on two-component bacterial regulatory systems that may be possible targets in the search of new antibacterial drugs because of their universality and the role in regulation of pathogenicity. We discuss (a) the structural-functional organization of two-component systems taking as example OmpR family (EnvZ/OmpR and PhoQ, PhoP) regulating a number of processes in bacteria faciliting adaptation to stress from changes in the environment and human host, providing virulence and formation of biofilms, these being a cause of many human chronic infections, and (b) the genes and functions regulated by two-component systems by EnvZ/OmpR system, OmpR protein in particular, and PhoQ/PhoP. The review speculates on possibilities of searching for inhibitors of the proteins of two-component systems and their possible role in virulence attenuation of pathogenic bacteria.     

The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

The adaptation of Pseudomonas aeruginosa to its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance of P. aeruginosa as well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence of P. aeruginosa is unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of the oprH-phoP-phoQ operon and its target, the arn lipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, a phoQ mutant cocultured on epithelial cells produced less secreted protease and lipase and, like the phoQ mutant, piv, lipH, and lasB mutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation in phoQ also resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation of phoQ results in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.     

In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by S. enterica serovar Typhimurium: the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to S. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.     

Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pallidum.

Defensins, which are peptides with broad antimicrobial activity, are major constituents of rabbit neutrophils and certain macrophages. We tested six rabbit defensins, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, for activity against Treponema pallidum subsp. pallidum. Mixtures of T. pallidum and defensin in 10% normal rabbit serum (NRS) or heat-inactivated NRS (HI-NRS) were incubated anaerobically for various time periods ranging between 0 and 16 h and then examined by dark-field microscopy for treponemal motility or inoculated intradermally into rabbits to assess treponemal virulence. Immobilization of T. pallidum by NP-1 (400 micrograms/ml) occurred after 4 and 8 h of coincubation in mixtures containing NRS and HI-NRS, respectively. Similarly, neutralization of T. pallidum by NP-1 occurred more rapidly and was complete when incubations were performed in NRS as compared with that in HI-NRS. Endpoint titration confirmed the augmentation of NP-1 antitreponemal activity by heat-labile serum factors; NP-1 showed neutralizing activity at 4 micrograms/ml (about 1 microM) in NRS and at 40 micrograms/ml in HI-NRS. When NP-1 was tested in serum that was deficient in C6, the T. pallidum neutralizing activity of NP-1 was reduced to levels slightly greater than that observed in HI-NRS. NP-1 that had been reduced and alkylated was inactive against T. pallidum. When NP-2, NP-3a, NP-3b, NP-4, and NP-5 were tested at 400 micrograms/ml, all exerted potent treponemicidal activity, manifested by abrogation or delayed development of cutaneous lesions relative to that of controls. These data suggest that defensins may equip certain macrophages and neutrophils to participate in host defense against T. pallidum, that the direct activity of defensins against T. pallidum is enhanced by heat-labile serum factors (presumably complement), and that conformational factors influence the biological activity of the defensin molecule.     

Synergistic activity of rabbit granulocyte peptides against Candida albicans.

Rabbit granulocytes contain six antimicrobial peptides that are structurally homologous to the human neutrophil "defensins." NP-5, a rabbit defensin, lacks significant activity against Candida albicans. Nevertheless, its addition to submicromolar concentrations of rabbit NP-1, NP-2, or NP-3a potentiates their candidacidal effect. Thus, granulocyte defensins can act synergistically against potential pathogens.     

The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence

Yersinia pestis and Yersinia pseudotuberculosis are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for Y. pestis survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for Y. pseudotuberculosis pathogenesis was investigated. Y. pseudotuberculosis phoP mutants were unable to replicate in low-Mg(2+) medium or in macrophages. phoP(+) Y. pseudotuberculosis strains initiated replication in macrophages after a lag period of approximately 5 h, as shown by fluorescence microscopy and viable count assays. Y. pseudotuberculosis phoP mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing phoP(+) or phoP mutant Y. pseudotuberculosis was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing phoP mutant Y. pseudotuberculosis acquired cathepsin D at a higher rate than phagosomes containing phoP(+) bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident approximately 5 h after infection, suggesting that phoP mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a Y. pestis phoP mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A Y. pseudotuberculosis phoP mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for Y. pseudotuberculosis pathogenesis.     

Contribution of rabbit leukocyte defensins to the host response in experimental syphilis.

In the companion paper (L. A. Borenstein, M. E. Selsted, R. I. Lehrer, and J. N. Miller, Infect. Immun. 59:1359-1367, 1991), we report that rabbit alveolar macrophage and neutrophil derived defensins possess antimicrobial activity against Treponema pallidum subsp. pallidum, the etiologic agent of syphilis. In this study, antisera specific for NP-1 and NP-2 (defensins present in certain macrophages and polymorphonuclear leukocytes) and NP-5 (a defensin produced only in neutrophils) were used to detect these peptides by immunoperoxidase staining in testicular lesions from infected rabbits. Profound amounts of cell-free and cell-associated defensins were detected in the tunica albuginea and interstitial spaces during the first 24 h of infection. The presence of defensins was transient and almost undetectable by day 4. Interstitial defensins were detected again at day 10 and increased through day 16, at which time lesion healing was evident by hematoxylin and eosin staining. The appearance and increase in detectable defensins between days 10 and 16 of infection correlated with a reduction in numbers and disappearance of T. pallidum, as demonstrated by using silver staining. The extent and pattern of immunostaining for NP-1 and NP-2 corresponded with immunostaining for NP-5 and identified neutrophils as the cellular source of the defensins. These findings indicate that defensins may contribute to the control of local T. pallidum infection and suggest a role for acute inflammatory processes in the resolution of early experimental syphilis.     

Increased Pho regulon activation correlates with decreased virulence of an avian pathogenic Escherichia coli O78 strain

Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.