The PhoP virulence regulon and live oral Salmonella vaccines

The PhoP virulence regulon and live oral Salmonella vaccines Samuel I. Miller*, Wendy P. Loomis, Celia Alpuche-Aranda, Irmgard Behlau and Elizabeth Hohmann The PhoP virulence regulon is essential to S a l m o n e l l a t y p h i m u r i u m mouse typhoid fever pathogenesis and survival within macrophages. This virulence regulon is composed of the PhoP (transcriptional regulator) and PhoQ (environmental sensor)proteins and the genetic loci the), positively (pags .for PhoP activated genes) and negatively (prgs for PhoP repressed genes) regulate. Three regulated loci pagC, pagD, and p r g H , when singly mutated, affect the virulence ofS. typhimurium for mice. Strains with p h o P locus mutations are £fJ'ective as live vaccines in mice, and strains with a constitutive regulator)' mutation, a point mutation in PhoQ, can protect mice against typhoid fever when only very.few organisms are administered. The addition of various PhoP regulon mutations further attenuates a r o A mutants o f S. t y p h i m u r i u m , suggesting that these mutations wouM be useful in further attenuating vaccine strains with metabolic path~?ay mutations. The p h o P , p h o Q , pagC, and p a g D genes are highly conserved between S. t y p h i m u r i u m and S. typhi and may be valuable as mutations in live vaccines for human t),phoidfever. A plasmid suicide vector that allows deletion o f the p a g C gene and stable insertion of heterologous antigen genes within the deleted p a g C locus has been constructed and used successfully in S. t y p h i m u r i u m and S. typhi. Keywords: Salmonella; PhoP virulence regulon; live oral vaccines; gene regulation

Enteric fevers are major causes of morbidity and mortality in man and animals 1. Although it requires the development of cell-mediated as well as humoral immunity, typhoid fever can be prevented by vaccination z. Purified bacterial components and acetone inactivated or heat/phenol treated organisms administered parenterally, as well as live attenuated bacteria administered orally, have been used clinically with approximately 70% efficacy as vaccines against Salmonella typhi 2 7. Unfortunately, none of the currently available typhoid vaccines are broadly used as a public health tool. Though they are moderately effective, parenteral dead vaccines have unacceptable side effects, including fever, malaise, local pain and induration 3 6. Additionally, the patient population most in need of vaccines for typhoid fever may be culturally resistant to parenteral inoculation. Experiments in animals have demonstrated that live vaccines provide more complete and superior protection as compared to dead organisms ~ lo. Lastly, the hope of using Salmonella as a live carrier vaccine of heterologous antigens that would be delivered directly to the macrophage and intestinal lymphoid tissue antigen processing cells has led to a worldwide research effort to develop better live oral vaccines for enteric fever. A major advance in typhoid fever immunization was the development of the live oral vaccine Ty21a by Infectious Disease Unit, Massachusetts General Hospital, Boston, MA 02114, USA. *To whom correspondence should be addressed 0264410x/93/020122~)4 1993 Butterworth-HeinemannLtd 122

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Germanier and colleagues 7'11. This multidose vaccine, which has been approved by the Food and Drug Administration in the United States, is safe but has not been efficacious in all studies; it provided no protection in a group of Swiss travellers 12. The only moderate efficacy of Ty21a may be related to vaccine preparation (it may not always reconstitute after lyophilization), or to the properties of the organism itself. Ty21a was generated by chemical mutagenesis, and it has poor growth characteristics in vitro, due to a number of defined (leucine and isoleucine) and undefined auxotrophies, which may account for its low level of intestinal colonization and requirement for multiple oral doses 7.13,14. Additionally, the molecular basis of attenuation of Ty21 a is unknown is. These properties make it less than ideal as a carrier of heterologous antigens. |n the last decade, work in the mouse model of typhoid fever has documented that many defined mutations affect the virulence of S. typhimurium for mice. These have included various auxotrophic mutations in aromatic amino acid, purine and histidine biosynthesis and regulatory mutations that affect global regulons 16 21. The aromatic amino acid pathway mutations, in particular, have been a focus of interest for live vaccines, and S. typhimurium, S. dublin and S. cholera suis strains with aroA mutations have been found to be safe and effective as live vaccines in mice, pigs, and cows 16"22'23. Recently, defined aroC aroD mutants of S. typhi were fed to human volunteers 24. These organisms were safe at low doses but significant symptoms of typhoid fever were noted at doses

The PhoP regulon and Salmonella vaccines: S.I. Miller et al.

of 5 × 107 organisms, implying that these vaccines may need other attenuating mutations. Additionally, mutations in genes encoding biosynthetic pathways (pathways similar to those of the normal commensal flora) may not be as desirable for vaccine attenuation as mutations in essential virulence factors unique to Salmonella, because of the possibility of complementation by homologous recombination with environmental organisms. Therefore our approach has been to focus on investigations of the mechanism of virulence of Salmonella species and to use this information to develop defined live Salmonella vaccines based on Salmonella-specific virulence factors. As we will discuss below, PhoP regulon mutations investigated in our laboratory would appear to be good candidates for both de novo vaccine development as well as for addition to existing auxotrophic mutant vaccines. A fundamental feature of mouse typhoid fever pathogenesis is the ability of S. typhimurium to survive within macrophages, and given the pathophysiology of human typhoid this is likely to be important to S. typhi pathogenesis 2'21. A large body of evidence from several laboratories has documented that the PhoP regulon is essential to mouse typhoid fever pathogenesis and survival within macrophageslV'25'26. The PhoP regulon is comprised of two regulatory genes phoP and phoQ and the genes they positively and negatively regulate (Figure 1) 1"7'27 29. Positively activated gene products encoded by pags ( P h o P activated genes) require the PhoP and PhoQ proteins for expression. PhoP (224 amino acids) and PhoQ (487 amino acids) are similar to a family of two-component regulatory systems that sense environmental change and perform signal transduction by a phosphotransferase mechanism that activates transcription of a number of unlinked genes 17. When simulation of signal transduction activation is conferred

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Figure 1 Schematic representation of the PhoP regulon. The oval bounded by a double membrane represents a Salmonella organism. PhoQ is shown with a periplasmic domain. On stimulation, such as occurs within macrophages PhoP is phosphorylated by PhoQ. PhoP-P is shown binding to the bacterial chromosome and activing phoP activated genes (pags). PagC and PagD are shown as envelope proteins promoting survival within macrophages. In a different environment than that in which pags are expressed phoP repressed genes (prgs) are activated. PrgH is shown as an envelope protein essential to epithelial cell invasion. Other loci that are part of the PhoP regulon are shown under either activated (pags) or repressed (prgs)

on strains with a mutation in PhoQ that results in the unregulated constitutive expression of pags (phenotype PhopC), other gene products encoded by prgs for PhoP repressed genes are repressed 29. By two-dimensional protein gel analysis of whole cell proteins from strains with mutations in phoP and phoQ genes, it has been estimated that approximately 40 different proteins are regulated by these proteins. Mutations in phoP, phoQ, pagC, pagD and prgH all affect the virulence of S. typhimurium for Balb/c mice (Refs 17 and 27 and C.A. Alpuche and S. Miller, unpubfished observations). Strains with phoP, phoQ, pagC and paqD mutations are all defective in survival within cultured macrophages (Ref. 17 and unpublished observations). All of these genes have been cloned, and, with the exception of pagD, the DNA sequence and deduced amino acid sequence are known, pagC encodes a 188 amino acid outer membrane protein essential to survival within macrophages 3°. Transcrip-tional activation of PhoP activated genes occurs specifically in response to the intracellular environment of the macrophage 31. We have shown that these genes are not increased in transcription on Salmonella invasion of cultured epithelial cells, but approximately 5 hours after phagocytosis by macrophages, pa9 transcription increases 100-fold as compared to extracellular organisms 31. The delay in pa9 transcription appears to be related to the time required for full acidification of the phagolysosome to a pH < 5.0. The need for intact pa9 loci for survival and the specificity of pa9 gene expression within macrophages underscores the notion that deletions of these genes may create vaccine strains which will be able to effectively colonize and transcytose the intestinal epithelia, enter macrophages and be processed, but then die within these antigenpresenting cells. Recently, we have also defined a number of S. typhimurium loci (unpublished observations) encoding PhoP repressed genes. These loci are unlinked and encode envelope proteins. Since PhoP activated genes are not maximally expressed until phagolysosomal acidification occurs, some of these repressed genes may be important to survival within macrophages before full acidification occurs. One of these loci, prgH, is essential for full virulence in mice and encodes a protein essential to epithelial cell invasion. The addition of a prgH mutation to strains with phoP mutations further attenuates S. typhimurium. When more fully characterized, these PhoP repressed genes may be useful as additional mutations in live vaccines. The phoP constitutive mutation (phenotype PhoP c) may be important in increasing the immunogenicity of live Salmonella vaccines. This mutation dramatically decreases S. typhimurium virulence and survival within macrophages 29. As few as 15 PhoP c organisms injected intraperitoneally can protect against greater than 105 wild type organisms administered parenterally to mice 29. This suggests that these organisms are either expressing important protective antigens such as pags at a higher level, or that the surface of the constitutive strain is in some way more immunogenic than the unmutated strain. Recently, we have characterized this constitutive mutation as a single point mutation in the periplasmic domain of PhoQ (unpublished observations). This mutation probably simulates a conformational change that occurs in response to the macrophage environment. Although this constitutive mutation can revert to a wild type phenotype,

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characterization of other similar mutations should allow the development of mutants with multiple point mutations that result in the same phenotype. Eventually such mutations may be useful in live vaccines. This array of P h o P regulated genes raises the question of which P h o P regulon mutations would be the first choice for vaccine development? Regulatory mutations, although they may cause greater attenuation, seem less appropriate because it is possible that virulence factor promoters can undergo spontaneous mutational reversion to an unregulated constitutive phenotype. The ideal choice seems deletion of the virulence factors themselves. The first choice would appear to be pagC, as it contributes significantly to mouse virulence, and is well characterized at the molecular level. This locus has the further advantage that its DNA is unique to Salmonella species by blot hybridization and it is conserved in all Salmonella strains examined including S. typhi and S. paratyphi (unpublished observations). We have recently constructed a vector that allows the deletion of the pagC gene in Salmonella and the stable insertion of heterologous 'antigen genes onto the chromosome (unpublished observations) (Figure 2). One advantage of this approach is that additional safety could be imparted to any existing Salmonella vaccine strains by deletion of pagC; in addition, the possible useful heterologous antigen genes which could be placed on the Salmonella chromosome are legion. Also, the DNA surrounding the heterologous antigen gene would be unique to Salmonella discouraging transfer to mobile genetic elements and possibly to other Enterobacteriaciae. Although antigens can be delivered on plasmid replicons that do not require antibiotic resistance to be maintained, chromosomal insertion should be more stable genetically and have much lower likelihood of transfer to other pathogenic organisms s2. Our vector (pVAC468) is a derivative of the suicide vectors pGP704 and pCVD442

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that utilize marker exchange through a single homologous recombination event to integrate the entire plasmid 33'34. Selection on media containing sucrose utilizing the sacB gene of Bacillus subtilis allows identification of strains with second homologous recombination events that result in replacement of the wild type pagC locus with the deleted locus containing the heterologous antigen gene 35. pVAC468 has convenient restriction endonuclease sites which allow the easy insertion of heterologous antigen genes. We have used pVAC468 to make deletions in S. typhi and to insert the Shiga-like toxin gene into the S. typhimurium aroA deleted vaccine strain SL3261. This vector may have wide applicability for insertion of heterologous antigen genes into S. typhi vaccines. Our knowledge of S. typhimurium mouse typhoid pathogenesis has increased markedly over the last several years. Future studies of the above vaccines in man, as well as the development of human in vitro models of Salmonella infection should contribute even more to this knowledge. It is clear that the P h o P regulon is essential to virulence and that multiple P h o P regulated virulence genes exist, contributing in various degrees to immunogenicity and pathogenicity. Balancing safety and immunogenicity is likely to be a difficult tightrope to tread in the case of both S. typhimurium and S. typhi. Overattenuated vaccines, such as the purine mutants ls.36, will not lead to acceptable immunity, yet adequate attenuation must be present for safety. Theoretically, deletion of virulence factors may limit protective immunity, if an immune response to these proteins is essential for protection. However, unlike mucosal pathogens such as Vibrio cholerae, in which a few major virulence factors are central to pathogenesis, the pathophysiology of typhoid fever is complex, and influenced by the multiple environments the organism experiences within the host cells it encounters. It seems likely, therefore, that multiple virulence factors are operative apd immunogenic, and that the correct balance between attenuation and immunity can be achieved by the use of Salmonella strains with several mutations in different virulence genes. Ultimately, as our knowledge of the mechanisms by which these genes contribute to virulence increases, we may be able to delete portions of these genes essential to virulence functions while retaining those portions essential to immunogenicity. The technology and knowledge to create rational multivalent vaccines that can protect against multiple enteric diseases is close at hand and the next decade should see development of such vaccines as public health tools. ACKNOWLEDGEMENTS

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This work was supported by National Institutes of Health grants RO1 AI30479 and KI 1 AI00917 to S.I.M. C.A.A. was supported by the Fundacion Aaron Saenz, Hospital Infantil de Mexico. I.B. is supported by a National Research Service Award AI08280 and a grant from the Infectious Disease Society of America. E.H. is supported by an institutional Physician Scientist Award from the National Institutes of Health to the Massachusetts General Hospital. REFERENCES 1 Edelman, R. and kevine, M.M. Summary of an international workshop on typhoid fever. Rev. Infect. Dis. 1986, 8, 329-349

The P h o P r e g u l o n a n d S a l m o n e l l a v a c c i n e s : S.I. M i l l e r et al. 2

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