- Email: [email protected]
Typhoid Fever: The More We Learn, the Less We Know (Apologies, Albert Einstein)
Cell Host & Microbe
Previews pools. Among features of this AMP-based regeneration system, it is less efficient than ISC-based systems. In particular, reconstitution of depleted AMP pools without increasing proliferation requires a considerable extension of developmental time, which leads to developmental delays at the organism level. Although the present study describes this developmental delay as an essential component of larval EC regeneration, the mechanism by which infection-induced AMP depletion promotes the developmental delay remains unexplored. Similar developmental delays have been described in Drosophila larvae and were critical for coordinating growth programs in response to growth disturbances such as tissue damage. Moreover, these delays were dependent on a secretory Drosophila insulin-like peptide 8 (DILP8), which was shown to delay the entry of metamorphosis by inhibiting ecdysone
biosynthesis (Colombani et al., 2012; Garelli et al., 2012). Whether similar DILP8ecdysone hormonal processes are involved in larval developmental delays and coordinate the growth program in response to enteric infection remains an intriguing subject for future studies. ACKNOWLEDGMENTS The authors are supported by a grant (no. 2015R1A3A2033475) from the National Research Foundation of Korea. REFERENCES Buchon, N., Broderick, N.A., Chakrabarti, S., and Lemaitre, B. (2009). Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev. 23, 2333–2344. Buchon, N., Broderick, N.A., Kuraishi, T., and Lemaitre, B. (2010). Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection. BMC Biol. 8, 152.
Colombani, J., Andersen, D.S., and Le´opold, P. (2012). Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science 336, 582–585. Garelli, A., Gontijo, A.M., Miguela, V., Caparros, E., and Dominguez, M. (2012). Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation. Science 336, 579–582. Houtz, P., Bonfini, A., Bing, X., and Buchon, N. (2019). Recruitment of adult precursor cells underlies limited repair of the infected larval midgut. Cell Host Microbe 26, this issue, 412–425. Jiang, H., and Edgar, B.A. (2009). EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors. Development 136, 483–493. Mathur, D., Bost, A., Driver, I., and Ohlstein, B. (2010). A transient niche regulates the specification of Drosophila intestinal stem cells. Science 327, 210–213. Micchelli, C.A., and Perrimon, N. (2006). Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439, 475–479. Ohlstein, B., and Spradling, A. (2006). The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439, 470–474.
Typhoid Fever: The More We Learn, the Less We Know (Apologies, Albert Einstein) Nancy Wang,1 Leigh A. Knodler,1,2 and Richard A. Strugnell1,* 1Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia 2Washington State University Paul G. Allen School for Global Animal Health, Pullman, WA, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.08.012
In this issue of Cell Host & Microbe, Karlinsey et al. (2019) combine TraDIS with humanized mice to identify genes required for early replication of Salmonella Typhi in vivo. Surprisingly, some expected virulence traits and genes appear dispensable in the replication of S. Typhi, supporting findings from a recent human challenge study by Gibani et al. (2019). William Osler, the Canadian physician who revolutionized medical education, wrote what is considered to be the first definitive textbook for training medical doctors. Osler thought enough of typhoid fever to make the disease chapter 1 of this landmark 1892 book (Osler, 1892), and to devote 38 pages to a description of the infection succinctly characterized by hyperplasia and ulceration of the intestines, lymphadenopathy, and fever, all of which are marked by complexity and inconsis-
tency. Osler’s detailed clinical description still holds today. Importantly, he noted that the third of Koch’s laws, ‘‘the requirement for the disease to be experimentally produced by pure cultures,’’ had not been met and held that this occurred because ‘‘.the animals used for experimentation are not susceptible to typhoid fever’’ (page 3). Most perceptibly, Osler also described the production of a ptomaine by the pathogen, which he termed a ‘‘typhotoxin.’’
Estimates of the current incidence of typhoid fever place the disease burden at 10–20 million cases per year, with a mortality rate of approximately 1%, predominantly in low- and low-to-middle income countries (LICs and LMICs). There are three vaccines in use, a conjugated and non-conjugated polysaccharide vaccine, and a live-attenuated vaccine—all have efficacy in the range 50%–70%, albeit with maximal protection that is thought to last for 1–3 years.
Cell Host & Microbe 26, September 11, 2019 ª 2019 Elsevier Inc. 303
Cell Host & Microbe
Previews
temperature (qF)
human challenge system 104 103 102 101 100 99 98 97
treatment
humanized mice perforaon neurological signs
0
7
14
21
me post challenge (days)
Challenge Blood culture
+
+
Figure 1. Infection with Salmonella Typhi in Humanized Mice and in the Human Challenge System Model the Acute Phase of Typhoid Fever Typical temperature chart from a case of typhoid fever in a human volunteer orally infected with S. Typhi. The challenge system in human volunteers (Gibani et al., 2019) requires treatment at onset of clinical typhoid, or at day 14. Studies of clinical typhoid in patients suggest that many of the infection-related sequele, e.g., perforation, do not manifest until the end of week 2 post-infection, or later (Osler, 1892). This necessity to treat volunteers means that a role for typhoid toxin in these later signs cannot be excluded. The humanized mouse model resembles the early bacteremic phase when there are interactions between S. Typhi and the innate immune system, but likely limited engagement of the bacterium with the adaptive immune system, which may be responsible for the later sequele.
Much of what we know about the molecular pathogenesis of typhoid fever has been extrapolated from studies of Salmonella enterica serovar Typhimurium (S. Typhimurium) in the inbred murine model. There are significant caveats to interpreting this work, including the many obvious genetic differences between S. Typhimurium and the human-specific Salmonella enterica serovar Typhi (S. Typhi), not the least the presence of the Vi capsule and toxin in S. Typhi. Host adaptation and restriction appear to have driven the formation of more than 100 pseudogenes in S. Typhi (Holt et al., 2009), a feature shared with other human-adapted and specific pathogens such as Mycobacterium leprae. It is generally felt that a deeper understanding of the pathogenic processes used by S. Typhi could lead to the development of a live-attenuated vaccine with increased, and longer lasting, protection.
The development of an animal model using S. Typhi as the pathogen has been a long-term aim of those working in the field. Despite several ‘‘false starts’’ wherein S. Typhi replication in mice was difficult to reproduce, the development of ‘‘humanization’’ technology wherein severely immunodeficient (e.g., SCID or Rag2 / gc / ) mice were reconstituted with a human immune system has provided an experimental animal model in which molecular pathogenic mechanisms of S. Typhi can be explored (Libby et al., 2010; Song et al., 2010). In this complementation process, the mice adopt the human hemopoietic compartment, which can better support pathogens that have evolved to engage with human cells. This murine humanization technique has now been combined with a highthroughput, genomics-based method for identifying bacterial virulence determinants. In this issue of Cell Host & Microbe, Karlinsey et al. (2019) used a very
304 Cell Host & Microbe 26, September 11, 2019
high-density transposon library and genomic sequencing, the technique known as transposon-directed insertion site sequencing (TraDIS), to identify the genes that were essential to the growth of S. Typhi in humanized SCID mice after intraperitoneal (i.p.) infection. The paper reveals that in early infection (24 h) (Figure 1), the replication of S. Typhi in the liver and spleen of humanized mice is independent of the Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), phoPQ, and expression of cdtB, a component of the typhoid toxin. This was inferred by the relative fitness of S. Typhi carrying inactivating insertions in these pathogenicity islands, or in specifically targeted genes. PhoPQ is a twocomponent regulatory system that regulates SPI-2 and other genes required for S. Typhimurium virulence in mice. SPI-2 is an important pathogenicity island for S. Typhimurium in highly sensitive C57BL/6 mice where SPI-2-delivered
Cell Host & Microbe
Previews type III effectors perform key roles in protecting the bacterium against killing by the murine reticuloendothelial system. The lack of full dependence of S. Typhi on SPI-2 might have been inferred by a recent review of SPI-2 effector proteins in different serovars of S. enterica that found that S. Typhi had lost (or never acquired) 50% of the SPI-2 effectors that were analyzed (Jennings et al., 2017). The approach of Karlinsey et al. (2019) validated some earlier findings in naturally and experimentally infected humans, and tissue culture models of S. Typhi infection. Vi encapsulation by S. Typhi was essential for survival in the humanized murine model; Wain et al. found that Vi-negative S. Typhi were rarely observed in the blood of humans with natural typhoid infections (Wain et al., 2005). The TraDIS study also revealed a role for LPS in virulence, as S. Typhi with inactivating insertions in the glycosyltransferase genes responsible for assembling the outer core of the S. Typhi LPS and the O-antigen were also highly attenuated. Mutations affecting LPS biogenesis have a checkered history in typhoid research. Among many other point mutations, the vaccine strain of S. Typhi Ty21a carries a conditional ‘‘rough’’ LPS phenotype through mutation of galE, which was thought to attenuate the bacterium, especially in the gut. However, a defined galE mutant of S. Typhi caused a typhoid fever-like illness in human volunteers orally challenged with a high dose of the organism (Hone et al., 1988). Iron acquisition genes were also essential for S. Typhi growth in the humanized mice, demonstrating a key role for iron in the humanized mouse model. Insertions into genes that encode salmochelin were highly attenuating, an observation that corroborated studies showing essentiality of iron acquisition in S. Typhimurium infections in mice. S. Typhi carrying mutations in the pre-chorismate pathway (e.g., aroA and aroC) were growth attenuated in humanized mice, recapitulating an observation made in human volunteers infected with a defined aromatic mutant more than 20 years ago. S. Typhi purine auxotrophs were likewise attenuated, similar to their S. Typhimurium counterparts in sensitive mice. The technical limitations with the humanized mouse model are well known— the organisms in this example were
delivered i.p., possibly circumventing the need for SPI-1. The acute time point (24 h) highlights the importance of genes in early interaction with the host, but genes required for longer term adaptation in the host may have been missed (Figure 1). Animal numbers were necessarily small and there is some variation between animals, depending on the source and degree of engraftment of the donor tissue. Only hematopoietic cells were transferred, i.e., epithelial and stromal cells remained murine. While there were histological similarities between the human and mouse infections, mice lacking an immune system from early development (e.g., SCID), even if engrafted at birth, are likely to have lymphoid tissue ultrastructure that is abnormal (Libby et al., 2010). The absence of a phenotype of the cdtB mutant of S. Typhi in acute infection of humanized mice is a disappointment for the field and may limit prosecution of a typhoid toxin-based toxoid as a new vaccine for typhoid fever. The observation made in the humanized mice recapitulates findings from a very recent study conducted in human volunteers where a typhoid toxin-negative variant of S. Typhi appeared fully virulent in the early phases of typhoid fever (Gibani et al., 2019). In this study by Gibani et al., human volunteers were challenged with wild-type S. Typhi or an isogenic toxin deletion mutant and were observed until a definitive diagnosis of typhoid fever was made, or until day 14, when they were treated with antibiotics (Figure 1). Analysis of the two groups of volunteers did not reveal differences in the incidence of blood infection, i.e., typhoid fever, but did see an increase in the duration of bacteremia in the volunteers challenged with the toxin-negative mutant. Increased infection by the toxinnegative strain was mirrored in a growth competition study in mice lacking bloc3. Mice lacking the Rab32 nucleotide exchange factor BLOC3 support the growth of S. Typhi when delivered i.p. (Spano` et al., 2016), likely because a Rab32/ BLOC3 trafficking pathway normally delivers antimicrobial cargo to the S. Typhicontaining vacuole. Upon examining the spleens of bloc3 / mice, the toxin-negative mutant of S. Typhi outcompeted the growth of the S. Typhi parent strain by a factor of 2-fold over 5 days (Spano` et al., 2016).
As Osler carefully documented, the clinical picture of typhoid fever is complex. The blood infection is often missed in LMICs. The intestinal hyperplasia can result in a severe form of disease, sometimes fatal, where it is thought that the immune response in the gut-associated lymphoid tissue drives ulceration of the Peyer’s patches and then perforation (Osler, 1892), leading to egress of the intestinal contents into the peritoneal cavity and potentially fatal peritonitis. Allied with these immunopathological changes, a high and prolonged fever is sometimes associated with neurological signs simulating meningitis. Neither of these sequelae, which happen two or more weeks after initial natural exposure, are examined in the human volunteer challenge, or in the humanized mouse model (Figure 1). It is possible that the typhoid toxin has a role in both of these complications of typhoid fever. CdtB appears to be a target for the human immune system. Studies by Napolitani et al. compared peripheral blood mononuclear cells from bacteremic and non-bacteremic volunteers challenged with S. Typhi, and there was higher frequency of CdtB-specific, Th1-type T cells in those subjects that developed a bacteremia (Napolitani et al., 2018). This observation leaves open the possibility that the typhoid toxin is an immune target resulting in the hyperplasia associated with a severe form of the disease. The quest continues to understand the pathogenesis of this complex host-specific pathogen that has lost many nonessential genes through evolution that have remained intact in host ‘‘generalists’’ like S. Typhimurium. The fact that S. Typhi has independently acquired and now retains typhoid toxin and SPI-2 (though not all the SPI-2 effectors) would suggest that both have a role in human infection. The experimental systems we have to explore their function, while representing important and exciting advances, may not yet be complete.
REFERENCES Gibani, M.M., Jones, E., Barton, A., Jin, C., Meek, J., Camara, S., Galal, U., Heinz, E., RosenbergHasson, Y., Obermoser, G., et al. (2019). Investigation of the role of typhoid toxin in acute typhoid fever in a human challenge model. Nat. Med. 25, 1082–1088.
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Previews Holt, K.E., Thomson, N.R., Wain, J., Langridge, G.C., Hasan, R., Bhutta, Z.A., Quail, M.A., Norbertczak, H., Walker, D., Simmonds, M., et al. (2009). Pseudogene accumulation in the evolutionary histories of Salmonella enterica serovars Paratyphi A and Typhi. BMC Genomics 10, 36.
M.A., Greiner, D.L., Schultz, L.D., Gallagher, L.A., Bawn, M., et al. (2019). Genome-wide analysis of Salmonella enterica serovar Typhi in humanized mice reveals key virulence factors. Cell Host Microbe 26. Published online August 22, 2019. https://doi.org/10.1016/j.chom.2019.08.001.
Hone, D.M., Attridge, S.R., Forrest, B., Morona, R., Daniels, D., LaBrooy, J.T., Bartholomeusz, R.C., Shearman, D.J., and Hackett, J. (1988). A galE via (Vi antigen-negative) mutant of Salmonella typhi Ty2 retains virulence in humans. Infect. Immun. 56, 1326–1333.
Libby, S.J., Brehm, M.A., Greiner, D.L., Shultz, L.D., McClelland, M., Smith, K.D., Cookson, B.T., Karlinsey, J.E., Kinkel, T.L., Porwollik, S., et al. (2010). Humanized nonobese diabetic-scid IL2rgammanull mice are susceptible to lethal Salmonella Typhi infection. Proc. Natl. Acad. Sci. USA 107, 15589–15594.
Jennings, E., Thurston, T.L.M., and Holden, D.W. (2017). Salmonella SPI-2 type III secretion system effectors: molecular mechanisms and physiological consequences. Cell Host Microbe 22, 217–231. Karlinsey, J.E., Stepien, T.A., Mayho, M., Singletary, L.A., Bingham-Ramos, L.K., Brehm,
Napolitani, G., Kurupati, P., Teng, K.W.W., Gibani, M.M., Rei, M., Aulicino, A., Preciado-Llanes, L., Wong, M.T., Becht, E., Howson, L., et al. (2018). Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses. Nat. Immunol. 19, 742–754.
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Osler, W. (1892). Specific infectious diseases - I. Typhoid fever. In A Text-Book on the Practice of Medicine, pp. 2–39. Song, J., Willinger, T., Rongvaux, A., Eynon, E.E., Stevens, S., Manz, M.G., Flavell, R.A., and Gala´n, J.E. (2010). A mouse model for the human pathogen Salmonella typhi. Cell Host Microbe 8, 369–376. Spano`, S., Gao, X., Hannemann, S., Lara-Tejero, M., and Gala´n, J.E. (2016). A bacterial pathogen targets a host Rab-family GTPase defense pathway with a GAP. Cell Host Microbe 19, 216–226. Wain, J., House, D., Zafar, A., Baker, S., Nair, S., Kidgell, C., Bhutta, Z., Dougan, G., and Hasan, R. (2005). Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates from Pakistan. J. Clin. Microbiol. 43, 1158–1165.
Previews pools. Among features of this AMP-based regeneration system, it is less efficient than ISC-based systems. In particular, reconstitution of depleted AMP pools without increasing proliferation requires a considerable extension of developmental time, which leads to developmental delays at the organism level. Although the present study describes this developmental delay as an essential component of larval EC regeneration, the mechanism by which infection-induced AMP depletion promotes the developmental delay remains unexplored. Similar developmental delays have been described in Drosophila larvae and were critical for coordinating growth programs in response to growth disturbances such as tissue damage. Moreover, these delays were dependent on a secretory Drosophila insulin-like peptide 8 (DILP8), which was shown to delay the entry of metamorphosis by inhibiting ecdysone
biosynthesis (Colombani et al., 2012; Garelli et al., 2012). Whether similar DILP8ecdysone hormonal processes are involved in larval developmental delays and coordinate the growth program in response to enteric infection remains an intriguing subject for future studies. ACKNOWLEDGMENTS The authors are supported by a grant (no. 2015R1A3A2033475) from the National Research Foundation of Korea. REFERENCES Buchon, N., Broderick, N.A., Chakrabarti, S., and Lemaitre, B. (2009). Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev. 23, 2333–2344. Buchon, N., Broderick, N.A., Kuraishi, T., and Lemaitre, B. (2010). Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection. BMC Biol. 8, 152.
Colombani, J., Andersen, D.S., and Le´opold, P. (2012). Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science 336, 582–585. Garelli, A., Gontijo, A.M., Miguela, V., Caparros, E., and Dominguez, M. (2012). Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation. Science 336, 579–582. Houtz, P., Bonfini, A., Bing, X., and Buchon, N. (2019). Recruitment of adult precursor cells underlies limited repair of the infected larval midgut. Cell Host Microbe 26, this issue, 412–425. Jiang, H., and Edgar, B.A. (2009). EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors. Development 136, 483–493. Mathur, D., Bost, A., Driver, I., and Ohlstein, B. (2010). A transient niche regulates the specification of Drosophila intestinal stem cells. Science 327, 210–213. Micchelli, C.A., and Perrimon, N. (2006). Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439, 475–479. Ohlstein, B., and Spradling, A. (2006). The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439, 470–474.
Typhoid Fever: The More We Learn, the Less We Know (Apologies, Albert Einstein) Nancy Wang,1 Leigh A. Knodler,1,2 and Richard A. Strugnell1,* 1Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia 2Washington State University Paul G. Allen School for Global Animal Health, Pullman, WA, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.08.012
In this issue of Cell Host & Microbe, Karlinsey et al. (2019) combine TraDIS with humanized mice to identify genes required for early replication of Salmonella Typhi in vivo. Surprisingly, some expected virulence traits and genes appear dispensable in the replication of S. Typhi, supporting findings from a recent human challenge study by Gibani et al. (2019). William Osler, the Canadian physician who revolutionized medical education, wrote what is considered to be the first definitive textbook for training medical doctors. Osler thought enough of typhoid fever to make the disease chapter 1 of this landmark 1892 book (Osler, 1892), and to devote 38 pages to a description of the infection succinctly characterized by hyperplasia and ulceration of the intestines, lymphadenopathy, and fever, all of which are marked by complexity and inconsis-
tency. Osler’s detailed clinical description still holds today. Importantly, he noted that the third of Koch’s laws, ‘‘the requirement for the disease to be experimentally produced by pure cultures,’’ had not been met and held that this occurred because ‘‘.the animals used for experimentation are not susceptible to typhoid fever’’ (page 3). Most perceptibly, Osler also described the production of a ptomaine by the pathogen, which he termed a ‘‘typhotoxin.’’
Estimates of the current incidence of typhoid fever place the disease burden at 10–20 million cases per year, with a mortality rate of approximately 1%, predominantly in low- and low-to-middle income countries (LICs and LMICs). There are three vaccines in use, a conjugated and non-conjugated polysaccharide vaccine, and a live-attenuated vaccine—all have efficacy in the range 50%–70%, albeit with maximal protection that is thought to last for 1–3 years.
Cell Host & Microbe 26, September 11, 2019 ª 2019 Elsevier Inc. 303
Cell Host & Microbe
Previews
temperature (qF)
human challenge system 104 103 102 101 100 99 98 97
treatment
humanized mice perforaon neurological signs
0
7
14
21
me post challenge (days)
Challenge Blood culture
+
+
Figure 1. Infection with Salmonella Typhi in Humanized Mice and in the Human Challenge System Model the Acute Phase of Typhoid Fever Typical temperature chart from a case of typhoid fever in a human volunteer orally infected with S. Typhi. The challenge system in human volunteers (Gibani et al., 2019) requires treatment at onset of clinical typhoid, or at day 14. Studies of clinical typhoid in patients suggest that many of the infection-related sequele, e.g., perforation, do not manifest until the end of week 2 post-infection, or later (Osler, 1892). This necessity to treat volunteers means that a role for typhoid toxin in these later signs cannot be excluded. The humanized mouse model resembles the early bacteremic phase when there are interactions between S. Typhi and the innate immune system, but likely limited engagement of the bacterium with the adaptive immune system, which may be responsible for the later sequele.
Much of what we know about the molecular pathogenesis of typhoid fever has been extrapolated from studies of Salmonella enterica serovar Typhimurium (S. Typhimurium) in the inbred murine model. There are significant caveats to interpreting this work, including the many obvious genetic differences between S. Typhimurium and the human-specific Salmonella enterica serovar Typhi (S. Typhi), not the least the presence of the Vi capsule and toxin in S. Typhi. Host adaptation and restriction appear to have driven the formation of more than 100 pseudogenes in S. Typhi (Holt et al., 2009), a feature shared with other human-adapted and specific pathogens such as Mycobacterium leprae. It is generally felt that a deeper understanding of the pathogenic processes used by S. Typhi could lead to the development of a live-attenuated vaccine with increased, and longer lasting, protection.
The development of an animal model using S. Typhi as the pathogen has been a long-term aim of those working in the field. Despite several ‘‘false starts’’ wherein S. Typhi replication in mice was difficult to reproduce, the development of ‘‘humanization’’ technology wherein severely immunodeficient (e.g., SCID or Rag2 / gc / ) mice were reconstituted with a human immune system has provided an experimental animal model in which molecular pathogenic mechanisms of S. Typhi can be explored (Libby et al., 2010; Song et al., 2010). In this complementation process, the mice adopt the human hemopoietic compartment, which can better support pathogens that have evolved to engage with human cells. This murine humanization technique has now been combined with a highthroughput, genomics-based method for identifying bacterial virulence determinants. In this issue of Cell Host & Microbe, Karlinsey et al. (2019) used a very
304 Cell Host & Microbe 26, September 11, 2019
high-density transposon library and genomic sequencing, the technique known as transposon-directed insertion site sequencing (TraDIS), to identify the genes that were essential to the growth of S. Typhi in humanized SCID mice after intraperitoneal (i.p.) infection. The paper reveals that in early infection (24 h) (Figure 1), the replication of S. Typhi in the liver and spleen of humanized mice is independent of the Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), phoPQ, and expression of cdtB, a component of the typhoid toxin. This was inferred by the relative fitness of S. Typhi carrying inactivating insertions in these pathogenicity islands, or in specifically targeted genes. PhoPQ is a twocomponent regulatory system that regulates SPI-2 and other genes required for S. Typhimurium virulence in mice. SPI-2 is an important pathogenicity island for S. Typhimurium in highly sensitive C57BL/6 mice where SPI-2-delivered
Cell Host & Microbe
Previews type III effectors perform key roles in protecting the bacterium against killing by the murine reticuloendothelial system. The lack of full dependence of S. Typhi on SPI-2 might have been inferred by a recent review of SPI-2 effector proteins in different serovars of S. enterica that found that S. Typhi had lost (or never acquired) 50% of the SPI-2 effectors that were analyzed (Jennings et al., 2017). The approach of Karlinsey et al. (2019) validated some earlier findings in naturally and experimentally infected humans, and tissue culture models of S. Typhi infection. Vi encapsulation by S. Typhi was essential for survival in the humanized murine model; Wain et al. found that Vi-negative S. Typhi were rarely observed in the blood of humans with natural typhoid infections (Wain et al., 2005). The TraDIS study also revealed a role for LPS in virulence, as S. Typhi with inactivating insertions in the glycosyltransferase genes responsible for assembling the outer core of the S. Typhi LPS and the O-antigen were also highly attenuated. Mutations affecting LPS biogenesis have a checkered history in typhoid research. Among many other point mutations, the vaccine strain of S. Typhi Ty21a carries a conditional ‘‘rough’’ LPS phenotype through mutation of galE, which was thought to attenuate the bacterium, especially in the gut. However, a defined galE mutant of S. Typhi caused a typhoid fever-like illness in human volunteers orally challenged with a high dose of the organism (Hone et al., 1988). Iron acquisition genes were also essential for S. Typhi growth in the humanized mice, demonstrating a key role for iron in the humanized mouse model. Insertions into genes that encode salmochelin were highly attenuating, an observation that corroborated studies showing essentiality of iron acquisition in S. Typhimurium infections in mice. S. Typhi carrying mutations in the pre-chorismate pathway (e.g., aroA and aroC) were growth attenuated in humanized mice, recapitulating an observation made in human volunteers infected with a defined aromatic mutant more than 20 years ago. S. Typhi purine auxotrophs were likewise attenuated, similar to their S. Typhimurium counterparts in sensitive mice. The technical limitations with the humanized mouse model are well known— the organisms in this example were
delivered i.p., possibly circumventing the need for SPI-1. The acute time point (24 h) highlights the importance of genes in early interaction with the host, but genes required for longer term adaptation in the host may have been missed (Figure 1). Animal numbers were necessarily small and there is some variation between animals, depending on the source and degree of engraftment of the donor tissue. Only hematopoietic cells were transferred, i.e., epithelial and stromal cells remained murine. While there were histological similarities between the human and mouse infections, mice lacking an immune system from early development (e.g., SCID), even if engrafted at birth, are likely to have lymphoid tissue ultrastructure that is abnormal (Libby et al., 2010). The absence of a phenotype of the cdtB mutant of S. Typhi in acute infection of humanized mice is a disappointment for the field and may limit prosecution of a typhoid toxin-based toxoid as a new vaccine for typhoid fever. The observation made in the humanized mice recapitulates findings from a very recent study conducted in human volunteers where a typhoid toxin-negative variant of S. Typhi appeared fully virulent in the early phases of typhoid fever (Gibani et al., 2019). In this study by Gibani et al., human volunteers were challenged with wild-type S. Typhi or an isogenic toxin deletion mutant and were observed until a definitive diagnosis of typhoid fever was made, or until day 14, when they were treated with antibiotics (Figure 1). Analysis of the two groups of volunteers did not reveal differences in the incidence of blood infection, i.e., typhoid fever, but did see an increase in the duration of bacteremia in the volunteers challenged with the toxin-negative mutant. Increased infection by the toxinnegative strain was mirrored in a growth competition study in mice lacking bloc3. Mice lacking the Rab32 nucleotide exchange factor BLOC3 support the growth of S. Typhi when delivered i.p. (Spano` et al., 2016), likely because a Rab32/ BLOC3 trafficking pathway normally delivers antimicrobial cargo to the S. Typhicontaining vacuole. Upon examining the spleens of bloc3 / mice, the toxin-negative mutant of S. Typhi outcompeted the growth of the S. Typhi parent strain by a factor of 2-fold over 5 days (Spano` et al., 2016).
As Osler carefully documented, the clinical picture of typhoid fever is complex. The blood infection is often missed in LMICs. The intestinal hyperplasia can result in a severe form of disease, sometimes fatal, where it is thought that the immune response in the gut-associated lymphoid tissue drives ulceration of the Peyer’s patches and then perforation (Osler, 1892), leading to egress of the intestinal contents into the peritoneal cavity and potentially fatal peritonitis. Allied with these immunopathological changes, a high and prolonged fever is sometimes associated with neurological signs simulating meningitis. Neither of these sequelae, which happen two or more weeks after initial natural exposure, are examined in the human volunteer challenge, or in the humanized mouse model (Figure 1). It is possible that the typhoid toxin has a role in both of these complications of typhoid fever. CdtB appears to be a target for the human immune system. Studies by Napolitani et al. compared peripheral blood mononuclear cells from bacteremic and non-bacteremic volunteers challenged with S. Typhi, and there was higher frequency of CdtB-specific, Th1-type T cells in those subjects that developed a bacteremia (Napolitani et al., 2018). This observation leaves open the possibility that the typhoid toxin is an immune target resulting in the hyperplasia associated with a severe form of the disease. The quest continues to understand the pathogenesis of this complex host-specific pathogen that has lost many nonessential genes through evolution that have remained intact in host ‘‘generalists’’ like S. Typhimurium. The fact that S. Typhi has independently acquired and now retains typhoid toxin and SPI-2 (though not all the SPI-2 effectors) would suggest that both have a role in human infection. The experimental systems we have to explore their function, while representing important and exciting advances, may not yet be complete.
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