Typhi genes expressed during infection or involved in pathogenesis

Salmonella enterica serovar Typhi (Typhi), the aetiologic agent of typhoid fever, is a human restricted pathogen. Elucidation of the interactions between the infected host and this pathogen is critical to understand infectious diseases but is deterred by a lack of in vivo infection assays, since Typhi uniquely infects humans and there is no suitable animal model. Macrophages can be used as an alternative model, as the ability to survive and replicate within these cells is thought to be one of the major pathogenesis determinants for Salmonella. Typhi genes that are expressed within human macrophages have been identified, as well as Typhi immunogenic proteins expressed in humans with typhoid. Known virulence factors of Salmonella are expressed during infection of macrophages, such as SPI-2 encoded genes, supporting the validity of the model; however, many genes of unknown functions are also expressed. The importance of these genes should be investigated during future studies aimed at elucidating the intracellular lifestyle of this human-specific pathogen. This review describes Typhi genes expressed during infection or involved in cell interaction.


Introduction
Understanding Typhi pathogenesis is deterred by a lack of in vivo infection assays since Typhi only infects humans; thus there are no suitable animal models available.Because Typhi is restricted to humans, Salmonella enterica serovar Typhimurium (Typhimurium), a serovar with a high degree of genome homology (>90%) [1,2], has been used for many years to study typhoid fever pathogenesis using a murine infection model in which Typhimurium causes a systemic infection.This model has been crucial in understanding systemic infections by Salmonella.However, it has also been demonstrated that the mouse model does not always reflect the human disease.For example, the ability to disseminate from the bowel and establish extraintestinal niches is promoted by the spv locus located on the virulence plasmid in Typhimurium.The absence of the spv genes from Typhi is a strong indication that the pathogenesis of typhoid fever is fundamentally different [3].Moreover, we need to confirm that what we have learned using the murine model with Typhimurium is applicable to Typhi.Many Salmonella virulence factors have already been identified and studied, but there are inevitably many more to discover.
Macrophages represent an important host defense mechanism in humans infected with Salmonella and the ability to survive and replicate within these cells is thought to be one of the major pathogenesis determinants for Salmonella [4,5].Different molecular approaches have been developed and used to identify Salmonella genes expressed during infection.One way to understand molecular mechanisms of pathogenesis is to study the transcriptional response of the bacteria during infection, as virulence factors may be specifically expressed during the course of typhoid infection.Global expression profiling using microarrays can help to define the mechanisms required by the bacteria to cause disease and the host responses required to defeat bacterial infection.However, technical issues currently impede transcriptional profiling of bacterial genes during host infection because of the relatively small number of bacteria present in an infected host, the short half-life of RNA, and the scarcity of polyadenylation on transcripts.Two transcriptomic studies of Typhi have been performed: one that focused on in vitro conditions that could be encountered in vivo, such as peroxide induced stress [6], and one during infection of human macrophages [7].Proteomic profiling is another approach that has been used to obtain information regarding bacterial metabolism or mechanisms used to survive within various cell types by identifying proteins produced within host cells [8,9].Proteomics methods have been based on the use of different mass spectrometry-based methods to identify bacterial proteins produced within host cells.A proteomics analysis of Typhi grown in low pH, low magnesium minimal media (MgM or LPM) was recently reported [10].MgM is designed to approximate the phagosome of infected macrophages and is known to induce expression of SPI-2 virulence genes and other genes related to virulence and intramacrophage survival [11].Another strategy that has recently been developed identifies bacterial antigens that are immunogenic and produced during infection, a technique called in vivo induced antigen technology (IVIAT) [12].This technique was successfully used to identify Typhi proteins produced in typhoid patients [13].
In this review, in vivo gene expression of Typhi is described, focusing mainly on Typhi genes expressed within macrophages.

Salmonella pathogenicity islands (SPI)
SPIs are insertions of large regions of DNA containing virulence genes, located on the bacterial chromosome.These gene blocks are often inserted near a tRNA gene and generally display distinct codon usage and a different overall base composition from the core bacterial chromosome, suggesting that they were acquired from a foreign source.Thus far, 15 SPIs have been identified in Typhi [1,14].SPI-1 and SPI-2, which are present in all S. enterica serovars, represent 2 major pathogenesis determinants that encode type III secretion systems (T3SS).SPI-1 and SPI-2 T3SS have distinct roles in Salmonella pathogenesis.SPI-1 effectors are injected into host cells via the T3SS and are required for invasion of epithelial cells [15], whereas SPI-2 contributes to Salmonella survival inside macrophages [16,17].Although these systems are important and even crucial for Typhimurium, very little information is available concerning Typhi.SPI-1.Many Typhi genes involved in invasion identified so far are homologous genes present in Typhimurium, including SPI-1 genes (invC, invA, invE, invG, prgH, iagAB, sipEBCDA) [18][19][20][21][22][23] (Table 1).There should be distinct invasion genes that remain unidentified as Typhi adheres, invades, and migrates through human epithelial cells better than Typhimurium [20,24].Moreover, optimal adherence occurs at high osmolarity, when the Vi capsule is produced at its lowest levels [20,25].Some SPI-1 genes are upregulated following uptake by macrophages, but most genes are not differentially expressed when compared to bacteria present in the supernatant of macrophages [7].This finding may be different if another growth condition is used as the comparator.Thus, as for Typhimurium, a key role for SPI-1 T3SS in invasion is predicted for Typhi.SPI-2.The SPI-2 locus of S. enterica is 40 kb in size and is divided into two functional entities.A portion of 25 kb, important for virulence, contains the T3SS and several translocated effectors.In contrast, the 15-kb portion encodes the tetrathionate reduction (ttr) system and proteins of unknown function and was not required for virulence [26].Although a crucial role in virulence for SPI-2 in Typhimurium has been demonstrated and its importance has been well established, the data concerning Typhi SPI-2 genes are almost nonexistent.A Typhi strain bearing a double mutation in ssaV (a SPI-2 gene) and in aroC (aromatic biosynthesis) was previously shown to survive less efficiently in human macrophages compared to the wild-type parent strain [27].In human volunteers, this doubly mutated strain was attenuated [28] (Table 1).Within human macrophages, many of the Typhi SPI-2 genes that belong to the 25 kb locus (18 out of 31) are up-regulated following bacterial uptake, and the majority of SPI-2 encoded genes are up-regulated 2 h post-infection (29 out of 31).Most of the SPI-2 encoding genes are also up-regulated until 24 hours post-infection (26 out of 31) [7].The Typhi SPI-2 genes located on the 15 kb locus are not differentially expressed.Moreover, Typhi may not use the ttr system as ttrS is a pseudogene in CT18 [1].SPI-7.SPI-7 is a large 134 kb segment which is absent from Typhimurium and encodes the Vi antigen as well as the Type IV pili (see adhesins) and the SopE prophage.The virulence of Typhi is associated with the presence of the Vi antigen, which is needed for Typhi to survive inside phagocytes and necessary for serum resistance, a characteristic required for systemic dissemination [29][30][31].The Vi polysaccharidic capsule is encoded by the viaB locus, which is composed of 11 genes and contains two regions: one involved in biosynthesis, including tviA-E, and the other required for translocation of the polysaccharide to the cell surface, vexA-E [32].The cDNA corresponding to the tviB gene has been captured and cloned from human macrophages 2 hours post infection but is not detected 24 hours post-infection [33].A transcriptomic study of Typhi genes in macrophages did not demonstrate up-regulation of the viaB locus; however, the tviABCD region was not present on the microarray and expression of these genes could not be assessed [7].However, a Typhi strain harboring a deletion in the viaB locus was shown to be highly invasive, which can be explained by the production of the SPI-1 T3SS [23].Similarly, by using a tviB mutant, the presence of the Vi capsule has been shown to inhibit adhesion as well as invasion of epithelial cells [34] (Table 1).
Other SPIs.The role of genes that belong to other pathogenicity islands in Typhi pathogenesis has not been investigated in depth yet.The magnesium transport system mgtBC located on SPI-3 is strongly induced intracellularly by Typhi during infection of macrophages, as well as pipD located on SPI-5, and pagC, pagD, and envE of SPI-11 [7].PagC is produced in typhoid patients [13].On SPI-10, the prpZ locus encoding for proteins with homology to eukaryotic-type Ser/Thr protein phosphatase and kinases has been found to promote survival in macrophages [35] (Table 1).

Adhesins
The genome of serovar Typhi contains 13 putative operons corresponding to fimbrial gene sequences termed bcf, csg (agf), fim, saf, sef, sta, stb, stc, std, ste, stg, sth and tcf, and pil, the type IV pili [1].Five of these operons, sef, sta, ste, stg, and tcf, and the Type IV pili are not detected in serovar Typhimurium [1] and may be important for Typhi host specificity.In typhoid fever patients, antibody to 3 fimbrial systems, TcfB, StbD, and CsgEFG, has been detected [13].The csg operon is also up-regulated 2 hours post infection inside macrophages [7].A Typhi strain carrying a deletion of the fim genes, encoding for type 1 fimbriae, is more invasive than the wild-type strain [36].In absence of Type 1 fimbriae, different bacterial surface proteins may become available to interact with host cells, causing a higher level of invasion.The pil genes encoding for type IV pili facilitate Typhi entry into human intestinal epithelial cells and macrophages [37,38].The deletion of stg reduced adherence of Typhi to epithelial cells but increased uptake by human macrophages, although survival inside macrophages was similar to the wild-type parent [39] (Table 1).
Lysosyme is another component produced by the host to eliminate the bacterial invader by attacking the bacterial cell wall, but some bacteria produce lysosyme inhibitors to evade antibacterial enzymes.A novel family of lysozyme inhibitors was recently discovered, mliC (ydhA) (membrane bound lysosyme inhibitor of ctype lysozyme) [50] and seem to promote macrophage survival of Typhi [33].The phage-shock-protein (Psp) system responds to stresses and may be involved in antimicrobial resistance [51].PspC, PspD and PspE were identified by IVIAT during human infection with Typhi [13] and are also up-regulated in human macrophages [7] (Table 1).

Metal transport
It has been shown that a Typhi mutant defective for enterochelin synthesis and transport has a lower ability to enter and proliferate in epithelial cells and macrophages [52][53][54].Some proteins involved in heavy metal transport, MerP and STY0909, were identified by IVIAT during human infection with Typhi [13].However, during infection of human macrophages, Typhi genes involved in iron acquisition and transport (such as fes, fhu, feo, ent, iro) are down-regulated intracellularly [7].It is possible that these genes are already up-regulated in the supernatant of infected cells or the conditions that were used as a comparator.Alternatively, it may be a bias of the cultured THP-1 model as intracellular pathogens such as Mycobacterium tuberculosis and Typhi do not seem to face iron limiting conditions in these cells; their transcriptional profiles did not correspond with a predicted low-iron environment [7,55].

Other genes
The sigma factor RpoS is a global stationary phase regulator, controlling expression of many virulence associated systems, including Vi synthesis [56], and is required for virulence of Salmonella [57].A mutation in rpoS renders Typhi less cytotoxic to THP-1 macrophages and although the mutant survives similarly to the wild-type strain [58], the mutant was less invasive on epithelial cells [59] (Table 1).Transcription of rpoS was shown to increase in macrophages [7,58].Moreover, Ty2 and the live oral vaccine Ty21a strains are rpoS mutants [60].
Typhi flagellar mutants (flhDC or fliA) are deficient in cell invasion, and result in a reduction of SPI-1 gene expression, which is more pronounced in Typhi than Typhimurium [18].Macrophage cytotoxicity is also reduced in flagellar mutants [18].The motility defect cannot be restored by centrifugation as observed with Typhimurium [61].

Conclusion
Host-pathogen interactions are very complex and considerable effort is required for their elucidation.Studying interactions between the infected host and Typhi should improve our understanding of typhoid fever.Typhi has developed remarkable persistence mechanisms within the host that help ensure its survival and transmission.However, data on human typhoid collected by using modern immunological and molecular techniques are scarce since Typhi uniquely infects humans, and there are no suitable animal models available.As survival within macrophages is an essential step for Salmonella pathogenesis, macrophages represent a useful model to study Typhi.Typhi gene expression during infection was monitored in human macrophages.In effect, the transcriptome of Typhi from infected macrophages at 2 hours, 8 hours, and 24 hours post-infection has been obtained [7].Interestingly, 117 genes are up-regulated at all intracellular time points.Of these, 32 genes belong to SPIs or have been previously associated with pathogenicity, including 17 genes from SPI-2, involved in intracellular survival.Twenty genes that belong to the membrane lipoproteins and porins class were identified, suggesting important adaptations and modifications at the outer membrane level.Many genes (30) encoding for hypothetical or unknown proteins were identified and await further investigation to determine their possible roles as novel virulence factors.Similarly, 19 of the constitutively up-regulated genes in macrophages are absent in Typhimurium, suggesting such Typhi-specific genes may be involved in survival in macrophages.Because of a lack of accumulated data, it may be too premature to compare gene expression between Typhi and Typhimurium.Moreover, the model used is often different.However, Typhi is less adherent, invasive and cytotoxic than Typhimurium [4,34] suggesting that many differences are left to be discovered.
In order to circumvent the limitations associated with in vitro models, proteins that are immunogenic and expressed uniquely in humans with typhoid were identified using IVIAT [13].Of the 30 identified antigens encoded on the chromosome, 16 were also upregulated at least at one time point during infection of macrophages.It should be stressed that no single approach will provide all of the information necessary for the desired level of understanding of the infectious processes.Each approach, sometimes in combination with one or another approach, will provide superior results in some cases but not in others.Thus, it will be the collective efforts of many investigators, using the diversity of established as well as novel approaches, that we will achieve our ultimate goal to fully understand the mechanisms of Salmonella persistence, transmission, infectivity and pathogenesis.Elucidating the bacterial genes expressed in the host and those underlying typhoid pathogenesis should lead to the development of new strategies including novel antibacterial treatments and identification of novel vaccine candidates to control the disease.

Table 1 .
Typhi genes involved in pathogenesis.