Salmonella enterica serovar Typhimurium is a Gram-negative, facultative bacillus and a leading cause of human gastroenteritis, which is often associated with nontyphoid symptoms such as diarrhoea and abdominal pain [1,2]. In the United States, there are estimated to be 1.4 million illnesses related to nontyphoid salmonella infections each year . In Europe, Salmonella Typhimurium is one of the two serotypes (the other being Salmonella Enteritidis) accounting for the majority of cases of salmonellosis . Although humans and animals have high-efficient immune systems, Salmonella Typhimurium has evolved overtime to develop a large number of resistant genes or gene clusters through horizontal gene transfer to avoid or act against the immune system. In this review, we aim to clarify the relationships among the ancestral two-component regulatory system PhoP/PhoQ with virulence-associated genes, gene clusters (pathogenicity islands) and other regulatory systems.
The main pathogenetic gene clusters of Salmonella Typhimurium
The pathogenicity of Salmonella Typhimurium is related to many virulence-related gene clusters. More specifically, the ability of Salmonella Typhimurium to invade the host intestinal epithelial cell was mediated by a special gene cluster called Salmonella pathogenicity island 1 (SPI1), which is located at 63 centisome of the entire genome . The SPI1 of Salmonella Typhimurium encoded a type three secretion system (T3SS), which is made out of a large number of proteins and played important roles in delivering translocon and effector proteins into the host cell cytoplasm, resulting in rearrangement of actin in the cytoskeleton, formation of membrane ruffles and finally internalization through macropinocytosis [6,7]. Entering into intestinal epithelial cells, Salmonella Typhimurium could also be captured directly by dendritic cells from the intestinal lumen and transported into the Peyer's patches. After passing through the intestinal epithelial cells and entering into Peyer's patches, Salmonella Typhimurium was ingested by phagocytic cells such as macrophages. In this case, the pathogenicity island 2 (SPI2) is activated. The SPI2 cluster is located at 31 centisome of the whole chromosome. Genetic analysis indicated that SPI2 was a unique virulence cluster within S. enterica species, not belonging to Salmonella bongori or other bacterial species [8–10]. Further studies demonstrated that the SPI2 of Salmonella Typhimurium also harbours a T3SS (SPI2-T3SS). It consisted of 31 genes which served as components of type three secretion apparatus (ssa), effector proteins (sse), chaperones (ssc) and another two-component regulation system SsrA/SsrB; all are involved in intracellular survival, proliferation and systemic infection .
The features of the PhoP/PhoQ two-component regulator system
The two-component operon PhoP/PhoQ is a major virulence regulatory system in Salmonella Typhimurium. It is composed of a response regulator (Phop) and an environmental sensor kinase (PhoQ) [12,13]. The Phop protein is a member of the OmpR family and consists of a conserved N-terminal domain that comprises the essential aspartate residue, which constitutes the phosphorylation and DNA binding sites at the C-terminus . The PhoQ protein is found to have two transmembrane regions, the one at the N-terminus spanning the inner membrane with a periplasmic sensor domain and a cytoplasmic domain. The cytoplasmic domain harbours a HAMP linker next to the membrane, a conserved autophosphorylation site, and a C-terminal ATP binding domain. When PhoQ senses the environmental signals such as low Mg2+ and pH, it autophosphorylates and becomes activated. Subsequently, phosphate is transferred to the aspartate residue of Phop protein. The phosphorylated Phop protein could activate or repress the expression of the corresponding genes [15,16].
The functions of PhoP/PhoQ regulated genes
As a response regulator, Phop protein plays two roles. On one hand, when Phop protein is triggered by PhoQ protein, it could promote the transcription levels of itself and subsequently activate the target genes designated Phop-activated genes (pags). On the other hand, the phosphorylated Phop protein could also reduce or depress the expression levels of the Phop-repressed genes (prgs) . To date, there are more than 120 different genes that are found under the control of PhoP/PhoQ system . The functions of pags are various; some such as pagC are active in intramacrophage survival  and some such as MgtA and MgtCB mediate bacterial magnesium ion transport [19,20]. Resistance to cationic antimicrobial peptides was also related to pags, especially the pagP protein, which causes modification of lipid A, a major cell surface molecule of Gram-negative bacilli, and increases resistance to polymyxin . The roles of prgs include stimulating macropinocytosis and entering into nonphagocytic eucaryotic cells by a ruffling mechanism , signalling epithelial cells to endocytose Salmonella Typhimurium and formation of the supramolecular SPI1-T3SS needle complex such as the prgHIJK operon [23–25].
The relationship between the PhoP/PhoQ regulator system and pathogenicity island 1
As the ability to invade intestinal epithelial cells of Salmonella Typhimurium is mediated by SPI1-T3SS, the transcription of itself was triggered by many factors in vitro, such as osmolarity, growth phase, and pH. At the molecular level, the expression of SPI1 genes is controlled by a complicated regulatory network; at its core is the HilA protein, which is a transcriptional activator with a DNA binding domain belonging to the OmpR/ToxR family [26,27]. The expression of HilA is directly controlled by three AraC-like activators: HilC, HilD, and RtsA. HilC and HilD are encoded by SPI1, whereas RtsA is encoded in an operon with RtsB located at 93.9 centisomes. All three genes have been proven to directly combine to the upstream region of HilA and deletion of each of them results in a lower level of HilA expression . In addition to this, HilA was indirectly controlled by HilE and other regulators. Two-hybrid analysis indicates that HilE can interact with HilD and control the expression of HilD. The PhoP/PhoQ system was considered to control the expression of HilE and finally regulated the expression of SPI1 genes through a HilA-dependent cascade effect .
The regulatory network among PhoP/PhoQ locus, pathogenicity island 2 expressed genes and SsrA/SsrB two-component regulator
As mentioned above, the SPI2 encoded genes are required for intracellular survival and proliferation. Previous studies had indicated that many PhoP/PhoQ null mutants were neither virulent nor capable of surviving within macrophages [29–31]. Recently, Yoon et al. used a quantitative real-time PCR (qRT-PCR) method to monitor the transcription level of SPI2 encoded genes and they found 100-fold decreases in transcription of all SPI2 genes in a PhoP/PhoQ mutant background. This evidence strongly implies that there may be some intrinsic relationship between the PhoP/PhoQ regulatory system and SPI2 expressed genes. Deiwick et al. believed that the regulatory network between PhoP/PhoQ locus and SPI2 encoded genes was mediated by SsrA/SsrB two-component regulatory system. The SsrA/SsrB locus is encoded within the SPI2 gene cluster, and like the PhoP/PhoQ operon, the SsrA/SsrB two-component regulatory system also consists of a putative cognate sensor SsrA (also referred to as spiR) and a response regulator, SsrB. However, the arrangement of SsrA/SsrB is different from PhoP/PhoQ; the SsrA/SsrB is not an integral operon. The response regulator SsrB is located downstream of the sensor SsrA and separated by a 30 bp intergenic region . When SsrB regulator is triggered by SsrA, it subsequently elicits transcription of genes encoding the components of the SPI2-TTSS, as well as genes encoding SPI2 effectors . Within macrophages, the SsrA/SsrB locus can be activated by the PhoP/PhoQ operon. More specifically, expression of SsrA could not be induced in PhoP mutants. In addition, upstream sequence analysis of the SsrB start codon identified a putative Phop binding site. Additional in-vivo chromatin immunoprecipitation experiments demonstrated that the Phop protein could bind to the SsrB promoter when Salmonella bacilli are inside macrophages . All of these findings support the concept that SsrA/SsrB system is under the control of PhoP/PhoQ two-component regulatory system. However, participation of PhoP/PhoQ in SPI2 expression has also been disputed – using a plasmid-based fusion of the SsaG and SsrA promoters to a promoterless green fluorescent protein gene, fluorescence was found inside macrophages using a Phop mutant strain . Later investigation also showed that the defect of the PhoP/PhoQ two-component regulatory system did not reduce the maximum expression level of the genes in the SsrA/SsrB system, but only delayed expression .
The relativity of the PhoP/PhoQ regulation system with the PmrA/PmrB two-component regulation system
In S. enterica serovar Typhimurium, the PhoP/PhoQ system not only acts as a direct regulator, but also controls other two component regulatory systems. PmrA/PmrB is one such system identified in 1993 from a spontaneous mutation (PmrA) associated with increased polymyxin B (PMB) and cationic antibacterial proteins resistance . So far, more than 20 genes have been shown to be under the control of PmrA/PmrB locus. Gunn  estimated that a total of over 100 genes were regulated by the PmrA/PmrB system. The PmrA-PmrB regulator can be triggered by the Phop-PhoQ system via the PmrD pathway. PmrD is one of the pags which regulates the activity of PmrA by stabilizing the phosphorylation state of PmrA at a posttranscriptional level .
The two-component regulatory system PhoP/PhoQ controls the transcription of a large number of virulence-associated genes via direct or indirect pathways. However, there are still many unsolved problems such as the possible relationship between PhoP/PhoQ and another ancestral two-component regulatory system, OmpR/EnvZ. The detailed roles of the PhoP/PhoQ locus in forming the type three secretion apparatus and the functions of many pags and prgs are still unclear. However, as more attention is directed towards the PhoP/PhoQ system of Salmonella Typhimurium, more and more answers to such questions will be revealed.
Conflicts of interest
There are no conflicts of interest.
This research was supported by the Changjiang Scholars and Innovative Research Team in University (PCSIRT0848), China Agricultural Research System (CARS-43–8) and National Science and Technology Support Program for Agriculture (2011BAD34B03).
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