Molecular Significance of lon and cpxR Genes in the Pathogenicity of Salmonella

The important foodborne zoonotic pathogen Salmonella causes gastroenteritis. The dynamics of host-pathogen Salmonella interaction and infection might enhance the development of novel targeted preventative measures and drug regimens. The lon and cpxR are virulence associated genes, which have an important role in the Salmonella pathogenesis. However, the deletions of lon and cpxRlead to the construction of genetically engineered live Salmonella vaccine candidate. In this review, lon and cpxR genes are focused for their involvement in Salmonella pathogenesis. Furthermore, the importance of these genes was briefly emphasized during the construction of Salmonella vaccine candidate.


Introduction
Worldwide, salmonellosis is a major public health concern, which frequently causes gastroenteritis and zoonotic infections [1] [2].In the United States, Salmonella spp.lead approximately 1.2 million human illnesses annually [3].These infections are mainly acquired by exposure of contaminated food or infected animals [3] [4].An initial step in the Salmonella pathogenesis is bacterial penetration of the intestinal epithelium.Penetration requires the expression of invasion genes, which are generally found in Salmonella pathogenicity island 1 (SPI1) [5].SPI1 invasion genes encode a bacterial type III secretion apparatus and several effectors, which are important for interaction with eukaryotic proteins in pathogenesis [6] [7].
The understanding of within-host population dynamics of Salmonella infections is important for allowing delivery of targeted interventions.Among the strategies that have been used to control Salmonella, vaccination represents one of the most suitable strategies [8].An understanding of within-host dynamics of Salmonella enterica interactions with eukaryotic cells could shape the development of vaccines.Comparative analysis of live and killed vaccines revealed that killed vaccines were unable to afford desired protection, while the suitable live vaccines were efficient in protection [9].But, the potential for virulence reversal through horizontal gene transfer remains an important concern for live vaccines [10].In Salmonella, significant involvement of lon and cpxR genes in the pathogenic mechanisms has been reported in the studies [11]- [15].This review focuses on the importance of lon and cpxR genes in the Salmonella pathogenesis.In addition, the possibility of utilizing lon and cpxR genes for the construction of live vaccines was proposed due to their considerable involvement in pathogenic mechanisms [13]- [15].

lon Gene
Lon protease is a cytoplasmic protein in prokaryotes and a mitochondrial matrix protein in eukaryotes [16].Lon is a member of four families of ATP-dependent proteases-including the Clp family (ClpAP and ClpXP), HslVU, and FtsH-which have been well characterized in bacteria [17]- [19].Lon has four identical 87-kDa subunits, each consisting of a highly charged N-terminal domain, a centrally located ATP binding domain, and a proteolytically active C-terminal domain [20] [21].Lon has been known as a powerful negative regulator for the expression of invasion genes encoded on Salmonella pathogenicity island 1 (SPI-1) through degradation of HilC and HilD.In addition, the invasive phenotype of Salmonella is negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis in Escherichia coli.Lon protein negatively regulates the ability of the bacterium to invade epithelial cells.It also affects macrophage survival, and is essential to cause systemic infection by Salmonella [5] [11] [22].Lonis an evolutionarily conserved stress protein induced by multiple stressors.It assists to remove damaged and abnormal proteins during stress, and contributes to the cell division, cell morphology and DNA maintenance [5] [11] [23]- [26].In addition, Lon participates in controlling multiple pathways: post-translational quality control [27], capsule synthesis through degradation of RcsA, which is a transcriptional activator of the biosynthetic genes [28], sporulation [29], cell cycle progression [30], lateral flagellar biosynthesis [31], negative regulation of type III secreted protein [32], ribosomal protein degradation after amino acid starvation [33], antitoxin protein degradation in toxin-antitoxin systems [34], bacterial fimbria and extra-cellular polysaccharide production [15].

cpxR Gene
The cell envelope of Gram-negative bacteria is composed of the inner membrane, the periplasmic space and the outer membrane.It is also exposed by flagella, porins, secretion systems and adhesions [35].Different signal transduction systems permit Salmonellae to perceive alterations in the external environment or damage to their cellular components.After these alterations, physiology of Salmonella undergoes several changes in order to prolong survival.The response to alterations in the cell envelope is regulated by at least three extra cytoplasmic stress response (ESR) pathways in Salmonella spp., including the alternative sigma factor σ E (RpoE) [36] [37], the two-component regulator CpxAR [38], and the two-component regulator BaeSR [39] [40].CpxA/CpxR is two component (a sensor kinase/a response regulator) signal transduction pathway.CpxA (Sensor Kinase) is found in the cytoplasmic membrane, where it senses diverse signals, including alkaline pH, altered membrane lipid composition, interaction with hydrophobic surfaces, and misfolded pilin subunits.Subsequently, CpxAautophosphorylates and donates its phosphoryl group to activate CpxR, (Response Regulator).CpxR composed of an N-terminal receiver domain (REC) with an aspartate (D51) at the site of phosphorylation, and a C-terminal effector domain, which mediates the output response as a transcriptional regulator of target genes [41].Interestingly, the balance between phosphorylated and dephosphorylated CpxR is crucial for the initiation and durability of a specific genetic response to the external stimulus [42] [43].CpxAR also directly and indirectly inhibits the formation of the P pili [44].CpxAR also governs the protein expressions such as DsbA and PpiA, which help in pilin assembly in the periplasm.CpxAR could be associated with negatively regulation of the expression of curli in Salmonella.Activated CpxR regulates part of the envelope stress response system, pilus assembly, type III secretion, motility and chemotaxis, adherence, and biofilm development.So, CpxR can be related to both adhesion and invasion of epithelial cells [45].

Genetically Constructed Vaccine Candidate after lon and cpxR Gene Deletion
After deletion of lon gene, the increased invasiveness can result from the accumulation of HilC and HilD, leading to overexpression of the SPI-1 genes, which are important for infective Salmonella to cross the small intestinal barrier [22].A lon mutant can efficiently invade cultured epithelial cells, and enhanced production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD.The expression of SPI1 proteins is also regulated in response to several environmental conditions.The disruption of the lon gene can affect its replication in the host cell and its capability to cause overwhelming systemic disease [11].The lon mutant can reach extraintestinal sites but unable to proliferate efficiently within the spleen of mice.Thus, Lon protease is essentially involved in the lethal systemic infection with Salmonella in mice.However, the lon mutant can not survive and proliferate within macrophage cells, suggesting that the Lon protease of Salmonella is involved in the withstanding of the killing mechanism of macrophage and in growth intracellularly.The reduced capability of the lon mutant to survive and grow in macrophage could be due to the enhanced susceptibility to the oxidative killing mechanism associated with respiratory burst and the low phagosomal pH.The overexpression of SPI1 genes by Londepletion leads rapid and massive macrophage apoptosis through a mechanism including caspase-1 and -3 [46].In addition, CpxR mutant can develop protection against exposure to alkaline pH 8.0 during growth in broth.However, the nature of this process remains unknown [45].The cpxR mutants were more efficiently internalized in the eukaryotic cells than the wild type strain After deletions of lon and cpxR genes, the mutants showed more fimbria and capsular productions than those of the wild type Salmonella [13]- [15].Thus, the mutant strains constructed with deletions of lon and cpxR showed increase capability for adhesion or invasion, but decreased survival, replication and systemic infection in the host cell, resulting in easy eradication from host cells without causing side effects.In addition, the chances of reversion to the wild-type phenotype are less because of the complete deletion of two virulence-associated genes, lon and cpxR.Since, capsular polysaccharides are major antigenic components, which can induce strong immune responses for protection against pathogens [13].In this way, the lon and cpxR gene deleted Salmonella mutant showed effective vaccine candidate against Salmonella serovars [13]- [15].

Significance of Developed Vaccine Candidate
The high productions of fimbria and capsular polysaccharides by lon and cpxR deleted Salmonella mutants showed elevated immune responses, which can subsequently protect against Salmonella infections [13]- [15].In addition, the developed mutant vaccine candidate is used for delivery of heat-labile enterotoxin B subunit protein (LTB) of E. coli as an adjuvant to enhance immune responses and protection efficacy against Salmonellosis [48]- [50].Development of a reliable vaccine is critical, as salmonellosis has global effects on human health.The lon and cpxR genes deleted veterinary vaccines against Salmonella in poultry and swine industries are an important step in preventing the spread of infection to humans through consumption of contaminated meat and poultry eggs [48] [51] [52].